Modeling human alcohol abuse in mice with a common SLC6A3 regulatory variant Project Abstract The purpose of this R21 research is to model for stress-regulated alcohol consumption through generating a clinically-related SLC6A3 regulatory mutation in mice. Such work will not only facilitate investigations of causality recapitulation and signaling pathway delineation for gene-environment interaction, but also precipitate disease progression prediction and genome editing-based cure development. Collaboratively among eight groups in four countries, we recently uncovered a stress-sensitive common variant in the human dopamine transporter (DAT) gene (SLC6A3) which is implicated in a clinical symptom of alcohol use disorder (AUD). Understanding such a genetic disorder may indeed provide novel insights into the pathogenic mechanisms underlying AUD. How does a common variant regulate alcohol consumption in a stress-dependent manner? It is well known that AUD is a phenotypically complex disease but the central theme is excessive consumption of alcohol. AUD has not only a polygenic etiology but also environmental attribution so that to clarify the genetics-stress interaction in regulation of alcohol consumption remains a key in delineating the disease mechanism. In our pilot studies, we find that a common variant, DNPiB, in SLC6A3 is associated with reduced levels of alcohol consumption among different populations and among patients with AUD. At a molecular level, DNPiB is recognized by a stress-responsive transcription factor. We hypothesize that individuals carrying DNPiB will consume more alcohol in the presence of stress than in the absence of stress. This makes it an attractive case for developing DNPiB-based precision medicine for AUD. The present project aims to generate and study knock-in mice carrying DNPiB vs DNPiA as a control, allowing us to answer questions regarding mechanisms as well as clinical utilities linking DAT?s environmental genomics with AUD that could not otherwise be answered. Expression pattern and level of DAT in the brain will be examined by immunohistochemistry with techniques ongoing in our lab. We will characterize the DNPiB vs DNPiA mice in general terms such as body weight and maternal behavior. Neurochemical assays of DAT function will measure DA uptake. Importantly, we will examine stress-related ethanol intake and preference. DAT plays a role in a spectrum of stress- and DA-related diseases such as PTSD, schizophrenia, autism spectrum disorder, attention deficit hyperactivity disorder, PD, depression and substance use disorders. By representing all humans, the DNPiB vs DNPiA model will flexibly allow a better understanding of how environment- regulated SLC6A3 activity is involved in these complex diseases, developmentally and environmentally, and more importantly, provide a platform for in vivo genomic repair.
The study in animal models of inherited alcohol use disorder can provide mechanistic insights into the pathogenesis. In the present project we will generate and study a mouse model with the same regulatory variants in the dopamine transporter that in humans are associated with varying alcohol consumption. This work has the potential to uncover critical functions of the dopamine transporter important for many common neuropsychiatric diseases (PTSD, schizophrenia, or addiction) and to develop a genomic cure for the genetic disease.
