Alcoholism is a serious health and socioeconomic problem in the U.S. Understanding how alcohol produces reward, motivates further consumption, and eventually leads to addiction is necessary to design effective treatments for alcohol use disorders (AUDs). Synaptic transmission mediates all of these behaviors, however, little is known about the effect of alcohol on synaptic transmission in the context of human neurons. The single nucleotide polymorphism (SNP) rs1799971 (OPRM1 A118G) produces a non-synonymous amino acid substitution in the mu-opioid receptor (MOR), in which Asparagine 40 (MOR N40) is replaced with Aspartate (MOR D40), and is associated with AUDs in specific ethnic groups. Importantly, Naltrexone, a nonselective MOR antagonist, has potent therapeutic effects in alcoholic individuals with MOR D40. We generated human neurons from 7 subjects carrying either homozygous MOR N40 or MOR D40. Our preliminary data suggest that human neurons carrying D40 show defective re-sensitization after MOR activation by DAMGO ([D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin), suggesting defective trafficking of MORs. Supporting this, bioinformatic analyses and mouse models of human MOR N40D suggest that D40 disrupts an N-glycosylation site on MOR. However, the mechanism by which MOR protein trafficking defects affect the interaction between ethanol and opioids is not known. It is particularly important to reveal the molecular mechanisms underlying the function of N40D MOR variants in their native neuronal context because previous studies performed in heterologous systems have revealed inconclusive and confusing results. Moreover, a species-specific trafficking mechanism of MORs has been suggested. The objective of this proposal is to study the impact of alcohol and opioid signaling in both mouse and human neurons carrying both the N40 and D40 MOR allelic variants, focusing on the synaptic mechanisms that likely underlie behavior. The central hypothesis is that defective D40 MOR trafficking results in an altered effect of the interaction between alcohol and opioids on synaptic function in the reward neurocircuitry. We will first examine the effect of alcohol on synaptic function in a defined neurocircuitry composed of human neurons carrying these gene variants. Next, we will use a mouse model of human N40D, and study the synaptic mechanism in the reward neurocircuitry, i.e. the midbrain ventral tegmental area (VTA), in relation to MOR function and ethanol. The proposed research is innovative, because we will combine recent developments in stem cell biology, the state-of-the-art synaptic physiology, and novel microfabrication technologies to directly probe the impact of alcohol and opioid signaling on synaptic function in both mouse and human neuronal networks carrying OPRM1 gene variants. We expect to unravel a species- and cell type-specific mechanism of MOR N40D variants that may provide novel information for understanding AUDs.
It is not understood why specific gene variants contribute to alcohol use disorders (AUDs) or how they may affect central nervous system function. We have prepared stem cells from people with addiction-associated gene variations, converted them to neurons, and we are now examining how these human neurons respond to alcohol. We propose to use these human neurons, as well as mouse neurons carrying the same gene variants, to ask specific questions about these cellular processes and interactions with the long-term goal of finding methods to help people to control drinking behavior, since alcohol abuse causes severe health and socioeconomic consequences in the U.S.
|Fantuzzo, Joseph A; De Filippis, Lidia; McGowan, Heather et al. (2017) ?Neurocircuitry: Establishing in vitro models of neurocircuits with human neurons. Technology (Singap World Sci) 5:87-97|
|Liu, Ji; Conde, Kristie; Zhang, Peng et al. (2017) Enhanced AMPA Receptor Trafficking Mediates the Anorexigenic Effect of Endogenous Glucagon-like Peptide-1 in the Paraventricular Hypothalamus. Neuron 96:897-909.e5|
|Lim, Chae-Seok; Kang, Xi; Mirabella, Vincent et al. (2017) BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method. Genes Dev 31:537-552|
|Fantuzzo, J A; Mirabella, V R; Hamod, A H et al. (2017) Intellicount: High-Throughput Quantification of Fluorescent Synaptic Protein Puncta by Machine Learning. eNeuro 4:|
|Liu, Jing-Jing; Bello, Nicholas T; Pang, Zhiping P (2017) Presynaptic Regulation of Leptin in a Defined Lateral Hypothalamus-Ventral Tegmental Area Neurocircuitry Depends on Energy State. J Neurosci 37:11854-11866|
|De Filippis, Lidia; Halikere, Apoorva; McGowan, Heather et al. (2016) Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells. Mol Brain 9:51|
|Oni, Eileen N; Halikere, Apoorva; Li, Guohui et al. (2016) Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele. Sci Rep 6:34341|
|Carlson, Aaron L; Bennett, Neal K; Francis, Nicola L et al. (2016) Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds. Nat Commun 7:10862|