PP4‐dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure
2019; Springer Nature; Volume: 38; Issue: 13 Linguagem: Inglês
10.15252/embj.2018101032
ISSN1460-2075
AutoresArnau Hervera, Luming Zhou, Ilaria Palmisano, Eilidh McLachlan, Guiping Kong, Thomas H. Hutson, Matt C. Danzi, Vance Lemmon, John L. Bixby, Andreu Matamoros‐Angles, Kirsi Forsberg, Francesco De Virgiliis, Dina P. Matheos, Janine L. Kwapis, Marcelo A. Wood, Radhika Puttagunta, José Antonio del Rı́o, Simone Di Giovanni,
Tópico(s)Signaling Pathways in Disease
ResumoArticle22 May 2019free access Source DataTransparent process PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure Arnau Hervera Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain Institute of Neuroscience, University of Barcelona, Barcelona, Spain Search for more papers by this author Luming Zhou Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Ilaria Palmisano Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Search for more papers by this author Eilidh McLachlan Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Search for more papers by this author Guiping Kong Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Thomas H Hutson Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Search for more papers by this author Matt C Danzi The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Vance P Lemmon orcid.org/0000-0003-3550-7576 The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author John L Bixby The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Andreu Matamoros-Angles Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain Institute of Neuroscience, University of Barcelona, Barcelona, Spain Search for more papers by this author Kirsi Forsberg Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Francesco De Virgiliis Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Dina P Matheos Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA Search for more papers by this author Janine Kwapis Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA Search for more papers by this author Marcelo A Wood Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA Search for more papers by this author Radhika Puttagunta Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Spinal Cord Injury Center, University Hospital Heidelberg, Heidelberg, Germany Search for more papers by this author José Antonio del Río Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain Institute of Neuroscience, University of Barcelona, Barcelona, Spain Search for more papers by this author Simone Di Giovanni Corresponding Author [email protected] orcid.org/0000-0003-3154-5399 Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Arnau Hervera Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain Institute of Neuroscience, University of Barcelona, Barcelona, Spain Search for more papers by this author Luming Zhou Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Ilaria Palmisano Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Search for more papers by this author Eilidh McLachlan Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Search for more papers by this author Guiping Kong Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Thomas H Hutson Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Search for more papers by this author Matt C Danzi The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Vance P Lemmon orcid.org/0000-0003-3550-7576 The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author John L Bixby The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA Search for more papers by this author Andreu Matamoros-Angles Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain Institute of Neuroscience, University of Barcelona, Barcelona, Spain Search for more papers by this author Kirsi Forsberg Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Francesco De Virgiliis Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Dina P Matheos Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA Search for more papers by this author Janine Kwapis Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA Search for more papers by this author Marcelo A Wood Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA Search for more papers by this author Radhika Puttagunta Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Spinal Cord Injury Center, University Hospital Heidelberg, Heidelberg, Germany Search for more papers by this author José Antonio del Río Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain Institute of Neuroscience, University of Barcelona, Barcelona, Spain Search for more papers by this author Simone Di Giovanni Corresponding Author [email protected] orcid.org/0000-0003-3154-5399 Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany Search for more papers by this author Author Information Arnau Hervera1,2,3,4,5,‡, Luming Zhou1,6,7,‡, Ilaria Palmisano1, Eilidh McLachlan1, Guiping Kong1,6, Thomas H Hutson1, Matt C Danzi8, Vance P Lemmon8, John L Bixby8, Andreu Matamoros-Angles2,3,4,5, Kirsi Forsberg6, Francesco De Virgiliis1,6,7, Dina P Matheos9, Janine Kwapis9, Marcelo A Wood9, Radhika Puttagunta6,10, José Antonio del Río2,3,4,5 and Simone Di Giovanni *,1,6 1Department of Medicine, Division of Brain Sciences, Molecular Neuroregeneration, Imperial College London, London, UK 2Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), Parc Científic de Barcelona, Barcelona, Spain 3Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain 4Department of Cell Biology, Physiology and Immunology, Universitat de Barcelona, Barcelona, Spain 5Institute of Neuroscience, University of Barcelona, Barcelona, Spain 6Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Tuebingen, Germany 7Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Tuebingen, Germany 8The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA 9Center for the Neurobiology of Learning & Memory, Department of Neurobiology & Behavior, University of California, Irvine, CA, USA 10Spinal Cord Injury Center, University Hospital Heidelberg, Heidelberg, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +44 020 759 43178; E-mail: [email protected] EMBO J (2019)38:e101032https://doi.org/10.15252/embj.2018101032 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure. Synopsis We found that calcium-dependent activation of PP4/2 signalling controls the axonal regenerative ability via HDAC3 dephosphorylation. This mechanism discriminates between axonal regeneration and regenerative failure Calcium activates PP4/2 that in turn dephosphorylates HDAC3 enhancing histone acetylation following a regeneration competent peripheral lesion. However, it fails to do so following a regenerative incompetent spinal cord injury. HDAC3 is a central regulatory signalling hub of the axonal regeneration programme. HDAC3 genetic or pharmacological inhibition promotes axonal regeneration of sensory axons following a non-regenerative spinal cord injury. Introduction Following a central nervous system (CNS) injury, such as stroke or spinal cord injury, axonal regeneration is highly restricted. In stark contrast, spontaneous albeit partial functional axonal regeneration is possible after a peripheral nervous system (PNS) injury. This is likely due to the differences of the CNS and PNS in both intrinsic properties of neurons and the surrounding cellular environment. Importantly, glial cell-dependent signalling can affect the intrinsic properties of neurons (Puttagunta et al, 2011), as is evident by the fact that the neuronal regenerative gene expression programme is only activated when axons lie within the PNS, but not in the CNS. This is typically modelled by the lumbar dorsal root ganglion (DRG) neurons (Neumann & Woolf, 1999; Teng & Tang, 2006), which project a peripheral axonal branch into the sciatic nerve (consisting of a permissive cellular environment) and a central axonal branch into the spinal cord (consisting of an inhibitory cellular environment). Strikingly, the peripheral branch of DRG mounts a robust regenerative response following a sciatic nerve injury, while the central branch fails to regenerate following a spinal injury (Neumann & Woolf, 1999; Neumann et al, 2002). Furthermore, regeneration of the CNS branch is greatly enhanced by prior injury to the peripheral branch (conditioning lesion; Neumann & Woolf, 1999; Neumann et al, 2002) that leads to an increase in gene expression of a number of axonal growth and regeneration-associated genes, which does not occur after spinal lesions alone (Chong et al, 1994; Tonra et al, 1998; Zhang et al, 2000; Mason et al, 2002; McGraw et al, 2004; Stam et al, 2007). Dynamic gene expression changes are controlled and coordinated by epigenetic regulation such as DNA methylation or histone post-translational modifications which are essential in stem cell reprogramming, development of tissues and organs, as well as cancer initiation (Kretsovali et al, 2012). Strategies that take advantage of broad histone deacetylase (HDAC) inhibitory drugs (classes I and II such as TSA or MS-275) have been poor in promoting axonal regeneration after an optic nerve crush (Gaub et al, 2011) or in enhancing regeneration past the lesion site after a spinal cord injury (Finelli et al, 2013). Recently, we found that ERK-dependent phosphorylation leads to acetylation of histone H3K9 at the promoters of select RAGs after sciatic injury (Puttagunta et al, 2014) and that viral overexpression of P300/CBP-associated factor (P/CAF) promotes axonal regeneration after spinal cord injury by reactivating a regenerative gene expression programme (Puttagunta et al, 2014). We also recently investigated DNA methylation in DRG following sciatic nerve versus spinal injury by methylation arrays including the CpG island-rich promoter region of 13,000 genes and found that DNA methylation was not differentially represented on regeneration-associated genes (Lindner et al, 2014). However, this study fell short of providing genomewide DNA methylation profiles; therefore, it did not allow ruling out a role for DNA methylation in axonal regeneration. However, whether specific axonal signalling mechanisms can discriminate between axonal regeneration and regenerative failure by differentially activating or restricting the regenerative programme remains elusive. Here, we hypothesize that critical axonal signals are conveyed to modify gene expression to restrict the regeneration programme after a non-regenerative spinal lesion, while these restrictions are lifted following a regenerative peripheral injury. Since regulation of gene expression relies on enzymes whose function is controlled by their activity, initially