PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure (2019)
Figure 8

Figure 8. Pharmacological inhibition of HDAC3 promotes DRG regenerative growth.

A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.

C-D. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).

E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.

F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).

H. Summary diagramme: following central nervous system (CNS) spinal injury, protein phosphatase 4/2 activity is not induced since calcium levels remain unchanged compared to uninjured conditions. HDAC3 remains phosphorylated and occupies deacetylated chromatin contributing to its compaction inhibiting gene expression. Following peripheral nervous system (PNS) sciatic injury, protein phosphatase 4/2 activity is induced by calcium. HDAC3 is dephosphorylated leading to its inhibition and release from chromatin sites contributing to increase in histone acetylation and in the expression of regeneration associated genes (RAGs).

Extended View Figures:

Extended View Figure 1. 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 Figure 1A. Only EZH2 inhibitor 503 showed a significant increase in DRG outgrowth when compared to vehicle (24 hours in culture). Data is 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 of H3K9ac after HDAC1 (233, 100nM) or HDAC1/2 (963, 100nM) inhibition on DRGs. (C) Data is 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 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 is expressed as mean fold change vs control AAV-GFP of fluorescent intensity of H3K9ac in V5/GFP positive or negative cells ± s.e.m. N= 6 biological replicates (***p<0.005, Student's t-test).

F. Immunofluorescence anti-TUJ1 following shRNA-mediated HDAC3 silencing vs scrambled (scr-shRNA) in cultured DRG cells shows an increase in neurite outgrowth after 48h in culture. Scale bar, 100 μm.

G. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus scrambled shRNA (ANOVA followed by Bonferroni test).

Extended View figure 2. 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 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 is expressed as mean fold change (band intensity) vs sham± s.e.m. N= 3 biological replicates (Student's t-test).

Extended View Figure 3. Dephosphorylation but not expression of HDAC3 is calcium dependent.

A. pHDAC3 immunostaining in primary DRG neurons after KCl stimulation shows HDAC3 dephosphorylation specifically in DRG cells where high levels of calcium (Fluo4AM) were detected (arrows)(compare a-b with c-d). Scale bars, 300 μm (A) and 10 μm (a-d).

B. Graphs show bursts of intracellular calcium during live imaging of Fluo4AM fluorescence after KCl administration. Data is expressed as single cell percentage of fluorescence variation versus basal fluorescence.

C. Significant negative Pearson correlation between calcium levels (Fluo4AM) and pHDAC3 (r2: 0.4022); data is expressed as single cell fluorescence levels, N =176 cells from 3 biological replicates.

D. HDAC3 immunostaining in primary DRG neurons after KCl stimulation shows similar HDAC3 levels in DRG cells where high and low levels of calcium (Fluo4AM) were detected (arrows). Scale bar, 300 μm and 10 μm (a-d).

E. HDAC3 and Fluo4AM intensities show no correlation (r2: 0.00014); data is expressed as single cell fluorescence levels, N =86 cells from 3 biological replicates.

Extended View Figure 4. Intrathecal pharmacological or genetic inhibition of HDAC3 activity increases H3K9 acetylation on promoters of regenerative genes and induces neurite outgrowth of DRG neurons on permissive and inhibitory substrates.

A. ChIP-qPCR data following 966-dependent inhibition of HDAC3 after DCA (24 hours) shows an increase in H3K9ac on promoters of regeneration associated genes. Data is expressed as 2-ΔΔCt normalized versus IgG and input for each sample ± s.e.m. N= 3-4 biological replicates (5 animals per sample). * p<0.05, ** p<0.01 indicate significant difference versus vehicle (Student's t-test).

B. AAV-mediated dead mutant HDAC3 in vivo infection of DRG neurons induced neurite outgrowth after 12h in culture, including above the level of a conditioning lesion. Scale bars, 100 μm.

C. Data is expressed as mean neurite length ± s.e.m. N= 4 biological replicates (3 technical replicates). (*p<0.05, **p<0.01) indicate significant difference versus sham+EV (Empty Vector) (ANOVA followed by Bonferroni test).

D. Intrathecal administration of RGFP966 (966) in vivo induced neurite outgrowth on PDL and myelin substrates after 12h in culture. Scale bar, 100 μm.

E. Data is expressed as mean neurite length ± s.e.m. N= 3 biological replicates (3 technical replicates). (*p<0.05) indicate significant difference versus respective vehicle (Student's t-test).

Extended View Figure 5. GFAP and CD11b positive immunoreactivity around the injury site.

(A-F) Immunofluorescence anti-GFAP (A, B) or anti-CD11b (D, E) shows that the GFAP positive scar (five weeks after SCI) or CD11b immunoreactivity around the injury site are not affected by intrathecal administration of RGFP966 (966) through osmotic minipump vs vehicle. Scale bar, 500 μm. (C) Data is expressed as fold change of GFAP+ area pixel intensity (dotted lines define boundaries of GFAP+ scar) between 966 vs vehicle ± s.e.m. N= 7 biological replicates. (Student's t-test). (F) Data is expressed as fold change of CD11b+ pixel intensity (within the glial scar area) between 966 vs vehicle ± s.e.m. N= 7 biological replicates (Student's t-test).