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. 2010 May;120(5):1603-16.
doi: 10.1172/JCI40000. Epub 2010 Apr 26.

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation

Bermans J Iskandar  1 , Elias Rizk, Brenton Meier, Nithya Hariharan, Teodoro Bottiglieri, Richard H Finnell, David F Jarrard, Ruma V Banerjee, J H Pate Skene, Aaron Nelson, Nirav Patel, Carmen Gherasim, Kathleen Simon, Thomas D Cook, Kirk J Hogan
Affiliations

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation

Bermans J Iskandar et al. J Clin Invest. 2010 May.

Abstract

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

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Figures

Figure 1
Figure 1. Summary of experimental interventions within the folate and methylation pathways.
Folic acid enters the cell through Folr1, which is upregulated with injury. It is then converted to the active tetrahydrofolate (THF) form by Dhfr. This allows eventual production of nucleotides and certain amino acids as well as transfer of the methyl group into the methionine methylation cycle. The latter occurs through the B12-dependent MS step. Subsequently, SAM is the substrate used by the methyltransferase enzymes for the methylation reactions. Inhibition of Folr1, Dhfr, MS, and Dnmt suppresses CNS regeneration. In turn, activation of Dnmt enhances CNS regeneration.
Figure 2
Figure 2. Combined spinal cord and peripheral nerve injury induces expression of Folr1 but not Rfc1.
4 days after sharp transection of both dorsal columns and the left sciatic nerve in rats, the left L5 and L6 DRGs were removed, sectioned, and mounted to undergo in situ hybridization (A, D) and immunohistochemistry (C, F) of the Folr1 and Rfc1 receptors. The spinal cord was removed for RT-PCR (B, E) and Western immunoblot (Figure 7, A and E). Note the significant upregulation of Folr1 with injury, with no change in the Rfc1 levels both in the spinal cord (SC) and DRG: in situ Folr1 n = 8 (SC), 8 (DRG); in situ Rfc1 n = 5 (SC), 5 (DRG); Folr1 RT-PCR n = 3 (SC), 3 (DRG); Rfc1 RT-PCR n = 3 (SC), 3 (DRG); immuno Folr1 n = 4 (SC), 4 (DRG); immuno Rfc1 n = 4 (SC), 4 (DRG). 2-tailed Student’s t test; mean ± SEM; *P < 0.05. (G, H) Folr1 in situ hybridization of DRG sections without (G) and with (H) preceding combined spinal cord and left sciatic nerve injury. (I, J) Folr1 immunostaining of DRG sections without (I) and with (J) preceding combined spinal cord and left sciatic nerve injury. (K) Confocal microscopy showing colocalization of the red Folr1 stain with the green neuronal (neurofilament) stain. Original magnification, ×40.
Figure 3
Figure 3. Mice with reduced expression of the Folr1 gene do not regenerate injured DRG axons in vitro.
Forty-eight hours after an in vivo sharp injury to the spinal cord (dorsal columns) and left sciatic nerve in mice, the left lumbar DRGs that contain the cell bodies of the injured axons were removed, dissociated, and placed in a culture medium that sustains growth. In the WT animals (A, C), a significant number of axons reached a length of 300 μm; conversely, neurons from the Folr1+/– mice were less able to extend long axons. Instead, these neurons were more likely to show sprouting of short axons (B, D), indicating a decreased ability of the neurons to regenerate, as previously described (4, 24). Whereas 41% of neurons were able to extend axons greater than 299 μm in the WT animals, only 12% of neurons showed such growth in the Folr1+/– mice. Original magnification, ×20.
Figure 4
Figure 4. Mice with reduced expression of the Folr1 gene do not regenerate injured spinal and optic axons in vivo.
(A) Mouse SCRM in which a sciatic nerve graft is implanted at the site of bilateral C3 dorsal column lesion. Removal of the left sciatic nerve of the animal effectively created a “conditioning” peripheral nerve injury on that side, which has been shown to enhance growth of injured spinal axons into the graft. At 2 weeks, a fluorescent tracer was placed at the free end of the graft and was detected in the conditioned (left) lumbar DRG neuron cell bodies of the axons that had grown into the graft. (B) Regeneration of spinal sensory axons into a peripheral nerve graft in WT and Folr1+/– mice with and without FA supplementation. n = 10 (WT); 10 (WT FA); 10 (Folr1+/–); 10 (Folr1+/–). One-way ANOVA with Bonferroni’s correction. (C) Fluorescent (i.e., regenerated) neurons in a DRG section. Original magnification, ×10. (D) Mouse retinal regeneration model in which a sciatic nerve graft was implanted to the proximal stump of an axotomized optic nerve. At 2 months, a fluorescent tracer was placed at the free end of the graft and was detected in the RGC cell bodies of the axons that had grown into the graft. (E) Regeneration of RGC axons into a peripheral nerve graft in WT and Folr1+/– mice with and without FA supplementation. (F) Fluorescent RGC neurons on a retinal flat mount. Original magnification, ×40. n = 10 (WT); 10 (WT FA); 10 (Folr1+/–); 8 (Folr1+/–). One-way ANOVA with Bonferroni’s correction; mean ± SEM; *P < 0.05.
