Methionine increases BDNF DNA methylation and improves memory in epilepsy

Abstract

Objective: Temporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus.

Methods: Using the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model.

Results: We found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade.

Interpretation: Our findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE.

Figure 1

Altered brain-derived neurotrophic factor (Bdnf) gene expression and DNA methylation levels in the epileptic hippocampus are associated with memory deficits. (a) Diagram of experimental setup. (b, c) Epileptic animals displayed hippocampus-dependent memory deficits on test day at 24 h posttraining in the contextual fear conditioning (CFC) and the object location (OL) memory paradigms (CFC:t₍₁₇₎ = 3.41, P < 0.01, n = 10–11; OL:t₍₉₎ = 3.37, P < 0.01, n = 5–6, t-test, *significance relative to non-epileptic group). (d) BdnfmRNA levels were significantly increased in the hippocampus of the non-epileptic, epileptic untrained, and epileptic CFC animals following CFC training compared to non-epileptic untrained controls. BdnfmRNA levels were significantly increased in the hippocampus of the epileptic CFC animals compared to all other groups (F_3,18 = 67.58, P < 0.001, n = 5–6, one-way analysis of variance [ANOVA] with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). (e) Bisulfite sequencing analysis of 12 CpG sites within promoter 4 and the noncoding exon IV of the Bdnf gene revealed a significant decrease in DNA methylation levels in the non-epileptic CFC-trained, epileptic untrained, and epileptic CFC-trained animals compared to the non-epileptic untrained controls. The epileptic CFC group had significantly decreased DNA methylation compared to all other groups (F_3,15 = 11.64, P < 0.001, n = 4–5, one-way ANOVA with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). Error bars are SEM.

Figure 2

Methyl supplementation with methionine (Met) alters hippocampal brain-derived neurotrophic factor (Bdnf) gene DNA methylation and mRNA levels in epileptic animals. (a) Diagram of experimental setup. (b) Methyl supplementation with Met significantly reduced BdnfmRNA levels in the hippocampus of the epileptic animals (F_3,24 = 9.99, P < 0.001, n = 6–8, one-way analysis of variance [ANOVA] with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). (c) Methyl supplementation with Met significantly increased BdnfDNA methylation in the epileptic hippocampus (F_3,12 = 4.26, P < 0.05, n = 4, one-way ANOVA with post hoc test, *significance relative to nonepileptic group; ^#significance between experimental groups). (d) Experimental design setup. (e) Decreased BdnfDNA methylation in the epileptic contextual fear conditioning (CFC) group was rescued by Met supplementation (F_3,22 = 15.06, P < 0.001, n = 5–9, one-way ANOVA with post hoc test, *significance relative to non-epileptic; ^#significance relative to non-epileptic CFC; ^§significance relative to epileptic CFC). (f) A schematic of transcription factor (TF)-binding regulatory elements within the rat Bdnf exon IV transcription start site (TSS). The cAMP response element-binding (CREB) TF is known to bind to the depicted sequence within the Bdnf gene while the depicted Sp1-binding site is considered to be a putative site. (g) Methyl supplementation with Met significantly reduced BdnfmRNA levels in the hippocampus of CFC-trained epileptic animals (F_3,19 = 48.69, P < 0.001, n = 5–6, one-way ANOVA with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). Error bars are SEM.

Figure 3

Methyl supplementation reverses hippocampal memory deficits associated with epilepsy. (a) Diagram of experimental setup. (b) Methyl supplementation rescued memory impairments in the epileptic animals in the contextual fear conditioning (CFC) paradigm (F_3,36 = 5.95, P < 0.01, n = 10–11, one-way analysis of variance [ANOVA] with post hoc test, *significance relative to non-epileptic group). (c) Methyl supplementation rescued memory deficits in the epileptic animals in the object location (OL) paradigm (F_3,19 = 7.06, P < 0.01, n = 5–6, one-way ANOVA with post hoc test, *significance relative to non-epileptic group). (d) Methyl supplementation had no effect on fear conditioning training (F_3,26 = 7.68, P < 0.01, n = 7–9, one-way ANOVA with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). (e) Methyl supplementation with Met did not significantly alter movement time in the open-field paradigm. Epileptic animals showed greater mobility compared to non-epileptic rats (F_3,24 = 5.53, P < 0.01, n = 6–8, one-way ANOVA with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). (f) Methyl supplementation with Met did not alter the time spent in the open arena during the open-field paradigm (F_3,24 = 1.46, P > 0.05, n = 6–8, one-way ANOVA with post hoc test). Error bars are SEM.

