Specifying Human iPSC-derived Medium spiny Neurons for Cocaine Abuse Research

描述（由申请人提供）：可卡因滥用仍然是美国的一个主要公共卫生问题。可卡因成瘾是一种慢性复发性神经系统疾病，与严重的医疗和心理社会并发症有关。可卡因成瘾的机制知之甚少，目前没有有效的治疗方法。更好地了解成瘾的发展对于创造有效的可卡因成瘾疗法至关重要。我们对可卡因成瘾的了解主要来自动物模型的研究，通过分析PET（正电子发射断层扫描）图像和晚期可卡因成瘾者的死后脑组织获得的人类神经元病理学信息的贡献有限。从可卡因成瘾者中获得分化的神经元用于分子分析一直是困难的。患者来源的诱导多能干细胞（iPSC）为探索可卡因成瘾机制提供了一个很好的平台。本研究的目的是利用诱导多能干细胞（iPSC）来源的人纹状体包括中脑核的中型棘神经元（MSNs）作为模型来研究可卡因成瘾的机制。纹状体MSNs在可卡因成瘾中起着关键作用，它们接受来自前额叶皮层的多巴胺能输入。体外培养的纹状体MSNs上的突触发生需要来自皮层神经元的谷氨酸能输入。目的1是从可卡因依赖性患者（CD）和未受影响的对照（UC）两者产生iPSC衍生的纹状体MSN和额叶皮质（FC）多巴胺能神经元。iPSC衍生的皮质神经元将被工程化以稳定表达GFP，使得它们可以在与纹状体MSN的共培养中被鉴定，而iPSC衍生的MSN将通过用抗体特异性DARPP 32、GABA和多巴胺受体（D1R和D2R）进行免疫染色来鉴定。目的2是比较CD和UC组之间iPSC衍生的纹状体MSNs的形态学、神经化学和电生理特性。这项研究将为可卡因成瘾研究提供重要工具，并可能揭示CD和UC之间的关键形态学，神经化学和电生理差异。因此，它可以作为阐明可卡因成瘾的分子和细胞机制的基础。 公共卫生相关性：可卡因滥用仍然是一个主要的公共卫生问题，其潜在机制尚不清楚。本研究利用多能干细胞探索可卡因成瘾的机制，其结果可能为可卡因成瘾的研究提供重要工具，并揭示可卡因成瘾的机制。

DESCRIPTION (provided by applicant): Cocaine abuse remains a major public health problem in the US. Cocaine addiction is a chronic relapsing neurological disorder associated with severe medical and psychosocial complications. The mechanisms of cocaine addiction are poorly understood, and no effective treatment is currently available. A better understanding of the development of addiction is essential for creating effective therapy for cocaine addiction. Our knowledge about cocaine addiction has been generated mostly from studies with animal models, with limited contributions from information about human neuronal pathology obtained by analyzing PET (Positron emission tomography) images and postmortem brain tissues of end-stage cocaine addicts. It has been difficult to obtain differentiated neurons from cocaine addicts for molecular analysis. Patient-derived induced pluripotent stem cells (iPSCs) provide an excellent platform for exploring the mechanisms of cocaine addiction. The purpose of this proposal is to use iPSC-derived the medium spiny neurons (MSNs) in human striatum including nucleus accumbens as a model to investigate the mechanisms of cocaine addiction. Striatal MSNs play key roles in cocaine addiction, and they receive glutamatergic input from prefrontal cortex. Glutamatergic inputs from cortical neurons are required for the synaptogenesis on the striatal MSNs in striatal culture in vitro. Aim 1 is to generate iPSC-derived striatal MSNs and frontal cortical (FC) glutamatergic neurons from both cocaine-dependent patients (CD) and unaffected controls (UC). The iPSC-derived cortical neurons will be engineered to stably express GFP so that they can be identified in co-culture with striatal MSNs, while the iPSC-derived MSNs will be identified by immunostaining with antibodies specific DARPP32, GABA and dopamine receptors (D1R and D2R). Aim 2 is to compare morphological, neurochemical and electrophysiological properties of the iPSC-derived striatal MSNs between the CD and UC groups. This study will generate important tools for cocaine addiction research, and may uncover key morphological, neurochemical and electrophysiological differences between CD and UC. Therefore, it may serve as the foundation for elucidating the molecular and cellular mechanisms of cocaine addiction. PUBLIC HEALTH RELEVANCE: Cocaine abuse remains a major public health problem and the underlying mechanisms are poorly understood. This study uses pluripotent stem cells to explore the mechanisms of cocaine addiction and the results of this study may generate important tools for research into cocaine addiction and uncover the mechanism of cocaine addiction.