Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration

Abstract

The peripheral nervous system (PNS) regenerates after injury. However, regeneration is often compromised in the case of large lesions, and the speed of axon reconnection to their target is critical for successful functional recovery. After injury, mature Schwann cells (SCs) convert into repair cells that foster axonal regrowth, and redifferentiate to rebuild myelin. These processes require the regulation of several transcription factors, but the driving mechanisms remain partially understood. Here we identify an early response to nerve injury controlled by histone deacetylase 2 (HDAC2), which coordinates the action of other chromatin-remodelling enzymes to induce the upregulation of Oct6, a key transcription factor for SC development. Inactivating this mechanism using mouse genetics allows earlier conversion into repair cells and leads to faster axonal regrowth, but impairs remyelination. Consistently, short-term HDAC1/2 inhibitor treatment early after lesion accelerates functional recovery and enhances regeneration, thereby identifying a new therapeutic strategy to improve PNS regeneration after lesion.

Figure 1. HDAC1/2 are robustly upregulated in SCs after lesion.

(a) Western blots of HDAC2 and HDAC1 in lysates of crushed (Cr) and contralateral (Co) nerves of adult mice at 1 dpl (n=5), 3 dpl (n=5), 5 dpl (HDAC2, n=6; HDAC1, n=7), 12 dpl (HDAC2, n=6; HDAC1, n=8), 30 dpl (HDAC2, n=6; HDAC1, n=8) and 60 dpl (n=6), and quantification normalized to GAPDH and compared to Co=1, showing HDAC1/2 upregulation after lesion. Of note: both bands detected by the HDAC2 antibody were quantified and added together in the graph. (b) Co-immunofluorescence of HDAC2 or HDAC1 (red) with CD68 (green=macrophages) and DAPI labelling (blue=nuclei, overlay appears pink) at 12 dpl, showing upregulation in SCs (CD68-negative cells with elongated nuclei, white arrows) of crushed compared to contralateral nerves. Note that macrophages (CD68-positive cells, blue arrowheads) express variable levels of HDAC1 and HDAC2. Scale bar, 10 μm. (c) Denaturing IP of SUMO-1, HDAC2 (H2) or control Flag (ctrl) in unlesioned (no lesion) adult mouse sciatic nerve lysates, and western blot of HDAC2, showing that the ∼75 kDa band detected by the HDAC2 antibody corresponds to a SUMOylated form of HDAC2. HDAC2 and GAPDH western blots on lysates used for IP show the inputs. Representative photos of three independent experiments are shown. One-tailed (HDAC2, 12 dpl and 60 dpl; HDAC1, 5 dpl) or two-tailed Student's t-tests, unpaired (HDAC2: 3 dpl) or paired, P values: *P<0.05, **P<0.01, ***P<0.001. Values, mean; error bars, s.e.m.

Figure 2. HDAC1/2 slow down axonal regrowth but promote remyelination.

(a) BrdU incorporation (green) and DAPI (blue) identifying earlier induction of proliferation in dKO compared to control nerves. Scale bar=10 μm. Electron micrographs (b,e) and toluidine-blue staining (f) and quantification showing in dKO compared to controls a decreased percentage of intact myelin rings (b, scale bar=10 μm) and increased number of axonal sprouts (e, Scale bar, 5 μm) at 5 dpl, and thinner remyelinating sheaths at 1 month post lesion (mpl) (higher g ratio in dKO, (f), Scale bar, 10 μm). (c) Z-series projections of whole-nerve Neurofilament immunofluorescence in control and dKO nerves, brightfield showing lesion site and axonal tracings (acquired using NeuronJ, Scale bar, 300 μm) quantifying axonal regrowth from the lesion site. White arrows, fragmented axons. Coloured boxes, magnifications (Scale bar, 100 μm). (d) Z-series projections of GAP-43 immunofluorescence (green) and DAPI labelling (blue=nuclei) showing strongly increased GAP-43 expression in dKO compared with control nerves at 3 dpl (20-μm thick cryosections) and 5 dpl (5-μm thick cryosections). Scale bar, 30 μm. Note that immunofluorescence staining of 3 dpl sections was not carried out at the same time as staining of 5 dpl sections, and imaging was also done separately with different exposure times. Unpaired two-tailed Student's t-tests, P values: *P<0.05, **P<0.01. Values, mean, error bars, s.e.m. Sample size: n=3 animals per group per time point for all graphs, electron microscopy and immunofluorescence. (a) Number of cells counted: 334 to 469 per animal at 3 dpl (per genotype: 1,218 for controls, 1,140 for dKO); 111 to 727 per animal at 5 dpl (per genotype: 1,779 for controls, 1,262 for dKO); 553 to 4,380 per animal at 12 dpl (per genotype: 5,440 for controls, 8,892 for dKO). (b) Number of myelin rings counted: 344 to 469 per animal. (c) Number of tracings: 10 to 25 per nerve. (d) Surface of nerve ultrathin section counted: 0.026 to 0.03 mm² per animal. (e) 60–70 myelinated axons (randomly chosen) counted per nerve. The g ratio (axon diameter: axon+myelin diameter) of both contralateral (0.71 for control and 0.72 for dKO nerves, P value=0.32: no significant difference between control and dKO) and crushed nerves was measured.