Figure 4

Methyl supplementation decreases interictal spiking. (a) Diagram of experimental setup. (b) Representative trace of interictal spikes observed in epileptic rats 3 weeks post-KA administration (* represents what was counted as a spike). (c) Methyl supplementation significantly reduced interictal spike number in the first 24 h postinjection. Interictal spiking rate showed a trend toward returning back to baseline at 24–48 h post-Met supplementation (F_2,6 = 4.06, P < 0.05, n = 7, one-way analysis of variance [ANOVA] with post hoc test, *significance relative to pre-Met). (d) Methyl supplementation did not have a significant effect on number of seizures per day (F_2,18 = 0.82, P > 0.05, n = 7, one-way ANOVA with post hoc test). (e) Methyl supplementation did no significantly affect duration of seizures (F_2,23 = 0.91, P > 0.05, n = 3–16, one-way ANOVA with post hoc test, ns = not significant). Error bars are SEM.

Figure 5

Methyl supplementation increased theta and alpha rhythm power. (a) Power spectrum density surrounding interictal spikes. (b) Methyl supplementation significantly increased theta rhythm power (F_2,5 = 12.30, P < 0.01, n = 6, one-way analysis of variance [ANOVA] with post hoc test, *significance relative to pre-Met). (c) Methyl supplementation significantly increased alpha rhythm power on day 2 post-Met treatment (F_2,5 = 5.90, P < 0.05, n = 6, one-way ANOVA with post hoc test, *significance relative to pre-Met). Error bars are SEM.

Figure 6

DNA methyltransferases (DNMT) inhibition prevents the effect of methyl supplementation on hippocampal brain-derived neurotrophic factor (Bdnf) DNA methylation, Bdnf expression and memory formation. (a) Diagram of experimental setup. (b) The increase in BdnfDNA methylation due to methyl supplementation is blocked by DNMT inhibition (F_4,18 = 6.93, P < 0.01, n = 3–6, one-way analysis of variance [ANOVA] with post hoc test, *significance relative to non-epileptic group; ^# significance relative to epileptic group; ^§significance relative to epileptic + Met group). (c) DNMT inhibition attenuated the effect of methyl supplementation with Met on BdnfmRNA levels in the epileptic hippocampus (F_4,24 = 11.96, P < 0.001, n = 5–8, one-way ANOVA with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). (d) BDNF protein expression was significantly reduced in Met-treated epileptic animals compared to nontreated epileptic animals. DNMT inhibition attenuated the decrease in BDNF protein expression due to methyl supplementation with Met. Duplicates of each sample were run and normalized to valosin-containing protein (VCP) and beta-actin 4 as within-lane loading controls. Representative blots of samples from each treatment group are shown (F_3,14 = 3.84, P < 0.05, n = 5–6, one-way ANOVA with post hoc test, *significance relative to epileptic group). (e) DNMT inhibition blocked Met-induced memory enhancement (F_4,31 = 7.81, P < 0.001, n = 7–8, one-way ANOVA with post hoc test, *significance relative to non-epileptic group; ^#significance between experimental groups). Error bars are SEM. (f) Potential mechanism of the effect of methionine treatment on memory restoration in epileptic animals. Shaded words represent the molecular mechanisms demonstrated in the present study. Unshaded words and dotted lines are potential pathways that could also be involved in the memory restoration process with methionine. Thus, methionine can be rapidly converted to S-adenosyl methionine (SAM), the universal methyl donor in the brain. SAM donates a CH3 group that leads to increased DNA methylation and gene transcription changes in the epileptic hippocampus. Once SAM donates a methyl group, it is converted to S-adenosyl-l-homocysteine (SAH), which leads to the production of adenosine. Adenosine production can lead to decreases in cell excitability that could subsequently decrease interictal spike activity in the epileptic hippocampus. Arginine is also a downstream by product of SAH hydrolysis that can lead to changes in nitric oxide production and can affect cell excitability.