Figure 5
Figure 5. Dhfr bioactivation is essential for folate-mediated CNS regeneration.
Using the SCRM model of afferent spinal regeneration in rats, i.p. MTX was used to inhibit Dhfr, thus preventing the conversion of FA into the bioactive form tetrahydrofolate, as demonstrated in the Dhfr activity assay (A) (n = 5 [uninjured]; n = 5 [injured]; n = 5 [injured and exposed to MTX]; Student’s t test; mean ± SEM; *P = 0.006), with only a small change in protein levels on Western immunoblot (C, D). N, normal (uninjured); Inj, combined spinal cord and sciatic nerve injury. Note that there is minimal or no increase in Dhfr protein and activity levels after spinal cord/sciatic nerve injury. The dose of MTX (400 μg/kg) that determined maximal inhibition with minimal toxicity was selected from a literature review (46, 51, 52). MTX suppressed the regeneration of spinal axons into a nerve graft to below baseline levels (B) (n = 10 [untreated controls]; n = 7 [FA]; n = 7 [MTX]; n = 6 [MTX and FA]). 1-way ANOVA with Bonferroni’s correction; mean ± SEM; *P < 0.05.
Figure 6
Figure 6. Combined spinal cord and peripheral nerve injury inhibits methyl cycling, SAM bioavailability, and MS activity; and MS inhibition abolishes folate-mediated axonal regeneration.
Injury to the rat spinal cord and left sciatic nerve causes a diminution in the SAM/SAH ratio (n = 6 [normal]; n = 6 [injured]. Student’s t test; mean ± SEM; *P < 0.05) (A), suggesting a direct effect of injury on DNA methylation. To confirm this observation, spinal axonal regeneration was measured in the SCRM model on the “conditioned” side after treatment with N2O. N2O is a specific inhibitor of the MS enzyme; thus it interferes with the entry of active folate into the methionine methylation cycle. N2O suppressed axon growth to below control levels. n = 10 (untreated); n = 7 (FA); n = 8 (N2O); n = 8 (FA). One-way ANOVA with Bonferroni’s correction; mean ± SEM; *P < 0.05 (B). To determine whether N2O suppresses MS, MS activity and SAH levels were measured in the spinal cord after N2O exposure. N2O suppressed MS activity (C) with no change in the enzyme protein levels by Western analysis (not shown) and caused a corresponding rise in SAH (D). Note the drop in MS activity after injury and after N2O treatment. Recovery from the N2O effect occurs by 3 days (C). N, no injury; S, spinal cord injury; N0 and S0, No N2O treatment; N1, N2O given the day before removing the spinal tissue; N2, N2O given 3 days before removing the spinal tissue; S1, N2O treatment every other day for 2 weeks; S2, N2O given 2 weeks before removing spinal tissue. n = 3 in each group; each specimen was run in triplicate. One-way ANOVA with Bonferroni’s correction; mean ± SEM, *P < 0.05.
Figure 7
Figure 7. Combined spinal cord and peripheral nerve injury suppresses the protein levels of the de novo methyltransferases Dnmt3a and Dnmt3b, but not the maintenance methyltransferase Dnmt1, and FA treatment restores the levels in a biphasic fashion that parallels the biphasic response to FA treatment seen with spinal regeneration and Folr1 expression.
(A–D) Combined spinal cord and peripheral nerve injury suppresses Dnmt3a and Dnmt3b protein levels in mouse spinal cords, with no effect on Dnmt1. Furthermore, FA treatment restores these levels in both Dnmt3a and Dnmt3b. When increasing doses of FA are given i.p., a biphasic response is observed in the de novo methyltransferases Dnmt3a and Dnmt3b, with maximal protein levels occurring at 80 μg/kg. (F) Folic acid supplementation was previously shown to affect rat CNS regeneration following injury in a dose-dependent manner that corresponds tightly to the de novo Dnmt response. These results, obtained from our laboratory and previously reported by Iskandar et al. (7), are confirmed here in a different group of animals. One-way ANOVA with Bonferroni’s correction; n (FA dose) = 9 (0 μg/kg); 9 (40 μg/kg); 5 (80 μg/kg); 9 (400 μg/kg); 9 (800 μg/kg). Mean ± SEM; P < 0.05 for all comparisons except 0 versus 400, 0 versus 800, and 40 versus 400. Conversely, treatment with MTX significantly decreases Dnmt3a and Dnmt3b protein levels, while treatment with N2O nearly eliminates Dnmt3a and Dnmt3b protein expression. Neither injury nor folate, MTX, nor N2O affect the levels of the maintenance methyltransferase Dnmt1 (A, E). Furthermore, although Folr1 expression increases after injury, its response to increasing doses of FA is similarly biphasic. (A) Western blots. All lanes were run on the same gel. Noncontiguous lanes are separated by a thin white line in the figure. (B–D, E) Plots of the band densities relative to the corresponding actin bands. One-way ANOVA with Bonferroni’s correction; n = 3 in each group; mean ± SEM; *P < 0.05.
