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Title: Axon regeneration genes identified by RNAi screening in C . elegans .
Authors: Nix P ; Hammarlund M ; Hauth L ; Lachnit M ; Jorgensen EM ; Bastiani M
Journal: J Neurosci
Year: 2014-01-08
Doc ID: WBPaper00044677
Bibliographic Information
Abstract
Matching Sentences
SECTION: abstract. To identify genes affecting axon regeneration in Caenorhabditis elegans , we performed both an RNAi-based screen for defective motor axon regeneration in unc-70 / -spectrin mutants and a candidate gene screen . [Field: abstract, subscore: 4.00]
SECTION: discussion. Proteins " positively " required for axon regeneration are shown in green ; those that " inhibit " axon regeneration are shown in red . [Field: discussion, subscore: 4.00]
SECTION: introduction. To identify genes affecting axon regeneration in Caenorhabditis elegans , we performed both an RNAi-based screen for defective motor axon regeneration in unc-70 / -spectrin mutants and a candidate gene screen . [Field: introduction, subscore: 4.00]
SECTION: results. , January 8 , 2014 34 ( 2 ) : 629 645 Nix et al . Genetics of Axon Regeneration KGB-1 MAPK pathway targets FOS-1 during axon regeneration The downstream target of PMK-3 signaling in regeneration is the MAPK-activated kinase MAK-2 , which influences stability of cebp-1 , the C . elegans CCAAT / enhancer-binding protein ( Yan et al . , 2009 ) . [Field: results, subscore: 4.00]
SECTION: results. Regeneration phenotype of MAPK pathway mutants Mammalian Family Gene Allele % Regeneration Wild-type oxIs12 70 MAP4K MST1 cst-1 / 2 basDf1 87b PAK max-2 nv162 37c PAK pak-1 ok448 75 MAP3K TAO kin-18 ok395 86b DLK dlk-1 ju476 0c MLK mlk-1 ok2471 23c MTK mtk-1 ok1382 75 ASK nsy-1 ok593 71 ZAK zak-1 km27 62 TAK Y105C5A0 . 24 km39 73 MLK C24A1 . 3 tm364 68 MLK Y53F4B0 . 1 km43 81 MAP2K MKK4 sek-6 ok1386 87b MKK4 mkk-4 ju91 0c MKK7 mek-1 ks54 27c MKK3 / 6 sek-1 km4 55 MKK4 sek-3 ok1276 70 STE7 sek-4 km42 66 STE7 sek-5 tm4028 69 MKK7 jkk-1 km2 59 MAPK JNK jnk-1 gk7 96b JNK kgb-2 gk361 86b p38 pmk-3 ok169 0c JNK kgb-1 um3 3c p38 pmk-1 km25 67 p38 pmk-2 gk21 ND JNK C49C3 . 10 tm3933 76 Targets FOS fos-1a ar105 90b FOS fos-1 km30 26c JUN jun-1 gk557 68 Regulators MKP7 vhp-1 sa366 90b MKP3 lip-1 zh15 90b DUSP ZK757 . 2 ok3597 98b DUSP F28C6 . 8 ok2013 91b JSAP unc-16 e109 93b DUSP F26A3 . 4 bas2 76 DUSP F13D11 . 3 tm5591 70 DUSP C04F12 . 8 tm5298 75 DUSP C16A3 . 2 tm5280 69 DUSP C24F3 . 2 tm5115 64 JIP jip-1 km18 73 SHC shc-1 ok198 58 aNumber of axotomized axons . bSignificantly improved regeneration . cSignificantly reduced regeneration . [Field: results, subscore: 4.00]
SECTION: results. D , Schematic drawing showing spontaneous growth cone formation in unc-70 and possible consequences to outgrowth : ( a ) the formerly intact axon breaks , ( b ) the new growth cone extends to a nearby , intact axon causing it to break , and ( c ) the migrating growth cone itself breaks and generates a new growth cone . [Field: results, subscore: 4.00]
SECTION: results. Candidate regeneration genes tested by axotomy Gene Allele Molecular function / ortholog Wild-type oxIs12 No phenotype arr-1 ok401 -Arrestin atf-6 ok551 ATF6 transcription factor ccpp-1 ok1821 Cytosolic carboxypeptidase family cdk-8 tm1238 CDK8 cyclin-dependent kinase ced-7 n1996 ABC transporter ced-10 n3246 RAC1 GTPase cpx-1 ok1552 Complexin daf-18 nr2037 PTEN tumor suppresor ; lipid phosphatase dhc-1 js319 Dynein heavy chain E02D9 . 1 tm4000 Predicted MAP2K ehs-1 ok146 Eps15 evl-9 ar121 Unknown evl-16 ar93 Unknown evl-17 ar94 Unknown F32B5 . 1 tm3359 Arginine kinase gska-3 ok970 Glycogen synthase kinase alpha subunit hrp-1 ok963 hnRNP A1 hmr-1b zu389 Neuronal cadherin kin-2 ce179 cAMP-dependent protein kinase subunit lin-7 e1413 LIN7 PDZ domain protein mir-2 gk259 MicroRNA mlp-1 ok832 Muscle LIM protein mtch-1 ok1800 Mitochondrial carrier homolog mtm-5 ok469 Myotubularin family nex-2 bas4 Annexin family nex-3 gk385 Annexin family nex-4 gk102 Annexin family pct-1 wy575 Pctaire class cell cycle kinase pek-1 ok275 PERK kinase ppfr-1 tm2180 Protein phosphatase four regulatory subunit ptl-1 ok621 Tau-like ; microtubule binding protein sir-2 . 1 ok434 Yeast SIR2-like snb-1 js124 Synaptobrevin snt-1 md290 Synaptotagmin snt-2 tm1711 Synaptotagmin snt-3 tm2426 Synaptotagmin snt-4 ok503 Synaptotagmin snt-6 tm3686 Synaptotagmin sta-1 ok587 STAT transcription factor family tom-1 ok285 Tomosyn unc-59 e261 Septin wwp-1 gk372 NEDD4-like E3 ubiquitin ligase Improved growth cone formation atfs-1 gk3094 ATF transcription factor cdka-1 tm648 CDK5 cyclin dependent kinase daf-2 e1370 Insulin / IGF Receptor hst-3 . 1 tm734 Heparan Sulphotransferase kal-1 gb503 KAL1 ; mutated in Kallmann syndrome lin-10 sy217 Mint ; PDZ domain protein mpk-1 ga119 MAP kinase ric-4 ok173 SNAP25 snt-5 ok3287 Synaptotagmin unc-26 s1710 Synaptojanin unc-57 ok310 Endophilin unc-104 e1265 Synaptic vesicle kinesin unc-116 e2310 Kinesin heavy chain Reduced growth cone formation aak-2 gt33 AMP-activated kinase daf-16 m26 FOXO transcription factor mtm-6 ok330 Myotubularin family mtm-9 ar479 Myotubularin family nex-1 gk148 Annexin family psr-1 ok714 Phosphatidylserine receptor stau-1 tm2266 Staufen ; dsRNA-binding protein unc-33 e204 CRMP ; collapsin response mediator protein unc-129 ev557 TGF family secreted growth factor aNumber of axotomized axons . [Field: results, subscore: 4.00]
SECTION: results. Growth cone extension in unc-70 can lead to several possible outcomes : ( 1 ) the intact axon sprouting a growth cone may itself fully break ; ( 2 ) the growth cone may extend along a nearby , intact commissure , causing it to break ; or ( 3 ) the newly regenerated axon behind the growth cone may break ( Fig . 2 D , E ) . [Field: results, subscore: 4.00]
SECTION: abstract. Through our analysis of mutants , we shed new light on certain aspects of regeneration , including the role of -spectrin and membrane dynamics , the antagonistic activity of MAP kinase signaling pathways , and the role of stress in promoting axon regeneration . [Field: abstract, subscore: 3.00]
SECTION: discussion. Conversely , jnk-1 overexpression may inhibit regeneration by preventing the release of organelles from the cell soma and axon initial segment , which would limit the amount of accessible membrane necessary for growth cone formation and regeneration . [Field: discussion, subscore: 3.00]
SECTION: introduction. Key words : axon regeneration ; MAP kinases ; C . elegans ; DLK ; laser axotomy ; neural regeneration Introduction [Field: introduction, subscore: 3.00]
SECTION: introduction. Through our analysis of mutants , we shed new light on certain aspects of regeneration , including the role of -spectrin and membrane dynamics , the antagonistic activity of MAP kinase signaling pathways , and the role of stress in promoting axon regeneration . [Field: introduction, subscore: 3.00]
SECTION: references. CrossRef Medline Bradke F , Fawcett JW , Spira ME ( 2012 ) Assembly of a new growth cone after axotomy : the precursor to axon regeneration . [Field: references, subscore: 3.00]
SECTION: results. Axon regeneration in ire-1 ; xbp-1 double mutants was normal , indicating that reduced growth cone formation in the xbp-1 mutant was a result of ire-1 misexpression ( Fig . 6 B ) . [Field: results, subscore: 3.00]
SECTION: results. The mutant rescued fos-1 ( km30 ) sterility , but failed to rescue regeneration , suggesting that activated KGB-1 phosphorylates FOS-1 to promote axon regeneration ( Fig . 5 D ) . [Field: results, subscore: 3.00]
SECTION: results. The mtm-1 growth cones are often large and unbranched ( left growth cone ) , but many fail ( right growth cone ) . [Field: results, subscore: 3.00]
SECTION: results. Membrane dynamics during axon regeneration As a result of our time-lapse analysis of unc-70 mutants , we identified a new phenotype associated with axon repair and regeneration . [Field: results, subscore: 3.00]
SECTION: results. 33 Uncharacterized conserved protein C35A5 . 8 aEach value represents the average number of commissures from an individual RNAi experiment . bRNAi denotes that the mutant allele was either lethal or unavailable and axotomy was performed in animals fed the RNAi clone . cNumber of commissures with growth cones / total number of commissures scored . dSignificantly improved regeneration . eSignificantly reduced regeneration . fThe mutation resulted in severe neuronal outgrowth and guidance defects , precluding axotomy . [Field: results, subscore: 3.00]
SECTION: results. UNC-70 / -spectrin is required to stabilize the axon during regeneration In designing and executing the screen , we expected to find genes required to either promote or inhibit regeneration , but instead , we only identified RNAi clones that inhibited regeneration in unc-70 mutants . [Field: results, subscore: 3.00]
SECTION: results. Mutations blocking regeneration reduced the likelihood of growth cone formation , but in most cases did not eliminate all growth cones ( Fig . 1 G ) . [Field: results, subscore: 3.00]
SECTION: results. , January 8 , 2014 34 ( 2 ) : 629 645 Nix et al . Genetics of Axon Regeneration showed improved regeneration in the mutant background and 15 of 31 mutants blocked regeneration ( Fig . 1E , F ) . [Field: results, subscore: 3.00]
SECTION: abstract. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention . [Field: abstract, subscore: 2.00]
SECTION: discussion. An important approach will be to determine whether elimination of particular pathways in C . elegans can also enhance regeneration in older animals or induce regeneration in neurons that do not normally regenerate . [Field: discussion, subscore: 2.00]
SECTION: discussion. We found that axon regeneration improved when animals were exposed to stress-inducing conditions such as heat , starvation , and possibly hypoxia . [Field: discussion, subscore: 2.00]
SECTION: discussion. Based on this model , jnk-1 and unc-16 mutants may show improved regeneration due the immediate availability of membranous compartments for axonal growth and repair after injury . [Field: discussion, subscore: 2.00]
SECTION: discussion. Loss of jnk-1 resulted in animals with improved regeneration , whereas jnk-1 overexpression completely inhibited regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. Through our analysis of these genes , we gained an understanding of the essential nature of two MAP kinase signaling modules in promoting axon regeneration ( Hammarlund et al . , 2009 ; Nix et al . , 2011 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. We also found that regeneration was inhibited in klf-1-overexpressing strains , indicating that KLF-1 function in regeneration is conserved . [Field: discussion, subscore: 2.00]
SECTION: discussion. As an independent validation , mammalian KLF family members were shown to be negative regulators of axon regeneration ( Moore et al . , 2009 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. In our screen , we found that the transcription factors FOS-1 , HSF-1 , and HIF-1 are required for GABA neuron regeneration , whereas UNC-130 , CEH-13 , and KLF-1 are inhibitors of regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. Indeed , bcc-1 and F16A11 . 2 mutants show enhanced regeneration , whereas overexpression of either gene or zbp-1 strongly inhibits regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. One model consistent with our results involves putative RNA-binding proteins ( bcc-1 , F16A11 . 2 , tdp-1 , zbp-1 ) binding to and / or sequestering mRNAs until they are necessary to promote axon regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. * p 0 . 05 ; * * p 0 . 01 ; * * * p 0 . 001 , NS , Fisher ' s exact test . Error bars indicate 95 % confidence interval . C , Example of candidates affecting axon regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. Stress response pathways are necessary for efficient axon regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. C . elegans Notch signaling was recently shown to function as an inhibitor of axon regeneration ( El Bejjani and Hammarlund , 2012 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. Through a combination of local protein synthesis , protein trafficking , and transcriptional regulation , the cell transitions to a developmental growth phase . [Field: discussion, subscore: 2.00]
SECTION: discussion. Successful growth cone formation and axonal regeneration requires a multitude of coordinated events within the cell ( Raiv- ich and Makwana , 2007 ; Abe and Cavalli , 2008 ; Bloom and Mor- gan , 2011 ; Bradke et al . , 2012 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. Discussion In this study , we completed an unbiased genetic screen for genes affecting axon regeneration . [Field: discussion, subscore: 2.00]
SECTION: introduction. Caenorhabditis elegans has emerged as an attractive model with which to study axon regeneration ( Wu et al . , 2007 ; Gabel et al . , 2008 ; Chen and Chisholm , 2011 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Many individual molecules have been implicated in the intrinsic mechanisms of axon regeneration ( Raivich and Makwana , 2007 ; Sun and He , 2010 ; Liu et al . , 2011 ; Bradke et al . , 2012 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention . [Field: introduction, subscore: 2.00]
SECTION: introduction. Development / Plasticity / Repair Axon Regeneration Genes Identified by RNAi Screening in C . elegans Paola Nix , 1 Marc Hammarlund , 2 Linda Hauth , 1 Martina Lachnit , 3 Erik M . Jorgensen , 1 , 4 and Michael Bastiani1 1Department of Biology , University of Utah , Salt Lake City , Utah 84112 ; 2Department of Genetics , Program in Cellular Neuroscience , Neurodegeneration and Repair , Yale University School of Medicine , New Haven , Connecticut 06510 ; 3Dresden University of Technology , 01307 Dresden , Germany ; and 4Howard Hughes Medical Institute , Chevy Chase , Maryland 20815 Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration . [Field: introduction, subscore: 2.00]
SECTION: materials. Regeneration was quantified by scoring the percentage of severed axons that formed a new growth cone and / or grew a distance of 5 m or more . [Field: materials, subscore: 2.00]
SECTION: references. CrossRef Medline Yiu G , He Z ( 2006 ) Glial inhibition of CNS axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Yan D , Wu Z , Chisholm AD , Jin Y ( 2009 ) The DLK-1 kinase promotes mRNA stability and local translation in C . elegans synapses and axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Williams W , Nix P , Bastiani M ( 2011 ) Constructing a low-budget laser axotomy system to study axon regeneration in C . elegans . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Tedeschi A ( 2011 ) Tuning the orchestra : transcriptional pathways controlling axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. Curr Opin Neurobiol Sun F , Park KK , Belin S , Wang D , Lu T , Chen G , Zhang K , Yeung C , Feng G , Yankner BA , He Z ( 2011 ) Sustained axon regeneration induced by codeletion of PTEN and SOCS3 . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Sun F , He Z ( 2010 ) Neuronal intrinsic barriers for axon regeneration in the adult CNS . [Field: references, subscore: 2.00]
SECTION: references. , January 8 , 2014 34 ( 2 ) : 629 645 Nix et al . Genetics of Axon Regeneration Shin JE , Cho Y , Beirowski B , Milbrandt J , Cavalli V , DiAntonio A ( 2012 ) Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Park KK , Liu K , Hu Y , Kanter JL , He Z ( 2010 ) PTEN / mTOR and axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Nix P , Hisamoto N , Matsumoto K , Bastiani M ( 2011 ) Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Moore DL , Blackmore MG , Hu Y , Kaestner KH , Bixby JL , Lemmon VP , Goldberg JL ( 2009 ) KLF family members regulate intrinsic axon regeneration ability . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Liu K , Tedeschi A , Park KK , He Z ( 2011 ) Neuronal intrinsic mechanisms of axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Hammarlund M , Nix P , Hauth L , Jorgensen EM , Bastiani M ( 2009 ) Axon regeneration requires a conserved MAP kinase pathway . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Gumy LF , Tan CL , Fawcett JW ( 2010 ) The role of local protein synthesis and degradation in axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Gotenstein JR , Swale RE , Fukuda T , Wu Z , Giurumescu CA , Goncharov A , Jin Y , Chisholm AD ( 2010 ) The C . elegans peroxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Gabel CV , Antoine F , Antonie F , Chuang CF , Samuel AD , Chang C ( 2008 ) Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C . elegans . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline El Bejjani R , Hammarlund M ( 2012 ) Notch signaling inhibits axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Chen L , Wang Z , Ghosh-Roy A , Hubert T , Yan D , O ' Rourke S , Bowerman B , Wu Z , Jin Y , Chisholm AD ( 2011 ) Axon regeneration pathways identified by systematic genetic screening in C . elegans . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Chen L , Chisholm AD ( 2011 ) Axon regeneration mechanisms : insights from C . elegans . [Field: references, subscore: 2.00]
SECTION: references. CrossRef Medline Bloom OE , Morgan JR ( 2011 ) Membrane trafficking events underlying axon repair , growth , and regeneration . [Field: references, subscore: 2.00]
SECTION: results. These results also support a shared role for proteins in both the mitochondrial UPR and axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. hif-1 also showed reduced regeneration , but a formal test of the effects of hypoxia on regeneration was not done . [Field: results, subscore: 2.00]
SECTION: results. To address this question , we characterized axon regeneration in hsf-1 ( heat-shock factor 1 ) and hif-1 ( hypoxia inducible factor ) mutants and under conditions of heat stress and starvation . [Field: results, subscore: 2.00]
SECTION: results. Stress response pathways are necessary for axon regeneration The p38 and JNK family of MAP kinases are also known as stressactivated protein kinases ( SAPKs ) . [Field: results, subscore: 2.00]
SECTION: results. We investigated whether phosphorylation at Thr-304 also influences axon regeneration by expressing a mutant transgene , fos-1 T304A , in which Thr-304 is mutated to alanine . [Field: results, subscore: 2.00]
SECTION: results. Growth cone formation was normal in jun-1 mutants , whereas fos-1-null mutants show significantly reduced growth cone formation ( Fig . 5 D ; Table 2 ) . [Field: results, subscore: 2.00]
SECTION: results. E , Summary of MAPK signaling components affecting axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. C , The MAP3K kin-18 may inhibit axon regeneration via JNK-1 . [Field: results, subscore: 2.00]
SECTION: results. B , The MAP2Ks jkk-1 and sek-6 may function upstream of JNK-1 to inhibit axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. MAPK signaling via JNK-1 inhibits axon regeneration and requires functional kinase activity . [Field: results, subscore: 2.00]
SECTION: results. A , dlk-1 and kgb-1 mutants represent two MAPK modules essential for axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. Multiple MAPK pathways antagonistically influence axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. In summary , we have reported previously the requirement for PMK-3 and KGB-1 MAPK signaling in promoting axon regeneration ( Hammarlund et al . , 2009 ; Nix et al . , 2011 ) . [Field: results, subscore: 2.00]
SECTION: results. Although none showed reduced regeneration , we found that three alleles , lip-1 ( zh15 ) , ZK757 . 2 ( ok3597 ) , and F28C6 . 8 ( ok2013 ) , improved growth cone formation . [Field: results, subscore: 2.00]
SECTION: results. % Regeneration na p 70 105 12 83 0 . 0001 58 24 0 . 3456 84 38 0 . 0898 98 43 0 . 0001 17 48 0 . 0001 82 88 0 . 0654 38 82 0 . 0001 37 52 0 . 0001 60 52 0 . 2819 83 77 0 . 0783 46 56 0 . 0063 49 86 0 . 0047 0 48 0 . 0001 33 79 0 . 0001 13 61 0 . 0001 11 96 0 . 0001 85 86 0 . 0161 72 76 0 . 7419 48 86 0 . 003 54 85 0 . 035 22 80 0 . 0001 70 77 1 32 121 0 . 0001 86 80 0 . 0084 33 123 0 . 0001 55 33 0 . 1479 77 35 0 . 5175 80 45 0 . 2322 57 67 0 . 103 14 85 0 . 0001 77 71 0 . 3014 96 68 0 . 0001 99 72 0 . 0001 98 53 0 . 0001 24 78 0 . 0001 Nix et al . Genetics of Axon Regeneration The dual-specificity MAPK phosphatases ( MKPs ) remove phosphate groups from activated MAPKs and represent an important control point for regulating MAPK activity . [Field: results, subscore: 2.00]
SECTION: results. kin-18 mutants had significantly improved growth cone initiation , although overexpression of kin-18 in GABA neurons did not affect regeneration ( Fig . 5 C ; Table 3 ) . [Field: results, subscore: 2.00]
SECTION: results. Overexpression of either construct failed to inhibit regeneration , indicating that kinase activity is essential to the function of JNK-1 in regeneration ( Fig . 5 A ) . [Field: results, subscore: 2.00]
SECTION: results. Bottom right , Failed growth cone collapsed around a large vacuole ( left arrowhead ) and a large growth cone remnant along the axon of a successful commissure ( right arrowhead ) . [Field: results, subscore: 2.00]
SECTION: results. Bottom left , right arrowhead , Successful growth cones often leave behind growth cone remnants . [Field: results, subscore: 2.00]
SECTION: results. Axon regeneration defects in myotubularin mutants by loss-of-function and overexpression . [Field: results, subscore: 2.00]
SECTION: results. Our previous work described two MAPK modules ( DLK-1- MKK-4-PMK-3 and MLK-1-MEK-1-KGB-1 ) that are both essential to promote axon regeneration ( Hammarlund et al . , 2009 ; Nix et al . , 2011 ) . [Field: results, subscore: 2.00]
SECTION: results. Multiple MAP kinase pathways influence axon regeneration The MAP kinase signal involves a threetiered kinase cascade consisting of a MAP kinase kinase kinase ( MAP3K ) , a MAP kinase kinase ( MAP2K ) , and a MAP kinase ( MAPK ) . [Field: results, subscore: 2.00]
SECTION: results. We found significant regeneration defects in mtm-6 and mtm-9 mutants , although regeneration was not further decreased in mtm-1 ; mtm-6 double mutants ( Fig . 4 A ) . [Field: results, subscore: 2.00]
SECTION: results. Microvesicle release is an early step in axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. Axon regeneration defects in unc-70 / -spectrin mutants . [Field: results, subscore: 2.00]
SECTION: results. , January 8 , 2014 34 ( 2 ) : 629 645 % Regeneration na p 70 105 72 105 0 . 7613 65 75 0 . 628 71 97 1 60 55 0 . 2909 68 75 0 . 871 61 66 0 . 2488 62 80 0 . 3477 71 58 1 54 67 0 . 051 76 66 0 . 4847 67 120 0 . 6697 83 24 0 . 2137 80 50 0 . 183 79 56 0 . 2669 70 64 1 56 86 0 . 0698 65 74 0 . 5212 62 76 0 . 3394 76 88 0 . 3344 75 51 0 . 5757 60 57 0 . 2267 75 88 0 . 4247 82 82 0 . 0813 63 91 0 . 3637 79 76 0 . 1752 76 104 0 . 3519 69 138 1 79 57 0 . 267 63 89 0 . 2694 63 56 0 . 3834 75 76 0 . 5037 84 61 0 . 0632 79 105 0 . 155 76 62 0 . 4767 63 101 0 . 378 70 81 1 76 93 0 . 338 56 81 0 . 0652 75 71 0 . 4991 74 54 0 . 5848 73 102 0 . 6489 70 88 1 95 75 0 . 0001 84 77 0 . 023 89 82 0 . 0013 83 78 0 . 0376 82 115 0 . 0405 90 59 0 . 0035 96 46 0 . 0002 95 88 0 . 0001 92 84 0 . 0002 95 65 0 . 0001 96 75 0 . 0001 94 62 0 . 0002 89 72 0 . 0031 53 72 0 . 0148 27 59 0 . 0001 26 106 0 . 0001 44 91 0 . 0005 39 105 0 . 0001 46 91 0 . 0013 46 87 0 . 0012 50 50 0 . 0214 25 102 0 . 0001 Nix et al . Genetics of Axon Regeneration sures in unc-130 ; unc-70 ) . [Field: results, subscore: 2.00]
SECTION: results. The eventual outcome is that , despite increased growth cone formation , by the end of an imaging period , there were fewer intact commissures in unc-130 ; unc-70 compared with unc-70 ( 40 / 65 commissures intact in unc-70 vs 23 / 65 commisunc-70 ; RNAi commissure counta Allele testedb % Regeneration after axotomyc 4 . 3 , 4 . 5 RNAi 46 / 80 ( 57 % ) 5 . 3 , 5 . 3 , 7 . 2 RNAi 47 / 58 ( 81 % ) 5 . 4 , 4 . 9 RNAi 56 / 75 ( 75 % ) 4 . 8 , 4 . 5 RNAi 48 / 68 ( 70 % ) 5 . 3 , 4 . 1 wy302 64 / 70 ( 91 % ) d 4 . 4 , 6 . 4 ok145 52 / 53 ( 98 % ) d 5 . 5 , 6 . 3 n1089 58 / 64 ( 91 % ) d 4 . 6 , 6 RNAi 34 / 67 ( 51 % ) e 5 . 4 , 5 RNAi 30 / 44 ( 68 % ) 5 RNAi 43 / 61 ( 70 % ) 3 . 5 , 4 . 4 ok423 NS defects 3 , 4 . 4 gk451 50 / 53 ( 94 % ) d 5 . 1 , 6 . 4 , 4 . 6 bas3 23 / 74 ( 31 % ) e 3 . 7 , 4 . 4 RNAi 34 / 62 ( 55 % ) J . Neurosci . [Field: results, subscore: 2.00]
SECTION: results. Improved regeneration increases the frequency of growth cone formation , which ultimately induces a greater number of breaks in mutants lacking -spectrin . [Field: results, subscore: 2.00]
SECTION: results. Nix et al . Genetics of Axon Regeneration Spontaneous growth cones do not occur in wild-type animals , but they do regularly appear along both severed and intact axons in unc-70 mutants . [Field: results, subscore: 2.00]
SECTION: results. Despite this , several candidates that showed a strong regeneration block in the unc-70 background showed improved regeneration when assayed by axotomy in the mutant strain alone . [Field: results, subscore: 2.00]
SECTION: results. The result of our screens described here includes at least 50 new genes implicated in C . elegans axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. Mutants showing significantly improved regeneration are in green and mutants showing significantly decreased regeneration are in red . [Field: results, subscore: 2.00]
SECTION: results. D , Wild-type axon regeneration after axotomy . [Field: results, subscore: 2.00]
SECTION: results. Axon regeneration genes identified by genetic screening . [Field: results, subscore: 2.00]
SECTION: results. Mutations improving regeneration result in an increased likelihood of growth cone formation and improved migration to the dorsal cord . [Field: results, subscore: 2.00]
SECTION: results. However , growth cone motility and guidance is inefficient at this stage and by 24 h postaxotomy few growth cones ( 510 % ) successfully reached targets in the dorsal nerve cord . [Field: results, subscore: 2.00]
SECTION: results. We exploited this phenotype of unc-70 / -spectrin mutants to screen for genes required for axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. As the animal ages , there is a progressive failure of regeneration with each cycle of axotomy and regeneration so that adults display a severely abnormal nervous system . [Field: results, subscore: 2.00]
SECTION: results. An RNAi-based screen for axon regeneration genes Embryonic neurons lacking the cytoskeletal component - spectrin develop normally . [Field: results, subscore: 2.00]
SECTION: title. Axon regeneration genes identified by RNAi screening in C . elegans . [Field: title, subscore: 2.00]
SECTION: abstract. The complex molecular events required for robust regeneration of mature neurons are not fully understood , particularly in vivo . [Field: abstract, subscore: 1.00]
SECTION: abstract. Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration . [Field: abstract, subscore: 1.00]
SECTION: discussion. Sun et al . demonstrated that CNS neurons in adult mice show improved and sustained regeneration in PTEN- and SOCS3-deleted double mutants compared with single mutants or the wild-type ( Sun et al . , 2011 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. Genes inhibiting regeneration are of particular interest because these pathways may be easier to eliminate via drug application rather than by upregulating growth-promoting pathways . [Field: discussion, subscore: 1.00]
SECTION: discussion. As an organism ages , it is less able to cope with errors in protein translation and folding and this can in part explain the age-dependent decline in regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Therefore , the success of regeneration to some extent becomes an issue of protein homeostasis . [Field: discussion, subscore: 1.00]
