Dynamic DNA Methylation Regulates Levodopa-Induced Dyskinesia

Abstract

Levodopa-induced dyskinesia (LID) is a persistent behavioral sensitization that develops after repeated levodopa (l-DOPA) exposure in Parkinson disease patients. LID is a consequence of sustained changes in the transcriptional behavior of striatal neurons following dopaminergic stimulation. In neurons, transcriptional regulation through dynamic DNA methylation has been shown pivotal to many long-term behavioral modifications; however, its role in LID has not yet been explored. Using a rodent model, we show LID development leads to the aberrant expression of DNA demethylating enzymes and locus-specific changes to DNA methylation at the promoter regions of genes aberrantly transcribed following l-DOPA treatment. Looking for dynamic DNA methylation in LID genome-wide, we used reduced representation bisulfite sequencing and found an extensive reorganization of the dorsal striatal methylome. LID development led to significant demethylation at many important regulatory areas of aberrantly transcribed genes. We used pharmacologic treatments that alter DNA methylation bidirectionally and found them able to modulate dyskinetic behaviors. Together, these findings demonstrate that l-DOPA induces widespread changes to striatal DNA methylation and that these modifications are required for the development and maintenance of LID.

Significance statement: Levodopa-induced dyskinesia (LID) develops after repeated levodopa (l-DOPA) exposure in Parkinson disease patients and remains one of the primary obstacles to effective treatment. LID behaviors are a consequence of striatal neuron sensitization due to sustained changes in transcriptional behavior; however, the mechanisms responsible for the long-term maintenance of this cellular priming remain uncertain. Regulation of dynamic DNA methylation has been shown pivotal to the maintenance of several long-term behavioral modifications, yet its role in LID has not yet been explored. In this work, we report a pivotal role for the reorganization of DNA methylation in the development of LID and show that modification of DNA methylation may be a novel therapeutic target for use in preventing or reversing dyskinetic behaviors.

Keywords: DNA methylation; dyskinesia; l-DOPA.

Figure 1.

Aberrant regulation of DNA methylation-modifying enzymes. a, Behavioral data demonstrating that repeated l-DOPA treatment induces a sensitized dyskinetic response, measured by ALO score (top) or rotational behavior (bottom). *p < 0.05 versus l-DOPA-treated day 1 (repeated-measures ANOVA). n = 8 per group. bi–iii, l-DOPA induces enhanced expression of immediate-early genes. ci–iii, DNA methyltransferase expression is not affected by dyskinesia development. di–iii, TET3 is aberrantly expressed in dyskinetic animals. ei–iii, Gadd45b and Gadd45 g expression is enhanced following dyskinesia development (two-way ANOVA; Tukey's post hoc tests; n = 6 or 7 per group). Error bars indicate SEM. *p < 0.05 for interaction between lesion and treatment.

Figure 2.

Aberrant regulation of DNA methylation following the development of LID is in a locus-specific manner. a, LID development has no effect on global methylation levels as measured by ELISA (two-way ANOVA; n = 7 per group). b, MeDIP enrichment is similar across all samples for a spiked-in positive control. ci–iii, MeDIP at the promoter regions of immediate early genes aberrantly transcribed in LID show lesion-dependent effects (two-way ANOVA). Brackets indicate p < 0.05 for a main effect of lesion (n = 3 or 4 per group). di–iii, MeDIP at regions previously shown to have dynamic methylation in other models of striatal plasticity display reductions in DNA methylation at promoter regions in a l-DOPA and lesion-dependent manner (two-way ANOVA; Tukey's post hoc tests; n = 7 per group) (Massart et al., 2015). Error bars indicate SEM. *p < 0.05 versus all other groups. ^#p < 0.05 versus both intact groups.

Figure 3.