we adopted a pharmacological small-molecule inhibitor screen of key epigenetic enzymes aiming to identify signalling pathways that might influence the regenerative growth potential of primary adult neurons and overcome regenerative failure. From this screen, we discovered that HDAC3 inhibition promoted neurite outgrowth on both growth-permissive and inhibitory substrates. Next, we found that a regeneration-competent sciatic nerve injury induces an increase in calcium that activates protein phosphatase 4 (PP4) and slightly increases the activity of protein phosphatase 2 (PP2), which in turn dephosphorylate HDAC3, inhibiting its activity. However, a spinal lesion does not elicit increases in calcium signalling nor PP activity therefore failing to dephosphorylate HDAC3. Inhibition of PP4/2 activity promotes DRG regenerative growth similarly to mimicking HDAC3 phosphorylation. Combined bioinformatics analysis of H3K9ac ChIPseq and RNAseq from DRG as well as HDAC3 protein–protein interaction databases suggested that HDAC3 might restrict the regenerative programme. Indeed, we found that inhibition of HDAC3 activity engages several regeneration-associated signalling pathways. Finally, we translated these findings to in vivo models of spinal cord injury where we found that either AAV-mediated overexpression of HDAC3 deacetylase-dead mutant or pharmacological HDAC3 inhibition overcomes regenerative failure by promoting axonal growth of sensory axons. In summary, we found that calcium activation of PP4 leading to HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure. Results Pharmacological inhibition and genetic manipulation show that HDAC3 inhibition selectively enhances neurite outgrowth on both growth-permissive and non-permissive substrates We performed a compound screen to identify whether inhibiting the activities of enzymes that modify key epigenetic marks could promote neurite outgrowth of adult neurons on both growth-permissive and non-permissive inhibitory substrates. All inhibitors were initially screened at various concentrations in a neurite outgrowth assay using adult mouse dorsal root ganglia (DRG) neurons cultured on a growth-permissive laminin–PDL substrate. The inhibitors that promoted significant outgrowth were further examined on a myelin growth inhibitory substrate. We screened 14 pharmacological inhibitors of enzymes affecting epigenetic marks (Table 1, Fig EV1A). These include inhibitors of DNMT1-3, DNMT3b, HDAC class II (HDAC4, 5, 7, 9), HDAC1, HDAC1-2, HDAC3, the HMT G9a for H3K9me3, EZH2 for H3K27me3, DOT1L for H3K79me3, the HKDM JMJD2 for H3K9me3, JMJD3 for H3K27me3 and the HDR PADI4 for H3R2-8-17cit. Inhibitors of bromodomain BRD2/3/4 and BAZ2 proteins that are linked to both permissive and repressive epigenetic marks were also examined. The EZH2 inhibitor 503 and the HDAC3 inhibitor RGFP966 significantly enhanced neurite outgrowth by more than twofold compared to vehicle. However, only RGFP966-dependent HDAC3 inhibition resulted in significant and extensive neurite outgrowth on both permissive laminin and inhibitory myelin substrates (Fig 1A–C), and enhanced H3K9 acetylation as expected (Fig 1D and E). In contrast, inhibitors of HDAC class II or HDAC1 (233) and HDAC1-2 (963) did not affect DRG outgrowth, although they increased overall histone acetylation as expected (Figs 1A and EV1B and C). Next, we infected cultured DRG neurons plated on PDL–laminin or myelin with an AAV-HDAC3 deacetylase-dead mutant (HDAC3mut), a control AAV-GFP or AAV-HDAC3wt. We found that while overexpression of wt HDAC3 restricted outgrowth, overexpression of mutant HDAC3 strongly promoted neurite outgrowth on both permissive and inhibitory substrates and enhanced histone acetylation (Figs 1F and G, and EV1D and E). Similarly, HDAC3 gene silencing with validated shRNAs promoted DRG outgrowth (Fig EV1F and G), supporting the HDAC3 pharmacological inhibition and deacetylase mutant overexpression data. This indicates that HDAC3 may be a repressor of axonal regeneration whose inhibition might allow gene expression-dependent regenerative reprogramming. Table 1. List of inhibitory drugs, target molecule and epigenetic mark employed in a screening for DRG neurite outgrowth Drug Enzyme Target Function 5-AZA DNMT1/3 DNMT 5′-Methylcytosine 13X DNMT3B DNMT 5′-Methylcytosine 69A HDAC class II (4/5/7/9) HDAC H3/4ac 233 HDAC1 HDAC H3/4ac 963 HDAC1/2 HDAC H3/4ac 966 HDAC3 HDAC H3/4ac 801 BAZ2A/BAZ2B HME/DNMT H3/4ac 51A BRD 2/3/4 S/T kin H3K79me3 17A DOT1L HMT H3K27me3 J4/77A JMJD3 HKDM H3K9me3 71B G9A HMT H3R(2/8/17)cit 484 PAD14 HDI/HRMD H3K9me3/36me3/4me3 67A JMJD2/JARID1 HKDM H3K9me3 503 EZH2 HMT H3K27me3 Click here to expand this figure. Figure EV1. Genetic silencing or inhibition of HDAC3 promotes neurite outgrowth of DRG neurons in vitro A. DRG outgrowth screening of small-molecule inhibitors of epigenetic enzymes (described in Table 1), excluding HDAC inhibitors displayed in Fig 1A. Only EZH2 inhibitor 503 showed a significant increase in DRG outgrowth when compared to vehicle (24 h in culture). Data are expressed as mean fold change of average neurite length ± s.e.m. N = 4 biological replicates. **P < 0.01 indicate significant difference vs. vehicle (ANOVA followed by Bonferroni test). B, C. Immunoblot showing the specific increase in