SECTION: discussion. Efficient regeneration requires an injured neuron to manage a profusion of new protein synthesis and recapitulate much of the developmental program . [Field: discussion, subscore: 1.00]
SECTION: discussion. Furthermore , loss of hsf-1 prevents regeneration even under nonstressed conditions , as does hif-1 . [Field: discussion, subscore: 1.00]
SECTION: discussion. The improved regeneration we observed in animals exposed to heat stress requires functional HSF-1 . [Field: discussion, subscore: 1.00]
SECTION: discussion. Other stress-activated proteins are also important for regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. The inhibitory effect of jnk-1 overexpression was dependent on kinase activity because overexpression of a kinase-dead form of the protein no longer blocked regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. We found that activation of the JNK-1 MAP kinase inhibits GABA neuron regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Because the assay primarily quantifies the incidence of growth cones , certain genes identified in the screen will require further analysis using additional criteria for validation . [Field: discussion, subscore: 1.00]
SECTION: discussion. This raises the possibility that loss of the spectrin cytoskeleton in unc-70 sensitizes the background to further defects in regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Several gene candidates identified in the screen did not show a regeneration phenotype when assayed by axotomy . [Field: discussion, subscore: 1.00]
SECTION: discussion. Both CEBP-1 and DLK-1 are essential for regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. , January 8 , 2014 34 ( 2 ) : 629 645 Nix et al . Genetics of Axon Regeneration lation of the CEBP-1 transcription factor ( Yan et al . , 2009 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. B , Increased IRE-1 activity in xbp-1 mutants and by ire-1 overexpression inhibits regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Regeneration in hif-1 is also reduced , but was not tested under hypoxic conditions . [Field: discussion, subscore: 1.00]
SECTION: discussion. A , Stress-inducing conditions improve regeneration outcomes and require hsf-1 . [Field: discussion, subscore: 1.00]
SECTION: discussion. Its main enzymatic activity is dephosphorylation of PI ( 3 ) P and PI ( 3 , 5 ) P2 , so mtm-1 could be involved in signaling membrane damage or in cytoskeletal dynamics necessary for growth cone formation . [Field: discussion, subscore: 1.00]
SECTION: discussion. Two lipid phosphatases suggest that membrane lipid composition and / or lipid signaling play an important role in regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. , January 8 , 2014 34 ( 2 ) : 629 645 641 The damaged membrane is repaired before growth cone formation can begin . [Field: discussion, subscore: 1.00]
SECTION: discussion. Nix et al . Genetics of Axon Regeneration J . Neurosci . [Field: discussion, subscore: 1.00]
SECTION: discussion. The total number of genes screened represents about one-quarter of the C . elegans genome and suggests that continued screening by this method may add to the collection of regeneration genes . [Field: discussion, subscore: 1.00]
SECTION: discussion. Twenty-two of the 65 mutants showed regeneration phenotypes after axotomy . [Field: discussion, subscore: 1.00]
SECTION: discussion. These include 15 positive regulators and 16 negative regulators of regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Of the 70 candidates isolated from the screen , 31 showed regeneration defects when assayed by axotomy in a mutant strain . [Field: discussion, subscore: 1.00]
SECTION: discussion. We identified 70 candidate regeneration genes by screening for reduced commissure number in unc- 70 / -spectrin mutants fed a total of 5076 RNAi clones . [Field: discussion, subscore: 1.00]
SECTION: introduction. Several candidate genes have been implicated previously in regeneration and others define new and conserved pathways of interest . [Field: introduction, subscore: 1.00]
SECTION: introduction. We used this phenotype as the basis for an RNAi screen for genes affecting regeneration and identified 70 candidate genes . [Field: introduction, subscore: 1.00]
SECTION: introduction. In the -spectrin mutant , this results in successive rounds of breakage and regeneration . [Field: introduction, subscore: 1.00]
SECTION: introduction. Once broken , the axon responds by forming a growth cone and extending the axon Received Sept . [Field: introduction, subscore: 1.00]
SECTION: introduction. However , unbiased genetic screens for regeneration genes have not been practical in any model system . [Field: introduction, subscore: 1.00]
SECTION: introduction. High-throughput screening of drug compounds influencing regeneration has also been performed ( Samara et al . , 2010 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. Although many pro-regenerative molecules are known , the essential mechanisms needed for successful regeneration of CNS neurons remains elusive . [Field: introduction, subscore: 1.00]
SECTION: introduction. Therapeutic interventions aimed toward treating axonal injury would ideally target the intrinsic mechanisms responsible for regeneration in preconditioned or embryonic neurons . [Field: introduction, subscore: 1.00]
SECTION: introduction. In the case of DRG neurons , treatment with a preconditioning lesion in the peripheral axon can overcome the barrier to CNS regeneration ( Neumann and Woolf , 1999 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. The capacity of neurons to regenerate rapidly decreases with age due to an inhibitory CNS environment and ineffective activation of the intrinsic regeneration program ( Yiu and He , 2006 ; Liu et al . , 2011 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. The complex molecular events required for robust regeneration of mature neurons are not fully understood , particularly in vivo . [Field: introduction, subscore: 1.00]
SECTION: references. CrossRef Medline Nix et al . Genetics of Axon Regeneration J . Neurosci . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Wu Z , Ghosh-Roy A , Yanik MF , Zhang JZ , Jin Y , Chisholm AD ( 2007 ) Caenorhabditis elegans neuronal regeneration is influenced by life stage , ephrin signaling , and synaptic branching . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Spira ME , Oren R , Dormann A , Gitler D ( 2003 ) Critical calpain-dependent ultrastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Samara C , Rohde CB , Gilleland CL , Norton S , Haggarty SJ , Yanik MF ( 2010 ) Large-scale in vivo femtosecond laser neurosurgery screen reveals smallmolecule enhancer of regeneration . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Raivich G , Bohatschek M , Da Costa C , Iwata O , Galiano M , Hristova M , Nateri AS , Makwana M , Riera-Sans L , Wolfer DP , Lipp HP , Aguzzi A , Wagner EF , Behrens A ( 2004 ) The AP-1 transcription factor c-Jun is required for efficient axonal regeneration . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Raivich G , Makwana M ( 2007 ) The making of successful axonal regeneration : genes , molecules and signal transduction pathways . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Patodia S , Raivich G ( 2012 ) Role of transcription factors in peripheral nerve regeneration . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Neumann S , Woolf CJ ( 1999 ) Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Kadoya K , Tsukada S , Lu P , Coppola G , Geschwind D , Filbin MT , Blesch A , Tuszynski MH ( 2009 ) Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Halder G , Johnson RL ( 2011 ) Hippo signaling : growth control and beyond . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Ghosh-Roy A , Wu Z , Goncharov A , Jin Y , Chisholm AD ( 2010 ) Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase . [Field: references, subscore: 1.00]
SECTION: references. CrossRef Medline Ben-Zvi A , Miller EA , Morimoto RI ( 2009 ) Collapse of proteostasis repre- Nix et al . Genetics of Axon Regeneration J . Neurosci . [Field: references, subscore: 1.00]
SECTION: results. In addition , we screened the atfs-1 ( gk3094 ) mutant by axotomy and found significantly improved regeneration in the mutant background ( Fig . 1 H ) . [Field: results, subscore: 1.00]
SECTION: results. We identified ubl-5 as a gene candidate in the unc-70 regeneration screen ( Table 1 ) . [Field: results, subscore: 1.00]
SECTION: results. We speculate that increased IRE-1 endonuclease activity might affect mRNAs present in the cytoplasm , causing inappropriate cleavage of the transcripts necessary for regeneration . [Field: results, subscore: 1.00]
SECTION: results. Indeed , overexpression of ire-1 in neurons also blocked regeneration ( Fig . 6 B ; Table 3 ) . [Field: results, subscore: 1.00]
SECTION: results. We found that loss of xbp-1 results in severely reduced regeneration , whereas the loss of ire-1 had no effect ( Fig . 6 B ) . [Field: results, subscore: 1.00]
SECTION: results. Alternatively , it suggests that the majority of the heat shock response occurs through HSF-1 , but that secondary pathways contributing to regeneration may also be activated by heat shock . [Field: results, subscore: 1.00]
SECTION: results. We observed a small but significant increase in growth cone initiation when hsf-1 mutants were heat shocked . [Field: results, subscore: 1.00]
SECTION: results. Furthermore , hsf-1 mutants showed severely reduced regeneration even in the absence of heat shock . [Field: results, subscore: 1.00]
SECTION: results. Regeneration was blocked in heatshocked hsf-1 mutants ( Fig . 6 A ) . [Field: results, subscore: 1.00]
SECTION: results. The improved regeneration we observed after a short period of heat shock was dependent on HSF-1 activity . [Field: results, subscore: 1.00]
SECTION: results. Similarly , growth cone formation was improved when axotomized animals were recovered on plates in the absence of food . [Field: results, subscore: 1.00]
SECTION: results. A 1 h period of heat stress at 33C , followed by recovery at 20C , resulted in significantly improved regeneration ( Fig . 6 A ) . [Field: results, subscore: 1.00]
SECTION: results. Does activating the stress response by other means alter regeneration ? [Field: results, subscore: 1.00]
SECTION: results. Overexpression of a genomic fos-1 transgene rescued the sterility associated with fos-1 ( km30 ) -null mutants and rescued the regeneration phenotype back to wildtype levels . [Field: results, subscore: 1.00]
SECTION: results. Expression of a dominantnegative form of fos-1 that encodes the DNA-binding and dimerization domains but lacks the transcriptional activation domain also inhibited regeneration when expressed specifically in the GABA neurons ( Fig . 5 D ) . [Field: results, subscore: 1.00]
SECTION: results. We examined the C . elegans Jun and Fos homologs for a potential role in regeneration . [Field: results, subscore: 1.00]
SECTION: results. In mammals , axonal activation and retrograde transport of JNK , followed by upregulation of c-jun , is a consistent marker of successful regeneration ( Raivich et al . , 2004 ; Raiv- ich and Makwana , 2007 ) . [Field: results, subscore: 1.00]
SECTION: results. D , FOS-1 transcription factor is necessary for regeneration and is a target of KGB-1 MAPK signaling . [Field: results, subscore: 1.00]
SECTION: results. Improved regeneration by loss of jnk-1 weakly suppresses loss of kgb-1 , but is not sufficiently to suppress the loss of dlk-1 . [Field: results, subscore: 1.00]
SECTION: results. , January 8 , 2014 34 ( 2 ) : 629 645 639 that additional MAPK signaling factors antagonistically influence the outcome of regeneration in the worm ( Fig . 5 E ) . [Field: results, subscore: 1.00]
SECTION: results. Here , we show na p Double-mutant combinations na % Regeneration 105 dlk-1 ; jnk-1 62 0 100 0 . 0038 kgb-1 jnk-1 72 36 82 0 . 0001 jkk-1 sek-1 57 58 73 0 . 4979 jkk-1 ; lpIn2 64 22 77 0 . 0132 sek-6 ; lpIn2 59 36 69 0 . 0001 jkk-1 sek-6 ; lpIn2 76 47 52 0 . 0001 kin-18 ; basEx23 113 56 59 0 . 5901 unc-16 ; basEx23 83 71 48 0 . 8825 55 0 . 3777 74 0 . 7384 74 0 . 8702 74 0 . 0861 133 0 . 0011 76 0 . 0001 46 0 . 0001 51 0 . 0659 71 1 62 0 . 7312 49 1 46 0 . 2618 68 0 . 0001 60 0 . 0144 69 0 . 0001 62 0 . 0001 73 0 . 7455 ND 59 0 . 3729 51 0 . 0046 109 0 . 0001 47 0 . 8518 62 0 . 0001 58 0 . 0036 61 0 . 0001 66 0 . 0011 40 0 . 0041 70 0 . 3956 104 1 61 0 . 4772 72 1 91 0 . 4477 59 0 . 7223 73 0 . 1127 J . Neurosci . [Field: results, subscore: 1.00]
SECTION: results. The regeneration phenotypes observed in the jnk-1 loss-of-function or overexpression strains may be explained by its role in vesicle transport . [Field: results, subscore: 1.00]
SECTION: results. Mutations in another JNK- interacting protein , jip-1 , did not show a regeneration phenotype in our assay ( Table 4 ) . [Field: results, subscore: 1.00]
SECTION: results. Loss of unc-16 also suppresses the blocked regeneration in jnk-1- overexpressing animals ( Fig . 5 C ) . [Field: results, subscore: 1.00]
SECTION: results. unc-16 mutants improve regeneration to the same extent as jnk-1 and kgb-2 MAPK mutants ( Table 2 ) . [Field: results, subscore: 1.00]
SECTION: results. Nix et al . Genetics of Axon Regeneration each other . [Field: results, subscore: 1.00]
SECTION: results. We predicted that loss of one of these might result in blocked regeneration due to increased activity of either JNK-1 or KGB-2 . [Field: results, subscore: 1.00]
SECTION: results. Loss of vhp-1 increases levels of activated PMK-3 and KGB-1 , improving regeneration . [Field: results, subscore: 1.00]
SECTION: results. We identified the MAP3K kin-18 as a candidate in the unc-70 regeneration screen . [Field: results, subscore: 1.00]
SECTION: results. However , neither overexpression of wild-type sek-6 nor the phosphomimetic sek-6 ( DD ) resulted in a regeneration defect ( Table 3 ) . [Field: results, subscore: 1.00]
SECTION: results. One MAP2K , sek-6 , did show improved regeneration in the mutant background and may function upstream of jnk-1 or kgb-2 . [Field: results, subscore: 1.00]
SECTION: results. The jkk-1 sek-1 double mutant was indistinguishable from either single mutant , indicating that these MAP2Ks are unlikely to function redundantly in regeneration . [Field: results, subscore: 1.00]
SECTION: results. jkk-1 mutants did not show a regeneration defect , suggesting that JNK-1 activity may be stimulated by a different MAP2K upon axotomy or that additional MAP2Ks may Table 3 . [Field: results, subscore: 1.00]
SECTION: results. Alternatively , JNK-1 kinase activity itself could inhibit regeneration . [Field: results, subscore: 1.00]
SECTION: results. One possibility is that JNK-1 overexpression competes with PMK-3 and / or KGB-1 for binding to an activator or substrate , thereby preventing proper activation and thus blocking regeneration . [Field: results, subscore: 1.00]
SECTION: results. JNK-1 and KGB-2 overexpression inhibited new growth cone formation ( Fig . 5 A ; Table 3 ) . [Field: results, subscore: 1.00]
SECTION: results. However , loss of jnk-1 moderately suppressed the regeneration phenotype of kgb-1 MAPK mutants ( Fig . 5 A ) . [Field: results, subscore: 1.00]
SECTION: results. Improved regeneration in the jnk-1 mutant was dependent on normal activation of the pmk-3 MAPK pathway , because dlk-1 ( MAP3K ) ; jnk-1 double mutants , like dlk-1 single mutants , failed to regenerate . [Field: results, subscore: 1.00]
SECTION: results. , January 8 , 2014 34 ( 2 ) : 629 645 637 Loss-of-function mutants in the MAPKs jnk-1 and kgb-2 show improved regeneration ( Fig . 5 A ) . [Field: results, subscore: 1.00]
SECTION: results. Nix et al . Genetics of Axon Regeneration J . Neurosci . [Field: results, subscore: 1.00]
SECTION: results. Overexpression of mtm-1 may cause failure to initiate growth cones ( lower left ) . [Field: results, subscore: 1.00]
SECTION: results. Arrowheads indicate the highly branched wild-type growth cones seen at L4 . [Field: results, subscore: 1.00]
SECTION: results. A , mtm-1 and other myotubularin family members are required for normal GABA neuron regeneration . [Field: results, subscore: 1.00]
SECTION: results. Here , we identify two additional MAPKs that function as negative regulators of regeneration . [Field: results, subscore: 1.00]
SECTION: results. Loss of lipid phosphorylation due to increased mtm-1 expression could alter membrane identity , resulting in increased endosomal structures and failure to deliver vesicles to the plasma membrane , where they are needed for growth cone formation . [Field: results, subscore: 1.00]
SECTION: results. Endocytosis is an important event in remodeling the axonal membrane during growth cone formation ( Bloom and Mor- gan , 2011 ) . [Field: results, subscore: 1.00]
SECTION: results. mtm-1 overexpression in GABA neurons led to dramatic defects in axonal morphology and decreased regeneration ( Fig . 4A , B ; Table 3 ) . [Field: results, subscore: 1.00]
SECTION: results. However , migration of the mutant growth cone is ineffective , eventually collapsing . [Field: results, subscore: 1.00]
SECTION: results. The growth cones often appear to be embryonic-like in nature with fewer filopodial extensions than wild-type ( Fig . 4 B ) . [Field: results, subscore: 1.00]
SECTION: results. Aberrant growth cones are visible after axotomy in mtm-1 ( op309 ) time-lapse experiments . [Field: results, subscore: 1.00]
SECTION: results. Regeneration was moderately decreased ; however , it is likely that mtm-1 ( op309 ) still retains some function because deletion of the mtm-1 gene causes lethality . [Field: results, subscore: 1.00]
SECTION: results. , January 8 , 2014 34 ( 2 ) : 629 645 Nix et al . Genetics of Axon Regeneration ulating membrane dynamics and membrane trafficking . [Field: results, subscore: 1.00]
SECTION: results. At 228 minutes , membrane blebs are visible and appear to interfere with normal growth cone formation . [Field: results, subscore: 1.00]
SECTION: results. D , nex-1 is required for normal GABA neuron regeneration . [Field: results, subscore: 1.00]
SECTION: results. The growth cone forms shortly after at 363 min . [Field: results, subscore: 1.00]
SECTION: results. Although nex-1 mutations affect growth cone initiation and may be involved in membrane repair at the site of damage , the trigger for microvesicle release from intact axons remains to be determined . [Field: results, subscore: 1.00]
SECTION: results. We interpret these blebs as regions of damaged membrane that are not removed from the injury site in the absence of nex-1 activity and think that the damaged membrane interferes with normal growth cone formation . [Field: results, subscore: 1.00]
SECTION: results. In some cases , a growth cone may form , but often collapses or fails to progress due to abnormal motility ( Fig . 3 E ) . [Field: results, subscore: 1.00]
SECTION: results. After axotomy , retraction bulb formation is normal , but excessive blebbing can be seen at the site of damage before the appearance of a growth cone . [Field: results, subscore: 1.00]
SECTION: results. Defective regeneration in nex-1 mutants is more apparent in time-lapse images . [Field: results, subscore: 1.00]
SECTION: results. We performed axotomy in the C . elegans annexin mutants nex-1 , nex-2 , nex-3 , and nex-4 and found that nex-1 results in a significant reduction in regeneration compared with wild-type animals ( Fig . 3 D ) . [Field: results, subscore: 1.00]
SECTION: results. Annexins are Ca2 -sensitive phospholipid-binding proteins implicated in membrane repair and therefore are good candidates to mediate this process in regeneration ( Draeger et al . , 2011 ) . [Field: results, subscore: 1.00]
SECTION: results. Nix et al . Genetics of Axon Regeneration J . Neurosci . [Field: results, subscore: 1.00]
SECTION: results. At 291528 minutes , both stumps regenerate new growth cones . [Field: results, subscore: 1.00]
SECTION: results. At 48 minutes , the growth cone has broken the adjacent axon and the proximal end rapidly retracts ( lower arrow ) , leaving behind fragments that are slowly cleared ( top arrow , 48 159min ) . [Field: results, subscore: 1.00]
SECTION: results. C , Quantification of spontaneous growth cone formation in unc-70 mutants per hour per commissure . [Field: results, subscore: 1.00]
SECTION: results. A , Timing of first growth cone ( GC ) appearance in unc-70 mutants compared with wild-type . [Field: results, subscore: 1.00]
SECTION: results. During this period , we observed the release of microvesicle particles from the tip of the severed axon ( the site of future growth cone formation ; Fig . 3 A ) . [Field: results, subscore: 1.00]
SECTION: results. This is followed by a quiescent period before growth cone formation and migration begins . [Field: results, subscore: 1.00]
SECTION: results. Together , our data suggest that the spectrin cytoskeleton is a barrier to growth cone formation and must be removed , but is later required to reform and stabilize the axon during outgrowth . [Field: results, subscore: 1.00]
SECTION: results. We observed a nearly threefold increase in the number of spontaneous growth cones in unc-130 ; unc-70 compared with unc-70 alone ( Fig . 2 C ) . [Field: results, subscore: 1.00]
SECTION: results. The increased number of growth cones in unc-130 ; unc-70 was evident by time-lapse microscopy . [Field: results, subscore: 1.00]
SECTION: results. Axotomy in unc-130 single mutants results in increased growth cone formation relative to the wild-type , whereas unc-130 RNAi in unc-70 led to a strong decrease in the number of intact commissures ( Table 1 ) . [Field: results, subscore: 1.00]
SECTION: results. Over time , this leads to the appearance of more failed regeneration attempts . [Field: results, subscore: 1.00]
SECTION: results. This observation accounts for the fact that we did not identify candidate genes with improved regeneration in the unc-70 mutant background . [Field: results, subscore: 1.00]
SECTION: results. In each case , the tension created by growth cone migration along its substrate is sufficient to induce new breaks . [Field: results, subscore: 1.00]
SECTION: results. A spontaneous growth cone appearing in an unc-70 mutant will either collapse and be resolved or it may extend for some distance , contributing to the axon ' s branched morphology . [Field: results, subscore: 1.00]
SECTION: results. This is supported by the observation that spontaneous growth cones are significantly less frequent when unc-70 is paired with the muscle myosin mutant unc-54 ( Fig . 2 C ) . [Field: results, subscore: 1.00]
SECTION: results. It is possible that small , mechanical stresses on the axon that are insufficient to cause a break may be enough to trigger a localized calcium increase and thus growth cone initiation . [Field: results, subscore: 1.00]
SECTION: results. Indeed , after axotomy , growth cone initiation occurred earlier in unc-70 mutants relative to the wildtype ( Fig . 2 A ) . [Field: results, subscore: 1.00]
SECTION: results. Given these observations , we predicted that unc-70 mutants lacking -spectrin would show early growth cone formation . [Field: results, subscore: 1.00]
SECTION: results. It has also been proposed that removal of spectrin facilitates vesicle fusion to the plasma membrane , which is necessary for growth cone expansion ( Spira et al . , 2003 ) . [Field: results, subscore: 1.00]
SECTION: results. Calpain activation , in turn , leads to degradation of cytoskeletal components and cellular restructuring essential for successful regeneration ( Spira et al . , 2003 ; Bradke et al . , 2012 ) . [Field: results, subscore: 1.00]
SECTION: results. Candidate regeneration genes identified by RNAi screening Clone Locus WormBase description Signaling T17E9 . 1 kin-18 Serine / threonine protein kinase F14H12 . 4 cst-1 STE20-like kinase ; Drosophila hippo ZK792 . 6 let-60 Ras GTP-binding protein H08M01 . 2 rga-5 Rho-GTPase activating protein C02F4 . 2 tax-6 Calcineurin ; serine / threonine protein phosphatase F02A9 . 6 glp-1 Notch receptor H39E23 . 1 par-1 Serine / threonine kinase reqired for establishing embryonic polarity F33E2 . 2 dlk-1 MAPKKK ; Eukaryotic protein kinase domain K11D12 . 10 mlk-1 MAPKKK ; tyrosine kinase specific for activated p21cdc42Hs F17E5 . 1 lin-2 MAGUK protein , calcium / calmodulin dependent protein kinase M02A10 . 3 sli-1 Cbl family E3 ubiquitin ligase , Ras and EGFR signaling F25H5 . 1 lim-9 Enables Wnt-directed planar cell polarity ; Lim domain protein T06D8 . 3 Lipid phosphate phosphatase , PAP2 family Y110A7A . [Field: results, subscore: 1.00]
SECTION: results. Errorbarsindicate95 % confidenceinterval . Nix et al . Genetics of Axon Regeneration J . Neurosci . [Field: results, subscore: 1.00]
SECTION: results. GH , Percentage of growth cone formation in candidate genes characterized by axotomy . [Field: results, subscore: 1.00]
SECTION: results. F , Axotomy in a mutant with blocked regeneration . [Field: results, subscore: 1.00]
SECTION: results. E , Axotomy in a mutant with improved regeneration . [Field: results, subscore: 1.00]
SECTION: results. C , unc-70 mutant fed an RNAi clone targeted against a candidate regeneration gene . [Field: results, subscore: 1.00]
SECTION: results. Thirty-one of 70 candidate genes we identified by screening displayed a regeneration phenotype upon axotomy ( Fig . 1 G ) . [Field: results, subscore: 1.00]
SECTION: results. In the wild-type , 70 % of severed axons initiated a growth cone and regrew beyond the site of axotomy ( Fig . 1 D ) . [Field: results, subscore: 1.00]
SECTION: results. We assayed regeneration in the GABA motor neurons of L4-stage animals . [Field: results, subscore: 1.00]
SECTION: results. We did not identify RNAi clones improving regeneration outcomes in unc-70 ; the reasons for this are described in Results and [Field: results, subscore: 1.00]
SECTION: results. RNAi of a small number of candidate genes led to improved regeneration in the unc-70 mutant background . [Field: results, subscore: 1.00]
SECTION: results. The 488 candidate clones resulting in a weak phenotype in the primary screen were not tested further , whereas strong or moderate candidates were repeated and confirmed in additional RNAi experiments , resulting in 70 candidate regeneration genes ( Table 1 ) . [Field: results, subscore: 1.00]
SECTION: results. Branched axons , visible growth cones , path-finding defects , and gaps in the dorsal nerve cord are all characteristics of axons that have broken and regenerated . [Field: results, subscore: 1.00]
SECTION: results. To assay regeneration , we selected animals at the L4 stage and quantified the number of commissures that contacted the dorsal nerve cord . [Field: results, subscore: 1.00]
SECTION: results. This screen provides an unbiased approach to identify novel gene candidates with a function that may not have been associated previously with neuronal regeneration . [Field: results, subscore: 1.00]
SECTION: results. The axons continue to break due to movement , however , regeneration would fail due to RNAi of the candidate gene . [Field: results, subscore: 1.00]
SECTION: results. We reasoned that RNAi knock-down of a gene required for regeneration would result in an increased number of broken axons in the unc-70 mutant . [Field: results, subscore: 1.00]
SECTION: results. After hatching , unc-70 / -spectrin mutant neurons undergo spontaneous , movement-induced axotomy followed by regeneration ( Hammarlund et al . , 2007 ) . [Field: results, subscore: 1.00]
Match: [Sentence(s) appears to be scrambled. Click to see (opens new window)] [Field: results, subscore: 4.00]
Supplemental links/files: reference in endnote reference in xml Wormbase reference
Score: 388.00
Title: Syndecan promotes axon regeneration by stabilizing growth cone migration .