Figure 3. Delayed Oct6 and earlier/higher cJun upregulation in dKO.

Western blots and quantification normalized to GAPDH of (a) Oct6, (b) cJun, (c) Pax3, and (d) Sox2 in crushed (Cr) and contralateral (Co=1) nerves of control (Ctrl) and dKO, showing delayed Oct6 upregulation and earlier/higher cJun upregulation in dKO. Of note: the double band at ∼52 kDa and the lower band at ∼42 kDa detected by the Oct6 antibody were quantified and added together in the graph. Dashed lines: samples run on the same gel, but not on consecutive lanes. In a–d, coloured graphs quantify protein levels in Cr compared to Co, and grey/white bar graphs quantify protein levels in dKO compared with Ctrl crushed nerves. (e,g) Co-immunofluorescence of Oct6 (red, e) at 5 dpl or of cJun (green, g) at 1 dpl with the macrophage marker F4/80 (green in e, red in g) and DAPI labelling (nuclei) in control and dKO nerves showing decreased Oct6 and increased cJun levels in SCs of dKO compared with control nerves. White arrows indicate SCs (F4/80-negative cells with elongated nuclei) and blue arrowheads indicate macrophages (F4/80-positive cells). (f) Oct6 in situ hybridization in control and dKO nerves at 5 dpl showing decreased Oct6 transcript levels in dKO. Sample size: (a–d) 5 animals per group for 1 dpl, 2 dpl and 3 dpl, and 6 or 9 animals per group for 5 dpl and 12 dpl; (e–g) 3 animals per group. One-tailed (grey asterisk; blue/red asterisks: Control Oct6 at 5 dpl, Control cJun at 5 dpl, dKO cJun at 12 dpl, Control Pax3 at 2 dpl, Control Sox2 at 3 dpl and 5 dpl) or two-tailed (black asterisks; blue/red asterisks: other values) Student's t-tests, unpaired (bar graphs, and cJun at 2 dpl in coloured graph) or paired (coloured graphs, except for cJun at 2 dpl), P values: *P<0.05, **P<0.01. Values, mean; error bars, s.e.m. Scale bar, 10 μm.

Figure 4. Decreased Oct6 expression accelerates axonal regrowth.

(a)Western blots of Oct6 and cJun in RSCs transduced with lentiviruses carrying control (Csh=100) or Oct6 (Oct6sh) shRNAs and quantification normalized to GAPDH showing increased cJun levels in Oct6-downregulated RSCs. Western blot of (c) Oct6 in contralateral (contra) and crushed nerves of Oct6 ΔSCE mice at 5 dpl (representative photos are shown) confirming absence of Oct6 upregulation in this mutant after lesion (as previously reported12), and of (d) cJun in crushed and contralateral nerves of adult control and Oct6 ΔSCE mice at 1 dpl and quantification of protein levels in crushed nerves normalized to β-actin or GAPDH and compared to contralateral nerves (=100). (b) EdU incorporation identifying increased proliferation in RSCs transduced with lentiviruses carrying Oct6sh compared with Csh. Oct6sh and Csh lentiviruses also carry a GFP reporter showing high transduction efficiency in RSCs. Scale bar, 10 μm. (e) Toluidine-blue staining of semithin sections of Oct6 ΔSCE and control mouse sciatic nerves at 5 dpl and quantification of intact myelin rings (pink arrowheads). Scale bar, 10 μm. (f) Z-series projections (confocal stacks) of whole-nerve Neurofilament immunofluorescence in Oct6 ΔSCE nerves, brightfield showing lesion site and Neurofilament tracings (Scale bar, 300 μm) used for quantification of axonal regrowth (for this graph, the ctrl represented is the same as for graph in Fig. 2c). In Oct6 ΔSCE nerves, cJun upregulation already occurred at 1 dpl, demyelination at 5 dpl was increased and axonal regrowth at 3 dpl was longer compared with controls, but these effects were less pronounced than in dKO nerves, possibly due to somewhat higher residual levels of Oct6 in the nerves of Oct6 ΔSCE hypomorph mutant mice compared with dKO nerves. Two-tailed (black asterisks) or one-tailed (grey asterisk and cross) paired (a;d: crushed versus contra) or unpaired (b,e,f;d: Oct6 ΔSCE crushed versus control crushed) Student's t-tests, P values: *P<0.05 or ⁺P<0.05; **P<0.01, ***P<0.001. Values, mean; error bars, s.e.m. In d, asterisk shows significance compared with contra and the cross compared with control. Sample size: (a) n=3, (b) n=9, (c) 3 animals per group, (d) 6 animals per group, (e) 3 animals per group, 227 to 435 myelin rings counted per animal. (f) 3 animals per group, 10 to 25 tracings per nerve.

