Heroin use disorder (HUD) represents an enduring public health issue resulting in significant socioeconomic burdens to the United States, with domestic opiate-related deaths quadrupling from 1999 to 2017. Despite this the neurobiological mechanisms underlying HUD remain poorly understood. All drugs of abuse modulate dopaminergic signaling and have long been thought of as disorders of dopamine (DA) signaling. However, pharmacotherapeutic interventions targeting receptor mediated DA-signaling have not resulted in efficacious treatments. Our laboratory recently identified a novel signaling moiety for DA, termed dopaminylation, whereby DA itself acts as a post-translational modification (PTM) on substrate proteins via transamidation by the Transglutaminase 2 (TGM2) enzyme. I sought, then, to unbiasedly identify additional synaptic substrates of dopaminylation in vivo, utilizing a novel chemical tagging approach coupled to mass spectrometry. I identified 164 novel putative synaptic substrates of dopaminylation in Nucleus Accumbens (NAc), both in the context of normal neural function and in response to abstinence from chronic heroin self-administration. Following validation of a number of putative substrates, I turned my focus to gCaMKII as: 1) it is highly abundant 2) it is dopaminylated at a single amino acid residue, located within it’s autoinhibitory helix (glutamine [Q]285), a site that exists only two amino acids away from the critical threonine (T) residue 287. T287 is phosphorylated to direct Calmodulin (CaM) sequestration and subsequent nuclear translocation – thus, this site, represents an exciting ‘test’ case for establishing the importance of this signaling moiety in post synaptic plasticity 3) it is upregulated in its dopaminylation state following abstinence from heroin SA, an effect that persists to AD14 and 4)represents a critical substrate involved in mediating long range signals from the synapse to the nucleus in brain, ultimately promoting CREB activation. gCaMKII is a synaptic protein demonstrated to be necessary for learning/memory, late-long-term-potentiation (LTP) and excitation transcription coupling. CREB is an important transcription factor in brain implicated in all types of SUD, where dynamic alterations in activation have been observed to affect drug-related behaviors. I have additionally demonstrated that monoamine molecules can infiltrate striatal and cortical neurons in vitro, as well as that treatment of these neurons with DA induces gCaMKII dopaminylation. I also observed the dynamic regulation of gCaMKII/CaM localization and subsequent downstream CREB signaling in response to DA treatment in primary cortical-striatal neuronal culture. I therefore hypothesize that gCaMKIIQ285dop may represent a novel dopaminergic signaling moiety in brain, and may play a direct role in mediating heroin relapse behaviors via aberrant modulation of CREB signaling in NAc.

Substance Use Disorders arise from a complex interaction of synaptic and epigenetic plasticity, whereby modulation of dopaminergic signaling result in long lasting aberrant plasticities and harmful behavioral outcomes. This project examines a novel mechanism for synapse-to-nucleus dopaminergic signaling, one which would likely illuminate a new conceptual understanding of how dopamine functions in the brain, and have possibly broad implications outside the field of substance abuse research. The studies proposed here hold the potential to explain unanswered questions in dopamine-mediated pathophysiology of substance use disorders, as well as novel therapeutic targets therein.