Bioinformatic Analysis of DNA Methylation in Neural Progenitor Cell Models of Alcohol Abuse.

Several recent publications sought to investigate the effects of ethanol treatment on models of central nervous system development, specifically through changes in DNA methylation. Regulation of DNA methylation causes a long-lasting, epigenetic change in the capacity of the genome to respond to developmental or metabolic stimuli. Changes in technologies for quantifying DNA methylation have increased the ability to identify and interpret potential effects of ethanol. Here, we review these recent studies in order to evaluate the detection technologies and bioinformatic analyses. Our evaluation finds that whole- or targeted-genome sequencing combined with bisulfite conversion of unmethylated G to U residues is now the standard for assessing genome-wide effects, and specific differentially methylated regions can be validated by one of several widely-available techniques. The acceptance of these technologies should help understand how ethanol leads to life-long developmental or behavioral deficits, and, perhaps, suggest therapies to reverse these effects.

Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele.

Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 µM) with greater frequency and amplitude but they also exhibited rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. This study offers a proof-of-principle for utilizing human neurons to study gene variants contribution to addiction.

A positive feedback mechanism that regulates expression of miR-9 during neurogenesis.

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.