H3K9ac after HDAC1 (233, 100 nM) or HDAC1/2 (963, 100 nM) inhibition on DRGs. (C) Data are expressed as mean fold change of immunoblot band intensity ± s.e.m. N = 3 biological replicates (**P < 0.01, ***P < 0.005, Student's t-test). D, E. Immunofluorescence of anti-V5 or GFP and H3K9ac after DRG infection with AAV-HDAC3mut-V5 (Y298H) shows increase in H3K9ac as compared to control AAV-GFP (arrowheads). (E) Data are expressed as mean fold change vs. control AAV-GFP of fluorescent intensity of H3K9ac in V5/GFP-positive or V5/GFP-negative cells ± s.e.m. N = 6 biological replicates (***P < 0.005, Student's t-test). F. Immunofluorescence of anti-Tuj1 following shRNA-mediated HDAC3 silencing vs. scrambled (scr-shRNA) in cultured DRG cells shows an increase in neurite outgrowth after 48 h in culture. Scale bar, 100 μm. G. Data are expressed as mean ± s.e.m. N = 3 biological replicates. *P < 0.05 indicate significant difference versus scrambled shRNA (ANOVA followed by Bonferroni test). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Pharmacological HDAC3 inhibition and HDAC3 dead mutant enhance DRG neurite outgrowth A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24 h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233, respectively. B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24 h in culture, Tuj1-positive neurons) on PDL–laminin or myelin substrates. Scale bars, 100 μm. D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs. vehicle. F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj1/V5 double-positive neurons) after 36 h in culture, which is repressed by wild-type HDAC3 overexpression. Scale bars, 100 μm. Data information: Data are expressed as mean fold change or average neurite length ± s.e.m. N = 3–9 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.005 indicate significant difference of 966 versus vehicle or V5 vs. AAV-GFP (C and E, Student's t-test) (A and G, ANOVA followed by Bonferroni test). Source data are available online for this figure. Source Data for Figure 1 [embj2018101032-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint HDAC3 phosphorylation and activity are inhibited by a peripheral sciatic but not by a central spinal injury Next, we investigated the post-injury regulation of HDAC3 in the pseudo-unipolar DRG system in vivo following a regeneration-competent peripheral sciatic nerve axotomy (SNA) injury versus a regeneration-incompetent central spinal axotomy (DCA). This allowed for the comparison of HDAC3 regulation in the same neuronal population undergoing a regenerative versus non-regenerative axonal lesion. HDAC3 can be regulated at the gene or protein expression level; it can be shuttled between cytoplasm and nucleus; and it can be phosphorylated (Zhang et al, 2005; Karagianni & Wong, 2007; Togi et al, 2009). First, we measured the gene expression of HDAC3 and of the other class I HDACs, HDAC1 and HDAC2 by qPCR, in sham versus axonal injury in DRG. We found that all HDACs are expressed in DRGs, but neither a peripheral nor a central injury modifies their gene expression level (Appendix Fig S1A and B). Protein analysis of HDAC3 expression revealed that it is highly expressed in DRG neurons, mainly in the nucleus, but again neither its protein expression nor its nuclear–cytoplasmic shuttling was regulated by either a sciatic or a spinal injury (Fig EV2A–E). However, we found that the nuclear phosphorylation of HDAC3 (Ser424) was strongly reduced by a sciatic but not by a central spinal injury (Fig 2A–E). Since phosphorylation of HDAC3 promotes its enzymatic activity, we measured the activity of HDAC3 in DRG after DCA or SNA to find that HDAC3 deacetylase activity was strongly reduced by SNA only (Fig 2F), in line with its reduced phosphorylation. Click here to expand this figure. Figure EV2. HDAC3 expression and subcellular localization in DRGs do not change after peripheral or central axonal injuries A, B. Immunohistochemistry analysis of HDAC3 expression revealed that HDAC3 is highly expressed in lumbar DRG neurons (Tuj1 positive) and it does not change after SNA vs. sham or DCA vs. laminectomy (lam) as shown by the bar graph after measurement of the HDAC3 immunofluorescent signal in Tuj1-positive neurons (B), N = 3 biological replicates, ± s.e.m. Scale bar (A) 100 μm. HDAC3 expression is mainly nuclear in DRG neurons, and its subcellular localization does not change after SNA. C. Immunoblot analysis shows that HDAC3 expression does not change after SNA or DCA. D, E. Immunoblot analysis of isolated nuclear and cytoplasmic DRG fractions shows that HDAC3 subcellular localization does not change after SNA. Data are expressed as mean fold change (band intensity) vs. sham ± s.e.m. N = 3 biological replicates (Student's t-test). Source data are available online for this figure. Download figure Download PowerPoint Figure 2. Regenerative sciatic nerve injury selectively decreases HDAC3 phosphorylation and activity in DRG neurons A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24 h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm. B, C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counterstaining was used to label nuclei N = 9–10 biological replicates. ***P < 0.005 indicate significant difference versus sham or lamine
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