Figure 5. Reduced Krox20 and P0 expressions in dKO.

(a) Western blot of Krox20 in crushed (Cr) and contralateral (Co) nerves of control mice at 12 dpl and quantification normalized to GAPDH and compared with Co=1. (b,f) Western blots of Krox20 and Sox10 at 12 dpl (b) and of P0 at 1 mpl (f) showing reduced Krox20 (but unaffected Sox10) and P0 levels in dKO compared with Ctrl crushed nerves at 12 dpl and 1 mpl, respectively. Dashed lines—samples run on the same gel, but not on consecutive lanes. (c) Co-immunofluorescence of Krox20 (red) with CD68 (green, macrophages) and DAPI labelling (blue, nuclei) in cryosections of control and dKO sciatic nerves at 12 dpl. White arrows indicate SCs (CD68-negative cells with elongated nuclei, blue arrowheads indicate macrophages (CD68-positive cells). Krox20 (d) and P0 (e) in situ hybridizations in control and dKO nerves showing decreased transcript levels in dKO nerves. Scale bar, 10 μm. One-tailed (grey asterisk) or two-tailed (black asterisks) Student's t-tests, unpaired (b) or paired (a,f), P values: *P<0.05, **P<0.01. Values, mean; error bars, s.e.m. Sample size: (a,b,f) 6 animals per group, (c–e) 3 animals per group.

Figure 6. HDAC2-dependent de-repression by JMJD2C and KDM3A.

(a,d) Relative luciferase activity of the full Oct6 SCE, the ΔHR1 (constructs #3 and #19, ref. 35) and the ΔHR2 SCE showing that overexpression of HDAC1, HDAC2, JMJD2C or KDM3A increases the activation of the full Oct6 SCE or the ΔHR1 SCE, but not of the ΔHR2 SCE, compared with control GFP (=1) in RSCs cultured under dedifferentiating conditions. The experiment carried out in the presence of the HDAC1/2 inhibitor Mocetinostat or its vehicle indicates that activation induced by JMJD2C or KDM3A is prevented by HDAC1/2 inhibition. (i) Similarly, activation of the Krox20 MSE induced by JMJD2C or KDM3A overexpression in RSCs cultured under redifferentiating conditions is prevented by HDAC1/2 inhibition. Chromatin immunoprecipitation of (b) HDAC2 and Flag (Neg=1) in unlesioned (no lesion) nerves or at 1 dpl, of (e) JMJD2C, KDM3A and Neg (GFP or flag) in control or dKO nerves at 1 dpl, of (f,g) H3K9me3, H3K9me2 and Neg (GFP or Flag) in unlesioned nerves and at 1 dpl in control (f) and dKO (g) on the Oct6 SCE HR2, and of (h) HDAC2, JMJD2C, KDM3A, H3K9me3, H3K9me2 and Neg on the Krox20 MSE in unlesioned nerves or at 12 dpl. (c) Oct6 mRNA fold increase in RSCs transfected with GFP (=1), HDAC2, JMJD2C or KDM3A expression constructs and cultured under dedifferentiating conditions. Asterisks show significance compared with no lesion (b,f–h), to control (e) or to GFP (a,c,d,i), and crosses compared with vehicle (d,i). One-tailed (grey asterisks) or two-tailed (black asterisks or crosses) Student's t-tests, unpaired (d,i: HDAC1/2 inhibitor versus Vehicle; b,e–h) or paired (d,i: JMJD2C or KDM3A versus GFP; a,c), P values: *P<0.05, ⁺P<0.05, **P<0.01, ***P<0.001. Values, mean; error bars, s.e.m. Sample size: (a) full Oct6 SCE, n=12; ΔHR1 and ΔHR2 SCE, n=4; (b) 3 animals per group no lesion and 1 dpl; (c) HDAC2, JMJD2C, n=3; GFP, KDM3A, n=6; (d) n=4; (e) n=3 animals per group control and dKO; (f,g) 3 animals per group no lesion and 1 dpl; (h) 3 animals per group no lesion and 12 dpl; (i) n=3.

Figure 7. Oct6 and Krox20 activation by Sox10 depends on HDAC1/2.