Authors: Edwards TJ ; Hammarlund M
Journal: Cell Rep
Year: 2014-07-10
Doc ID: WBPaper00045468.sup.3
Bibliographic Information
Abstract
Matching Sentences
SECTION: discussion. Importantly , while most genes affecting regeneration in C . elegans modulate the frequency of growth cone formation or the length of extension , the growth cone collapse defects we describe are unique among regeneration phenotypes in C . elegans and argue that genetic pathways exist to promote aspects of axon regeneration that are not yet fully appreciated . [Field: discussion, subscore: 6.00]
SECTION: references. We also assigned each regenerating axon into a general category : 1 ) growth cone formation and migration to 3 / 4 muscle attachment site , 2 ) dynamic growth that included filaments , branches , and growth cones that did not reach 3 / 4 muscles attachment sites , 3 ) misguided growth cone extension , 4 ) branch or filament growth , but no stable growth cone formation , and 5 ) miscellaneous ( see Table S1 ) . [Field: references, subscore: 6.00]
SECTION: references. Number of animals Total number of axons Active growth no growth cone Growth cone not to dorsal muscles Misguided growth cone Growth cone to dorsal muscles Miscellaneous Total growth cone formation Table S2 . [Field: references, subscore: 6.00]
SECTION: results. Syndecan Translates Growth Activity into Rapid Extension To determine whether decreased growth cone stability affects the speed and efficiency of migration , we examined the effective regeneration growth rate by measuring the dorsal progression of the axon during periods of growth activity ( Figures 3 A and 3B ) . [Field: results, subscore: 5.00]
SECTION: results. Syndecan Is Required for Regeneration of the GABAergic Motor Neurons In order to determine whether syndecan functions in axon regeneration in vivo , we examined axon regeneration in loss-of-function mutants in sdn-1 , the sole C . elegans syndecan gene . [Field: results, subscore: 5.00]
SECTION: discussion. Our work identifies growth cone maintenance as a critical choke point for axon regeneration , and it is consistent with the model that neuronal syndecan has an HS-independent mode of action in maintaining regenerating growth cones and promoting recovery of damaged neuronal circuits . [Field: discussion, subscore: 4.00]
SECTION: discussion. While a study of growth cone behavior in developing sdn-1 neurons is lacking , we presume that defects in growth cone stability are dramatically exacerbated during regeneration , as stunted , dysmorphic growth structures localized ventrally in uninjured sdn-1 mutants are relatively rare ( Rhiner et al . , 2005 ; unpublished data ) . [Field: discussion, subscore: 4.00]
SECTION: introduction. Our results define syndecan as a regeneration factor and highlight the importance of sustained growth cone migration for successful axon regeneration . [Field: introduction, subscore: 4.00]
SECTION: results. We focused only on axons that initiated some growth activity during the imaging window and examined various phenotypes , including initiation of activity in the ventral stump , growth cone behavior , and regeneration to the dorsal muscle boundary ( this anatomical boundary contains adhesion sites between the skin and muscle [ Francis and Waterston , 1991 ] that impede growth cone migration toward the dorsal nerve cord [ Knobel et al . , 1999 ] ) . [Field: results, subscore: 4.00]
SECTION: abstract. We provide evidence that syndecan has two distinct functions during axon regeneration : ( 1 ) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and ( 2 ) an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein , independently of HS . [Field: abstract, subscore: 3.00]
SECTION: abstract. Using single-neuron laser axotomy and in vivo time-lapse imaging , we show that syndecan , a heparan sulfate ( HS ) proteoglycan , is required for growth cone function during axon regeneration in C . elegans . [Field: abstract, subscore: 3.00]
SECTION: abstract. Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration . [Field: abstract, subscore: 3.00]
SECTION: discussion. Thus , regenerating axons must express syndecan to maintain growth cones , and failure to execute this genetic program results in growth cone collapse and failed regeneration ( Figure 7B ) . [Field: discussion, subscore: 3.00]
SECTION: discussion. Our data suggest that syndecan functions in neurons to promote growth cone stability during axon regeneration , whereas it functions in the hypodermis to promote axon guidance . [Field: discussion, subscore: 3.00]
SECTION: introduction. We conclude that syndecan has a function in growth cone stabilization during axon regeneration that is mechanistically distinct from its described role in axon guidance . [Field: introduction, subscore: 3.00]
SECTION: introduction. However , how syndecan contributes to regenerative growth is unknown , and whether heparan sulfate itself promotes ( Chau et al . , 1999 ) or inhibits ( Groves et al . , 2005 ) axon regeneration remains unclear . [Field: introduction, subscore: 3.00]
SECTION: introduction. In contrast to initial growth cone formation after injury ( Bradke et al . , 2012 ) , relatively little is known about the molecular mechanisms that support sustained growth cone migration during regeneration . [Field: introduction, subscore: 3.00]
SECTION: introduction. Axon regeneration is mediated by growth cone migration , often across increased distances and unfamiliar landscapes . [Field: introduction, subscore: 3.00]
SECTION: introduction. We provide evidence that syndecan has two distinct functions during axon regeneration : ( 1 ) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and ( 2 ) an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein , independently of HS . [Field: introduction, subscore: 3.00]
SECTION: introduction. Using single-neuron laser axotomy and in vivo time-lapse imaging , we show that syndecan , a heparan sulfate ( HS ) proteoglycan , is required for growth cone function during axon regeneration in C . elegans . [Field: introduction, subscore: 3.00]
SECTION: introduction. SUMMARY Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration . [Field: introduction, subscore: 3.00]
SECTION: materials. Categorical data ( full regeneration , partial regeneration , dysmorphic growth , etc . ) was compared using a Fisher ' s exact test . Continuous data ( neurite length , time to initiation , duration , etc . ) was analyzed with an unpaired t test . p values were calculated with GraphPad QuickCalcs ( http : / / www . graphpad . com / quickcalcs / ) . [Field: materials, subscore: 3.00]
SECTION: materials. Partial regeneration represented axons that extended growth cones dorsally close to the midline or beyond , including full regeneration . [Field: materials, subscore: 3.00]
SECTION: references. The average growth rates of productive ( > 5um ) and unproductive ( < 5um ) events in wild type and sdn-1 mutants were compared using an unpaired t-test . To analyze growth cone dynamics , we annotated the start and end times of all growth cones formed from the severed axon . [Field: references, subscore: 3.00]
SECTION: references. The regenerating axon shows long periods of filamentous growth before formation of a growth cone at the tip . [Field: references, subscore: 3.00]
SECTION: references. Assembly of a new growth cone after axotomy : the precursor to axon regeneration . [Field: references, subscore: 3.00]
SECTION: results. Overall , the regeneration phenotype of syndecan RNAi animals was similar to that of rib-2 mutants , suggesting that hypodermal syndecan promotes the guidance of regenerating neurons via its HS sugar chains , while the neuronal function of syndecan in growth cone stability during regeneration is mediated by the syndecan core protein . [Field: results, subscore: 3.00]
SECTION: results. sdn-1 Mutant Axons Display Dysmorphic Growth after Axotomy ( A ) Representative image of regenerating growth cone in a wild-type animal . ( B and C ) Images of dysmorphic growth in sdn- 1 ( zh20 ) mutants . [Field: results, subscore: 3.00]
SECTION: results. These experiments suggest that syndecan has two separable molecular roles : one in axon guidance , where syndecan functions via its modified HS chains ( and potentially chondroitin ) ( Bulow and Hobert , 2004 ; Rhiner et al . , 2005 ) , and one in growth cone stability during axon regeneration . [Field: results, subscore: 3.00]
SECTION: results. Thus , both heparan and chondroitin sugar chains appear to be required for guidance but dispensable ( at least zygotically ) for growth cone migration during axon regeneration . [Field: results, subscore: 3.00]
SECTION: results. We severed axons in rib-2 ( gk318 ) mutants isolated from balanced heterozygotes and observed normal rates of full regeneration to the dorsal cord as well as normal rates of partial regeneration , representing growth cone formation and migration close to the midline ( Figures 5 C and 5D ) . [Field: results, subscore: 3.00]
SECTION: results. Taken together , these data show that one of syndecan ' s primary roles is to stabilize growth cones and that decreased growth cone stability dramatically affects the final outcome of regeneration . [Field: results, subscore: 3.00]
SECTION: results. Finally , we observed that while the start time to filamentous growth activity after injury was unchanged in sdn-1 mutants ( Figure 2 E ) , initial growth cone formation was significantly delayed ( Figure 2F ) . [Field: results, subscore: 3.00]
SECTION: results. Finally , we tested the ability of previously described rescuing constructs ( which partially rescue some axon guidance phenotypes ) to restore regeneration and found that these also fail to rescue the axon regeneration defects ( Rhiner et al . , 2005 ) . [Field: results, subscore: 3.00]
SECTION: results. We assessed the ability of injured neurons to complete a relatively difficult and complex task : full regeneration back to the dorsal nerve cord , which requires growth cone initiation , sustained growth , and directed migration ( Fig- ure 1 B ) . [Field: results, subscore: 3.00]
SECTION: title. Syndecan promotes axon regeneration by stabilizing growth cone migration . [Field: title, subscore: 3.00]
SECTION: abstract. In the absence of syndecan , regenerating growth cones form but are unstable and collapse , decreasing the effective growth rate and impeding regrowth to target cells . [Field: abstract, subscore: 2.00]
SECTION: discussion. Our findings are confirmed by recent regeneration screens in distinct neuron types ( Chen et al . , 2011 ; Nix et al . , 2014 ) , supporting the idea that syndecan is generally required during regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. Other known regeneration factors affect growth cone formation or axon guidance . [Field: discussion, subscore: 2.00]
SECTION: discussion. Model for Syndecan Function during Development and Regeneration ( A ) Syndecan acts as a growth cone stabilizer to prevent collapse and drive efficient migration . [Field: discussion, subscore: 2.00]
SECTION: discussion. The formation and regulation of adhesive contacts may be an essential step in sustained growth cone migration , and it could explain the regeneration defects we observe in sdn-1 mutant worms . [Field: discussion, subscore: 2.00]
SECTION: discussion. We observe that growth cones form and retract on damaged axons in syndecan mutants , failing to sustain growth toward the dorsal nerve cord . [Field: discussion, subscore: 2.00]
SECTION: discussion. ( C and D ) The synthesizing enzyme rib-2 displays normal full regeneration and partial regeneration after axotomy . [Field: discussion, subscore: 2.00]
SECTION: discussion. HSPG-Related Genes Are Not Required for Growth Cone Stability and Migration during Regeneration ( A ) Schematic diagram of the syndecan protein with modified sugar residues ( colored hexagons ) formed by various modifying enzymes . [Field: discussion, subscore: 2.00]
SECTION: discussion. DISCUSSION We have uncovered a role for the HSPG syndecan in promoting axon regeneration ( Figure 7 A ) . [Field: discussion, subscore: 2.00]
SECTION: introduction. In C . elegans , a recent screen in posterior lateral microtubule mechanosensory axons identified syndecan as one of its strongest hits ( Chen et al . , 2011 ) , and syndecan ' s role in regeneration was further validated in a screen for GABAergic neuron regeneration ( Nix et al . , 2014 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. The dynamic regulation of syndecan after neuronal injury suggests that it may have important functions during axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. In particular , the intrinsic mechanisms that function within regenerating neurons to support stable and directed growth cone migration during regeneration are poorly understood . [Field: introduction, subscore: 2.00]
SECTION: introduction. In vivo time-lapse imaging reveals that regenerating growth cones are disorganized and take aberrant trajectories ( Kerschensteiner et al . , 2005 ; Pan et al . , 2003 ; Ylera et al . , 2009 ) , suggesting that lack of sustained and directed migration is a major barrier to successful regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. In the absence of syndecan , regenerating growth cones form but are unstable and collapse , decreasing the effective growth rate and impeding regrowth to target cells . [Field: introduction, subscore: 2.00]
SECTION: introduction. Cell Reports Article Syndecan Promotes Axon Regeneration by Stabilizing Growth Cone Migration Tyson J . Edwards 1 and Marc Hammarlund 1 , * 1Department of Genetics , Program in Cellular Neuroscience , Neurodegeneration and Repair , Yale University School of Medicine , BCMM 436E , 295 Congress Avenue , New Haven , CT 06510 , USA * Correspondence : marc . hammarlund @ yale . edu http : / / dx . doi . org / 10 . 1016 / j . celrep . [Field: introduction, subscore: 2.00]
SECTION: references. Growth was defined as at least 2 concurrent filaments from the stump , an extremely long filament extension , a branch , or a growth cone , which persisted for 3 consecutive frames . [Field: references, subscore: 2.00]
SECTION: references. The growth cone collapses and a new growth cone forms more ventrally on the proximal stump . [Field: references, subscore: 2.00]
SECTION: references. The regenerating axon forms a growth cone structure that moves first ventrally and then dorsally before collapsing and reforming growth on the proximal stump . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration genes identified by RNAi screening in C . elegans . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration in peripheral nerves is enhanced by proteoglycan degradation . [Field: references, subscore: 2.00]
SECTION: references. Notch signaling inhibits axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration pathways identified by systematic genetic screening in C . elegans . [Field: references, subscore: 2.00]
SECTION: results. Thus , our results are consistent with the idea that GABA syndecan is sufficient to mediate growth cone stability and migration during regeneration . [Field: results, subscore: 2.00]
SECTION: results. Alternatively , syndecan could be acting nonautonomously in the hypodermis , which is the substrate for GABA neuron growth during development and regeneration . [Field: results, subscore: 2.00]
SECTION: results. * p < 0 . 05 . acting in an HS-independent manner to promote growth cone migration during regeneration . [Field: results, subscore: 2.00]
SECTION: results. During regeneration , we observed normal levels of full regeneration in slt-1 ( eh15 ) mutants , which encodes a potential null allele of Slit ( Hao et al . , 2001 ) , compared to wild-type ( Figure 5F ) . [Field: results, subscore: 2.00]
SECTION: results. We propose that the syndecan core protein itself mediates growth cone stability during regeneration . [Field: results, subscore: 2.00]
SECTION: results. In sqv-5 homozygotes , we observed normal rates of full and partial regeneration 2 days after axotomy but also an increase in misguided regenerating growth cones ( Figure S1 ) . [Field: results, subscore: 2.00]
SECTION: results. The Effective Growth Rate Is Reduced in sdn-1 Mutants ( AD ) The growth rate was quantified by measuring the distance from the ventral nerve cord to the tip of the axon at the beginning and end of activity periods in wild-type ( A ; Movie S1 ) and sdn-1 mutants ( B ; Movie S2 ) . [Field: results, subscore: 2.00]
SECTION: results. Thus , we conclude that during regeneration , zygotically produced HS is required for axon guidance , but not for growth cone stability . [Field: results, subscore: 2.00]
SECTION: results. Since individual modifications to syndecan ' s HS chains are dispensable for regeneration , we asked whether the HS chains themselves are required for regeneration . [Field: results, subscore: 2.00]
SECTION: results. In order to determine whether individual heparan sulfate sugar modifications are important for syndecan ' s function in axon regeneration , we examined loss-of-function mutants that lack individual modifying enzymes . [Field: results, subscore: 2.00]
SECTION: results. Syndecan Stabilizes Growth Cones during Regeneration ( A ) Representative series of a regenerating axon in a wild-type worm from time-lapse analysis ( see Movie S1 ) . [Field: results, subscore: 2.00]
SECTION: results. These dysmorphic growth structures are consistent with growth cone collapse and other dynamic behaviors observed during our time-lapse analysis . [Field: results, subscore: 2.00]
SECTION: results. In contrast to wild-type axons , which usually exhibit a growth cone structure that migrates toward the dorsal nerve cord ( Figure 4 A ) , we observed a broad range of dysmorphic phenotypes in sdn-1 mutants , including small branches , long filaments , and malformed 274 Cell Reports 8 , 272283 , July 10 , 2014 2014 The Authors growth cone structures ( Figures 4 B and 4C ) . [Field: results, subscore: 2.00]
SECTION: results. Regenerating Axons in sdn-1 Mutants Display Dysmorphic Growth To confirm that the defects in sdn-1 mutants were not caused by lengthy paralysis or repeated imaging associated with time-lapse analysis , we asked whether endpoint analysis could identify the growth cone defects observed in our time-lapse images . [Field: results, subscore: 2.00]
SECTION: results. We conclude that in addition to promoting growth cone stability and inhibiting collapse , syndecan has an important role in translating growth activity into directed migration . [Field: results, subscore: 2.00]
SECTION: results. Unproductive events were defined as events in which essentially no net growth occurred during the activity period ( < 5 mm net dorsal growth ) . [Field: results, subscore: 2.00]
SECTION: results. Activity was characterized by the initiation of sustained filamentous growth from the cut axon and proceeded until activity stopped or the axon reached the dorsal muscles ( see Supple- mental Experimental Procedures [Field: results, subscore: 2.00]
SECTION: results. To deter- mine the effect of this increase in collapses on growth cone perdurance , we measured the duration of growth cones from the time of initiation until collapse or the end of acquisition . [Field: results, subscore: 2.00]
SECTION: results. In fact , half of regenerating sdn-1 axons that initially formed a growth cone exhibited at least one growth cone collapse , as compared to only 17 % in wild-type worms ( Figure 2 C ) . [Field: results, subscore: 2.00]
SECTION: results. By contrast , time-lapse analysis of regeneration in sdn-1 mutants revealed a surprising and unique defect : decreased stability of growth cones ( Figure 2 B ; Movies S2 , S3 , and S4 ) . [Field: results, subscore: 2.00]
SECTION: results. Thus , regeneration in wild-type animals is characterized by growth cone formation at the axon stump and robust migration . [Field: results, subscore: 2.00]
SECTION: results. Fifteen percent formed growth cones that did not reach the dorsal muscles , and 13 % exhibited some filament extension without forming a growth cone ( Table S1 ) . [Field: results, subscore: 2.00]
SECTION: results. Growth Cones Collapse in Regenerating sdn-1 Mutants Our endpoint analysis demonstrated that syndecan was required for long-distance migration during regeneration . [Field: results, subscore: 2.00]
SECTION: results. A Long-Distance Enhancer May Regulate Syndecan Expression during Regeneration We next attempted to rescue regeneration defects in sdn-1 mutants . [Field: results, subscore: 2.00]
SECTION: results. Thus , syndecan is required for axon regeneration , and animals that lack syndecan fail to restore circuit connectivity after nerve injury . [Field: results, subscore: 2.00]
SECTION: results. We found that this extra time increased the amount of regeneration to the dorsal cord in wild-type animals from 32 % to 52 % ( p < 0 . 0001 ) but did not enable any additional regeneration in syndecan mutants ( p = 0 . 1385 , alleles pooled ) . [Field: results, subscore: 2.00]
SECTION: results. To determine whether loss of syndecan blocks or merely delays regeneration , we assessed regeneration after 48 hr in all three alleles and in a sdn-1 transheterozygote ( Figure 1D ) . [Field: results, subscore: 2.00]
SECTION: discussion. Additionally , our time-lapse analysis demonstrates that it is possible to quantify distinct regeneration phenotypes that may remain hidden by single-endpoint assays . [Field: discussion, subscore: 1.00]
SECTION: discussion. We have shown that regeneration of individual GABAergic motor neurons requires the HSPG syndecan . [Field: discussion, subscore: 1.00]
SECTION: discussion. Syndecan also plays an autonomous role in neurons to promote growth cone stability and migration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Guidance during regeneration is dependent on hypodermal expression of sdn-1 and requires the HS synthesizing enzyme rib-2 . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( B ) Syndecan is important for the guidance of axons during development and regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Thus , syndecan can be regulated by a distant enhancer to specifically promote migration , suggesting that failed rescue of regeneration in sdn-1 mutants may be due to a missing expression element . [Field: discussion, subscore: 1.00]
SECTION: discussion. FiRE is activated by different growth factors in a cell-type-specific manner ( Jaakkola et al . , 1998a ; Rautava et al . , 2003 ) and is upregulated specifically in migrating , but not proliferating , keratinocytes in vivo ( Jaakkola et al . , 1998b ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. The inability to rescue the syndecan regeneration defects with tissue-specific and native promoters suggests that sdn-1 regulation is under tight transcriptional control . [Field: discussion, subscore: 1.00]
SECTION: discussion. Alternatively , syndecan could promote regeneration through adhesion pathways that modify actin filaments . [Field: discussion, subscore: 1.00]
SECTION: discussion. Microtubule remodeling at the growth cone is required for axon elongation and sustained migration ( Bradke et al . , 2012 ; Dent and Gertler , 2003 ; Erturk et al . , 2007 ; Vitriol and Zheng , 2012 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. One potential intracellular target of neuronal syndecan that might mediate growth cone stability is the axonal microtubule network . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( F ) Full regeneration is decreased in wild-type worms on sdn-1 RNAi . [Field: discussion, subscore: 1.00]
SECTION: discussion. Arrowhead shows the growth cone . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( D ) Image of a misguided growth cone in a wildtype worm on sdn-1 RNAi after laser axotomy . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( C ) Partial regeneration is significantly decreased in sdn-1 mutants ( zh20 and ev697 alleles pooled ) after 24 hr , whereas it is the same in control versus sdn-1 RNAi . [Field: discussion, subscore: 1.00]
SECTION: discussion. Growth Cone Formation and Migration Is Maintained in Syndecan-RNAi Animals ( A ) An example of a developmental misguidance error in a sdn-1 ( zh20 ) animal . The axon migrated toward the dorsal nerve cord but then turned prematurely and migrated toward the tail . [Field: discussion, subscore: 1.00]
SECTION: discussion. HS binds multiple extracellular signaling molecules ( Bernfield et al . , 1999 ) , including fibroblast growth factor ( FGF ) ( Schlessinger et al . , 2000 ; Yayon et al . , 1991 ) , Slit-Robo ( Hussain et al . , 2006 ) , and Anosmin-1 , a secreted protein involved in neurite outgrowth and branching ( Hu et al . , 2004 ; Soussi-Yanicostas et al . , 1996 , 1998 , 2002 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. Syndecan ' s neuronal role in stabilizing growth cones may be independent of HS sugar chains , in contrast to its HS-dependent function in axon guidance . [Field: discussion, subscore: 1.00]
SECTION: discussion. Thus , syndecan may have a distinct role in stabilizing growth cones postdevelopmentally when the extracellular environment has changed ( Morgan et al . , 2007 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. These defects in growth cone stability limit the ability of regenerating axons to reconnect with their postsynaptic targets . [Field: discussion, subscore: 1.00]
SECTION: discussion. * p < 0 . 05 , * * * p < 0 . 0005 . to initiate , endure for shorter time periods , and are more prone to collapse , indicating a primary role for syndecan in stabilizing growth cones . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( G and H ) sdn-1 ( zh20 ) slt-1 ( eh15 ) double mutants display reduced partial and full regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( F ) Full regeneration is not reduced in slt-1 ( eh15 ) animals . [Field: discussion, subscore: 1.00]
SECTION: discussion. ( B ) The modifying enzymes do not show deficits in full regeneration after axotomy . [Field: discussion, subscore: 1.00]
SECTION: discussion. Although growth cones can form in response to injury in the absence of syndecan , they take longer Figure 5 . [Field: discussion, subscore: 1.00]
SECTION: introduction. We find that severed neurons in syndecan mutants fail to regenerate due to decreased growth cone stability . [Field: introduction, subscore: 1.00]
SECTION: introduction. In order to address the role of syndecan after neuronal injury , we examined regeneration in C . elegans syndecan mutants using laser axotomy . [Field: introduction, subscore: 1.00]
SECTION: introduction. Additionally , heparin--a closely related polysaccharide--makes ternary complexes with both fibroblast growth factor ( FGF ) and its receptor ( Schlessinger et al . , 2000 ; Yayon et al . , 1991 ) and Slit / Robo ( Hussain et al . , 2006 ; Johnson et al . , 2004 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. Consistent with this idea , HS binds multiple signaling molecules , including the morphogens Sonic Hedgehog , Wnts , and BMPs , insoluble extracellular matrix components such as fibronectin and laminin , and growth factors ( Bernfield et al . , 1999 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. Regenerating growth cones face challenges not encountered during development , including extended migratory distances , altered extracellular environments , increased target selection complexity , and new physical barriers . [Field: introduction, subscore: 1.00]
SECTION: materials. We placed L2- to L3-stage worms on RNAi plates and then examined L4- or adult-stage animals from subsequent generations for developmental and regeneration phenotypes . [Field: materials, subscore: 1.00]
SECTION: materials. Only axons that exhibited growth activity from the injured neuron were analyzed . [Field: materials, subscore: 1.00]
SECTION: materials. Axons that exhibited growth were quantified and compared using an unpaired t test . Time-Lapse Imaging We immobilized three to five worms simultaneously on 5 % 7 % agarose pads , mounted in 1 ml of 50 nm polystyrene microbeads ( Fang-Yen et al . , 2012 ) spiked with 0 . 55 mM muscimol . [Field: materials, subscore: 1.00]
SECTION: materials. Misguided regenerating axons were counted as the number of misguided axons divided by the number of total regenerating axons and represented instances where the growth cone extended but in anterior , posterior , or ventral directions , thus not necessarily reaching the midline or the dorsal cord . [Field: materials, subscore: 1.00]
SECTION: materials. Full regeneration represented axons that regenerated to the dorsal nerve cord . [Field: materials, subscore: 1.00]
SECTION: materials. Regeneration was typically assessed 1 day ( approximately 1824 hr ) or 2 days ( approximately 4248 hr ) postaxotomy , with an internal control done on the same day . [Field: materials, subscore: 1.00]
SECTION: materials. rib-2 mutants survive only from balanced heterozygotes , and only homozygous progeny that lost the fluorescent balancer were analyzed for regeneration . [Field: materials, subscore: 1.00]
SECTION: materials. EXPERIMENTAL PROCEDURES C . elegans Strains Worm strains were maintained at 20 C or room temperature on nematode growth media ( NGM ) plates seeded with OP50 bacteria . [Field: materials, subscore: 1.00]
SECTION: references. For measurements of growth cone duration , we included only those axons that initiated at least 300 minutes before the movie end time . [Field: references, subscore: 1.00]
SECTION: references. A growth cone was defined as a protrusion that had a membrane thickening , or palm , with at least 2 filaments or branches , and persisted for at least 30 minutes . [Field: references, subscore: 1.00]
SECTION: references. We compared the mean growth rates differentially for axons that grew above or below 5 micrometers . [Field: references, subscore: 1.00]
SECTION: references. We measured each period of growth for every axon . [Field: references, subscore: 1.00]
SECTION: references. Regeneration Data , Related to Figures 1-6 . [Field: references, subscore: 1.00]
SECTION: references. We observed significant decreases in the number of axons that migrated to the dorsal muscle boundary in sdn-1 mutants , as well as a decrease in the total percentage of growth cones formed . [Field: references, subscore: 1.00]
SECTION: references. Regeneration Phenotypes Observed in Time-lapse Analysis , Related to Figures 2 and 3 . [Field: references, subscore: 1.00]
SECTION: references. The regenerating axon forms a growth cone and moves to the dorsal nerve cord , collapsing several times before reaching the three-quarter mark . [Field: references, subscore: 1.00]
SECTION: references. Once migrating approximately three-quarters of the distance to the dorsal nerve cord , the growth cone spreads out and moves posteriorly . [Field: references, subscore: 1.00]
SECTION: references. The regenerating axon forms a growth cone and migrates dorsally . [Field: references, subscore: 1.00]
SECTION: references. ( B ) Full regeneration at 48 hours is also not reduced compared to wild type ( p = 0 . 1185 ) . [Field: references, subscore: 1.00]
SECTION: references. ( A ) Partial regeneration after 48 hours is normal in sqv- 5 ( n3611 ) mutants . [Field: references, subscore: 1.00]
SECTION: references. sqv-5 mutants can sustain growth cone migration after injury , related to Figure 5 . [Field: references, subscore: 1.00]
SECTION: references. Cell surface , heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor . [Field: references, subscore: 1.00]
SECTION: references. Growth cone travel in space and time : the cellular ensemble of cytoskeleton , adhesion , and membrane . [Field: references, subscore: 1.00]
SECTION: references. Anosmin-1 underlying the X chromosomelinked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner . [Field: references, subscore: 1.00]
SECTION: references. SDN-1 / syndecan regulates growth factor signaling in distal tip cell migrations in C . elegans . [Field: references, subscore: 1.00]
SECTION: references. Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo . [Field: references, subscore: 1.00]
SECTION: references. Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans . [Field: references, subscore: 1.00]
SECTION: references. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord . [Field: references, subscore: 1.00]
SECTION: references. Wound reepithelialization activates a growth factor-responsive enhancer in migrating keratinocytes . [Field: references, subscore: 1.00]
SECTION: references. The activation and composition of FiRE ( an FGF-inducible response element ) differ in a cell type- and growth factor-specific manner . [Field: references, subscore: 1.00]
SECTION: references. Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor family members in mesenchymal cells . [Field: references, subscore: 1.00]
SECTION: references. Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration . [Field: references, subscore: 1.00]
SECTION: references. Growth factors induce 3T3 cells to express bFGF-binding syndecan . [Field: references, subscore: 1.00]
