Novel tool development for quantitative PharmacoSTORM super-resolution imaging of the nanoscale distribution of D3 dopamine receptors

摘要

Abstract Substance use disorders affect ~20 million American adults according to the National Survey on Drug Use and Health. About 8.5 million patients also suffer from a comorbid mental health disorder such as depression, anxiety, and schizophrenia. Moreover, the prevalence of illicit substance use, the misuse of legal substances, and other mental health illnesses are all rapidly increasing in association with the Covid-19 pandemic. Thus, mechanistic understanding of the pathological neurobiological processes underlying mental health and substance use disorders, and their co-morbidity is imperative for the development of more efficient therapies to reduce physical, emotional, and social harm. It is widely acknowledged that dopaminergic signaling is highly impaired in these brain disorders. While the dopaminergic system has been intensely investigated in the last decades, it’s tremendous molecular and cellular complexity represents a major challenge for the detailed understanding of why certain treatments affecting the dopaminergic system are therapeutically beneficial or cause adverse effects. For example, pharmacological tools such as partial agonists, positive and negative allosteric modulators, and antagonists of the D3 dopamine receptor have been proposed by the National Institute of Drug Abuse as highly promising therapeutic tools for the treatment of substance use disorders. However, our understanding of the adaptive and maladaptive plasticity mechanisms controlled by D3 dopamine receptors and how this G protein- coupled receptor is involved in substance use disorders and other psychiatric diseases have remained very limited. This is an important, because emerging therapeutic trends, for example the expanding use of the FDA- approved D3 receptor-preferring drug cariprazine (VraylarTM) in the treatment of depression and schizophrenia and its putative usefulness in the treatment of comorbid substance use disorders illuminates the clinical significance of D3 receptors. However, the generally very low copy numbers of D3 receptors in neurons and the current lack of sensitive and specific antibodies to visualize their distribution and reorganization in association with pathological processes renders studies aiming to better understand the function of D3 dopamine receptors very difficult. Therefore, the proposed work will deliver novel fluorescent tools that will enable cell-type- and subcellular compartment-specific quantification of D3 receptor distribution at the nanoscale level in intact brain circuits affected in substance use disorders. In preliminary studies, we developed the PharmacoSTORM super- resolution nanoscale pharmacology approach and applied fluorescent cariprazine to discover high cariprazine binding density in the Islands of Calleja. In the proposed R21 CEBRA, we will complete two specific aims: Aim 1. Develop photoaffinity-based fluorescent chemical probes for high-yield PharmacoSTORM super- resolution imaging of D3 dopamine receptors. Aim 2. Establish photoaffinity labeling for PharmacoSTORM imaging of low copy numbers of D3 dopamine receptors on specific neuronal types implicated in addiction.