Development of LID induces a global rearrangement of the striatal methylome. a, DMRs were identified between saline (n = 8) and l-DOPA (n = 7) treated striata following 6-OHDA lesion using MethylKit and filtered for a corrected p value of <0.01. Regions near genes with established roles in LID were highlighted with red dots to support the functional relevance of the identified changes. b, DMRs were found to be evenly distributed across the genome, unbiased to chromosome. c, A direct correlation was observed between absolute methylation difference and distance from the transcriptional start site (Pearson's correlation coefficient, r = 0.138209, p = 2.2 × 10⁻¹⁶). d, Changes in methylation levels at DMRs nonparametrically correlated with dyskinetic behavioral changes (Spearman's correlation). Regions near genes whose expression has been directly correlated with dyskinetic behaviors were emphasized with red dots to highlight their relevance in LID. ei–iv, Using MeDIP on an independent cohort of animals, regions identified by genome-wide analysis were confirmed to exhibit dynamic demethylation in a lesion- and l-DOPA-dependent manner near genes aberrantly transcribed in LID (two-way ANOVA; Tukey's post hoc tests; n = 7 per group). *p < 0.05 versus all other groups. ^#p < 0.05 versus both intact groups.

Figure 4.

Highly dynamic DMRs are involved in transcriptional regulation and synaptic plasticity. a, Heatmap of highly differentially methylated regions (at least 5% change) showing a significant enrichment of DMRs exhibiting active demethylation (Fisher's exact test, p < 0.0001). Color key represents the direction and percentage change in methylation identified. b, Highly dynamic DMRs are outside of CGIs or shores and instead enriched extragenically and intragenically. Left graphs, Distribution of all regions identified by our analysis. Right graphs, Those regions containing HDRs. c, When normalized for the genomic coverage following RRBS enrichment, the highest proportion of HDRs are in intragenic and extragenic regions, whereas few CGIs display dynamic change. d, Ontological analysis of genes associated with DMRs shows enrichment for processes shown previously relevant to neuronal plasticity and synaptic function. e, Candidate gene targets associated with the newly identified DMRs are enriched for genes previously shown to be aberrantly transcribed in LID (Fisher's exact test) (Heiman et al., 2014).

Figure 5.

Parkinsonism alters dorsal striatal methylation at genomic regions affected by LID development. a, DMRs were identified between intact (n = 8) and lesioned (n = 7) striata following 6-OHDA treatment using MethylKit and filtered for a corrected p value of <0.01. b, Highly dynamic DMRs (>5% change) are outside of CGIs or shores and instead enriched extragenically and intragenically when normalized for genomic coverage during RRBS enrichment. c, There is a significant overlap between the DMRs exhibiting dynamic DNA methylation following both dopaminergic lesion and l-DOPA treatment. d, A strong correlation was observed between l-DOPA and lesion-dependent methylation effects in the striatum (Pearson's correlation coefficient, r = 0.5590431, p = 2.2 × 10⁻¹⁶). However, a subset of regions (highlighted in blue) exhibited demethylation following both effects. e, Ontological analysis of genes near DMRs showing dynamic demethylation following l-DOPA treatment and 6-OHDA lesion shows enrichment for processes relevant to neuronal plasticity and function. f, Candidate gene targets associated with the DMRs demethylated by lesion- and l-DOPA-dependent effects are enriched for genes previously shown to be aberrantly transcribed in D1 neurons following LID development (Fisher's exact test) (Heiman et al., 2014).

Figure 6.

Global modifications of DNA methylation are able to bidirectionally influence LID development and expression. a, Twice daily administration of methionine (7.2 mmol/kg, s.c.), at doses known to enhance global methylation, hinders LID ALO development and expression. *p < 0.05 versus vehicle (two-way ANOVA; Tukey's post hoc tests). n = 7 per group. bi–iv, Methionine supplementation increased DNA methylation at regions undergoing LID-dependent demethylation (two-way ANOVA; Tukey's post hoc tests). n = 7 per group. c, Chronic striatal infusion of RG-108 (100 μm), a DNMT inhibitor, enhances LID behavioral sensitization. p < 0.05 versus vehicle (two-way ANOVA; Tukey's post hoc tests). n = 10 per group. di–iv, RG-108 decreased DNA methylation at regions undergoing LID-dependent demethylation (two-way ANOVA; Tukey's post hoc tests). n = 7 per group. *p < 0.05 versus all groups. ^#p < 0.05 versus all groups. Error bars indicate SEM.