SECTION: references. Cytoskeletal dynamics and transport in growth cone motility and axon guidance . [Field: references, subscore: 1.00]
SECTION: references. A novel function to control binding of midkine , pleiotrophin , and basic fibroblast growth factor . [Field: references, subscore: 1.00]
SECTION: references. Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors . [Field: references, subscore: 1.00]
SECTION: references. Heparan sulphates upregulate regeneration of transected sciatic nerves of adult guinea-pigs . [Field: references, subscore: 1.00]
SECTION: results. Thus , hypodermal syndecan is required to mediate syndecan ' s function in axon guidance ( during development and regeneration ) . [Field: results, subscore: 1.00]
SECTION: results. However , although regenerating axons in syndecan RNAi animals could sustain growth , they were often misguided in the anterior , Cell Reports 8 , 272283 , July 10 , 2014 2014 The Authors 277 posterior , or ventral directions ( Figure 6D and 6E ) , resulting in a decrease in the number of axons that regenerated fully to the dorsal nerve cord ( Figure 6 F ) . [Field: results, subscore: 1.00]
SECTION: results. We found that syndecan RNAi animals , in contrast to sdn-1 mutant alleles , were able to sustain wild-type levels of partial regeneration ( Figure 6C ) . [Field: results, subscore: 1.00]
SECTION: results. Next , we cut axons in syndecan RNAi animals and assessed regeneration . [Field: results, subscore: 1.00]
SECTION: results. Thus , syndecan could be acting directly in GABAergic motor neurons to promote growth cone stability . [Field: results, subscore: 1.00]
SECTION: results. Syndecan Acts in Regenerating Neurons to Promote Sustained Migration We next sought to determine where syndecan is functioning to promote the migration of regenerating growth cones . [Field: results, subscore: 1.00]
SECTION: results. Thus , we conclude that syndecan ' s effects on regeneration are not being mediated through Slit signaling . [Field: results, subscore: 1.00]
SECTION: results. Additionally , slt-1 did not suppress the regeneration defects of sdn-1 mutants ( Figures 5 G and 5H ) . [Field: results, subscore: 1.00]
SECTION: results. Additionally , heparin is required for Slit-induced increases in growth cone collapse in vitro ( Hussain et al . , 2006 ) . [Field: results, subscore: 1.00]
SECTION: results. While C . elegans is devoid of chondroitin sulfate ( Yamada et al . , 1999 ) , we investigated whether nonsulfated chondroitin plays a role in regeneration by examining the chondroitin synthase mutant sqv-5 ( Hwang et al . , 2003 ; Mizuguchi et al . , 2003 ) . [Field: results, subscore: 1.00]
SECTION: results. ( F ) The average growth rate for productive events is decreased by 47 % in sdn-1 mutants , whereas the average rate for unproductive events is unchanged from wild-type . [Field: results, subscore: 1.00]
SECTION: results. Dorsal progression ( red bars ) was divided by duration of growth to determine the effective migration rate . [Field: results, subscore: 1.00]
SECTION: results. However , it remains a possibility that maternally contributed rib-2 could persist to mediate syndecan ' s role in growth cone stability , but not in axon guidance . [Field: results, subscore: 1.00]
SECTION: results. Interestingly , we did observe an increase in misguided regenerating axons in the rib-2 mutants ( Figure 5 E ) , demonstrating that heparan sulfate is essential for axon guidance during regeneration , just as it is during neuronal development . [Field: results, subscore: 1.00]
SECTION: results. Thus , individual modifications to syndecan ' s heparan sulfate chains , though required for development of the nervous system , are not required to promote growth cone migration after injury of the GABAergic motor neurons . [Field: results, subscore: 1.00]
SECTION: results. We found that modifying enzyme mutants display wild-type levels of full regeneration ( Figure 5B ) . [Field: results, subscore: 1.00]
SECTION: results. N ( axons with growth cones ) R 30 . [Field: results, subscore: 1.00]
SECTION: results. ( F ) The average time to growth cone initiation is increased in sdn-1 mutants . [Field: results, subscore: 1.00]
SECTION: results. N ( growth cones ) > 20 . [Field: results, subscore: 1.00]
SECTION: results. ( D ) The total duration of the growth cone from initiation to collapse or completion is decreased in sdn-1 mutants . [Field: results, subscore: 1.00]
SECTION: results. N ( axons with growth cones ) R 30 . [Field: results, subscore: 1.00]
SECTION: results. ( C ) Approximately three times as many axons exhibit growth cone collapse in sdn-1 mutants . [Field: results, subscore: 1.00]
SECTION: results. Arrowheads represent growth cones , and scale bars are 5 mm . [Field: results, subscore: 1.00]
SECTION: results. It then grows actively toward the dorsal nerve cord but collapses ( * ) as new growth is initiated on the proximal stump . [Field: results, subscore: 1.00]
SECTION: results. A growth cone forms at the tip and then turns back onto the proximal stump ( # ) . [Field: results, subscore: 1.00]
SECTION: results. Once reaching the dorsal muscle boundary , the growth cone stalls and begins to branch out along the anterior-posterior axis but does not collapse . [Field: results, subscore: 1.00]
SECTION: results. The growth cone forms and rapidly migrates toward the dorsal nerve cord . [Field: results, subscore: 1.00]
SECTION: results. Syndecan ' s Role in Stabilizing Growth Cones May Not Require Sugar Chains Syndecan ' s glycosaminoglycan chains are heavily modified by a diverse group of heparan sulfate-modifying enzymes , including Figure 2 . [Field: results, subscore: 1.00]
SECTION: results. We conclude that syndecan defects can be observed at single time points after axotomy and that growth cone defects in sdn-1 mutants are independent of the imaging process . [Field: results, subscore: 1.00]
SECTION: results. Consistent with the dysmorphic growth and stunted migration in sdn-1 mutants , we observed a significant decrease in the lengths of severed axons in sdn-1 mutants ( Figure 4 E ) . [Field: results, subscore: 1.00]
SECTION: results. We recovered animals to normal conditions after surgery and analyzed regeneration approximately 24 hr later . [Field: results, subscore: 1.00]
SECTION: results. Productive events , in which net dorsal growth did occur ( > 5 mm ) , were of relatively longer duration ( WT mean = 197 min , p = 0 . 0003 versus WT unproductive ; sdn-1 mean = 284 min , p = 0 . 0008 versus sdn-1 unproductive ) . [Field: results, subscore: 1.00]
SECTION: results. We measured the distance from the ventral nerve cord to the dorsal-most point of the axon at the beginning and end of activity periods and then calculated the effective growth rate during that period ( Figures 3C and 3D ) . [Field: results, subscore: 1.00]
SECTION: results. Growth cone destabilization severely impaired dorsal progression , as only 17 % of sdn-1 mutant axons reached the dorsal muscles during the analysis period ( Table S1 ) . [Field: results, subscore: 1.00]
SECTION: results. We found that growth cones in wild-type persisted for an average of 480 min , whereas those in sdn-1 mutants lasted only 281 min ( Figure 2D ) . [Field: results, subscore: 1.00]
SECTION: results. In total , 70 % of wild-type axons migrated to the dorsal muscles within our imaging time window ( Table S1 ) , consistent with the robust growth observed in our 24 hr and 48 hr endpoint analysis ( Figure 1 ) . [Field: results, subscore: 1.00]
SECTION: results. These growth cones quickly migrated toward the dorsal side of the animal until reaching the dorsal muscles ( Figure 2A ; Movie S1 ) . [Field: results, subscore: 1.00]
SECTION: results. In wild-type animals , most severed axons that initiated activity formed growth cones at or near the tip of the stump . [Field: results, subscore: 1.00]
SECTION: results. In total , our analysis of 61 wild-type and 47 sdn-1 axons revealed a requirement for syndecan in preventing growth cone collapse and promoting high-speed migration . [Field: results, subscore: 1.00]
SECTION: results. Regenerating GABAergic motor neurons were imaged at 4 min intervals in the period after axotomy when regenerating axons were most likely to exhibit active growth . [Field: results, subscore: 1.00]
SECTION: results. We conclude that unidentified , long-distance regulatory elements or genomic positional requirements may be necessary for achieving proper sdn-1 expression levels during regeneration . [Field: results, subscore: 1.00]
SECTION: results. We also made a MosSci insertion expressing sdn-1 from a neuron-specific promoter ; this insertion only moderately rescued the guidance defects and also displayed no rescue of regeneration ( data not shown ) . [Field: results, subscore: 1.00]
SECTION: results. However , neither insertion significantly improved regeneration ( data not shown ) . [Field: results, subscore: 1.00]
SECTION: results. Syndecan transheterozygotes display reduced full regeneration 2 days after axotomy . [Field: results, subscore: 1.00]
SECTION: results. ( C and D ) Full regeneration is decreased in all three sdn-1 alleles at 1 day ( C ) and 2 days ( D ) after axotomy . [Field: results, subscore: 1.00]
SECTION: results. ( B ) Representative image of full regeneration after laser axotomy . [Field: results, subscore: 1.00]
SECTION: results. Syndecan Is Required for Axon Regeneration ( A ) Syndecan is the only transmembrane HSPG and consists of long heparan sulfate sugar chains attached to a protein core . [Field: results, subscore: 1.00]
Match: [Sentence(s) appears to be scrambled. Click to see (opens new window)] [Field: references, subscore: 51.00]
Supplemental links/files: reference in endnote reference in xml Wormbase reference
Score: 355.00
Title: Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth .
Authors: Chung SH ; Awal MR ; Shay J ; McLoed MM ; Mazur E ; Gabel CV
Journal: Proc Natl Acad Sci U S A
Year: 2016-04-12
Doc ID: WBPaper00049460
Bibliographic Information
Abstract
Matching Sentences
SECTION: acknowledgments. No significant difference in regeneration rates between various trx-1 alleles and trx-1 ( + ) for ( A ) DLK-independent regeneration , ( B ) ectopic outgrowth , and ( C ) conventional regeneration ( 32 n 68 for regeneration data , except n 18 for pgpa-9 : : gfp ; 170 n 235 for ectopic outgrowth data ) . [Field: acknowledgments, subscore: 4.00]
SECTION: acknowledgments. For instance , the JNK pathway in C . elegans critically underlies conventional motor axon regeneration ( 14 ) but does not underlie conventional ASJ regeneration or DLK- independent regeneration ( Fig . 2A ) . [Field: acknowledgments, subscore: 4.00]
SECTION: acknowledgments. Thus , we observe several forms of regeneration , including a conventional DLK- mediated regeneration triggered by axon cut only , a DLK- independent regeneration triggered by axon + dendrite cut , and Chung et al . www . pnas . org / cgi / content / short / 1600564113 2 of 8 an enhanced DLK-mediated regeneration also triggered by axon + dendrite cut . [Field: acknowledgments, subscore: 4.00]
SECTION: introduction. In a previous study , we sought to dissect the subcellular components of the C . elegans ASJ sensory neurons by femtosecond laser surgery to establish their role in development and behavior Significance By laser surgery , genetics , and pharmacology , we demonstrate that neurons of the nematode Caenorhabditis elegans undergo a novel form of regeneration that is largely independent of defined regeneration pathways , including DLK , which underlies axon regeneration from C . elegans to mammals . [Field: introduction, subscore: 4.00]
SECTION: results. These results underscore the dendrite lesion ' s crucial role in enhancing axon regeneration and provide another phenotypic similarity between DLK-independent regeneration and mammalian lesion-conditioned regeneration . [Field: results, subscore: 4.00]
SECTION: results. , results are consistent with ASJ findings indicating two parallel regenerative pathways that are at dlk-1 ( - ) , axon cut regeneration dlk-1 ( + ) , no axon cut ectopic outgrowth dlk-1 ( - ) , no axon cut ectopic outgrowth downstream signaling loss-of-function sensory signaling none * * * * * * * * * * * * * * * * * * % regeneration in dlk-1 100 80 60 40 20 0 100 80 60 40 20 0 % ectopic outgrowth at 25 C outgrowth mutation unc-43 ( gf ) + dend cut * * unc-43 ( lf ) * * sax-1 ( lf ) * * daf-1 1 * * unc-36 * * osm-6 * * tax-2 * tax-4 * * egl-19 dend cut * * * * control B axon + dendrite cut axon cut 100 80 60 40 20 0 % regeneration fos-1 ns dlk-1 ; fos-1 ns dlk-1 ; kgb-1 * dlk-1 ; mlk-1 ns dlk-1 ; cebp-1 ns cebp-1 ns dlk-1 ( km12 ) ns dlk-1 ( ju476 ) dlk-1 ; kgb-1 ns dlk-1 ; mlk-1 ns dlk-1 ; cebp-1 ns dlk-1 ( km12 ) ns dlk-1 ( ju476 ) cebp-1 * * fos-1 * * kgb-1 ns mlk-1 ns wt conventional regeneration in motor / amphid neurons cebp-1 pmk-3 dlk-1 fos-1 kgb-1 mlk-1 A Fig . 2 . [Field: results, subscore: 4.00]
SECTION: results. Although DLK-independent regeneration is distinct from previously defined axon regeneration , we observe striking similarities to another type of amphid growth called activity-dependent ectopic axon outgrowth , which occurs primarily at 25 C cultivation . [Field: results, subscore: 4.00]
SECTION: acknowledgments. ( G ) Multiple pathways mediate regeneration in ALM neuron ; dlk-1 and unc-43 ( gf ) independently reduce length of ALM axon regeneration in dlk-1 ( + ) and dlk-1 backgrounds ( 20 n 29 ) . [Field: acknowledgments, subscore: 3.00]
SECTION: acknowledgments. Chung et al . www . pnas . org / cgi / content / short / 1600564113 3 of 8 gain-of-function wild-type loss-of-function unc-43 allele : ALM neuron dlk-1 ( + ) dlk-1 ( ju476 ) 200 150 100 50 0 length regenerated ( m ) * * ns * * * * ns G dlk-1 ; kgb-1 a + d ns dlk-1 ; tax-2 axon ns 15 C 20 C 25 C Tcult % regeneration 100 80 60 40 20 0 F % regeneration 100 80 60 40 20 0 stage at reimaging dauer * * L4 * * L2 dlk-1 ; daf-11 L1 surgery axon cut E # outgrowths per neuron 1 . 5 1 . 0 0 . 5 0 days after surgery 5 4 * * * * 3 * * * * 2 * 1 dlk-1 ; daf-11 axon dlk-1 a + d total D none not to ring to ring regen type : days after surgery 4 * * 3 * * 2 ns 1 % regeneration 100 80 60 40 20 0 C dlk-1 ; daf-11 axon cut 100 80 60 40 20 0 length regenerated ( m ) * * dlk-1 a + d * * dlk-1 axon wt axon ASH neuron B dendrite posterior anterior iii ii 10 m i A Fig . S1 . [Field: acknowledgments, subscore: 3.00]
SECTION: acknowledgments. Regeneration rates of dlk-1 ; unc-36 , although statistically higher than dlk-1 regeneration rates , belie an abortive regeneration phenotype . [Field: acknowledgments, subscore: 3.00]
SECTION: discussion. In mammals , DLK underlies preconditioning of peripheral axon regeneration ( 21 ) , a conditioning effect on DRG neurons in vitro ( 43 ) , and retinal ganglion cell regeneration after optic nerve crush ( 44 ) . [Field: discussion, subscore: 3.00]
SECTION: discussion. They are all axon outgrowth and regeneration unc-43 / CAMKII , sax-1 / NDR kinase electrical activity egl-19 / L-VGCC , tax-4 / CNGA1 , tax-2 / CNGB1 intact sensory dendrite cell structure ( osm-6 / IFT52 ) sensory transduction ( daf-11 ) environmental stimuli axon outgrowth inhibition ( activity-dependent ) DLK-independent regeneration dlk-1 / DLK ( motor , amphid ) kgb-1 / JNK ( motor ) cellular damage response ( e . . , Ca2 + influx ) axon injury conventional regeneration Fig . 4 . [Field: discussion, subscore: 3.00]
SECTION: introduction. We also note numerous similarities with lesion-conditioned regeneration , in which reduction of sensory activity triggers robust axon regeneration in the mammalian CNS . [Field: introduction, subscore: 3.00]
SECTION: introduction. Taken as a whole , our study unites disparate forms of neuronal outgrowth to uncover fresh molecular insights into activity-dependent control of the adult nervous system ' s intrinsic regenerative capacity . lesion conditioning | axon regeneration | femtosecond laser ablation | DLK-1 | activity-dependent ectopic axon outgrowth One of the principal goals of modern neuroscience is the comprehensive understanding and therapeutic application of neuronal regeneration ( 1 ) . [Field: introduction, subscore: 3.00]
SECTION: results. In a classic lesion conditioning experiment , peripheral sensory axotomy before central axotomy , known as preconditioning , more strongly improves central axon regeneration than concomitant axotomies and vice versa , suggesting a cellular intrinsic growth capacity that is enhanced by peripheral axotomy ( 39 ) . [Field: results, subscore: 3.00]
SECTION: results. Full-length regeneration included in regeneration " growth along ring " category . [Field: results, subscore: 3.00]
SECTION: results. Despite the limited DLK-independent regeneration at earlier reimaging times shown in Fig . 1D , reimaging at later times confirms continued regeneration that , although slower than DLK-mediated regeneration , eventually reaches and extends along the nerve ring ( Figs . 1 C and D and 3D for length measurement ) . [Field: results, subscore: 3.00]
SECTION: results. Both conventional regeneration after axotomy alone in WT animals and DLK-independent regeneration generate similar outgrowths of three basic morphologies , although the dynamics of regeneration are distinct ( see below ) . [Field: results, subscore: 3.00]
SECTION: abstract. Moreover , we note numerous similarities betweenC . elegansDLK-independent regeneration and lesion conditioning , a phenomenon producing robust regeneration in the mammalian CNS . [Field: abstract, subscore: 2.00]
SECTION: abstract. Here , we identify a novel form of neuronal regeneration , which is remarkably independent of DLK-1 / DLK , KGB-1 / JNK , and other MAPK signaling factors known to mediate regeneration inCaenorhabditis elegans , Drosophila , and mammals . [Field: abstract, subscore: 2.00]
SECTION: acknowledgments. trx-1 is not crucially involved in DLK-independent regeneration , ectopic outgrowth , and conventional regeneration . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Chung et al . www . pnas . org / cgi / content / short / 1600564113 6 of 8 trx-1 ( ok1449 ) ; ptrx-1 : : gfp trx-1 ( - ) pgpa-9 : : gfp or ptrx-1 : : gfp trx-1 ( + ) ptrx-1 : : trx-1 : : gfp trx-1 ( + + ) gain-of-function wild-type loss-of-function 100 80 60 40 20 0 % regeneration trx-1 ( + + ) ns trx-1 ( + ) ptrx-1 : : gfp C background trx-1 ( + + ) trx-1 ( + ) trx-1 ( - ) trx-1 ( + + ) trx-1 ( + ) trx-1 ( - ) ptrx-1 : : gfp ptrx-1 : : gfp trx-1 ( + ) pgpa-9 : : gfp % regeneration 100 80 60 40 20 0 100 80 60 40 20 0 % ectopic outgrowth at 25 C ns ns no outgrowth mutation ns ns tax-2 ( p691 ) B ns ns ns dlk-1 , axon + dendrite cut ns ns ns dlk-1 , axon cut A Fig . S4 . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Second , simple binary scoring of regeneration is sufficient in most cases to demonstrate triggering of DLK-independent regeneration , and practically , it is more feasible and reliable over the large number of experiments performed here . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Thus , our observations not only demonstrate a novel form of regeneration but also begin to illuminate a greater underlying complexity in regeneration processes . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Fourth , although L-VGCCs mediate sensory signaling that inhibits DLK-independent regeneration , in other contexts , they mediate beneficial calcium signals during the damage response : blocking L-VGCCs with nemadipine-A results in decreased calcium response and reduced conventional regeneration after PLM axotomy ( 16 ) . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Moreover , even after surgery in young adults , when DLK-independent regeneration is active , dendrite lesions increase WT-regenerated length beyond what might be predicted from strictly adding DLK-independent regenerated length , suggesting that dendrite injury enhances both DLK-independent and DLK-mediated regeneration pathways . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. First , the data in Fig . 1D indicate that dendrite lesions in WT animals enhance regeneration before the L4 stage , when DLK-independent regeneration from dendrite lesion is first observed . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. However , the regeneration rate in all outgrowing strains is significantly greater than the ectopic outgrowth rate , and therefore , there is significant regeneration triggered by outgrowth mutations . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. These data indicate that trx-1 is not crucially involved in DLK-independent regeneration , ectopic outgrowth , or conventional regeneration , permitting the use of ptrx-1 : : trx-1 : : gfp for ASJ visualization . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. We find that , although dlk-1 mutation completely eliminates conventional regeneration in some other neuron types , such as motor neurons ( 12 ) and the ASJ , dlk-1 reduces ALM regeneration by only 30 % ( Fig . S1G ) , confirming results of our previous study ( 15 ) . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. The ALM is a well-established model neuron for axon regeneration in Caenorhabditis elegans ( 15 ) . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Thus , the ASH results confirm ASJ findings that dlk-1 reduces conventional regeneration and dendrite lesion enhances DLK-independent regeneration . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. The total regeneration length is similar , regardless of the position of damage or regeneration . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Gabel CV , Antoine F , Chuang CF , Samuel AD , Chang C ( 2008 ) Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C . elegans . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Li C , Hisamoto N , Matsumoto K ( 2015 ) Axon regeneration is regulated by Ets-C / EBP transcription complexes generated by activation of the cAMP / Ca2 + signaling pathways . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Qiu J , et al . ( 2002 ) Spinal axon regeneration induced by elevation of cyclic AMP . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Nix P , et al . ( 2014 ) Axon regeneration genes identified by RNAi screening in C . elegans . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Nix P , Hisamoto N , Matsumoto K , Bastiani M ( 2011 ) Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Yan D , Wu Z , Chisholm AD , Jin Y ( 2009 ) The DLK-1 kinase promotes mRNA stability and local translation in C . elegans synapses and axon regeneration . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Hammarlund M , Nix P , Hauth L , Jorgensen EM , Bastiani M ( 2009 ) Axon regeneration requires a conserved MAP kinase pathway . [Field: acknowledgments, subscore: 2.00]
SECTION: acknowledgments. Liu K , Tedeschi A , Park KK , He ZG ( 2011 ) Neuronal intrinsic mechanisms of axon regeneration . [Field: acknowledgments, subscore: 2.00]
SECTION: discussion. As such , our study establishes a framework for defining the mechanisms that modulate a neuron ' s intrinsic growth capacity and ultimately enable robust regeneration within the adult nervous system . [Field: discussion, subscore: 2.00]
SECTION: discussion. The striking similarities between DLK-independent regeneration in C . elegans and lesionconditioned regeneration in the mammalian CNS suggest a conserved mechanism . [Field: discussion, subscore: 2.00]
SECTION: discussion. Physical damage of the axon leads to conventional regeneration through specific damage-induced calcium signaling that initiates dlk-1 and kgb-1 mediated regeneration ( orange pathway in Fig . 4 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. Importantly , DLK-independent and lesion-conditioned regeneration also exhibit a similar regeneration phenotype dependent on the sequence and timing of neurite lesions , including a preconditioning effect . [Field: discussion, subscore: 2.00]
SECTION: discussion. DLK-independent regeneration and ectopic outgrowth in C . elegans also share numerous similarities with lesion-conditioned regeneration observed in the mammalian CNS . [Field: discussion, subscore: 2.00]
SECTION: discussion. Although a recent study showed that dendrite regeneration in Drosophila dendritic arborization neurons is also independent of both DLK and JNK ( 40 ) , an analogous mechanism initiated by axon surgery has not been defined . [Field: discussion, subscore: 2.00]
SECTION: discussion. This novel regeneration is largely independent of DLK-1 / DLK , KGB-1 / JNK , and other known MAPK signaling factors underlying neuronal regeneration in C . elegans and other organisms . [Field: discussion, subscore: 2.00]
SECTION: introduction. As such , our study establishes a novel regeneration pathway in C . elegans , where physical , genetic , or pharmacological lesion of a sensory neurite or sensory signaling removes activity-dependent developmental inhibitors to permit robust regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. Finally , we note similarities between DLK-independent regeneration in C . elegans and regeneration after lesion conditioning in mammals , including shared phenotypes , role of L-type voltage-gated calcium channel ( L-VGCC ) , and mediation by cyclic adenosine monophosphate ( cAMP ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. First , regeneration occurs after severing both the axon and sensory dendrite , indicating that sensory dendrite cuts trigger robust DLK- independent regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. Remarkably , although dlk-1 ( ju476 ) completely prevents regeneration after axotomy , we discovered that strong DLK-independent regeneration returns under two conditions , which we explore in this study . [Field: introduction, subscore: 2.00]
SECTION: introduction. ASJ axons rapidly regenerate to the vicinity of their original synaptic targets , but regeneration is prevented by mutation of dlk-1 , which plays a crucial role in neuronal regeneration in C . elegans ( 12 ) , Drosophila ( 19 ) , and mammals ( 20 , 21 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Our study unites disparate forms of neurite outgrowth to uncover the molecular mechanisms that modulate regeneration in the adult CNS and suggests that ectopic outgrowth might represent a powerful gene discovery platform for regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. A striking exception to this paradigm is lesion conditioning , a phenomenon exemplified by the dorsal root ganglion ( DRG ) , where peripheral axon damage enables robust central axon regeneration ( 4 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Studies demonstrate that axon regeneration recruits or recapitulates mechanisms involved in a diverse range of biological processes , including synapse formation , stress response , apoptosis , and development . [Field: introduction, subscore: 2.00]
SECTION: introduction. Efforts to enhance axon regeneration generally fall into two broad categories : eliminating or blocking nonpermissive extrinsic inhibitors or promoting a neuron ' s intrinsic regenerative capacity ( 2 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Moreover , we note numerous similarities between C . elegans DLK-independent regeneration and lesion conditioning , a phenomenon producing robust regeneration in the mammalian CNS . [Field: introduction, subscore: 2.00]
SECTION: introduction. Here , we identify a novel form of neuronal regeneration , which is remarkably independent of DLK-1 / DLK , KGB-1 / JNK , and other MAPK signaling factors known to mediate regeneration in Caenorhabditis elegans , Drosophila , and mammals . [Field: introduction, subscore: 2.00]
SECTION: introduction. Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth Samuel H . Chunga , b , c , Mehraj R . Awalb , c , James Shayb , c , Melissa M . McLoedb , c , Eric Mazura , d , and Christopher V . Gabelb , c , 1 aSchool of Engineering and Applied Sciences , Harvard University , Cambridge , MA 02138 ; bDepartment of Physiology and Biophysics , Boston University School of Medicine , Boston , MA 02118 ; cBoston University Photonics Center , Boston , MA 02215 ; and dDepartment of Physics , Harvard University , Cambridge , MA 02138 Edited by Cornelia I . Bargmann , Rockefeller University , New York , NY , and approved March 1 , 2016 ( received for review January 15 , 2016 ) During development , a neuron transitions from a state of rapid growth to a stable morphology , and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury . [Field: introduction, subscore: 2.00]
SECTION: materials. Although a report noted AWB regeneration after dendrite surgery ( 54 ) , we do not observe regeneration after ASJ dendrite surgery ( 23 ) . [Field: materials, subscore: 2.00]
SECTION: results. In mammals , reduced L-VGCC calcium current contributes to axon regeneration of DRG neurons ( 5 ) , suggesting a possible mechanistic link to the outgrowth mechanisms that we observe here . [Field: results, subscore: 2.00]
SECTION: results. Although endogenous ( i . . , resting ) activity of mammalian DRG neurons is not significantly changed by neurite lesions , the loss of sensory-evoked activity appears to be an important signal to trigger central axon regeneration by lesion conditioning . [Field: results, subscore: 2.00]
SECTION: results. ( D ) Dendrite cuts enhance axon regeneration in a time-dependent manner . [Field: results, subscore: 2.00]
SECTION: results. Interestingly , the DRG full length along ring to ring not to ring none regeneration type : % neurons 100 50 0 150 100 50 0 length regenerated ( m ) -24 24 0 24 48 72 0 72 -4 . 5 7 0 7 10 24 0 24 tdend ( h ) treimage * * dlk-1 pre * * dlk-1 post * wt pre * wt post D taxon = 0 h 50 40 30 20 10 0 % ectopic outgrowth at 25 C cAMP mutation dlk-1 ( + ) dlk-1 ( - ) kin-2 * * * * pde-4 * * * acy-1 * * * wt nema * * DMSO gf wt lf egl-19 ad695 ns + n2368 ns ad695 ns ad1006 * * n582 * * N2 pgpa-9 : : gfp DiO pharma egl-19 background dlk-1 ( + ) C cAMP mutation kin-2 * * pde-4 * * acy-1 * * pharmacological treatment nema * * DMSO egl-19 background n582 * * + dlk-1 , axon cut % regeneration 100 80 60 40 20 0 B dlk-1 , axon cut gf wt lf egl-19 surgery type maximum % F / F bef aft bef aft bef aft bef aft 400 300 200 100 0 mock axon cut dend cut a + d cut * * * ns ns 200 % F / F A Fig . 3 . [Field: results, subscore: 2.00]
SECTION: results. In summary , the results above demonstrate many connections between DLK-independent regeneration and ectopic outgrowth , suggesting that they share a common activity-dependent mechanism for triggering and control of neuronal growth . [Field: results, subscore: 2.00]
SECTION: results. ( D ) Dendrite cuts enhance axon regeneration in the WT . [Field: results, subscore: 2.00]
SECTION: results. These % neurons 100 50 0 100 80 60 40 20 0 length regenerated ( m ) dlk-1 a + d * * wt a + d * * wt axon young adult cut , 24 h reimage dlk-1 a + d * * wt a + d * wt axon L2 cut , 24 h reimage dlk-1 a + d * * wt a + d * * wt axon L2 cut , 12 h reimage D full length along ring to ring not to ring none regeneration type : days after surgery 5 4 3 2 1 % neurons 100 80 60 40 20 0 C a + d axon 100 80 60 40 20 0 % regeneration dlk-1 * * dlk-1 * * wt B cell body , dendrite original axon new fiber 5 4 3 2 1 A 10 m Fig . 1 . [Field: results, subscore: 2.00]
SECTION: results. First , dendrite cuts trigger DLK-independent axon regeneration , and we find that dendrite lesions alone can trigger ectopic axon outgrowth ( Fig . 2B , left gray bars ) under conditions required for ectopic outgrowth ( i . . , 25 C cultivation and surgery on two successive generations--see SI Re- sults for discussion of maternal effect ) . [Field: results, subscore: 2.00]
SECTION: results. In addition , both ectopic outgrowth ( 10 ) and ASJ DLK-independent regeneration appear in the later stages of the animal ' s lifecycle , well after the amphid sensory neurons have developed their final morphology : regeneration in animals with dlk-1 usually does not emerge until the L4 larval stage or later ( Fig . 1D and Fig . S1E ) . [Field: results, subscore: 2.00]
SECTION: results. These results suggest that DLK-independent regeneration involves a novel mechanism largely independent of both the dlk-1 and kgb-1 MAPK pathways shown to mediate most C . elegans regeneration . [Field: results, subscore: 2.00]
SECTION: results. Thus , unlike motor neuron regeneration , ASJ conventional regeneration ( Fig . 2A , Inset ) does not require mlk-1 or kgb-1 . [Field: results, subscore: 2.00]
SECTION: results. As shown in Fig . 2A , Inset , conventional axon regeneration in motor neurons requires a coordinated activation of the DLK-1 / PMK-3 / CEBP-1 p38 and the parallel but coregulated MLK-1 / KGB-1 / FOS-1 JNK MAPK pathways ( 14 , 25 ) . [Field: results, subscore: 2.00]
SECTION: results. Second , we tested MAPK genes that mediate conventional motor neuron regeneration in C . elegans ( 24 ) for a role in conventional ASJ regeneration . [Field: results, subscore: 2.00]
