Proteomic Profiling of Glutamate Neurotransmission Alterations

Addiction is a chronic relapsing disease with devastating consequences to the health and well-being of patients and their families. Addiction is frequently not restricted to one drug: cannabis and alcohol are amongst the two most concomitantly used and abused drugs. Addiction vulnerability has been associated with altered prefrontal cortex (PFC) activity, leading to a more impulsive executive function. Repeated drug exposure increases glutamate release by PFC glutamatergic neurons projecting to NAc, increasing the sensitivity of the reward circuit to prior drug use-associated environmental cues. Previous in vivo studies identified important signaling networks involved in drug-seeking behaviors, but focused on VTA and NAc rather than PFC, and did not clarify whether the observed changes map directly onto changes in the protein machinery of the neurons. Moreover, addiction has a strong inheritable component, but animal models cannot reproduce the genetic environment of individuals with addiction or that are vulnerable to develop substance abuse. Using human iPSC-derived glutamatergic neurons as a model system of human cortical excitatory neurons is a suitable strategy to overcome the limitations of animal models.

Combining GWAS summary statistics and addiction related traits, our collaborators from the Collaborative Studies on Genetics of Alcoholism (COGA) have calculated polygenic risk scores (PRS) for a cohort of healthy individuals, as well as for cannabis and alcohol addicted individuals, generating an addiction-PRS distribution. hiPSCs from these individuals will be differentiated into excitatory neurons using the NGN2-induction protocol and chronically exposed to THC, ethanol, a combination of both drugs, or vehicle. The goal of the present study is to identify protein changes in the synaptic machinery of the neurons due to chronic THC and/or ethanol exposure using cutting-edge iTRAQ proteomics, and compare this changes between individuals with high and low addiction-PRS. The findings of this study will be further integrated with transcriptomic and functional datasets that have been generated in this cohort of addiction-PRS individuals, to converge on drug-induced disruptions in glutamatergic signaling in human hiPSC-derived excitatory neurons. Elucidating the whole synaptosomal proteome alterations due to chronic THC and/or ethanol exposure in these hiPSC-derived glutamatergic neurons and evaluating the potential differences in these drug-associated changes between individuals with high and low PRS for addiction will markedly advance our understanding of the molecular underpinnings behind genetic predisposition to addiction.