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

摘要 根据全国药物使用调查，药物使用障碍影响约2000万美国成年人， 健康大约850万患者还患有共病的心理健康障碍，如抑郁症，焦虑症， 和精神分裂症此外，非法使用药物、滥用法律的药物和其他药物的流行， 与新冠肺炎大流行相关的心理健康疾病都在迅速增加。因此，机械 了解心理健康和物质使用背后的病理神经生物学过程 疾病，以及它们的共病对于开发更有效的治疗以减少身体， 情感和社会伤害广泛认为，多巴胺能信号传导在这些神经元中高度受损。 大脑紊乱虽然多巴胺能系统在过去的几十年里得到了深入的研究，但它 巨大的分子和细胞的复杂性是一个重大的挑战，详细了解 为什么影响多巴胺能系统的某些治疗在治疗上是有益的或引起不良反应。 例如，药理学工具，如部分激动剂、正和负变构调节剂，和 D3多巴胺受体的拮抗剂已经由国家药物滥用研究所提出， 用于治疗物质使用障碍的有前途的治疗工具。然而，我们对 由D3多巴胺受体控制的适应性和适应不良可塑性机制，以及这种G蛋白- 偶联受体与物质使用障碍和其他精神疾病有关， 有限公司这一点很重要，因为新兴的治疗趋势，例如FDA的广泛使用- 获批的D3受体首选药物卡利拉嗪（Vraylar ™）治疗抑郁症和精神分裂症 其在治疗共病物质使用障碍中的推定有用性阐明了临床 D3受体的重要性然而，神经元中D3受体的拷贝数通常很低， 目前缺乏敏感和特异性抗体来可视化它们的分布和重组 与病理过程的研究，旨在更好地了解D3多巴胺受体的功能， 非常困难。因此，拟议的工作将提供新的荧光工具，使细胞类型和 完整脑中纳米水平D3受体分布的亚细胞区室特异性定量 物质使用障碍中受影响的电路。在初步研究中，我们开发了PharmacoSTORM超级， 分辨率纳米级药理学方法和应用荧光卡利拉嗪发现高卡利拉嗪 卡列哈群岛的结合密度。在拟议的R21 CEBRA中，我们将完成两个具体目标： 目标1.开发基于光亲和性的荧光化学探针用于高产PharmacoSTORM super- D3多巴胺受体的分辨率成像。 目标2.建立低拷贝数D3多巴胺的PharmacoSTORM成像的光亲和标记 与成瘾有关的特定神经元类型上的受体。

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.