SECTION: results. First , we tested dlk-1 ( km12 ) , which produces regeneration rates not statistically different from dlk-1 ( ju476 ) after either type of surgery and confirms that the dlk-1 regeneration phenotypes are not limited to one dlk-1 allele . [Field: results, subscore: 2.00]
SECTION: results. To further define the mechanisms underlying ASJ regeneration , we tested additional known genetic modulators by noting the frequency of regeneration after the axon or axon + dendrite surgery types ( Fig . 2A ) . [Field: results, subscore: 2.00]
SECTION: results. In this context , mutation of dlk-1 eliminates DLK-mediated regeneration , revealing the weaker but distinct DLK-independent regeneration . [Field: results, subscore: 2.00]
SECTION: results. These observations suggest that , in WT animals , both DLK-mediated regeneration and a novel DLK-independent regeneration are active in a single neuron and enhanced by dendrite surgery . [Field: results, subscore: 2.00]
SECTION: results. Although DLK-mediated regeneration appears to initiate within about 12 h after surgery or less if enhanced by dendrite cut , DLK-independent regeneration initiates from 24 h to several days after surgery ( Fig . 1C ) . [Field: results, subscore: 2.00]
SECTION: results. In contrast , DLK-independent regeneration resulting from surgery in L1 or L2 typically begins during the L4 larval stage : reimaging before L4 ( about 24 h after L2 ) reveals little regeneration in dlk-1 mutants ( Fig . 1D and Fig . S1E ) . [Field: results, subscore: 2.00]
SECTION: results. Thus , DLK-independent regeneration is morphologically similar to conventional regeneration , and both are clearly directed toward the original axon targets in the nerve ring . [Field: results, subscore: 2.00]
SECTION: results. ( Fig . S1 C and D ) details additional observations of this regeneration , illustrating and quantitatively comparing regeneration in single backgrounds . [Field: results, subscore: 2.00]
SECTION: results. These data include regeneration from dlk-1 axon + dendrite cuts as well as axon cuts in double mutants , with dlk-1 and mutations triggering regeneration listed in Fig . 2B ( see below ) . [Field: results, subscore: 2.00]
SECTION: results. As shown in Fig . 1C , we quantified the regeneration extent and found that > 80 % of DLK-independent regeneration successfully grows into the nerve ring by 5 d postsurgery ( e . . , Fig . 1 A2 , A4 , and A5 ) . [Field: results, subscore: 2.00]
SECTION: results. Remarkably , we find that concomitantly ( t < 1 min ) severing both the ASJ axon and the sensory dendrite ( axon + dendrite , a + d ) restores regeneration rates in dlk-1 animals ( Fig . 1 A4 and B ) , suggesting that severing the sensory dendrite triggers a nonconventional ( i . . , DLK-independent ) regeneration pathway that is not activated by axotomy alone . [Field: results, subscore: 2.00]
SECTION: results. Consistent with previous studies of several neuron types in C . elegans ( 12 , 13 , 18 ) , the dlk-1 ( ju476 ) mutation eliminates conventional DLK- 1mediated axon regeneration after axotomy alone , with < 5 % of postsurgery neurons regenerating ( Fig . 1 A3 and B ) . [Field: results, subscore: 2.00]
SECTION: results. In wildtype ( WT ) C . elegans , in vivo laser severing of ASJ axons results in robust regeneration , with > 95 % of neurons displaying regenerative outgrowth within several days ( Fig . 1 A2 and B ) , whereas dendrite transection results in little or no growth , even after 4 days ( 18 , 23 ) . [Field: results, subscore: 2.00]
SECTION: abstract. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP . [Field: abstract, subscore: 1.00]
SECTION: abstract. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration . [Field: abstract, subscore: 1.00]
SECTION: abstract. This DLK-independent regeneration inC . eleganshas direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type . [Field: abstract, subscore: 1.00]
SECTION: abstract. During development , a neuron transitions from a state of rapid growth to a stable morphology , and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury . [Field: abstract, subscore: 1.00]
SECTION: acknowledgments. Regeneration trx-1 ( + + ) data are replicated from Fig . 1B . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( B ) Ectopic outgrowth and DLK-independent regeneration rates are correlated . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( B ) Maximum projection of the z stack in A . Chung et al . www . pnas . org / cgi / content / short / 1600564113 5 of 8 100 80 60 40 20 0 % ectopic outgrowth at 25 C % regeneration 70 80 90 100 110 B = 0 . 79 * * stage and Tcult adult 25 C adult L4 L2 / L3 L1 15 C 100 80 60 40 20 0 % ectopic outgrowth posterior ring outgrowth type : * * * * ns A Fig . S3 . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Percentage regeneration of various backgrounds under different temperatures ( n = 27 , 27 , 12 , and 19 from left to right ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( F ) DLK-independent regeneration is not temperature-sensitive . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( E ) DLK-independent regeneration occurs late in development . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. P values are calculated comparing percentage of neurons with regeneration reaching ring ( 53 n 141 ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Percentage of neurons with indicated regeneration extent defined by fiber with farthest outgrowth . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( C ) Regenerations are aimed at nerve ring . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( B ) ASH total regeneration lengths for various surgeries and backgrounds . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. ( A ) Fundamental regeneration types . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Extended characterization of DLK-independent regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Third , binary scoring increases our ability to compare regeneration with published and current ectopic outgrowth measurements . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. In both cases , we rely on length measurements to discern differences in regeneration and interpret results . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Data are ambiguous only for unc-36 ( see above ) and in the WT background , where ASJ regeneration is highly robust ( Fig . 1D ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Several backgrounds exhibit slow development , delayed regeneration , or generally poor health , which complicates quantitative comparisons of regenerated length . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. First , regenerated length depends on the genetic background and the time between surgery and reimaging , which must be long because of the slow rate of DLK-independent regeneration ( Fig . 1D ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. We primarily scored individual neurons for the presence of regeneration rather than measuring length for several reasons . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Scoring Neuronal Growth . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Second , overlapping but distinct sets of genes and molecules mediate various forms of regeneration in different neuron types . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. However , it is important to note that our results also suggest additional complexities in neuronal outgrowth and regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Fig . 4 presents a generalized picture of the DLK-independent regeneration that we define in this study . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Complexity in Neuronal Regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. DLK-independent regeneration is not temperature-sensitive ( Fig . S1F ) , and nearly all neurons regenerate after surgery in the first generation . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Importantly , DLK-independent regeneration in C . elegans does not seem to exhibit the same maternal effect . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Our experiments confirming that axon transections farther from the cell body lead to new projection at the axon tip and growth along the ring ( see above ) , thus , raise the possibility that there exists a corresponding morphology of ectopic outgrowth . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Our study demonstrates that DLK-independent regeneration and ectopic outgrowth share many characteristics . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. We did not note any significant changes in regeneration at different temperatures ( Fig . S1F ) , although ectopic outgrowth rates can change by over 50 % ( Fig . S5 ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Regeneration Vs . Ectopic Outgrowth . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Together with results from Fig . 3D , these data indicate that the gain-of-function allele ad695 does not trigger ectopic outgrowth , consistent with our findings that reductions of egl- 19 function trigger DLK-independent regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Taken together , the above results strongly indicate that our interventions ( mutations , dendrite surgery , and nemadipine ) , which trigger ectopic outgrowth and DLK-independent regeneration , also decrease sensory-evoked activity in the ASJ . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. By eye , we also did not notice any gross differences in morphology or extent of neurite growths . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Finally , conventional regeneration rates between trx-1 ( + ) and trx-1 ( + + ) are not statistically different ( Fig . S4C ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. After axon + dendrite cuts and after only axotomy , regeneration rates are not statistically different between trx-1 ( + ) WT animals and either mutants with null allele trx-1 ( ok1449 ) ( 57 ) or animals expressing the translational fusion GFP ( Fig . S4A ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Because this transgene overexpresses functional TRX-1 thioredoxin , we examined the role of trx-1 in the relevant neurite growth mechanisms ( Fig . S4 ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Role of trx-1 in Neurite Growth Mechanisms . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. We performed regeneration assays in the ALM mechanosensory neuron to test the existence of parallel conventional and DLK-independent pathways in another neuron type . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Confirm unc-43 Regeneration Phenotype . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. As summarized in Fig . S3B , we note a correlation between ectopic outgrowth and DLK-independent regeneration rates . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. As shown in Fig . 2B , excepting unc-36 , each of the mutations that trigger ectopic outgrowth also triggers DLK-independent regeneration without dendrite lesion . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. We tested mutations shown to block sensory signaling in the ASJ neurons for a role in triggering DLK- independent regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. In contrast to the ASJ , where only 1 of 68 neurons regenerated in dlk-1 , about one-half the ASH neurons in dlk-1 animals regenerated , although the regeneration was significantly shorter than in the WT . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. In contrast to ectopic outgrowth , we observe no temperature dependence in DLK-independent regeneration rates at 15 C , 20 C , or 25 C ( Fig . S1F ) . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Similar to ectopic outgrowth ( 10 ) , we also noted a late time of DLK-independent regeneration after L1 life stage surgery as shown in Fig . S1E . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. As mentioned in the text , ASJ regeneration generally consisted of three morphologies , which are shown in Fig . S1A . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. For these reasons , despite the possibility of mechanistic differences between regeneration after complete and distal axotomies , we chose to sever axons close to the cell body . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. This regeneration roughly follows the original axon path , making discrimination between normal and regenerated neurites difficult . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. In dlk-1 , dendrite cuts together with axon cuts in the nerve ring lead to clear regeneration from the ring in 4 of 11 neurons . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. As noted in previous studies ( 56 ) , we confirmed that axon transections near the cell body lead to regenerative outgrowth from the cell body but that transections farther from the cell body lead more often to regeneration from the severed axon stump . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. We conducted extensive sets of experiments to characterize cellautonomous , DLK-independent regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Additional Characterization of DLK-Independent Regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Wu Z , et al . ( 2007 ) Caenorhabditis elegans neuronal regeneration is influenced by life stage , ephrin signaling , and synaptic branching . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Valakh V , Frey E , Babetto E , Walker LJ , DiAntonio A ( 2015 ) Cytoskeletal disruption activates the DLK / JNK pathway , which promotes axonal regeneration and mimics a preconditioning injury . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Stone MC , Albertson RM , Chen L , Rolls MM ( 2014 ) Dendrite injury triggers DLK- independent regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Neumann S , Woolf CJ ( 1999 ) Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Blesch A , et al . ( 2012 ) Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration : Superiority to campmediated effects . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Neumann S , Bradke F , Tessier-Lavigne M , Basbaum AI ( 2002 ) Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Raivich G , et al . ( 2004 ) The AP-1 transcription factor c-Jun is required for efficient axonal regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Shin JE , et al . ( 2012 ) Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Sun L , et al . ( 2014 ) Neuronal regeneration in C . elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Ghosh-Roy A , Wu Z , Goncharov A , Jin Y , Chisholm AD ( 2010 ) Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Pinan-Lucarre B , et al . ( 2012 ) The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Enes J , et al . ( 2010 ) Electrical activity suppresses axon growth through Ca ( v ) 1 . 2 channels in adult primary sensory neurons . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Richardson PM , Issa VMK ( 1984 ) Peripheral injury enhances central regeneration of primary sensory neurones . [Field: acknowledgments, subscore: 1.00]
SECTION: acknowledgments. Case LC , Tessier-Lavigne M ( 2005 ) Regeneration of the adult central nervous system . [Field: acknowledgments, subscore: 1.00]
SECTION: discussion. Removal of this inhibition triggers a novel regeneration pathway largely independent of DLK and JNK signaling . [Field: discussion, subscore: 1.00]
SECTION: discussion. Within this context , our work establishes the ASJ neuron in C . elegans as a powerful experimental model for studying DLK-independent regeneration and suggests ectopic outgrowth as a tractable gene discovery platform . [Field: discussion, subscore: 1.00]
SECTION: discussion. Taken together , our findings suggest that the same activity-dependent pathways restricting neurite outgrowth during development through activation of SAX-1 / NDR kinase and UNC-43 / CaMKII can also constitutively block regeneration after axonal injury in the adult . [Field: discussion, subscore: 1.00]
SECTION: discussion. SAX-1 and its Drosophila homolog Tricornered negatively regulate dendritic growth during development to establish correct sensory neurite tiling in worms and flies ( 45 , 46 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. Our study identifies key negative regulators of regeneration , sax-1 and unc-43 , which also restrict outgrowth during neuronal maturation . [Field: discussion, subscore: 1.00]
SECTION: discussion. Thus , this novel DLK-independent regeneration ( gray area in Fig . 4 ) requires both a central axon injury signal and the elimination of activity-dependent developmental blocks mediated by the peripheral sensory neurite . [Field: discussion, subscore: 1.00]
SECTION: discussion. Alternatively , axon injury ( blue line in Fig . 4 ) can activate DLK-independent regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Disruption of this pathway at any point by dendrite lesion , genetic mutation , or pharmacology removes the suppression , enabling neurite growth within two contexts . [Field: discussion, subscore: 1.00]
SECTION: discussion. ) , our findings suggest a simple working model for regeneration and outgrowth in C . elegans ( Fig . 4 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. Nonetheless , the similarities with the C . elegans DLK- independent regeneration observed here suggest the possibility of modeling aspects of CNS lesion conditioning in genetically accessible systems . [Field: discussion, subscore: 1.00]
SECTION: discussion. The shaded pathways comprise a DLK-independent regeneration , which shares characteristics with mammalian lesion conditioning . [Field: discussion, subscore: 1.00]
SECTION: discussion. The orange and green pathways represent conventional regeneration and ectopic outgrowth , respectively . [Field: discussion, subscore: 1.00]
SECTION: discussion. Simplified working model of neurite growth mechanisms in C . elegans . [Field: discussion, subscore: 1.00]
SECTION: discussion. Neurite growth mechanisms . [Field: discussion, subscore: 1.00]
SECTION: discussion. Information gained from these models may be combined with results in neuronal regeneration to inform multiple areas of study . [Field: discussion, subscore: 1.00]
SECTION: discussion. Our work suggests that examining nematode ectopic outgrowth could provide a fruitful source of insight into other vertebrate and invertebrate regeneration mechanisms and vice versa . [Field: discussion, subscore: 1.00]
SECTION: discussion. Our results indicate a direct link between DLK-independent regeneration and activity-dependent ectopic axon outgrowth , with comparable morphologies , late time of outgrowth , overlapping set of inducing mutations and surgeries , and at least partial DLK independence . [Field: discussion, subscore: 1.00]
SECTION: discussion. Discussion Our work describes regeneration in C . elegans amphid neurons that is triggered by lesion of both the axon and sensory dendrite . [Field: discussion, subscore: 1.00]
SECTION: introduction. We find numerous direct links between this DLK-independent regeneration and ectopic outgrowth in the same neurons . [Field: introduction, subscore: 1.00]
SECTION: introduction. Here , we demonstrate that this novel type of regeneration is largely independent of both the DLK-1 / DLK and the KGB-1 / JNK pathways . [Field: introduction, subscore: 1.00]
SECTION: introduction. Second , even without a dendrite cut , regeneration occurs under mutations that also trigger ectopic axon outgrowth . [Field: introduction, subscore: 1.00]
SECTION: introduction. Our results indicate genetic and molecular connections between DLK-independent regeneration and a previously studied activity-dependent ectopic axon outgrowth in C . elegans . [Field: introduction, subscore: 1.00]
SECTION: introduction. Thus , C . elegans , by virtue of its facile genetics and amenability to precise laser surgery , is an excellent system for studying axon outgrowth and regeneration . [Field: introduction, subscore: 1.00]
SECTION: introduction. More recently , the application of advanced subcellular laser ablation techniques ( 11 ) to C . elegans has permitted quantitative in vivo study of single-neuron regeneration in this genetically tractable system . [Field: introduction, subscore: 1.00]
SECTION: introduction. The nematode Caenorhabditis elegans is a well-established and powerful model system ( 6 ) for the genetic and molecular study of both activity-dependent neuronal development and regeneration . [Field: introduction, subscore: 1.00]
SECTION: introduction. These observations suggest that activity-dependent inhibitors of neurite outgrowth may represent a potent therapeutic target for enhancing neuronal regeneration in the face of injury or disease . [Field: introduction, subscore: 1.00]
SECTION: introduction. Subsequently , a neuron transitions from a state of rapid growth to a stable morphology , and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury . [Field: introduction, subscore: 1.00]
SECTION: introduction. Although much research in previous decades focused on the extrinsic angle , recent encouraging developments , particularly in invertebrate models , increasingly examine the cell intrinsic control of regeneration . [Field: introduction, subscore: 1.00]
SECTION: introduction. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP . [Field: introduction, subscore: 1.00]
SECTION: introduction. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration . [Field: introduction, subscore: 1.00]
SECTION: introduction. This DLK-independent regeneration in C . elegans has direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type . [Field: introduction, subscore: 1.00]
SECTION: materials. We calculated P values for these data by Fisher ' s exact test . For regenerated length measurements , we calculated P values by the unpaired , unequal variance , two-tailed t test . For calcium imaging data , we calculated P values by the paired , two-tailed t test . For ALM regeneration length data , we conducted a one-way analysis of variance ( ANOVA ) to compare the effect of genetic background on regenerated length in WT and mutant dlk-1 and unc-43 conditions . [Field: materials, subscore: 1.00]
SECTION: materials. Most of the ASJ regeneration and ectopic outgrowth data are binary : we score whether or not neurons regrow or outgrow . [Field: materials, subscore: 1.00]
SECTION: materials. For most experiments , we scored individual neurons for the presence of regeneration ( greater than 2 m ) rather than measuring outgrowth length . [Field: materials, subscore: 1.00]
SECTION: materials. Following standard methods ( 6 ) , we cultured strains on nematode growth medium ( NGM ) plates inoculated with OP50 bacteria ( i . . , seeded plates ) , the primary food source for laboratory C . elegans , and maintained animals at 20 C unless otherwise stated . [Field: materials, subscore: 1.00]
SECTION: results. Comparing the regenerated axon length in both WT and dlk-1 animals , we find that dendrite surgery after axon surgery ( tdend > 0 h ; " post " comparison ) produces less regeneration than concomitant surgeries ( tdend = 0 h ) , whereas dendrite surgery before axon surgery ( tdend < 0 h ; " pre " comparison ) produces more ( Fig . 3D ) . [Field: results, subscore: 1.00]
SECTION: results. Azide anesthesia likely reduces regeneration , and therefore , we processed each sequential surgery set concurrently with a concomitant surgery set , treating these animals alike except for laser surgery . [Field: results, subscore: 1.00]
SECTION: results. These results indicate that , similar to lesion conditioning , elevated cAMP signaling enhances DLK-independent regeneration and ectopic outgrowth . [Field: results, subscore: 1.00]
SECTION: results. Likewise , constitutive elevation of the cAMP signaling effector protein kinase A by mutation of its regulatory subunit kin-2 ( 37 ) also enables DLK-independent regeneration after axotomy alone . [Field: results, subscore: 1.00]
SECTION: results. Gain-of-function adenylate cyclase acy-1 ( md1756gf ) ( 37 ) and loss-of-function phosphodiesterase PDE4 pde-4 ( ce268 ) ( 38 ) both increase neuronal cAMP signaling and enable robust regeneration of the ASJ after axotomy alone in a dlk-1 mutant background ( Fig . 3B ) . [Field: results, subscore: 1.00]
SECTION: results. Therefore , we tested if elevated cAMP signaling can induce DLK-independent regeneration in the ASJ neuron . [Field: results, subscore: 1.00]
SECTION: results. Increased cAMP signaling enhances regeneration in C . elegans as well as the mammalian CNS ( 16 , 3336 ) . [Field: results, subscore: 1.00]
SECTION: results. These results demonstrate that reductions of EGL-19 function trigger ectopic outgrowth and DLK-independent regeneration . [Field: results, subscore: 1.00]
SECTION: results. Likewise , the EGL-19 antagonist nemadipine-A ( 32 ) also triggers robust DLK-independent regeneration without dendrite cut ( Fig . 3B ) : we find significantly more neurons regenerated under nemadipine-A than its solvent vehicle dimethyl sulfoxide ( DMSO ) . [Field: results, subscore: 1.00]
SECTION: results. As noted above , genetic disruption of the CACNA1C homolog egl-19 triggers DLK- independent regeneration without dendrite cut ( Fig . 3B ) . [Field: results, subscore: 1.00]
SECTION: results. We performed experiments to further define the channel ' s role in DLK-independent regeneration and ectopic outgrowth . [Field: results, subscore: 1.00]
SECTION: results. ( Lower ) Percentage of neurons with indicated regeneration extent defined by farthest outgrowth . [Field: results, subscore: 1.00]
SECTION: results. ( Upper ) Total regeneration lengths for various surgeries and backgrounds . [Field: results, subscore: 1.00]
SECTION: results. ( B and C ) Reduction of EGL-19 function and elevation of cAMP signaling trigger DLK-independent regeneration and ectopic outgrowth . [Field: results, subscore: 1.00]
SECTION: results. Reduction of Sensory and Calcium Activity and Elevation of cAMP Signaling Trigger DLK-Independent Regeneration and Ectopic Outgrowth . [Field: results, subscore: 1.00]
SECTION: results. Gray-shaded results : Outgrowthinducing mutations and dendrite cuts trigger DLK-independent regeneration . [Field: results, subscore: 1.00]
SECTION: results. ( B ) Comparison of DLK-independent regeneration and ectopic outgrowth . [Field: results, subscore: 1.00]
SECTION: results. ( A ) MAPK genes are not critical for DLK-independent regeneration . [Field: results, subscore: 1.00]
SECTION: results. In a complementary manner , the gain-of-function ( gf ) mutation unc-43 ( n498 ) successfully inhibits regeneration after axon + dendrite surgery , decreasing it by 20 % . [Field: results, subscore: 1.00]
SECTION: results. We found that the sax-1 ( ky211 ) and unc-43 ( n1186 ) loss-of-function ( lf ) mutations generate DLK- independent regeneration without a dendrite cut ( Fig . 2B , gray bars ) . [Field: results, subscore: 1.00]
SECTION: results. Finally , we asked if ectopic outgrowth and DLK-independent regeneration might share the same underlying regulation . [Field: results, subscore: 1.00]
SECTION: results. Chung et al . PNAS Early Edition | 3 of 9 NEUROSCI ENCE PNAS PLUS data suggest that both DLK-mediated and DLK-independent processes underlie ectopic outgrowth , similar to regeneration . [Field: results, subscore: 1.00]
SECTION: results. ( Lower ) Percentage of neurons with indicated regeneration extent defined by farthest outgrowth as in C . Data are represented as average SD . a + d , axon + dendrite . [Field: results, subscore: 1.00]
SECTION: results. ( Upper ) Total regeneration lengths for various surgeries and backgrounds . [Field: results, subscore: 1.00]
SECTION: results. Percentage of neurons with indicated regeneration extent defined by fiber with farthest outgrowth . [Field: results, subscore: 1.00]
SECTION: results. ( B ) dlk-1 eliminates and dendrite cut restores regeneration . [Field: results, subscore: 1.00]
SECTION: results. ( A5 ) Regeneration directed along ring in dlk-1 ; tax-4 after axon surgery . [Field: results, subscore: 1.00]
SECTION: results. ( A4 ) Regeneration in dlk-1 after axon + dendrite surgery . [Field: results, subscore: 1.00]
SECTION: results. ( A3 ) Lack of regeneration in dlk-1 after axon surgery . [Field: results, subscore: 1.00]
SECTION: results. ( A2 ) WT regeneration . [Field: results, subscore: 1.00]
SECTION: results. White arrows indicate regenerative growth , white arrowheads indicate cut dendrites , and black arrowheads indicate typical axotomy location . [Field: results, subscore: 1.00]
SECTION: results. Sensory dendrite cuts enhance ASJ regeneration in C . elegans WT and dlk-1 . [Field: results, subscore: 1.00]
SECTION: results. Dendrite cuts enhance regeneration under DLK-mediated and DLK-independent processes ( Fig . 1D ) , and , therefore , we next asked if ectopic outgrowth can occur independent of dlk-1 . [Field: results, subscore: 1.00]
SECTION: results. The results above strongly suggest that the same activity-dependent mechanism that triggers ectopic outgrowth also triggers DLK-independent regeneration after axon damage . [Field: results, subscore: 1.00]
SECTION: results. The rates of ectopic outgrowth and regeneration are strongly correlated ( Fig . S3B ) . [Field: results, subscore: 1.00]
SECTION: results. As shown in Fig . 2B , gray bars , most outgrowth mutations also successfully trigger DLK-independent regeneration after axotomy without a dendrite lesion at lower temperatures that generate less ectopic outgrowth ( additional details are in SI Results [Field: results, subscore: 1.00]
SECTION: results. Second , we tested an array of mutations that reduce sensory activity and trigger ectopic outgrowth to assess their role in triggering DLK-independent regeneration . [Field: results, subscore: 1.00]
SECTION: results. Based on these phenotypic similarities , we asked if the same physical or genetic lesions might trigger both ectopic outgrowth and DLK-independent regeneration . [Field: results, subscore: 1.00]
SECTION: results. 1600564113 Chung et al . DLK-Independent Regeneration Shares Phenotypic and Genetic Links with Ectopic Axon Outgrowth . [Field: results, subscore: 1.00]
SECTION: results. Double mutants of dlk-1 and these genes ( Fig . 2A ) do not exhibit significantly different regeneration rates from dlk-1 after either type of surgery ( only kgb-1 shows a mild 20 % reduction after a + d ) . [Field: results, subscore: 1.00]
SECTION: results. Third , we tested MAPK genes for roles in DLK-independent regeneration . [Field: results, subscore: 1.00]
SECTION: results. Although mutation of cebp-1 and fos-1 , which encode C / EBP and Fos basic region-leucine zipper transcription factors , significantly reduces conventional regeneration , mutation of mlk-1 and kgb-1 does not ( Fig . 2A , Left ) . [Field: results, subscore: 1.00]
SECTION: results. DLK-Independent Regeneration after ASJ Axon + Dendrite Lesion Is Independent of Defined MAPK Signaling Pathways . [Field: results, subscore: 1.00]
SECTION: results. Axon + dendrite surgeries have a similar effect in young adult animals , resulting in relatively modest regeneration in dlk-1 compared to WT after 24 h . [Field: results, subscore: 1.00]
SECTION: results. Enhancement occurs both at relatively short regeneration times of 12 and 24 h after surgery during the L2 larval stage and after surgery during the later young adult stage . [Field: results, subscore: 1.00]
SECTION: results. As shown in Fig . 1D , in WT ASJ neurons the addition of a dendrite cut generates significantly longer regeneration with greater success penetrating into the nerve ring compared to axon lesion alone . [Field: results, subscore: 1.00]
SECTION: results. After surgery , most ASJ neurons in WT animals substantially regenerate ; therefore , to quantify regeneration after different surgeries , we measured regenerated outgrowth length and penetrance into the nerve ring ( Materials and Methods and Fig . S2 ) . [Field: results, subscore: 1.00]
SECTION: results. We , therefore , examined the impact of dendrite cuts on regeneration in WT animals . [Field: results, subscore: 1.00]
SECTION: results. Given that a dendrite cut enhances regeneration in dlk-1 , we next sought to determine if this enhancement exists in the WT or arises by disruption of dlk-1 . [Field: results, subscore: 1.00]
SECTION: results. As shown in Fig . 1A and Fig . S1A , regeneration projects from the cell body anteriorly to the ring and follows the ring posteroventrally ( Fig . 1 A2 , A4 , and A5 ) , projects posteriorly from the cell body ( Fig . 1A2 ) , or occasionally projects off the dendrite and follows the ring posteroventrally ( Fig . 1 A2 and A4 ) . [Field: results, subscore: 1.00]
SECTION: results. Transection of the Axon and Sensory Dendrite Triggers DLK-Independent Regeneration in the ASJ Neuron . [Field: results, subscore: 1.00]
SECTION: title. Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth . [Field: title, subscore: 1.00]
Supplemental links/files: reference in endnote reference in xml Wormbase reference
Score: 343.00
Title: Signaling pathways that regulate axon regeneration .
