Locus-Specific DNA Methylation Assays to Study Glutamate Receptor Regulation.

Recent findings indicate that glutamate receptors are regulated at the epigenetic level through the posttranslational modification of histones and through DNA methylation. Furthermore, dysregulation of these marks in the context of neurological disease has been shown to influence glutamate receptor function. Over the past two decades, an appreciation for the essential role epigenetic mechanisms play in nervous system function has led to the development of many methods and tools to map, quantitate, and manipulate these chromatin marks. Here we describe two popular methods used to quantitate DNA methylation levels at the gene or nucleotide level. The first, cloning-based bisulfite sequencing involves modification of DNA samples using the chemical sodium bisulfite (BS) , which deaminates all unmethylated cytosines to form uracil. Subsequent PCR amplification converts the uracils to thymine, leaving any cytosines in the PCR product representative of methylation. Fragments are then cloned and sequenced to quantitate the percentage of methylation at each cytosine. The second technique, methyl-binding domain capture (MBDCap), involves shearing the genomic DNA into fragments via sonication. Samples are then incubated with magnetic beads conjugated to methyl-binding domain (MBD) peptides to bind and enrich fragments containing methylated CpGs. Quantitation of DNA methylation levels are then measured indirectly using qRT-PCR with primers specific to the region of interest. Because these methods do not require advanced technical knowledge and can be performed with common laboratory equipment, they are great options for interrogating DNA methylation patterns at the level of the gene, the regulatory region, or in the case of bisulfite sequencing, the nucleotide.

Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic.

Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder (Williamson, 1982). This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. In the present study, we identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference-mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. We also demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Our electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, our findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior.

Tet1 Oxidase Regulates Neuronal Gene Transcription, Active DNA Hydroxy-methylation, Object Location Memory, and Threat Recognition Memory.

A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the CNS. In this study, we used Tet1 gene knockout (Tet1KO) mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to: altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning) and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation.

Dynamic changes in 5-methylcytosine (5mC) have been implicated in the regulation of gene expression critical for consolidation of memory. However, little is known about how these changes in 5mC are regulated in the adult brain. The enzyme methylcytosine dioxygenase TET1 (TET1) has been shown to promote active DNA demethylation in the nervous system. Therefore, we took a viral-mediated approach to overexpress the protein in the hippocampus and examine its potential involvement in memory formation. We found that Tet1 is a neuronal activity-regulated gene and that its overexpression leads to global changes in modified cytosine levels. Furthermore, expression of TET1 or a catalytically inactive mutant (TET1m) resulted in the upregulation of several neuronal memory-associated genes and impaired contextual fear memory. In summary, we show that neuronal Tet1 regulates DNA methylation levels and that its expression, independent of its catalytic activity, regulates the expression of CNS activity-dependent genes and memory formation.

Interindividual Variability in Stress Susceptibility: A Role for Epigenetic Mechanisms in PTSD.

Post-traumatic stress disorder (PTSD) is a psychiatric condition characterized by intrusive and persistent memories of a psychologically traumatic event that leads to significant functional and social impairment in affected individuals. The molecular bases underlying persistent outcomes of a transient traumatic event have remained elusive for many years, but recent studies in rodents have implicated epigenetic modifications of chromatin structure and DNA methylation as fundamental mechanisms for the induction and stabilization of fear memory. In addition to mediating adaptations to traumatic events that ultimately cause PTSD, epigenetic mechanisms are also involved in establishing individual differences in PTSD risk and resilience by mediating long-lasting effects of genes and early environment on adult function and behavior. In this review, we discuss the current evidence for epigenetic regulation of PTSD in human studies and in animal models and comment on ways in which these models can be expanded. In addition, we identify key outstanding questions in the study of epigenetic mechanisms of PTSD in the context of rapidly evolving technologies that are constantly updating and adjusting our understanding of epigenetic modifications and their functional roles. Finally, we discuss the potential application of epigenetic approaches in identifying markers of risk and resilience that can be utilized to promote early intervention and develop therapeutic strategies to combat PTSD after symptom onset.