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Inhibiting Poly(ADP-Ribosylation) Improves Axon Regeneration

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Inhibiting Poly(ADP-Ribosylation) Improves Axon Regeneration SHORT REPORT Inhibiting poly(ADP-ribosylation) improves axon regeneration Alexandra B Byrne1,2,3†, Rebecca D McWhirter4,5, Yuichi Sekine3,6, Stephen M Strittmatter2,3,6, David M Miller III4,5, Marc Hammarlund1,2,3* 1Department of Genetics, Yale University School of Medicine, New Haven, United States; 2Department of Neuroscience, Yale University School of Medicine, New Haven, United States; 3Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, United States; 4Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States; 5Program in Neuroscience, Vanderbilt University, Nashville, United States; 6Department of Neurology, Yale University School of Medicine, New Haven, United States Abstract The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to *For correspondence: marc. proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical [email protected] neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of Present address: †Department the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across of Neurobiology, University of Massachusetts Medical School, species, and that chemical inhibition of PARPs can elicit axon regeneration. Worcester, United States DOI: 10.7554/eLife.12734.001 Competing interests: The authors declare that no competing interests exist. Introduction Funding: See page 12 Unlike damaged peripheral nerves, the central nervous system does not successfully regenerate after Received: 01 November 2015 injury. Failure to regenerate has been attributed to two components of the regeneration response: Accepted: 03 September 2016 intrinsic and extrinsic factors. While extrinsic inhibitory factors such as the glial microenvironment Published: 04 October 2016 can be modulated with some success, regeneration potential is still substantially hindered, providing Reviewing editor: Kang Shen, evidence that intrinsic factors play a significant role in modulating the ability of an axon to regener- Howard Hughes Medical ate (Richardson et al., 1980; Neumann and Woolf, 1999; GrandPre´ et al., 2000; Fournier et al., Institute, Stanford University, 2001; Qiu et al., 2002; Yiu and He, 2006; Park et al., 2008; Smith et al., 2009; Wang et al., United States 2011). Developing an understanding of the intrinsic mechanisms that regulate regeneration will pro- vide insight into the treatment of neurological injury and disease. Copyright Byrne et al. This DLK-1 (Dual Leucine Zipper Kinase) is a mitogen activated protein kinase kinase kinase (MAPKKK) article is distributed under the identified in C. elegans that functions intrinsically to regulate regeneration of adult axons in the cen- terms of the Creative Commons Attribution License, which tral and peripheral nervous systems across species, including flies and mammals permits unrestricted use and (Hammarlund et al., 2009; Yan et al., 2009; Xiong et al., 2010; Shin et al., 2012; Wang et al., redistribution provided that the 2013; Watkins et al., 2013; Byrne et al., 2014). Activation of dlk-1 enhances axon regeneration original author and source are and loss of dlk-1 function inhibits axon regeneration in young and aged animals (Hammarlund et al., credited. 2009; Yan et al., 2009; Byrne et al., 2014). In worms, flies, and mice, the function of DLK signaling Byrne et al. eLife 2016;5:e12734. DOI: 10.7554/eLife.12734 1 of 14 Short report Neuroscience eLife digest Neurons carry information around the body along slender projections known as axons. An injury that crushes or cuts an axon can lead to permanent disability if the axon fails to regenerate. While some damaged neurons in the body can repair themselves, typically those present in the brain and spinal cord cannot regenerate successfully after injury. The ability of a neuron to regenerate its axon depends in part on factors present inside the neuron itself. By understanding how these internal mechanisms regulate axon regeneration, researchers hope to develop new ways to boost the repair of damaged neurons. A protein called DLK acts inside neurons to promote regeneration of injured axons across a range of species including worms and mammals. In the absence of DLK, regeneration is impaired. The DLK signaling pathway is activated in damaged neurons and is thought to promote repair by altering the activity of genes and proteins that control the regeneration process. Byrne et al. have now identified genes that are activated by the DLK signaling pathway in the roundworm, Caenorhabditis elegans. The experiments show that DLK signaling