Authors: Saijilafu ; Zhang BY ; Zhou FQ
Journal: Neurosci Bull
Year: 2013-08-11
Doc ID: WBPaper00043898
Bibliographic Information
Abstract
Matching Sentences
SECTION: introduction. Saijilafu , et al . Signaling pathways that regulate axon regeneration 7 peripheral axotomy induces tubulin deacetylation and therefore increases microtubule dynamics in axons at the proximal injury site [ 55 ] . inhibition of tubulin deacetylation with a pharmacological inhibitor of histone deacetylase ( HDAC ) blocks sensory axon regeneration in vivo , suggesting that maintaining microtubule dynamics is necessary for axon regeneration . importantly , using the Campenot chamber culture system , it was shown that inhibition of HDAC locally at the axon is sufficient to block axon growth , confirming that HDAC acts locally on axonal microtubules to control axon growth rather than at the soma to control gene transcription . [Field: introduction, subscore: 8.00]
SECTION: introduction. In contrast to that of developing DRG neurons , regenerative axon growth from adult DRG neurons does not require Pi3K and ERK activity for local axon assembly [ 40 , 41 ] , indicating growth factorindependent pathways for PNS axon regeneration . indeed , Neurosci Bull July , 2013 6 application of a function-blocking antibody to NGF is unable to inhibit peripheral nerve regeneration [ 49 ] , providing solid evidence that in vivo peripheral axon regeneration does not require NGF . [Field: introduction, subscore: 7.00]
SECTION: introduction. Additional studies in C . elegans have shown that SVH-1 , a homolog of mammalian hepatocyte growth factor and its receptor SVH-2 function upstream of the MLK-1 pathway to promote axon regeneration [ 29 ] , whereas SVH-3 , a homolog of mammalian fatty acid amide hydrolase , inhibits axon regeneration through antagonizing the MLK-1 pathway [ 30 ] . in summary , two MAP kinase pathways function in C . elegans to regulate naturally-occurring axon regeneration , and both pathways mainly act locally at the axon to control microtubule reorganization and growth-cone remodeling upon axotomy ( Fig . 2 ) . [Field: introduction, subscore: 7.00]
SECTION: introduction. Because the manipulation of gene expression ( transcription or translation ) has proven effective in promoting CNS axon regeneration [ 1-5 ] , elucidating the signaling pathways that regulate gene expression during naturally-occurring axon regeneration in the mammalian PNS should become a major focus of axon regeneration research . [Field: introduction, subscore: 6.00]
SECTION: introduction. Signaling Pathways That Regulate Mammalian Axon Regeneration Adult Dorsal Root Ganglion ( DRG ) as a Model System for Mammalian Axon Regeneration Among adult PNS neurons , DRG neurons provide Saijilafu , et al . Signaling pathways that regulate axon regeneration 5 a favorable model for studying mammalian axon regeneration . [Field: introduction, subscore: 6.00]
SECTION: introduction. Because the major phenotype of the DLK-1 mutant is a defect in growth-cone formation after axotomy [ 9 ] and the temporal requirement of DLK-1 function for regeneration is very soon after the axotomy , it has been suggested that DLK-1 mainly functions locally at the growth cone to control axon regeneration . in support , a different study has shown that MAK-2 ( MAP kinase activated kinase , MAPKAP ) acts downstream of DLK-1 to control axon regeneration via controlling the local translation of CEBP-1 in the axon [ 16 ] . [Field: introduction, subscore: 6.00]
SECTION: introduction. As discussed earlier , in C . elegans , DLK-1 regulates axon regeneration mainly by acting at the growth cone ( Fig . 2 ) . in contrast , recent studies of DLK knockout mice have shown that mammalian DLK controls axon regeneration via the activation of c-Jun and Stat3 [ 59 , 60 ] , suggesting a role of DLK in controlling gene expression in the soma ( Fig . 3 ) . [Field: introduction, subscore: 5.00]
SECTION: introduction. SVH-1 , a homolog of the mammalian hepatocyte growth factor and its receptor SVH-2 function upstream of the MLK-1 pathway to promote axon regeneration , whereas SVH-3 , a homolog of mammalian fatty-acid amide hydrolase , inhibits axon regeneration by antagonizing the MLK-1 pathway . [Field: introduction, subscore: 5.00]
SECTION: abstract. Here , recent studies in which signaling pathways regulating naturally-occurring axon regeneration that have been identified are reviewed , focusing on how these pathways control gene expression and growth-cone function during axon regeneration . [Field: abstract, subscore: 4.00]
SECTION: abstract. Understanding the molecular mechanisms by which these neurons support axon regeneration will help us find ways to enhance mammalian CNS axon regeneration . [Field: abstract, subscore: 4.00]
SECTION: introduction. Because the Pi3K pathway is not required for local axon assembly during sensory axon regeneration [ 40 , 41 ] , this result suggests that Pi3K regulates mammalian sensory axon regeneration by controlling gene expression in the soma . indeed , in such p110 mutant mice , the expression level of Sprr1A , a well-known regeneration-associated gene , is significantly diminished . [Field: introduction, subscore: 4.00]
SECTION: introduction. Notably , either downregulation of CLASPs which reduces tubulin acetylation [ 52 ] , or inhibition of HDAC which increases tubulin acetylation [ 55 ] , blocks sensory axon regeneration , suggesting that a balance of microtubule stabilization and dynamics is required for optimal axon regeneration . [Field: introduction, subscore: 4.00]
SECTION: introduction. Down-regulation of CLASPs in adult DRG neurons reduces tubulin acetylation / microtubule stability and inhibits sensory axon regeneration in vivo [ 52 ] , providing strong evidence that regulation of axonal microtubule dynamics is important for successful axon regeneration . in addition to microtubule-binding proteins , microtubule dynamics are also regulated by post-translational modification , in particular acetylation , which is associated with increased microtubule stabilization [ 54 ] . [Field: introduction, subscore: 4.00]
SECTION: introduction. Consistent with this , adult DRG neurons undergoing peripheral axotomy are able to extend long axons in culture in the absence of growth factors [ 41 ] . instead , activation of integrin signaling by laminin is sufficient to induce robust axon growth from these pre-axotomized DRG neurons , suggesting that integrin signaling plays a key role in peripheral nerve regeneration . indeed , laminin isoforms are highly enriched in peripheral nerve pathways [ 50 ] , and loss of integrin subtype alpha 7 impedes facial nerve regeneration in vivo after a lesion [ 51 ] . [Field: introduction, subscore: 4.00]
SECTION: introduction. The promoting effect of Ca2 + signaling on axon regeneration is mediated by activation of adenyl cyclase ( AC ) and the subsequent elevation of cAMP , which in turn activates PKA [ 19 ] . importantly , the Ca2 + / AC / cAMP / PKA pathway acts upstream of DLK-1 to promote axon regeneration . [Field: introduction, subscore: 4.00]
SECTION: introduction. Saijilafu , et al . Signaling pathways that regulate axon regeneration 3 kinase ) as downstream mediators of DLK-1 to control axon regeneration . [Field: introduction, subscore: 4.00]
SECTION: introduction. Recent efforts have been made to induce axotomy and assess axon regeneration in adult neurons of C . elegans and Drosophila [ 6 ] , thus making them attractive new model systems for dissecting axon regeneration pathways . [Field: introduction, subscore: 4.00]
SECTION: introduction. Therefore , promoting axon regeneration is a major approach to treating these symptoms . one important reason for CNS regeneration failure is the irreversible loss of the intrinsic axon growth capacity of CNS neurons after maturation . [Field: introduction, subscore: 4.00]
SECTION: introduction. Here , recent studies in which signaling pathways regulating naturally-occurring axon regeneration that have been identified are reviewed , focusing on how these pathways control gene expression and growthcone function during axon regeneration . [Field: introduction, subscore: 4.00]
SECTION: introduction. Understanding the molecular mechanisms by which these neurons support axon regeneration will help us find ways to enhance mammalian CNS axon regeneration . [Field: introduction, subscore: 4.00]
SECTION: references. [ 18 ] Chen L , Wang Z , Ghosh-Roy A , Hubert T , Yan D , o ' Rourke S , et al . Axon regeneration pathways identified by systematic Saijilafu , et al . Signaling pathways that regulate axon regeneration 9 genetic screening in C . elegans . [Field: references, subscore: 4.00]
SECTION: introduction. Most of the axon regeneration pathways identified to date are located at the growth cone ( see Figs . 2 and 3 ) , and much less is known about signaling pathways functioning at the soma to control gene expression . [Field: introduction, subscore: 3.00]
SECTION: introduction. The emergence of nonmammalian injury and regeneration models has provided new opportunities to identify signaling pathways that regulate naturally-occurring axon regeneration . [Field: introduction, subscore: 3.00]
SECTION: introduction. Together , the evidence to date suggests that DLK signaling regulates mammalian axon regeneration by controlling gene expression in the Neurosci Bull July , 2013 8 neuronal soma and / or microtubule dynamics in the growth cone . [Field: introduction, subscore: 3.00]
SECTION: introduction. Does the Pi3K pathway play any role in the regulation of intrinsic axon growth during naturallyoccurring PNS axon regeneration ? [Field: introduction, subscore: 3.00]
SECTION: introduction. Signaling pathways regulating naturally-occurring mammalian axon regeneration in the peripheral nervous system . [Field: introduction, subscore: 3.00]
SECTION: introduction. Together , this evidence suggests that peripheral axotomy-induced regenerative axon growth and a culture condition without axon-promoting growth factors should be the model of choice for studying regeneration signaling pathways in vitro . [Field: introduction, subscore: 3.00]
SECTION: introduction. Therefore , to study regeneration pathways , it is important to use an in vitro model that mimics in vivo peripheral axotomy-induced axon regeneration . if an in vivo peripheral nerve injury is performed prior to culture , the cultured DRG neurons are able to extend long axons overnight . [Field: introduction, subscore: 3.00]
SECTION: introduction. A novel in vivo electroporation technique has recently been developed that allows specific manipulation of multiple gene expression in adult DRG neurons simultaneously [ 38 ] , providing a potentially useful tool for the in vivo dissection of pathways regulating mammalian axon regeneration . in contrast to the in vivo model , adult DRG neurons can readily be cultured and therefore have been widely used to study axon growth from mature neurons in vitro [ 39 ] . [Field: introduction, subscore: 3.00]
SECTION: introduction. Because the central branches of the DRG neurons share the same environment as corticospinal axons , it is believed that activation of a similar regeneration program in adult CNS neurons would enhance CNS axon regeneration . [Field: introduction, subscore: 3.00]
SECTION: introduction. These results indicate that peripheral axotomy increases the intrinsic ability of DRG neurons to support axon regeneration by activation of a transcription-dependent regeneration program and the subsequent expression of a group of regenerationassociated genes in the soma [ 37 ] . [Field: introduction, subscore: 3.00]
SECTION: introduction. Because JNK is well known to regulate axonal microtubules in mammalian neurons during axon growth [ 25-28 ] , it is likely that the MLK-1 / MEK-1 / KGB-1 pathway in C . elegans also targets microtubules for controlling axon regeneration . [Field: introduction, subscore: 3.00]
SECTION: introduction. To further support the role of DLK-1 in the growth cone , a later study showed that KLP-7 ( kinesin-13 ) and CCPP-6 ( cytosolic carboxipeptidase ) , two proteins directly involved in the regulation of microtubule dynamics , act downstream of DLK-1 to control axon regeneration [ 17 ] . [Field: introduction, subscore: 3.00]
SECTION: introduction. Signaling at the Growth Cone Growth cones are highly specialized structures at the distal tips of axons that control the rate and direction of axon growth during development . [Field: introduction, subscore: 3.00]
SECTION: introduction. Using such in vivo axotomy approaches combined with large-scale genetic screening , recent studies have identified several signaling pathways that control axon regeneration in vivo either at the growth cone or at the soma ( Fig . 2 ) . [Field: introduction, subscore: 3.00]
SECTION: introduction. Here , we review these pathways , focusing on where they act ( soma or growth cone ) to regulate axon regeneration . [Field: introduction, subscore: 3.00]
SECTION: introduction. The powerful genetic approaches available in these species have made them attractive model systems for dissecting the signaling pathways controlling axon regeneration . indeed , recent studies using these systems , in particular C . elegans , have revealed several regeneration signaling pathways in vivo [ 6-8 ] . [Field: introduction, subscore: 3.00]
SECTION: introduction. However , the degree of axon regeneration promoted by these molecular interventions is still rudimentary compared with the regeneration that occurs after injury to the mammalian peripheral nervous system ( PNS ) or in non-mammals . [Field: introduction, subscore: 3.00]
SECTION: introduction. Keywords : axon regeneration ; axonal growth ; signal transduction Introduction [Field: introduction, subscore: 3.00]
SECTION: references. [ 45 ] Hur EM , Yang iH , Kim DH , Byun J , Saijilafu , Xu WL , et al . Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules . [Field: references, subscore: 3.00]
SECTION: references. [ 32 ] Veldman MB , Bemben MA , Thompson RC , Goldman D . Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration . [Field: references, subscore: 3.00]
SECTION: references. [ 29 ] Li C , Hisamoto N , Nix P , Kanao S , Mizuno T , Bastiani M , et al . The growth factor SVH-1 regulates axon regeneration in C . elegans via the JNK MAPK cascade . [Field: references, subscore: 3.00]
SECTION: references. Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration . [Field: references, subscore: 3.00]
SECTION: references. Growing the growth cone : remodeling the cytoskeleton to promote axon regeneration . [Field: references, subscore: 3.00]
SECTION: references. Assembly of a new growth cone after axotomy : the precursor to axon regeneration . [Field: references, subscore: 3.00]
SECTION: introduction. Because the genetic approaches used in the non-mammalian models are not available for mammalian models , it is not practical to perform similar large-scale genetic screening in mice to identify mammalian axon regeneration pathways . [Field: introduction, subscore: 2.00]
SECTION: introduction. Summary and Future Directions Successful axonal regeneration in the injured nervous system is a complex process that involves coordinated regulation of gene expression in the soma and cytoskeleton assembly at the growth cone . [Field: introduction, subscore: 2.00]
SECTION: introduction. Moreover , a new study [ 62 ] found that Stat3 controls microtubule stability locally in the axon independent of its transcriptional activity , raising the possibility that DLKStat3 activated at the injury site after peripheral axotomy may also play a local role in the regulation of axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. Whether DLKJNK signaling also regulates axonal microtubules during mammalian axon regeneration awaits further study . [Field: introduction, subscore: 2.00]
SECTION: introduction. To date , however , there is no direct evidence showing that DLK regulates peripheral axotomy-induced gene expression , and future genetic-profiling studies of DLK knockout mice may provide an answer . it should be noted that besides regulating transcription factors , DLKJNK has also been shown to regulate developmental axon growth by controlling microtubule stability at the growth cone [ 61 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Future study using a compartmentalized culture system is required to provide a more definitive answer regarding the role of Pi3K in the regulation of gene expression during mammalian axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. in a mutant mouse line , where p110 , the endogenous catalytic subunit of Pi3K , is replaced with an inactive mutation , sensory axon regeneration is greatly impaired [ 58 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. The regeneration-associated genes identified by genetic profiling include many transcription factors , such as c-Jun , Stat3 , ATF3 , Smad1 , p53 , and SoX11 ( Fig . 3 ) . it is well recognized that these factors control the transcription of genes that support peripheral axotomyinduced axon regeneration [ 56 , 57 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Together , these studies highlight the important role of axonal microtubule dynamics in the regulation of mammalian axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. The DLKStat3 / JNK pathways may also be involved in the regulation of microtubule assembly during axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. In the soma , peripheral axotomy upregulates and activates many regeneration-associated transcription factors , such as p53 , Stat3 , Sox11 , ATF3 , Smad1 , and c-Jun , which control gene expression that supports axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. However , an interesting observation of peripheral axotomyinduced regenerative axon growth from adult DRG neurons is that active gene transcription is not required for axon growth in the first 24 h [ 41 , 48 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Using such approaches , previous studies have shown that Pi3K and ERK , two major signaling molecules mediating NGF- induced axon growth [ 46 ] , are both required locally at the axon to control axon growth from developing sensory neurons [ 47 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Signaling pathways regulating mammalian axon regeneration in the PNS are summarized in Fig . 3 . [Field: introduction, subscore: 2.00]
SECTION: introduction. However , such NGF-induced growth is regulated by a different pathway from that of peripheral axotomy-induced axon growth ( see below ) and the processes show a highly-branched morphology ( sprouting mode ) [ 39 , 41 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Addition of growth factors ( e . . NGF or GDNF ) induces robust axon growth . [Field: introduction, subscore: 2.00]
SECTION: introduction. Such peripheral axotomy-induced regenerative axon growth often starts within a few hours after cell plating and the axons extend with few branches ( elongation mode ) [ 41 ] . in contrast , nave ( uninjured ) DRG neurons cultured at low density grow few axons in the first 24 h in the absence of growth factors . [Field: introduction, subscore: 2.00]
SECTION: introduction. Therefore , to date no signaling pathways ( consisting of multiple upstream / downstream signaling molecules ) that regulate naturally-occurring mammalian axon regeneration have been delineated in vivo . [Field: introduction, subscore: 2.00]
SECTION: introduction. Future studies using these non-mammalian models should focus on somatic signaling to expand our knowledge of how gene expression is regulated during successful axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. How Notch signaling and KLF6 / 7 are regulated by upstream signaling pathways is not known . in sum , very few signaling pathways have been shown to control gene expression during axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. A subsequent study showed that KLF6 / 7 regulate axon regeneration by controlling the transcription of the tubulin isoform Tuba1a [ 33 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. By comparing gene expression in injured and uninjured neurons , Kreppel-like factors ( KLFs ) 6 / 7 have been shown to regulate the axon regeneration of zebrafish retinal ganglion cells [ 32 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Downstream of Notch , ADAM metalloprotease and -secretase in the canonical pathway cleave the Notch to generate the Notch intracellular domain ( NCiD ) , which translocates into the nucleus to control gene expression . importantly , direct ectopic expression of GFP-NCiD is sufficient to allow entry into the nucleus and inhibit axon regeneration , confirming the role of Notch signaling in the regulation of gene expression . [Field: introduction, subscore: 2.00]
SECTION: introduction. Specifically , Notch acts as a negative regulator of axon regeneration independent of extracellular Notch ligands . [Field: introduction, subscore: 2.00]
SECTION: introduction. However , no detailed study of these molecules has been reported yet . one unexpected player that may function to control gene expression during C . elegans axon regeneration is the protein Notch [ 31 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Signaling at the Neuronal Soma The large-scale genetic screen in C . elegans [ 18 ] has also identified several transcription factors that may regulate axon regeneration by modulating gene expression in the soma . [Field: introduction, subscore: 2.00]
SECTION: introduction. JNK signaling is also upregulated in Drosophila upon axonal injury in a brain needle-injury model [ 22 ] and a crushed larval ventral nerve cord model [ 23 ] . in a Drosophila whole-brain culture model , expression of dominant-negative JNK blocks axon regeneration , whereas expression of the constitutively active form of Drosophila JNK kinase induces robust regrowth of the severed axons , one-third of which even extend to re-enter the target area [ 24 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. In the same RNAi screen using a -spectrin mutant that identified DLK-1 [ 9 ] , another MAPKKK , MLK-1 , was shown to control axon regeneration in parallel with the DLK-1 pathway [ 21 ] ( Fig . 2 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Second , a very recent study [ 20 ] has shown that the core apoptotic executioner protein CED-3 ( caspase ) and its activator CED-4 ( Apaf-1 ) play surprising roles in controlling axon regeneration by acting upstream of DLK-1 . interestingly , in the same study , the CED-4 / CED-3 pathway was also placed downstream of axotomy-induced local Ca2 + influx . [Field: introduction, subscore: 2.00]
SECTION: introduction. First , an axotomy-induced Ca2 + increase through voltage-gated Ca2 + channels or internal Ca2 + stores is necessary for axon regeneration in C . elegans [ 19 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Because the loss-of-function mutant of EFA-6 partially bypasses the requirement of DLK-1 for axon regeneration , it is likely that EFA-6 is also a downstream mediator of DLK-1 . [Field: introduction, subscore: 2.00]
SECTION: introduction. Moreover , another genetic screen study has identified EFA-6 , an Arf guanine nucleotide exchange factor , as a negative regulator of axon regeneration via destabilizing axonal microtubules [ 18 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Lastly , the expression of the tubulin isoform Tuba1a regulated by KLF6 / 7 may contribute to the control of growth-cone remodeling during axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. In the soma , axotomy activates the transcription factors KLF6 / 7 to control the expression of genes supporting axon regeneration , whereas Notch signaling acts as a negative regulator . [Field: introduction, subscore: 2.00]
SECTION: introduction. Signaling pathways regulating axon regeneration in non-mammalian models . [Field: introduction, subscore: 2.00]
SECTION: introduction. How does the DLK-1 / MKK-4 / PMK-3 MAP kinase pathway regulate axon regeneration ? [Field: introduction, subscore: 2.00]
SECTION: introduction. Axon growth is achieved by coordinated regulation of gene expression in the soma , axonal transport along the shaft , and axon assembly at the growth cone . [Field: introduction, subscore: 2.00]
SECTION: introduction. A large-scale RNAi-based genetic screen has identified dual leucine zipper-bearing kinase 1 ( DLK-1 ) as a key regulator of axon regeneration [ 9 ] . [Field: introduction, subscore: 2.00]
SECTION: introduction. Formation of a growth cone after axotomy , which is achieved via reorganization of the cytoskeleton at the proximal axonal stump , is the first step for successful regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. Pathways that are ectopically manipulated to enhance axon regeneration are not discussed . [Field: introduction, subscore: 2.00]
SECTION: introduction. Recently , several signaling pathways that regulate naturally-occurring axon regeneration have been identified in non-mammalian and mammalian PNS models . [Field: introduction, subscore: 2.00]
SECTION: introduction. Axon extension , during either development or regeneration , comprises several highly-coordinated processes , including gene transcription / expression in the soma , transport of synthesized molecules along the axon , and axon assembly at the growth cone ( Fig . 1 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. As a result , we know much less about the signaling pathways that regulate naturally-occurring mammalian axon regeneration in vivo . [Field: introduction, subscore: 2.00]
SECTION: introduction. The first step would be to elucidate the underlying signaling pathways that regulate naturally-occurring axon regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. This fact indicates that understanding the molecular mechanisms by which naturally-occurring axon regeneration is regulated should be a major focus of investigation . [Field: introduction, subscore: 2.00]
SECTION: introduction. Neurosci Bull July , 2013 . http : / / www . neurosci . cn Doi : 10 . 1007 / s12264-013-1357-4 1 Review Signaling pathways that regulate axon regeneration Saijilafu1 , Bo-Yin Zhang1 , Feng-Quan Zhou1 , 2 1Department of Orthopaedic Surgery , 2The Solomon H . Snyder Department of Neuroscience , The Johns Hopkins University , Baltimore , Maryland , USA Corresponding author : Feng-Quan Zhou . [Field: introduction, subscore: 2.00]
SECTION: references. [ 58 ] Eickholt BJ , Ahmed Ai , Davies M , Papakonstanti EA , Pearce W , Starkey ML , et al . Control of axonal growth and regeneration of sensory neurons by the p110delta Pi 3-kinase . [Field: references, subscore: 2.00]
SECTION: references. [ 56 ] Tedeschi A . Tuning the orchestra : transcriptional pathways controlling axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. [ 55 ] Cho Y , Cavalli V . HDAC5 is a novel injury-regulated tubulin deacetylase controlling axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules . [Field: references, subscore: 2.00]
