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

描述(申请人提供)：在美国，可卡因滥用仍然是一个主要的公共卫生问题。可卡因成瘾是一种慢性复发性神经疾病，与严重的医疗和心理社会并发症有关。人们对可卡因成瘾的机制知之甚少，目前也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是至关重要的。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)图像和终末期可卡因成瘾者的死后脑组织获得的关于人类神经元病理的信息贡献有限。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一件困难的事情。患者来源的多能干细胞(IPSCs)为研究可卡因成瘾的机制提供了一个很好的平台。本研究的目的是以IPSC衍生的人纹状体(包括伏隔核)中棘神经元(MSN)为模型，研究可卡因成瘾机制。纹状体MSN在可卡因成瘾中起关键作用，它们接受来自前额叶皮质的谷氨酸能输入。在体外纹状体培养中，纹状体MSN的突触发生需要皮质神经元的谷氨酸能输入。目的1是从可卡因依赖患者(CD)和未受影响的对照组(UC)中产生IPSC来源的纹状体MSN和额叶皮质(FC)谷氨酸能神经元。IPSC来源的皮质神经元将被设计为稳定表达GFP，以便它们可以在与纹状体MSN共同培养时被鉴定，而IPSC来源的MSN将通过抗体DARPP32、GABA和多巴胺受体(D1R和D2R)的免疫染色进行鉴定。目的2比较CD组和UC组IPSC来源的纹状体MSN的形态、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在形态、神经化学和电生理方面的关键差异。因此，它可能为阐明可卡因成瘾的分子和细胞机制奠定基础。 与公共卫生有关：可卡因滥用仍然是一个主要的公共卫生问题，其根本机制还不清楚。本研究利用多能干细胞探索可卡因成瘾的机制，研究结果可能为可卡因成瘾的研究和揭示可卡因成瘾的机制提供重要工具。

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.