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, 94 (2), 337-346.e6

An Intrinsic Epigenetic Barrier for Functional Axon Regeneration

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An Intrinsic Epigenetic Barrier for Functional Axon Regeneration

Yi-Lan Weng et al. Neuron.

Abstract

Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons' intrinsic axonal growth competence are not well understood. Here, we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.

Keywords: DNA demethylation; TDG; Tet1; Tet3; axon regeneration; dorsal root ganglion; epigenetic DNA modification.

Figures

Figure 1
Figure 1
SNL up-regulates Tet3 and 5hmC levels in adult DRG neurons in vivo. (A) Sample confocal images of Tet3 in situ, GFP immunostaining and DAPI of L4 DRGs in adult Pirt-GCaMP3 neuronal reporter mice under naïve conditions and at 1 day upon sciatic nerve lesion (SNL). Scale bar: 20 μm. (B) Time-course of Tet3 induction in axotomized DRGs by Q-PCR analysis. Values represent mean ± SEM (n = 3 for each group; *P < 0.05; two-way ANOVA). (C) Sample confocal image of immunostaining for GFP, 5hmC, Glutamine Synthetase, a marker for glia, and DAPI in DRGs of adult Pirt-GCaMP3 neuronal reporter mice under naïve conditions and at SNL D1. Scale bar: 50 μm. (D) Quantification of 5hmC levels at different time points after SNL. The signal intensity in NeuN+ neuronal nuclei of naïve L4 DRGs was set as 1.0 and 100–180 neuronal nuclei from each condition in three independent experiments were quantified. Values represent mean ± SEM (n = 3 for each group; ***P < 0.001; two-way ANOVA). (E-F) Immunohistochemical analysis of 5hmC levels in Ctrl and Tet3 KD DRG neurons under naïve conditions and at SNL D1. Shown are sample images (E; scale bar: 20 μm) and quantification (F). Similar to (D). Values represent mean ± SEM (n = 3 for each group; ***P < 0.001; two-way ANOVA).
Figure 2
Figure 2
Tet3 is required for functional axon regeneration of adult DRG neurons upon SNL in vivo. (A-B) Analysis of regeneration of sensory axons by SCG10 immunostaining at SNL D3. Shown are sample images of regenerating sensory axons identified by SCG10 (A; scale bar: 500 μm) and quantification (B). SCG10 immunofluorescence intensity was measured at different distal distances and normalized to that at the lesion site as the regenerative index. Values represent mean ± SEM (n = 5 for each group; *P < 0.05; two-way ANOVA). (C-E) Analysis of regenerating axons visualized by GFP labeling at SNL D7. Cross-sections of sciatic nerves at −1 to 6 mm distal to the lesion site from AAV-Ctrl and AAV-Tet3 KD treated animals were analyzed. Shown are sample images of GFP and Tuj1 (C; scale bar: 300 μm) and quantification (D-E). Values represent mean ± SEM (n = 3–4 for each group; *P < 0.05; two-way ANOVA). (F-G) Assay of re-innervation of epidermal area of the hindpaw by regenerating sensory axons. Shown in (F) are the schematic diagram and sample images of cross sections of hindpaw glabrous skin of Ctrl and Tet3 KD mice immunostained with the pan neuronal marker PGP9.5. The dotted line indicates the border between dermis and epidermis. Scale bar: 20 μm. Also shown are quantifications of the number of intraepidermal nerve fibers in a 1 mm segment of different epidermal areas (G). Values represent mean ± SEM (n = 4 for each group; **P < 0.01; *P < 0.05; n.s. P > 0.1; two-way ANOVA). (H) Assessment of thermal sensory recovery after SNL in AAV-Ctrl and AAV-Tet3 KD treated animals. Values represent mean ± SEM (n = 9 – 12 animals per group; **P < 0.01; ***P < 0.001; two-way ANOVA).
Figure 3
Figure 3
Tet3 regulates the expression of multiple injury-induced RAGs and mediates active DNA demethylation of ATF3 genomic regions. (A) Analysis of expression of some known RAGs. The mRNA expression was assessed by Q-PCR at SNL D1 and compared to the Ctrl naive group. Values represent mean ± SEM (n = 3 for each group; ***P < 0.001; *P < 0.05; n.s. P > 0.1; two-way ANOVA). (B-D) Assessment of ATF3 induction in Tet3 KD DRGs at SNL D1 and D7. Shown are sample images of immunostaining for GFP, ATF3, and Glutamine Synthetase (G.S.) in Pirt-GCAMP3 neuronal reporter mice (B) and for ATF3 and GFP in normal mice (C) and quantifications (D). Scale bars: 20 μm. Values represent mean ± SEM (n = 4 for each group; ***P < 0.001; two-way ANOVA). (E-F) Methylation status of the ATF3 distal enhancer region 1 (DE1) in Ctrl and Tet3 KD DRG neurons. AAV transduced (GFP+) and non-transduced (GFP-) NeuN+ neurons from L4 and L5 DRGs at SNL D1 were isolated by FACS and subjected to bisulfite sequencing analysis. Shown in (E) are sample reads of individual alleles. Open circles indicate unmethylated cytosines and closed circles indicate methylated cytosines. Shown in (F) is a summary from three independent biological replicates with at least 20 alleles each. Values represent mean + SEM (n = 3 for each group; *P < 0.05; two-way ANOVA). (G) ChIP-Q-PCR analysis of Tet3 binding to different genomic regions that were also examined for DNA methylation levels (as shown in Figure S3C). Values represent mean ± SEM (n = 3 for each group; *P < 0.05; two-way ANOVA).
Figure 4
Figure 4
TDG is required for SNL-induced axon regeneration and ATF3 expression in adult DRG neurons. (A-D) In vivo axon regeneration assay. Similar to Figure 2C-D, shown are sample images (A; scale bar: 300 μm) and quantification (B) at SNL D7 with expression of control-shRNA or TDG-shRNA. The same data from Ctrl-shRNA in Figure 2C is replotted for comparison. Similar to Figure 2A-B, also shown are sample images of regenerating sensory axons identified by SCG10 (C; scale bar: 500 μm) in TDGf/f mice expressing GFP (Ctrl), or GFP and Cre (TDG-KO) and quantifications (D). Values represent mean ± SEM (n = 4 for each group; *P < 0.05; two-way ANOVA). (E-F) Assessment of ATF3 induction in TDG KD DRGs. Similar to Figure 3B, shown are sample images (E; scale bar: 20 μm) and quantifications (F). Values represent mean ± SEM (n = 4 for each group; **P < 0.01; two-way ANOVA). (G) TDG-dependent expression of multiple SNL-induced RAGs. The mRNA expression was assessed by Q-PCR at SNL D1 and compared to the Ctrl naive group. Values represent mean ± SEM (n = 3 for each group; ***P < 0.001; **P < 0.01; *P < 0.05; n.s. P > 0.1; two-way ANOVA).
Figure 5
Figure 5
Tet1 is required for Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult mouse. Adult Ptenf/f mice were injected with AAVs to co-express GFP, Cre, and control shRNA, or shRNA against Tet1, Tet2, or Tet3 in the eye, followed by optic nerve crush 2 weeks later. RGC axons were anterogradely labeled by cholera toxin β subunit 12 days after injury. Shown are sample images of labeled axons (A; scale bar: 20 μm) and quantifications (B). Values represent mean ± S.E.M. (n = 3 for each group; * P < 0.05; two-way ANOVA).

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