SECTION: references. [ 49 ] Diamond J , Coughlin M , Macintyre L , Holmes M , Visheau B . Evidence that endogenous beta nerve growth factor is responsible for the collateral sprouting , but not the regeneration , of nociceptive axons in adult rats . [Field: references, subscore: 2.00]
SECTION: references. Development of sympathetic neurons in compartmentalized cultures . il Local control of neurite growth by nerve growth factor . [Field: references, subscore: 2.00]
SECTION: references. Genetic study of axon regeneration with cultured adult dorsal root ganglion neurons . [Field: references, subscore: 2.00]
SECTION: references. Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons . [Field: references, subscore: 2.00]
SECTION: references. Sustaining intrinsic growth capacity of adult neurons promotes spinal cord regeneration . [Field: references, subscore: 2.00]
SECTION: references. [ 31 ] El Bejjani R , Hammarlund M . Notch signaling inhibits axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. [ 30 ] Pastuhov Si , Fujiki K , Nix P , Kanao S , Bastiani M , Matsumoto K , et al . Endocannabinoid-Goalpha signalling inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqalpha-PKC-JNK signalling . [Field: references, subscore: 2.00]
SECTION: references. [ 21 ] Nix P , Hisamoto N , Matsumoto K , Bastiani M . Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways . [Field: references, subscore: 2.00]
SECTION: references. [ 16 ] Yan D , Wu Z , Chisholm AD , Jin Y . The DLK-1 kinase promotes mRNA stability and local translation in C . elegans synapses and axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. [ 9 ] Hammarlund M , Nix P , Hauth L , Jorgensen EM , Bastiani M . Axon regeneration requires a conserved MAP kinase pathway . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration mechanisms : insights from C . elegans . [Field: references, subscore: 2.00]
SECTION: references. [ 6 ] Wang Z , Jin Y . Genetic dissection of axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. [ 4 ] Sun F , Park KK , Belin S , Wang D , Lu T , Chen G , et al . Sustained axon regeneration induced by co-deletion of PTEN and SoCS3 . [Field: references, subscore: 2.00]
SECTION: references. [ 3 ] Moore DL , Blackmore MG , Hu Y , Kaestner KH , Bixby JL , Lemmon VP , et al . KLF family members regulate intrinsic axon regeneration ability . [Field: references, subscore: 2.00]
SECTION: references. [ 1 ] Park KK , Liu K , Hu Y , Smith PD , Wang C , Cai B , et al . Promoting axon regeneration in the adult CNS by modulation of the PTEN / mToR pathway . [Field: references, subscore: 2.00]
SECTION: title. Signaling pathways that regulate axon regeneration . [Field: title, subscore: 2.00]
SECTION: introduction. Genetic deletion of Pten and the subsequent activation of the Pi3KAkt pathway have recently been shown to drastically promote the intrinsic axon growth of mature mammalian CNS neurons by controlling the mTor pathway [ 1 , 2 , 4 ] . [Field: introduction, subscore: 1.00]
SECTION: introduction. Signaling at the Neuronal Soma Gene expression at the soma , which provides the raw materials for axon assembly , is necessary for sustained axon growth . [Field: introduction, subscore: 1.00]
SECTION: introduction. Further studies showed that axotomyinduced Ca2 + influx and the subsequent activation of PKC act upstream of HDAC5 to regulate tubulin acetylation and axon growth . [Field: introduction, subscore: 1.00]
SECTION: introduction. The coordination of the two pathways controls the balance of microtubule dynamics and stabilization to achieve optimal microtubule assembly and axon growth . [Field: introduction, subscore: 1.00]
SECTION: introduction. At the growth cone , the integrinGSK3CLASP / APC pathway stabilizes axonal microtubules , whereas the Ca2 + PKCHDAC5 pathway maintains microtubule dynamics . [Field: introduction, subscore: 1.00]
SECTION: introduction. Downstream of integrin signaling , regenerative axon growth is mediated by the inactivation of glycogen synthase 3 ( GSK3 ) [ 41 ] , which in turn controls growth-cone microtubule assembly via the microtubule-binding protein adenomatous polyposis coli ( APC ) and CLiP-associating proteins ( CLASPs ) [ 52 ] . [Field: introduction, subscore: 1.00]
SECTION: introduction. To our knowledge , no similar study has been reported for regenerative axon growth from adult neurons . [Field: introduction, subscore: 1.00]
SECTION: introduction. Signaling at the Growth Cone one way to spatially dissect signaling pathways in neurons is the two-compartmental culture system that separates somata from axons both physically and chemically , such as the traditional Campenot chamber [ 42 , 43 ] and the recentlydeveloped microfluidic-based chambers [ 44 , 45 ] . [Field: introduction, subscore: 1.00]
SECTION: introduction. Depending on the experimental procedure or the culture conditions , in vitro axon growth from adult DRG neurons can be stimulated and regulated by distinct mechanisms and signaling pathways [ 40 , 41 ] . [Field: introduction, subscore: 1.00]
SECTION: introduction. However , if axotomy of the peripheral branch occurs prior to central branch axotomy ( a pre-conditioning lesion ) , regeneration of the central axons is greatly enhanced [ 34 ] . [Field: introduction, subscore: 1.00]
SECTION: introduction. At the growth cone , axotomy triggers Ca2 + influx , which subsequently activates the apoptotic Ced3 / 4 pathway and / or the second messenger AC / cAMP / PKA pathway , both of which converge onto the DLK-1 pathway . [Field: introduction, subscore: 1.00]
SECTION: introduction. However , based on downstream effectors and the time-courses in observed phenotypes , it seems that the regeneration pathways identified in C . elegans to date act mostly in the axon to control growth-cone formation after axotomy ( Fig . 2 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. Currently , there is no experimental approach in C . elegans to spatially distinguish signaling events during axon growth . [Field: introduction, subscore: 1.00]
SECTION: introduction. Assembly of the cytoskeletal elements and membrane components at the growth cone drives its advancement and the subsequent axon extension . [Field: introduction, subscore: 1.00]
SECTION: introduction. Signaling Pathways That Regulate Axon Regeneration in Non-mammalian Models Non-mammalian organisms , including C . elegans , Drosophila , and zebrafish , are invaluable model systems for studying neuronal morphogenesis during development . [Field: introduction, subscore: 1.00]
SECTION: introduction. For instance , pathways regulating axon assembly at the growth cone Neurosci Bull July , 2013 2 mainly target cytoskeletal proteins , especially microtubules , to control axon extension , whereas pathways regulating gene expression in the soma mainly target transcription factors . [Field: introduction, subscore: 1.00]
SECTION: introduction. To address this issue , recent attempts to augment the growth potential by regulating transcriptional or translational machinery have provided promising results [ 1-5 ] . [Field: introduction, subscore: 1.00]
SECTION: references. [ 61 ] Hirai S , Cui de F , Miyata T , ogawa M , Kiyonari H , Suda Y , et al . The c-Jun N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex . J Neurosci 2006 , 26 : 11992 12002 . [Field: references, subscore: 1.00]
SECTION: references. [ 59 ] Shin JE , Cho Y , Beirowski B , Milbrandt J , Cavalli V , DiAntonio A . Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration . [Field: references, subscore: 1.00]
SECTION: references. Coordinating gene expression and axon assembly to control axon growth : potential role of GSK3 signaling . [Field: references, subscore: 1.00]
SECTION: references. [ 51 ] Werner A , Willem M , Jones LL , Kreutzberg GW , Mayer U , Raivich G . impaired axonal regeneration in alpha7 integrindeficient mice . [Field: references, subscore: 1.00]
SECTION: references. A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth . [Field: references, subscore: 1.00]
SECTION: references. The TrkB-Shc site signals neuronal survival and local axon growth via MEK and Pi3-kinase . [Field: references, subscore: 1.00]
SECTION: references. Raf and akt mediate distinct Neurosci Bull July , 2013 10 aspects of sensory axon growth . [Field: references, subscore: 1.00]
SECTION: references. Local control of neurite survival by nerve growth factor . [Field: references, subscore: 1.00]
SECTION: references. Different signaling pathways mediate regenerative versus developmental sensory axon growth . [Field: references, subscore: 1.00]
SECTION: references. [ 37 ] van Kesteren RE , Mason MR , Macgillavry HD , Smit AB , Verhaagen J . A gene network perspective on axonal regeneration . [Field: references, subscore: 1.00]
SECTION: references. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury . [Field: references, subscore: 1.00]
SECTION: references. [ 33 ] Veldman MB , Bemben MA , Goldman D . Tuba1a gene expression is regulated by KLF6 / 7 and is necessary for CNS development and regeneration in zebrafish . [Field: references, subscore: 1.00]
SECTION: references. [ 25 ] Barnat M , Enslen H , Propst F , Davis RJ , Soares S , Nothias F . Distinct roles of c-Jun N-terminal kinase isoforms in neurite initiation and elongation during axonal regeneration . [Field: references, subscore: 1.00]
SECTION: references. [ 24 ] Ayaz D , Leyssen M , Koch M , Yan J , Srahna M , Sheeba V , et al . Axonal injury and regeneration in the adult brain of Drosophila . [Field: references, subscore: 1.00]
SECTION: references. [ 20 ] Pinan-Lucarre B , Gabel CV , Reina CP , Hulme SE , Shevkoplyas SS , Slone RD , et al . The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans . [Field: references, subscore: 1.00]
SECTION: references. Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase . [Field: references, subscore: 1.00]
SECTION: references. Global up-regulation of microtubule dynamics and polarity reversal during regeneration of an axon from a dendrite . [Field: references, subscore: 1.00]
SECTION: references. [ 10 ] Yanik MF , Cinar H , Cinar HN , Chisholm AD , Jin Y , Ben-Yakar A . Neurosurgery : functional regeneration after laser axotomy . [Field: references, subscore: 1.00]
SECTION: references. [ 8 ] El Bejjani R , Hammarlund M . Neural regeneration in Caenorhabditis elegans . [Field: references, subscore: 1.00]
SECTION: references. [ 5 ] Smith PD , Sun F , Park KK , Cai B , Wang C , Kuwako K , et al . SoCS3 deletion promotes optic nerve regeneration in vivo . [Field: references, subscore: 1.00]
Supplemental links/files: reference in endnote reference in xml Wormbase reference
Score: 342.00
Title: Insulin / IGF1 signaling inhibits age-dependent axon regeneration .
Authors: Byrne AB ; Walradt T ; Gardner KE ; Hubbert A ; Reinke V ; Hammarlund M
Journal: Neuron
Year: 2014-02-05
Doc ID: WBPaper00044735
Bibliographic Information
Abstract
Matching Sentences
SECTION: discussion. However , the effect of PTEN and TOR on axon regeneration differs from that of daf-16 / FOXO in that daf-18 / PTEN regulates axon regeneration in both young and aged adult animals ( Figures 5 A and 5B ) . [Field: discussion, subscore: 4.00]
SECTION: introduction. Our findings demonstrate that age-related decline in adult axon regeneration is an intrinsic , neuron-specific , and genetically regulated process and define separable functions for the INSR / IGF1R-FOXO and the PTEN-TOR pathways in regulating axon regeneration an aged animals . [Field: introduction, subscore: 4.00]
SECTION: results. DLK-1 Is Required for DAF-2- and DAF-18-Mediated Regeneration The strong and cell-autonomous regulation of regeneration by the transcription factor daf-16 / FOXO suggests that transcriptional regulation of one or more neuronal genes accounts for the DAF-16-dependent increase in axon regeneration we observe in aged daf-2 ( ) mutants . [Field: results, subscore: 4.00]
SECTION: results. This lack of interaction between daf-18 / PTEN and daf-16 / FOXO in regulating axon regeneration was also observed in animals that lacked daf-2 / INSR / IFG1R : we observed no difference in axon regeneration between aged daf-2 ( e1370 ) ; daf-18 ( mg198 ) and aged daf-2 ( e1370 ) ; daf-18 ( mg198 ) ; daf-16 ( mu86 ) animals ( Figure 5F ) . [Field: results, subscore: 4.00]
SECTION: results. Although neuron-specific expression of daf-16 improved axon regeneration in aged daf-2 ( ) ; daf-16 ( ) mutants , it was not as robust as axon regeneration in aged daf-2 ( ) mutants ( compare Figures 3B and 4 D ) . [Field: results, subscore: 4.00]
SECTION: results. However , not all manipulations that affect lifespan also affect axon regeneration ( see sir-2 . 1 and eat-2 , above ) , as would be expected if the decline in axon regeneration were secondary to senectitude . [Field: results, subscore: 4.00]
SECTION: results. Together , these deficiencies result in an overall failure of axon regeneration in 5-day wildtype adults , in stark contrast to the robust axon regeneration observed in wild-type adults that are 4 days younger . [Field: results, subscore: 4.00]
SECTION: discussion. Thus , PTEN and TOR signaling have emerged as major conserved regulators of axon regeneration , and increasing TOR activity may be useful for improving regeneration , particularly in combination with other proregenerative manipulations ( Sun et al . , 2011 ) . [Field: discussion, subscore: 3.00]
SECTION: discussion. Conserved Regeneration Mechanisms in C . elegans PTEN and TOR also regulate GABA axon regeneration . [Field: discussion, subscore: 3.00]
SECTION: discussion. We identify two independent neuronal mechanisms that regulate axon regeneration in the GABA neurons of aged animals : activity of the transcription factor DAF-16 / FOXO , potentially via the dlk-1 / DLK regeneration pathway , and activity of the lipid phosphatase DAF-18 / PTEN , via regulation of TOR . [Field: discussion, subscore: 3.00]
SECTION: discussion. DISCUSSION INSR / IGFR1 and FOXO Function Independently of PTEN and TOR to Regulate Axon Regeneration in Aged Neurons Axon regeneration in adult C . elegans declines dramatically with age . [Field: discussion, subscore: 3.00]
SECTION: references. Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration . [Field: references, subscore: 3.00]
SECTION: results. DAF-18 / PTEN , but Not Insulin , Regulates Regeneration via TOR Signaling ( A ) Representative images of axon regeneration wild-type and daf-18 ( mg198 ) animals aged on DMSO or rapamycin plates . [Field: results, subscore: 3.00]
SECTION: results. In contrast to daf-2 mutants , which increase regeneration ( and lifespan ) in a daf-16-dependent manner ( Kenyon et al . , 1993 ) , we found that axon regeneration in PTEN mutants was independent of daf- 16 . [Field: results, subscore: 3.00]
SECTION: results. Further , the finding that daf-18 and daf-2 both increase regeneration despite having opposite effects on lifespan suggests that they may regulate axon regeneration by different mechanisms . [Field: results, subscore: 3.00]
SECTION: results. DAF-18 / PTEN Does Not Regulate Regeneration via DAF-16 / FOXO in Aged Animals ( A and B ) ( A ) Axon regeneration frequency of young and ( B ) aged animals . [Field: results, subscore: 3.00]
SECTION: results. To determine the role of PTEN in axon regeneration in C . elegans , we assessed regeneration in young and aged adult daf-18 / PTEN mutants . [Field: results, subscore: 3.00]
SECTION: results. However , unlike daf-16 , which positively regulates regeneration in daf-2 ( ) mutants ( Figure 3 A ) , PTEN inhibits axon regeneration in mice and flies ( Christie et al . , 2010 ; Liu et al . , 2010 ; Park et al . , 2008 ; Song et al . , 2012b ; Sun et al . , 2011 ) . [Field: results, subscore: 3.00]
SECTION: results. Together , our data demonstrate that age-related decline of regeneration is not a secondary consequence of lifespan-related aging and define a new function for daf-16 in regulating axon regeneration cell-autonomously in the aging nervous system . [Field: results, subscore: 3.00]
SECTION: results. daf-16 Mediates Age-Dependent Regeneration Downstream of daf-2 ( A and B ) ( A ) Comparison of axon regeneration frequencies in aged adult insulin pathway mutants binned into categories ( B ) described in Figure 1 . [Field: results, subscore: 3.00]
SECTION: results. DAF-16 / FOXO Regulates Regeneration Independently of Its Role in Lifespan Determination The effects we observed of daf-2 and daf-16 on axon regeneration correlate with their effects on lifespan ( Figures 3A and 3C ) . [Field: results, subscore: 3.00]
SECTION: results. Axon regeneration is a complex , multistep process in which the injured neuron must respond to injury by initiating a growth cone and then sustain axon extension toward the appropriate target . [Field: results, subscore: 3.00]
SECTION: results. Therefore , axon regeneration declines in adulthood , in large part due to failure in aged animals of growth cone initiation in response to nerve injury . [Field: results, subscore: 3.00]
SECTION: results. Insulin / IGF1 Signaling Inhibits Axon Regeneration and Does so Specifically in Aged Animals We investigated the relationship between axon regeneration and age using single-neuron pulsed-laser surgery in adult C . elegans GABA motor neurons ( Figure 1A ) . [Field: results, subscore: 3.00]
SECTION: abstract. Finally , DLK-1 , a conserved regulator of regeneration , is downregulated by insulin / IGF1 signaling , bound by DAF-16 in neurons , and required for both DAF-16- and DAF-18-mediated regeneration . [Field: abstract, subscore: 2.00]
SECTION: abstract. Here we show that axon regeneration in aging C . elegans motor neurons is inhibited by the conserved insulin / IGF1 receptor DAF-2 . [Field: abstract, subscore: 2.00]
SECTION: abstract. The ability of injured axons to regenerate declines with age , yet the mechanisms that regulate axon regeneration in response to age are not known . [Field: abstract, subscore: 2.00]
SECTION: discussion. Therefore , our results demonstrate that agerelated decline in axon regeneration can be decoupled from lifespan , and these two effects of time are functionally distinct . [Field: discussion, subscore: 2.00]
SECTION: discussion. First , DAF-2 / INSR / IGF1R inhibits axon regeneration in older animals by modulating neuronal DAF-16 / FOXO function , and this function is independent of lifespan . [Field: discussion, subscore: 2.00]
SECTION: discussion. These functions include nutrient sensing , cell growth , actin organization , cytoskeletal dynamics , autophagy , and stress response ( Guertin and Sabatini , 2007 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. 019 Filbin , 2004 ; Ziv and Spira , 1997 ) , and microtubule stability ( Ghosh-Roy et al . , 2012 ; Hellal et al . , 2011 ; Sengottuvel et al . , 2011 ) --suggests that axon regeneration itself , and the intrinsic molecular mechanisms that regulate it , are of ancient origin and are functionally conserved across highly divergent species . [Field: discussion, subscore: 2.00]
SECTION: discussion. Our data establish C . elegans as a new model for studying the function of PTEN , TOR , and FOXO in axon regeneration . [Field: discussion, subscore: 2.00]
SECTION: discussion. In Drosophila , disrupting PTEN promotes axon regeneration of the class IV dendritic arborization ( da ) neurons ( Song et al . , 2012b ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. Further , at least in the CNS , the full effect of PTEN deletion on axon regeneration requires mTOR activation ( Liu et al . , 2010 ; Park et al . , 2008 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. In mice , PTEN disruption promotes axon regeneration of retinal ganglion cells ( Park et al . , 2008 ) , peripheral sensory ( sciatic ) neurons ( Christie et al . , 2010 ) , and corticospinal neurons ( Liu et al . , 2010 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. Like daf-16 / FOXO , the effect of PTEN and TOR on axon regeneration is intrinsic to the nervous system ( Figure 5 G ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. We found that insulin signaling acts at an early stage of adult life--between 1 and 5 days--to inhibit daf-16 and reduce regeneration of GABA neurons , with regeneration declining 10-fold in this time period ( Figure 2D ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. Finally , although in aged animals both axon regeneration and maintenance of morphology are both regulated by insulin signaling and daf-16 , the effects of aging occur at different rates . [Field: discussion, subscore: 2.00]
SECTION: discussion. Further , regeneration in aged animals is dependent on the dlk-1 MAP kinase pathway ( Figure 7 ) , while aberrant growth is independent of the dlk-1 pathway ( Tank et al . , 2011 ) . [Field: discussion, subscore: 2.00]
SECTION: discussion. The functions of DAF-16 in aging neurons are likely complex . Activated daf-16 enhances regenerative growth after laser injury ( Figure 3 A ) but suppresses aberrant growth in neurons that have not received an external injury ( Pan et al . , 2011 ; Tank et al . , 2011 ; Toth et al . , 2012 ) . [Field: discussion, subscore: 2.00]
SECTION: introduction. Finally , C . elegans is a powerful system for studying axon regeneration ( Chen and Chisholm , 2011 ; El Bejjani and Hammarlund , 2012 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. To characterize a neuron ' s response to age and injury , we investigated age-related decline in axon regeneration in C . elegans . [Field: introduction, subscore: 2.00]
SECTION: introduction. In animals with a mature nervous system , the various effects of age on axon regeneration could be a secondary consequence of aging ' s deleterious effects on the organism as a whole or a result of interactions between neurons and an aging cellular environment . [Field: introduction, subscore: 2.00]
SECTION: introduction. In C . elegans , axon regeneration also declines during adulthood ( Gabel et al . , 2008 ; Hammarlund et al . , 2009 ; Nix et al . , 2011 ; Wu et al . , 2007 ; Zou et al . , 2013 ) . [Field: introduction, subscore: 2.00]
SECTION: introduction. Axon regeneration has the potential to repair damaged neurons in response to injury . [Field: introduction, subscore: 2.00]
SECTION: introduction. Finally , DLK-1 , a conserved regulator of regeneration , is downregulated by insulin / IGF1 signaling , bound by DAF-16 in neurons , and required for both DAF-16- and DAF-18-mediated regeneration . [Field: introduction, subscore: 2.00]
SECTION: introduction. Here we show that axon regeneration in aging C . elegans motor neurons is inhibited by the conserved insulin / IGF1 receptor DAF-2 . [Field: introduction, subscore: 2.00]
SECTION: introduction. 019 SUMMARY The ability of injured axons to regenerate declines with age , yet the mechanisms that regulate axon regeneration in response to age are not known . [Field: introduction, subscore: 2.00]
SECTION: references. The DLK-1 kinase promotes mRNA stability and local translation in C . elegans synapses and axon regeneration . [Field: references, subscore: 2.00]
SECTION: references. Sustained axon regeneration induced by co-deletion of PTEN and SOCS3 . [Field: references, subscore: 2.00]
SECTION: references. Taxol facilitates axon regeneration in the mature CNS . [Field: references, subscore: 2.00]
SECTION: references. Spinal axon regeneration induced by elevation of cyclic AMP . [Field: references, subscore: 2.00]
SECTION: references. Promoting axon regeneration in the adult CNS by modulation of the PTEN / mTOR pathway . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways . [Field: references, subscore: 2.00]
SECTION: references. Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration requires a conserved MAP kinase pathway . [Field: references, subscore: 2.00]
SECTION: references. Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C . elegans . [Field: references, subscore: 2.00]
SECTION: references. Axon regeneration mechanisms : insights from C . elegans . [Field: references, subscore: 2.00]
SECTION: results. Insulin Signaling Regulates Expression of DLK-1 / DLK ( A ) Axon regeneration frequency of aged animals of the indicated genotypes . [Field: results, subscore: 2.00]
SECTION: results. dlk-1 / DLK-1 is a conserved dual-leucine zipper kinase MAPKKK that functions in axon regeneration by activating mkk-4 / MAPKK and pmk-3 / MAPK ( Hammarlund et al . , 2009 ; Nakata et al . , 2005 ; Yan et al . , 2009 ) . [Field: results, subscore: 2.00]
SECTION: results. These findings strengthen our conclusion that , despite functioning in the same pathway to regulate lifespan , DAF-18 / PTEN and insulin signaling function independently of one another to regulate axon regeneration : DAF-18 / PTEN signaling functions via regulation of TOR and insulin signaling functions via regulation of DAF-16 activity . [Field: results, subscore: 2.00]
SECTION: results. Next , we tested whether activated TOR might also mediate the effect of loss of DAF-2 on axon regeneration in aged animals . [Field: results, subscore: 2.00]
SECTION: results. Thus , the PTEN-TOR axis represents a conserved mechanism that regulates axon regeneration across species . [Field: results, subscore: 2.00]
SECTION: results. TOR has previously been shown to mediate the effect of PTEN on axon regeneration in mammals ( Christie et al . , 2010 ; Liu et al . , 2010 ; Park et al . , 2008 ; Song et al . , 2012b ; Sun et al . , 2011 ) . [Field: results, subscore: 2.00]
SECTION: results. By contrast , rapamycin treatment abolished the increased axon regeneration phenotype of daf-18 / PTEN mutants ( Figure 6 ) . [Field: results, subscore: 2.00]
SECTION: results. We inhibited TOR with rapamycin and found that had no effect on control animals , suggesting that TOR signaling is not a major component of axon regeneration in aged wild-type animals . [Field: results, subscore: 2.00]
SECTION: results. ( * p < 0 . 05 , relative to daf-18 ( mg198 ) DMSO , using Fisher ' s exact test ) . increased TOR activity might account for the effect of loss of PTEN on axon regeneration in aged C . elegans GABA neurons . [Field: results, subscore: 2.00]
SECTION: results. ( B ) Axon regeneration frequencies of aged adult animals placed on rapamycin or DMSO as indicated . [Field: results, subscore: 2.00]
SECTION: results. Therefore , daf-18 functions cell intrinsically to inhibit axon regeneration , independently of insulin signaling and FOXO . [Field: results, subscore: 2.00]
SECTION: results. To determine whether enhanced regeneration in daf-18 / PTEN is due to the same mechanism as enhanced regeneration in daf- 2 mutants , we determined its dependence on daf-16 . [Field: results, subscore: 2.00]
SECTION: results. Since daf-18 / PTEN mutants have shorter lives than wild-type animals ( Dorman et al . , 1995 ; Larsen et al . , 1995 ) ( Figure 5 E ) but retain the ability to regenerate axons when aged ( Figure 5 B ) , the loss of PTEN is another way ( besides daf-16 mosaics--see Figure 4 ) to decouple lifespan and axon regeneration in aged animals . [Field: results, subscore: 2.00]