Figure 8
Figure 8. Combined spinal cord and peripheral nerve injury suppress spinal cord DNA methylation, and FA restores methylation levels to baseline.
A global methylation assay (23) was performed to measure methyl group incorporation into DNA from rat spinal cord tissue in response to dorsal column/left sciatic nerve injury and FA supplementation. Global methylation in the spinal cord tissue was measured by a scintillation counter indicating the amount of radioactively labeled methyl groups incorporated into the sequence. The cpm values of each experimental group correspond inversely to the extent of global DNA methylation in the spinal cord in vivo. We show that global methylation is suppressed by injury, and the methylation levels are restored in response to the 80 μg/kg dose of FA. N, uninjured control spinal cord; I, spinal cord/sciatic nerve injury; F, spinal cord/sciatic nerve injury and treatment with FA at 80 μg/kg. n = 20 (N); n = 32 (I); n = 33 (F). One-way ANOVA with Bonferroni’s correction; mean ± SEM; *P < 0.05.
Figure 9
Figure 9. Global DNA methylation in the injured spinal cord follows a biphasic curve in response to increasing doses of FA.
This curve corresponds to the biphasic Dnmt and Folr1 protein levels as well as the spinal regeneration response. We measured global methylation in the spinal cords of animals with combined spinal cord and peripheral nerve injuries, which were subjected to varying doses of i.p. FA given daily starting 3 days prior to the injury and continuing 4 days. n = 12 (uninjured control animals); n (FA dose) = 32 (0 μg/kg); 33 (20 μg/kg); 32 (40 μg/kg); 33 (80 μg/kg); 34 (160 μg/kg); 32 (400 μg/kg); 32 (800 μg/kg). The quadratic term in the dose-response model was statistically significant (P = 0.001), confirming that there is a U-shaped dose response to the folate dose on a log scale. There are no differences between the injured/untreated animals and the animals treated with 20 μg/kg FA (P = 0.99), nor between the animals treated with the 400 μg/kg and 800 μg/kg doses (P = 0.85). Note the tight correlation in the biphasic FA dose effects between global DNA methylation, spinal regeneration (Figure 7F), and the Dnmt3a and Dnmt3b as well as Folr1 protein levels (Figure 7, A–C, and E). The FA dose of 80 μg/kg, at which both DNA methylation and Dnmt3a and Dnmt3b and Folr1 expression levels were maximal, was found to be the most effective in promoting regeneration.
Figure 10
Figure 10. Dnmt agonist promotes, and Dnmt antagonist inhibits, the regeneration of injured sensory spinal axons into a peripheral nerve graft.
Spinal axonal regeneration was measured in the SCRM model on the “conditioned” side after i.p. treatment with a Dnmt agonist 3-ABA (15 mg/kg) or antagonist 5-AzaC (0.1 mg/kg) with and without FA at 80 μg/kg. Treatment with the antagonist suppresses the proregenerative effect of FA (A). Conversely, treatment with the agonist at this dose significantly improves spinal regeneration into a nerve graft in vivo. The effect of 3-ABA at 15 mg/kg is equivalent to the 60 μg/kg dose of FA (7) (B). n = 7 (FA); n = 9 (FA and 5-AzaC); n = 11 (vehicle control); n = 9 (3-ABA); n = 7 (FA); n = 9 (FA and 3-ABA). One-way ANOVA with Bonferroni’s correction; mean ± SEM; *P < 0.05.
Figure 11
Figure 11. Combined spinal cord and peripheral nerve injury increases methylation of the Gadd45a promoter, and FA increases methylation further in a biphasic fashion in 12 of 18 CpG sites.
The spinal cords of animals subjected to combined spinal cord and left sciatic nerve injury (SCSN) and increasing i.p. concentrations of FA were removed for analysis. The percentage of methylation at each of 18 CpG sites of the Gadd45a promoter was measured in triplicate using pyrosequencing. Note the biphasic response to FA in 12 of the sites studied. Most of the missing data on the graphs are caused by failed analysis by both software and manual analysis. For the samples with no data on any of the CpG sites, this is caused by failed PCR. Overall, the quadratic term in the dose-response models was statistically significant (P = 0.0001; n = 3), confirming that there is a U-shaped dose-response to the folate dose. There is no evidence of differential dose responses among sites (P = 0.8), although power is low for identifying specific CpG sites with differing dose responses.
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Comment in

  • Neuronal injury: folate to the rescue?
    Kronenberg G, Endres M. Kronenberg G, et al. J Clin Invest. 2010 May;120(5):1383-6. doi: 10.1172/JCI40764. Epub 2010 Apr 26. J Clin Invest. 2010. PMID: 20424316 Free PMC article.

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