increases the activity of genes encoding enzymes known as PARGs, which in turn enhance axon regeneration. PARG enzymes remove chain-like molecules called poly(ADP-ribose) that are attached to target proteins. Further experiments showed that other enzymes known as PARPs, which add the poly(ADP- ribose) markers to proteins, act to inhibit axon regeneration in both Caenorhabditis elegans and in injured neurons from mice. Consistent with this, Byrne et al. found that drugs that inhibit PARP enzymes improved axon regeneration when they were given to C. elegans with injured neurons. These results suggest that a critical role of the DLK signaling pathway is to regulate poly(ADP- ribose) levels and that reducing the amount of poly(ADP-ribose) added to proteins can promote axon regeneration. The next step is to understand exactly how poly(ADP-ribose) regulates axon regeneration and to identify the other factors – besides poly(ADP-ribose), PARGs and PARPs – that act downstream of DLK signalling to regulate regeneration. DOI: 10.7554/eLife.12734.002 in regeneration depends on gene transcription (Xiong et al., 2010; Shin et al., 2012; Yan and Jin, 2012; Watkins et al., 2013; Stone et al., 2014). These data suggest that specific targets of DLK transcriptional regulation may mediate the ability of DLK signaling to promote regeneration. Further, these targets may identify novel aspects of the cell biology of axon regeneration. Finally, modulation of these targets might increase the intrinsic regenerative potential of injured axons. Results and discussion To identify targets of DLK transcriptional regulation in neurons, we took advantage of a recently developed method that uses FACS to isolate C. elegans neurons and compare their gene expression profiles (Spencer et al., 2014). We sorted GABA motor neurons from animals with activated DLK signaling (dlk-1(OE), conferred by overexpression of DLK-1L [Hammarlund et al., 2009; Yan and Jin, 2012]) and compared them to wild-type GABA neurons. To control for potential off-target effects of DLK activation, we also analyzed neurons that contained both dlk-1(OE) and a loss of func- tion mutation in pmk-3, the MAP kinase at the end of the canonical DLK signaling pathway (Nakata et al., 2005; Hammarlund et al., 2009; Yan and Jin, 2012). RNA sequencing and analysis suggested the parg genes as candidates for further evaluation. The gene parg-2 (poly(ADP-ribose) glycohydrolase-2) was significantly upregulated in neurons with activated DLK signaling (187-fold upregulated in dlk-1(OE) vs wild type, p<0.01, two-way ANOVA, Bonferroni post-test) (Figure 1A). Further, examination of RNA-Seq results for the parg-2 paralog, parg-1, detected a 2.5-fold increase in the dlk-1(OE) background compared to wild type (p<0.05, two-way ANOVA, Bonferroni post-test, Figure 1A) (See Materials and methods). Elevated expression of parg-1 and parg-2 by DLK signaling depended on the canonical DLK MAP kinase pathway since up-regulation was eliminated in neurons that over-expressed dlk-1 but lacked its downstream effector pmk-3 (Figure 1A). These data sug- gested that regulation of PARG function might be a major effect of DLK signaling. Overall, up-regu- lation (>two fold, p<0.05) (See Materials and methods) of gene expression by DLK signaling was Byrne et al. eLife 2016;5:e12734. DOI: 10.7554/eLife.12734 2 of 14 Short report Neuroscience A D uninjured GABA motor neurons *** wildtype 40 *** dlk-1(OE) D 35 dlk-1(OE); P A 30 * pmk-3(-) severed severed V * not regenerating regenerating 25 E 20 wild type wild type parg-1(-) parg-2(-) 15 ns 10 ns Gene Expression (RPKM) 5 0 parg-1 parg-2 rgef-1 B wild type dlk-1(OE) Punc-47::GFP Pparg-2::NLS::mCherry::NLS F H ** 100 100 80 80 Merge 60 * * 60 * 40 40 % Regeneration % 20 % Regeneration % 20 C 0 0 100 * wild typeparg-1(-)parg-2(-) wild typeparg-1(-); parg-2(-); 80 parg-1(-) parg-2(-) * Punc-47::parg-1Punc-47::parg-2 G I 60 parg-2 parg-1 * dlk-1 2.4 KB 31.3 KB 6.2 KB 40 parg-1 20 pmk-3 0 % Pparg-2::NLS::mCherry::NLS Pparg-2::NLS::mCherry::NLS % CRISPR gk120 -) E) ) -1(-) deletion deletion dlk-1( dlk dlk-1(- parg-1 / parg-2 wild type dlk-1(Owild type wild type parg-1(wp20) uncut in cut uncut in uninjured injured CRISPR Premature worm worm deletion stop codon axon regeneration Figure 1. PARG genes regulate axon regeneration. (A) dlk-1 overexpression upregulates parg-1 and parg-2 expression levels in GABA neurons. The upregulation is suppressed by loss of pmk-3 function. rgef-1 (a pan-neuronal Ras nucleotide exchange factor) expression levels are not affected by manipulations of the dlk-1 pathway (*p<0.05, ***p<0.01, two-way ANOVA, Bonferroni post-test).
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