SECTION: results. ( G ) Axon regeneration frequency of aged wild-type , daf-18 ( mg198 ) , and daf-18 ( mg198 ) animals with extrachromosomal Punc-47 : : daf-18cDNA , which is expressed in GABA neurons . [Field: results, subscore: 2.00]
SECTION: results. ( F ) Quantification of axon regeneration in aged animals of the indicated genotypes . [Field: results, subscore: 2.00]
SECTION: results. daf-16 Mediates Age-Dependent Regeneration Independently of Lifespan ( AE ) We assessed lifespan and regeneration in ( A ) aged daf-2 ( e1370 ) ; daf- 16 ( mu86 ) worms expressing daf-16 in the intestine ( Pges-1 : : daf-16 ) and in the nervous system ( Prgef-1 : : daf-16 ) . [Field: results, subscore: 2.00]
SECTION: results. Nonetheless , these data indicate that PTEN ' s role as an inhibitor of axon regeneration is conserved in C . elegans . [Field: results, subscore: 2.00]
SECTION: results. The increase in regeneration in aged daf-18 / PTEN mutants was not as large as that seen in aged daf-2 mutants ( Figures 5B and 2B ) , and regenerating axons in aged daf-18 / PTEN mutants often had a less robust growth structure ( Figures 5 D and 2C ) . [Field: results, subscore: 2.00]
SECTION: results. Alternatively , it may be an indication that while neuronal daf-16 is necessary and sufficient to promote regeneration , robust regeneration also requires daf-16 function outside the nervous system . [Field: results, subscore: 2.00]
SECTION: results. These results show that daf-16 function in neurons is sufficient to regulate axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. However , aged neuron : daf-16 * animals had improved axon regeneration compared to controls , even though they did not live any longer ( Figures 4B and 4C , Prgef-1 : : daf-16 ) . [Field: results, subscore: 2.00]
SECTION: results. Thus , daf-16 / FOXO could regulate axon regeneration by acting directly in neurons . [Field: results, subscore: 2.00]
SECTION: results. 019 lifespan is not sufficient to increase axon regeneration in aged animals . [Field: results, subscore: 2.00]
SECTION: results. We conclude that daf-16 and insulin / IGF1 signaling can regulate lifespan independently of regeneration and that increased Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals 4 Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 2.00]
SECTION: results. However , 5-day adult intestine : : daf-16 * animals showed a low level of axon regeneration that was similar to wild-type ( Figures 4C and 4D ) . [Field: results, subscore: 2.00]
SECTION: results. To test the idea that age-related loss of axon regeneration and lifespan are separately regulated , we attempted to decouple the two phenotypes using daf-16 ( ) mosaic animals ( Figure 4 ) . [Field: results, subscore: 2.00]
SECTION: results. One possible model to explain this finding is that increased axon regeneration in 5-day adult daf- 2 ( ) mutants is a secondary consequence of delayed aging ( Figure 3D , hatched arrow ii ) . [Field: results, subscore: 2.00]
SECTION: results. To test whether inhibition of regeneration by daf-2 also functions by inhibiting daf-16 , we assessed regeneration in mutants that lacked both daf-2 and daf-16 . [Field: results, subscore: 2.00]
SECTION: results. ( D ) Axon regeneration frequencies in wild-type and daf-2 ( e1370 ) animals of increasing age . [Field: results, subscore: 2.00]
SECTION: results. However , even in these old animals , some degree of axon regeneration was still present in daf-2 ( ) animals . [Field: results, subscore: 2.00]
SECTION: results. The frequency of axon regeneration in daf-2 ( ) mutants declined by 10 days postdevelopment ( Figure 2 D ) . [Field: results, subscore: 2.00]
SECTION: results. These results indicate that daf-2 inhibits both growth cone initiation and axon growth in aged animals . [Field: results, subscore: 2.00]
SECTION: results. Therefore , daf-2 specifically regulates an age-related decline in axon regeneration . [Field: results, subscore: 2.00]
SECTION: results. daf-2 regulates regeneration specifically in aged animals , as the frequency of regeneration in young adult daf-2 ( ) mutants was indistinguishable from that in young adult wild-type animals ( Figure 2D ) . [Field: results, subscore: 2.00]
SECTION: results. We found that in contrast to wild-type animals , 5-day adult daf-2 ( ) mutants exhibited no decline in axon regeneration ( Figures 2A and 2B ) . [Field: results, subscore: 2.00]
SECTION: results. However , modification of insulin signaling by mutating the insulin / insulin-like growth factor 1 receptor ( INSR / IGF1R ) DAF-2 enhanced regeneration in aged adults . [Field: results, subscore: 2.00]
SECTION: results. We tested axon regeneration in several well-characterized models that increase lifespan , including animals that have decreased insulin signaling ( daf-2 mutants ) ( Kenyon et al . , 1993 ; Kimura et al . , 1997 ) , animals that overexpress sirtuin and have an additional lifespanincreasing mutation ( sir-2 . 1OE mutants ) ( it issenbaum and Guarente , 2001 ; Viswanathan and Guarente , 2011 ) , and animals that are calorically restricted as a result of eating difficulty ( eat-2 mutants ) ( Avery , 1993 ; Lakowski and Hekimi , 1998 ) . [Field: results, subscore: 2.00]
SECTION: results. We hypothesized that the age-related decline in GABA axon regeneration might be suppressed by manipulations that delay aging and increase lifespan . [Field: results, subscore: 2.00]
SECTION: results. Regeneration categories were as follows : ML , indicating axons that did not extend past the midline ; ML , indicating axons that extended to the midline ; ML + , indicating axons that extended three-quarters of the distance between the ventral and dorsal cords ; and Full , indicating axons that fully regeneration to the dorsal cord . had regenerated toward their target , the dorsal nerve cord ( Figures 1 A1D ) . [Field: results, subscore: 2.00]
SECTION: results. ( D ) Comparison of axon regeneration frequency in young and aged wild-type animals . [Field: results, subscore: 2.00]
SECTION: results. Regeneration Declines in Aged Animals ( A ) GABA commissures are severed with a pulsed laser and scored for regeneration after 24 hr in young adult ( 1-day adult , L4 + 1 day at 25 C ) and aged adult ( 5-day adult , L4 + 5 days at 25 C ) animals . [Field: results, subscore: 2.00]
SECTION: results. 019 age ( rather than developmental changes ) on regeneration , we compared regeneration of severed axons at two adult time points : 1-day adults , aged 1 day past the final larval stage ( young adults ) and 5-day adults , aged 5 days past the final larval stage ( aged adults ) . [Field: results, subscore: 2.00]
SECTION: title. Insulin / IGF1 signaling inhibits age-dependent axon regeneration . [Field: title, subscore: 2.00]
SECTION: abstract. Together , our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR . [Field: abstract, subscore: 1.00]
SECTION: abstract. In addition , we found that DAF-18 / PTEN inhibits regeneration independently of age and FOXO signaling via the TOR pathway . [Field: abstract, subscore: 1.00]
SECTION: abstract. DAF-16 regulates regeneration independently of lifespan , indicating that neuronal aging is an intrinsic , neuron-specific , and genetically regulated process . [Field: abstract, subscore: 1.00]
SECTION: abstract. DAF-2 ' s function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16 / FOXO . [Field: abstract, subscore: 1.00]
SECTION: discussion. Moreover , lifespan and healthspan are distinct at the cellular level , as DAF-16 / FOXO functions in intestinal cells to regulate lifespan ( Libina et al . , 2003 ) and in neurons to regulate regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Second , despite functioning antagonistically to regulate lifespan ( Dorman et al . , 1995 ) , both loss of daf-18 / PTEN and loss of daf-2 result in increased regeneration . [Field: discussion, subscore: 1.00]
SECTION: discussion. Lifespan and Neuronal Healthspan Are Independent Phenotypes In addition to identifying molecular mechanisms that regulate GABA neuron regeneration in aging animals , our results demonstrate that the regulation of neuronal repair in aging animals is an essential component of healthspan , as opposed to lifespan . [Field: discussion, subscore: 1.00]
SECTION: discussion. Our data suggest that one important output of insulin-FOXO signaling is upregulation of the conserved dlk-1 regeneration pathway . [Field: discussion, subscore: 1.00]
SECTION: discussion. 9 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: discussion, subscore: 1.00]
SECTION: discussion. The conservation of PTEN and TOR functions in axon regeneration--along with conservation of the DLK-1 pathway ( Hammar- lund et al . , 2009 ; Itoh et al . , 2009 ; Shin et al . , 2012 ; Watkins et al . , 2013 ; Xiong et al . , 2010 ; Yan et al . , 2009 ) , calcium signaling ( Ghosh-Roy et al . , 2010 ; Pichichero et al . , 1973 ; Spencer and Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: discussion, subscore: 1.00]
SECTION: discussion. In mutant animals that lack daf-18 / PTEN , an increase in TOR activity results in increased regeneration ( Figure 6 ) . [Field: discussion, subscore: 1.00]
SECTION: discussion. DAF-16 Protects an Aging Nervous System Our results show that the insulin signaling pathway acts in aged animals to inhibit regeneration via regulation of daf-16 activity in the nervous system . [Field: discussion, subscore: 1.00]
SECTION: introduction. Moreover , regeneration in both daf-2 and daf-18 mutants requires the critical dlk-1 / DLK plasticity pathway . [Field: introduction, subscore: 1.00]
SECTION: introduction. The conserved phosphatase daf-18 / protein phosphatase and tensin homolog ( PTEN ) ( deleted on chromosome 10 ) also regulates regeneration yet does so in an age- and insulin-independent manner , via target of rapamycin ( TOR ) signaling . [Field: introduction, subscore: 1.00]
SECTION: introduction. daf-16 functions cell intrinsically in neurons to regulate regeneration independently of daf-16 ' s function in lifespan determination . [Field: introduction, subscore: 1.00]
SECTION: introduction. We show that this decline in regeneration is regulated by the insulin / IGF1 receptor daf-2 and the forkhead transcription factor daf-16 / FOXO . [Field: introduction, subscore: 1.00]
SECTION: introduction. We show that as adult animals age , these neurons exhibit decreased retraction , decreased growth response , and decreased extension in response to nerve injury . [Field: introduction, subscore: 1.00]
SECTION: introduction. Here we analyze how age affects regeneration in the GABA motor neurons of adult C . elegans . [Field: introduction, subscore: 1.00]
SECTION: introduction. Individual postdevelopmental C . elegans axons are readily amenable to pulsed laser axotomy , and their regeneration is easily visualized in vivo ( Byrne et al . , 2011 ; Gabel et al . , 2008 ; Hammarlund et al . , 2009 ; Rao et al . , 2008 ; Wu et al . , 2007 ; Yanik et al . , 2004 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. However , the mechanisms that mediate the decline of regeneration in adults are not well understood . [Field: introduction, subscore: 1.00]
SECTION: introduction. Understanding the mechanisms that regulate the decline of regeneration with age could help inform therapies for age-related changes in neuronal function , disease , and recovery . [Field: introduction, subscore: 1.00]
SECTION: introduction. For example , in the mammalian peripheral nervous system , where regeneration does occur in adults , regenerative potential is lower in old animals ( Pestronk et al . , 1980 ; Tanaka et al . , 1992 ; Verdu et al . , 1995 , 2000 ) . [Field: introduction, subscore: 1.00]
SECTION: introduction. Together , our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR . [Field: introduction, subscore: 1.00]
SECTION: introduction. In addition , we found that DAF-18 / PTEN inhibits regeneration independently of age and FOXO signaling via the TOR pathway . [Field: introduction, subscore: 1.00]
SECTION: introduction. DAF-16 regulates regeneration independently of lifespan , indicating that neuronal aging is an intrinsic , neuron-specific , and genetically regulated process . [Field: introduction, subscore: 1.00]
SECTION: introduction. DAF-2 ' s function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16 / FOXO . [Field: introduction, subscore: 1.00]
SECTION: introduction. Neuron Article Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration Alexandra B . Byrne , 1 , 2 Trent Walradt , 1 , 2 Kathryn E . Gardner , 1 Austin Hubbert , 1 , 2 Valerie Reinke , 1 and Marc Hammarlund 1 , 2 , * 1Department of Genetics , Yale University School of Medicine , New Haven , CT 06510 , USA 2Program in Cellular Neuroscience , Neurodegeneration and Repair , Yale University School of Medicine , New Haven , CT 06510 , USA * Correspondence : marc . hammarlund @ yale . edu http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: introduction, subscore: 1.00]
SECTION: materials. Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: materials, subscore: 1.00]
SECTION: materials. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals 10 Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: materials, subscore: 1.00]
SECTION: references. 13 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: references, subscore: 1.00]
SECTION: references. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: references, subscore: 1.00]
SECTION: references. Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers . [Field: references, subscore: 1.00]
SECTION: references. Localized and transient elevations of intracellular Ca2 + induce the dedifferentiation of axonal segments into growth cones . [Field: references, subscore: 1.00]
SECTION: references. Neurosurgery : functional regeneration after laser axotomy . [Field: references, subscore: 1.00]
SECTION: references. Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: references, subscore: 1.00]
SECTION: references. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals 12 Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: references, subscore: 1.00]
SECTION: references. Caenorhabditis elegans neuronal regeneration is influenced by life stage , ephrin signaling , and synaptic branching . [Field: references, subscore: 1.00]
SECTION: references. Influence of aging on peripheral nerve function and regeneration . [Field: references, subscore: 1.00]
SECTION: references. The effect of aging on efferent nerve fibers regeneration in mice . [Field: references, subscore: 1.00]
SECTION: references. Myelinated fiber regeneration after sciatic nerve crush : morphometric observations in young adult and aging mice and the effects of macrophage suppression and conditioning lesions . [Field: references, subscore: 1.00]
SECTION: references. A role for cAMP in regeneration of the adult mammalian CNS . [Field: references, subscore: 1.00]
SECTION: references. Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam . [Field: references, subscore: 1.00]
SECTION: references. Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration . [Field: references, subscore: 1.00]
SECTION: references. Effects of aging on nerve sprouting and regeneration . [Field: references, subscore: 1.00]
SECTION: references. Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation . [Field: references, subscore: 1.00]
SECTION: references. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury . [Field: references, subscore: 1.00]
SECTION: references. 11 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: references, subscore: 1.00]
SECTION: references. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: references, subscore: 1.00]
SECTION: references. Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase . [Field: references, subscore: 1.00]
SECTION: references. Neural regeneration in Caenorhabditis elegans . [Field: references, subscore: 1.00]
SECTION: results. Together , our data demonstrate that one notable difference between aged wild-type and daf-2 ( ) animals is expression of dlk-1 pathway genes and suggest that increased dlk-1 pathway activity in neurons--perhaps due to binding and direct regulation of dlk-1 by neuronal DAF-16--contributes to high regeneration in aged daf-2 animals . [Field: results, subscore: 1.00]
SECTION: results. Together with the evidence that daf-16 functions cell autonomously in neurons to regulate regeneration ( Figure 4 ) and that dlk-1 expression is regulated in an age- and daf-16- dependent manner ( Figures 7 B and S2 ) , these results suggest that one effect of DAF-16 activity in aged animals is to increase expression of dlk-1 in neurons . [Field: results, subscore: 1.00]
SECTION: results. Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. We used it issue-specific ChIP-seq data to investigate whether DAF-16 binds directly to dlk-1 in Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals 8 Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 1.00]
SECTION: results. With respect to regeneration , DAF-16 functions specifically in the nervous system ( Figure 4 ) . [Field: results, subscore: 1.00]
SECTION: results. Since dlk-1 is also absolutely necessary and sufficient for regeneration in aged wild-type animals , these results indicate that dlk-1 functions downstream or in parallel to daf-16 and daf-18 . [Field: results, subscore: 1.00]
SECTION: results. In addition , dlk-1 overexpression was sufficient to rescue regeneration in daf-16 mutants . [Field: results, subscore: 1.00]
SECTION: results. We found that dlk-1 is absolutely required for regeneration in aged daf-2 ( ) , and aged daf-18 ( ) mutants ( Figure 7A ) ( Hammarlund et al . , 2009 ) . [Field: results, subscore: 1.00]
SECTION: results. To investigate whether dlk-1 functions with daf-16 to regulate regeneration , we first performed genetic epistasis experiments between dlk-1 and daf-16 ( Figure 7 A ) . [Field: results, subscore: 1.00]
SECTION: results. Therefore , the dlk-1 MAP kinase pathway is a candidate target for daf-16-mediated regulation of age-related regeneration . [Field: results, subscore: 1.00]
SECTION: results. DLK-1 pathway function is required cell autonomously to regulate regeneration across species ( Hammarlund et al . , 2009 ; Itoh et al . , 2009 ; Shin et al . , 2012 ; Watkins et al . , 2013 ; Xiong et al . , 2010 ) . [Field: results, subscore: 1.00]
SECTION: results. 7 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. dlk-1 is also Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 1.00]
SECTION: results. Activated pmk-3 / MAPK promotes regeneration at least in part by posttranscriptional stabilization of the mRNA for the B-Zip protein cebp-1 / C / EBP1 , likely via mak-2 / MAPKAP2 ( Yan et al . , 2009 ) . [Field: results, subscore: 1.00]
SECTION: results. One potential target of such regulation is the genes of the dlk-1 MAP kinase pathway-- the best-characterized intrinsic regeneration pathway in C . elegans . [Field: results, subscore: 1.00]
SECTION: results. Therefore daf-2 / INSR / IGF1R functions independently of TOR to regulate regeneration , in contrast to daf-18 / PTEN . [Field: results, subscore: 1.00]
SECTION: results. We found that regeneration in aged daf-2 ( e1370 ) mutants is not affected by rapamycin ( Figure 6 ) . [Field: results, subscore: 1.00]
SECTION: results. These results suggest that daf-18 / PTEN ' s function in regeneration is mediated by TOR in C . elegans . [Field: results, subscore: 1.00]
SECTION: results. In addition , we found that GABA-specific daf-18 expression ( driven by the unc-47 [ vesicular GABA transporter ] promoter ) inhibits regeneration in aged daf-18 ( ) mutants ( Figure 5G ) . [Field: results, subscore: 1.00]
SECTION: results. Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals 6 Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 1.00]
SECTION: results. In contrast to the mechanisms of lifespan determination , daf-18 / PTEN regulates regeneration independently of daf-16 / FOXO and daf-2 / INSR / IGF1R . [Field: results, subscore: 1.00]
SECTION: results. * p < 0 . 05 , relative to daf-18 ( mg198 ) , using Fisher ' s exact test . ( H ) Model of interactions between components of the age-related regeneration response . [Field: results, subscore: 1.00]
SECTION: results. 5 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 1.00]
SECTION: results. ( C ) Regeneration of aged daf-2 ( e1370 ) ; daf-16 ( mu86 ) worms expressing daf- 16 in the nervous system ( Prgef-1 : : daf-16 ) and in the intestine ( Pges-1 : : daf-16 ) . [Field: results, subscore: 1.00]
SECTION: results. Although loss of daf-18 / PTEN and daf-2 / INSR / IGF1R both result in increased regeneration in aged adults , the two mutations have different effects on lifespan . [Field: results, subscore: 1.00]
SECTION: results. However , unlike daf-2 ( which had no effect on young animals ) , loss of daf-18 / PTEN also increased regeneration in young adults ( Figures 2 D and 5A ) . [Field: results, subscore: 1.00]
SECTION: results. We found that like loss of daf-2 , loss of daf-18 / PTEN significantly increased regeneration in aged adults ( Figures 2 B and 5 B ) . [Field: results, subscore: 1.00]
SECTION: results. DAF-18 / PTEN Inhibits Regeneration Independently of Age and DAF-16 / FOXO Like daf-16 / FOXO , daf-18 / PTEN ( protein phosphatase and tensin homolog deleted on chromosome 10 ) is required for lifespan extension in daf-2 / INSR / IFG1R mutants ( Dorman et al . , 1995 ; Larsen et al . , 1995 ) . [Field: results, subscore: 1.00]
SECTION: results. If so , we reasoned that neuron-specific daf-16 expression might rescue age-related regeneration in a daf-2 ( ) ; daf-16 ( ) background . [Field: results, subscore: 1.00]
SECTION: results. Indeed , the growth potential of injured axons depends in part on intrinsic factors ( Neumann et al . , 2002 ; Neumann and Woolf , 1999 ; Qiu et al . , 2002 ) . [Field: results, subscore: 1.00]
SECTION: results. These results suggested that daf-16 might function in neurons to increase regeneration in aged animals . [Field: results, subscore: 1.00]
SECTION: results. Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. Thus , despite increasing the lifespan of daf-2 ( ) ; daf-16 ( ) double mutants , intestinal DAF-16 expression did not affect age-related decline of regeneration ( Figures 4 B and 4C , + Pges-1 : daf-16 ) . [Field: results, subscore: 1.00]
SECTION: results. We reasoned that if daf-16 function in neurons--rather than daf- 16 function in lifespan extension--is required to increase regeneration in aged animals , the intestine : : daf-16 * animals should fail to regenerate when aged , even though they have increased lifespans . [Field: results, subscore: 1.00]
SECTION: results. Further , the steep decline in regeneration that we observe in 5-day-aged wild-type adults occurs well before significant age-related mortality ( Figure 3 C ) . [Field: results, subscore: 1.00]
SECTION: results. daf-16 regulates regeneration downstream of daf-2 in aged adults either ( i ) directly or ( ii ) indirectly . [Field: results, subscore: 1.00]
SECTION: results. Therefore , daf-2 inhibits regeneration in aged animals by preventing daf-16 function . [Field: results, subscore: 1.00]
SECTION: results. We found that eliminating daf-16 in daf-2 ( ) mutants abolished the high regeneration normally observed in aged daf-2 ( ) mutants ( Figures 3 A and 3B ) . [Field: results, subscore: 1.00]
SECTION: results. 019 DAF-2 Regulates Regeneration in Aged Animals via DAF-16 / FOXO daf-2 / INSR / IGF1R regulates lifespan by activating mechanisms that sequester the forkhead transcription factor daf-16 / FOXO in the cytoplasm , thus preventing daf-16 / FOXO from translocating to the nucleus and regulating transcription of effector genes ( Henderson and Johnson , 2001 ; Kenyon et al . , 1993 ; Lee et al . , 2001 ; Lin et al . , 2001 ) . [Field: results, subscore: 1.00]
SECTION: results. 3 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 1.00]
SECTION: results. Regeneration in 5-day adult daf-2 ( ) animals is significantly increased relative to 5-day adult wild-type animals ( p < 0 . 01 ; Fisher ' s exact test ) . [Field: results, subscore: 1.00]
SECTION: results. ( B ) Quantification of regeneration in 5-day adult wild-type and daf-2 ( e1370 ) animals 24 hr after axotomy . [Field: results, subscore: 1.00]
SECTION: results. In summary , our results demonstrate that the INS / IGF1 receptor DAF-2 regulates both growth cone initiation and axon extension in aged adult animals after injury . [Field: results, subscore: 1.00]
SECTION: results. By contrast , in wild-type animals 10 days postdevelopment , regeneration was never observed ( Figure 2D ) . [Field: results, subscore: 1.00]
SECTION: results. Therefore , daf-2 regulates growth rather than guidance of regenerating axons . [Field: results, subscore: 1.00]
SECTION: results. Further , these results show that the increased regeneration we observe in aged daf- 2 ( ) animals is not because the daf-2 ( ) axons retract less : in fact , daf-2 ( ) axons retract more and also regenerate farther . [Field: results, subscore: 1.00]
SECTION: results. To confirm that the failure of axons in aged wild-type animals to regenerate past the midline is due to defects in growth and not inaccurate axon guidance , we measured the length of severed axons . [Field: results, subscore: 1.00]
SECTION: results. Therefore , daf-2 inhibits growth cone initiation in aged axons . [Field: results, subscore: 1.00]
SECTION: results. We characterized the specific effects of daf-2 on age-related regeneration using the same categorical analysis as described above ( Figures 2A and 2C ) . [Field: results, subscore: 1.00]
SECTION: results. 019 models , neither sir-2 . 1 nor eat-2 significantly affected regeneration ( p > 0 . 3 ; Fisher ' s exact test ) . [Field: results, subscore: 1.00]
SECTION: results. Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
SECTION: results. Of these Neuron Insulin / IGF1 Inhibits Regeneration in Aged Animals 2 Neuron 81 , 113 , February 5 , 2014 2014 Elsevier Inc . [Field: results, subscore: 1.00]
SECTION: results. Regeneration in 5-day adult animals is significantly reduced relative to 1-day adult animals ( p < 0 . 01 , Fisher ' s exact test ) . [Field: results, subscore: 1.00]
SECTION: results. To characterize the effect of age on regeneration , we binned cut axons into four categories based on how far they Figure 1 . [Field: results, subscore: 1.00]
SECTION: results. By contrast , in aged adult wild-type animals , few axons ( 28 % ) initiated a regeneration response to injury ( Figures 1 C and 1D ) . [Field: results, subscore: 1.00]
SECTION: results. 1 Please cite this article in press as : Byrne et al . , Insulin / IGF1 Signaling Inhibits Age-Dependent Axon Regeneration , Neuron ( 2014 ) , http : / / dx . doi . org / 10 . 1016 / j . neuron . [Field: results, subscore: 1.00]
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