Elucidating the contributions of genetic variants associated with addiction phenotypes

Genome-wide association studies have identified several loci with variant sequences that are correlated with the risk of nicotine addiction and alcohol use disorders (AUD), however, little is known about the cellular mechanisms underlying these genetic variants. Drugs such as nicotine mediate addiction behaviors via the activation of nicotinic acetylcholine receptors (nAChR). We first prepared subject-specific induced pluripotent stem cells (iPSC) from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in the CHRNA5 gene encoding the nAChR 5 subunit. To understand the impact of the N398 variant in human derived cells, we differentiated iPSCs to midbrain-like dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Neurons from both variants demonstrated physiological and biochemical properties consistent with mature neuronal function. Results show that N398 variant midbrain-like DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. The N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both midbrain-like DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 ï­M, concentrations of nicotine similar to those of heavy smokers) with greater frequency and amplitude and rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. Additionally, to understand the impact of AUD variants in humans, we prepared iPSCs from four subjects diagnosed with AUD and carrying three SNPs within the KCNJ6 gene (minor alleles of rs702859, rs702860, and rs2835872) encoding the potassium inward rectifying GIRK2 channel. We differentiated iPSCs into neural stem cells (NSCs) and all were positive for NSC markers (SOX2, Nestin, and Musashi). To model acute alcohol exposure, we exposed NSCs to medium containing 75 mM ethanol. To test whether this concentration would alter NSC growth dynamics, we assessed the cell viability, size, and doubling time over 72 hrs of exposure to which there was no detectable change. From RNAseq gene expression experiments following a 24 hr treatment with or without ethanol, we have identified differential gene enrichment in genes involved in neural differentiation as well as predicted alternative splicing variants in the KCNJ6 gene. We have also begun to assess the neuronal activity using two sources of basal culture medium (BrainPhys [Stem Cell Technologies] and Neurobasal [Gibco]) following Neurogenin 2 lentiviral differentiation of iPSCs. We detected substantial calcium spiking in BrainPhys medium in comparison to Neurobasal medium though calcium imaging analysis. This suggests that BrainPhys medium provides a more sustainable culture environment for future experiments. Planned studies will utilize cultures grown in BrainPhys based medium to assess specific changes in neuronal activity for each genotype during ethanol exposure. Overall, the results of this work offer a proof-of-principle for utilizing human derived neurons to study gene variants contributing to addiction.Ph.D.Includes bibliographical referencesby Eileen N. On