Chromatin immunoprecipitation of Sox10 and GFP (Neg=1) in (a) unlesioned (no lesion) nerves or at 1 dpl on the Oct6 SCE HR2, or in (b) unlesioned nerves or at 12 dpl on the Krox20 MSE. Relative luciferase activity of (c) the full Oct6 SCE, the ΔHR1 (construct #3; ref. 35) and ΔHR2 SCE, or of (d) the Krox20 MSE, in primary RSCs transfected with GFP (=1) or Sox10 expression constructs and cultured under dedifferentiating (c) or redifferentiating (d) conditions for 1 day, in the presence of Mocetinostat (HDAC1/2 inhibitor) or its vehicle (c,d). One-tailed (grey asterisk or cross) or two-tailed (black asterisks or crosses) Student's t-tests, unpaired (a,b;c,d: HDAC1/2 inhibitor compared with Vehicle) or paired (c,d: Sox10 compared with GFP), P values: *P<0.05, ⁺P<0.05; **P<0.01, ⁺⁺P<0.01; and ***P<0.001. Values, mean; error bars, s.e.m. Asterisks show significance compared with no lesion (a,b) or to GFP (c,d), and crosses compared with Vehicle (c,d). Sample size: (a) three animals per group no lesion and 1 dpl; (b) 3 animals per group no lesion and 12 dpl; (c) n=3 or 4; (d) n=4.

Figure 8. Assembly of multifunctional protein complex.

(a–c) Non-denaturing IP of Sox10, HDAC2, JMJD2C, KDM3A or ctrl (GFP or Flag) in unlesioned (no lesion) adult mouse sciatic nerve lysates or at 1 dpl or 12 dpl, and western blot of HDAC2 (a), KDM3A (b) or Sox10 (c). Membranes where Sox10 and JMJD2C IPs were run together were first blotted with the HDAC2 antibody (a) and were re-blotted with the Sox10 antibody (c). GAPDH western blots on lysates used for IP show the inputs (in a, only one input for no lesion IP KDM3A and ctrl that were done on the same lysate divided by two). Sample size: each IP was done three times, using nerves of three different animals. One nerve was used per IP.

Figure 9. HDAC1/2 inhibitor treatment accelerates functional recovery.

Quantification of motor function recovery by Rotarod test at 4 and 10 dpl (a) and of sensory function recovery by toe pinch test at 10, 16, 18 and 29 dpl (b) in vehicle, 3-day or 5-day Mocetinostat-treated mice after sciatic nerve lesion. (c) Western blot of Oct6 and cJun and quantification normalized to GAPDH showing reduced Oct6 and increased cJun levels in nerves of mice treated with Mocetinostat (Moc.) for 1 day after lesion compared to vehicle (Veh.). (d) Electron micrographs of nerve ultrathin sections from vehicle, 3-day and 5-day Mocetinostat-treated mice and quantification showing increased density of sorted (mostly myelinated) axons and Remak bundle axons in both Mocetinostat-treated groups and increased myelin thickness at 30 dpl in the 3-day Mocetinostat-treated group, compared with vehicle (Veh.). Scale bar, 5 μm. One-tailed (grey asterisks) or two-tailed (black asterisks) unpaired Student's t-tests, P values: *P<0.05, **P<0.01, ***P<0.001. Values, mean; error bars, s.e.m. Asterisks show significance compared with the vehicle group. Sample size: (a,b) Vehicle, 9 animals; 3-day Mocetinostat, 8 animals; 5-day Mocetinostat, 8 animals; (c) 6 animals per group; (d) Vehicle, 5 animals; 3-day Mocetinostat, 3 animals; 5-day Mocetinostat, 3 animals.

Figure 10. Mechanism of Oct6 and Krox20 de-repression after lesion.

In myelinating SCs of adult peripheral nerves, low levels of Sox10 are bound to the Krox20 MSE, which is methylated at H3K9, and low levels of Krox20 are expressed. Oct6 is also weakly expressed, and KDM3A and low levels of Sox10 and HDAC2 are bound to the Oct6 SCE HR2, which is methylated at H3K9. In case of lesion, HDAC2, Sox10 and JMJD2C are recruited to the Oct6 SCE HR2 at 1 dpl to demethylate H3K9 and activate Oct6 expression. Upregulated Oct6 at 1 dpl slows down the upregulation of cJun, which induces the conversion of mature SCs into repair SCs and thereby promotes axonal regrowth. This mechanism can be inhibited by a short-term HDAC1/2 inhibitor treatment, which results in faster conversion into repair SCs and faster axonal regrowth. At 12 dpl, Sox10, HDAC2, KDM3A and JMJD2C are recruited to the Krox20 MSE to demethylate H3K9 and activate Krox20 expression. Upregulated Krox20 induces remyelination.