Derivation and dopaminergic differentiation of human drug addict-specific iPS cel

描述（由申请人提供）：成瘾可以定义为失去对物质使用的控制，尽管有不良后果。对合法和非法物质的成瘾破坏了成瘾者及其家庭的生活，给社会带来了巨大的成本和负担。导致成瘾发生和发展的分子和细胞机制仍有待阐明。据估计，40-60%的成瘾易感性可能归因于基因畸变。多个染色体区域与成瘾有关，包括那些含有多巴胺转运蛋白（DAT）和水疱单胺转运蛋白（VMAT2）基因的区域。目前，由于无法直接询问患者的神经细胞类型，了解这些单胺转运体的多态性如何促进成瘾的分子机制的努力受到严重阻碍。不存在携带特定遗传变异的患者特异性细胞来源，这些细胞能够稳健且可重复地分化为特定的神经谱系。我们建议开发一种基于细胞的系统，通过这种系统，可以对来自患病个体的神经细胞进行功能分析，以询问成瘾的分子机制。为了实现这一目标，我们开发了一个尖端的方案，它结合了多学科的技能和专业知识。在Aim 1中，我们将从携带单胺转运体多态性的成瘾患者和对照中获得并表征患者特异性的诱导多能干细胞（iPS）。由于中脑多巴胺能系统在自然和药物相关的奖励通路中发挥着重要作用，并且是药物滥用的共同底物，在Aim 2中，我们将区分患者特异性iPS细胞系为多巴胺能神经元，并对这些细胞进行详细的功能表征，以确定其分子特征（即A9， A10，中脑边缘或中脑皮层多巴胺能神经元）。在Aim 3中，我们将对这些患者特异性的iPS细胞衍生的多巴胺能神经元进行表征、比较和功能分析，这些神经元来自对照和成瘾患者，携带hDAT1和hVMAT2基因多态性。患者特异性iPS细胞技术有很大的潜力，通过提供基因上独特的、功能性的人类细胞来源，深刻影响我们对人类发育和疾病的理解。通过完成本提案中提出的目标，我们期望提供成瘾患者多巴胺能神经传递功能的详细特征，并深入了解这种复杂疾病的病理生理学以及单胺转运体基因变异对成瘾的贡献。我们已经建立了一个跨学科的团队，结合了吸毒成瘾者的民族志研究，神经分化和多巴胺能功能分析，以及多能性和iPS细胞的优势，来询问有关导致成瘾的细胞和分子功能障碍的新问题。我们期望我们的研究结果将与理解和治疗这种人类疾病直接相关。

DESCRIPTION (provided by applicant): Addiction can be defined as a loss of control of substance use despite adverse consequences. Addiction to legal and illegal substances destroys the lives of both addicted subjects and their families, exerting an enormous cost and burden on society. The molecular- and cellular-based mechanisms that contribute to the initiation and development of addiction remain to be elucidated. Estimates have suggested that 40-60% of the vulnerability to addiction may be attributable to genetic aberrations. Multiple chromosomal regions have been linked to addiction including those containing the dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) genes. Current efforts to understand how polymorphisms in these monoamine transporters contribute to the molecular mechanisms of addiction are severely hindered by the inability to directly interrogate neural cell types from the patients. Patient-specific sources of cells carrying specific genetic variants that are capable of robust and reproducible differentiation into specific neural lineages do not exist. We propose to develop a cell-based system whereby neural cells from afflicted individuals can be functionally assayed to interrogate the molecular mechanisms underlying addiction. To achieve this goal we have developed a cutting-edge proposal that that incorporates the skill and expertise of multiple disciplines. In Aim 1 we will derive and characterize patient-specific, induced pluripotent stem (iPS) cells from addiction patients and controls that carry monoamine transporter polymorphisms. Since midbrain dopaminergic system play a prominent role in natural and drug related reward pathways and represent a common substrate for drugs of abuse, in Aim 2 we will differentiate patient-specific iPS cells line into dopaminergic neurons and carry out a detailed and functional characterization of these cells to identify their molecular characteristics (i.e. A9, A10, mesolimbic or mesocortical dopaminergic neurons). In Aim 3, we will characterize, compare, and functionally assay these patient-specific, iPS cell-derived dopaminergic neurons from control and addiction patients that carry polymorphisms for hDAT1 and hVMAT2 gene. There is great potential for patient-specific iPS cell technology to profoundly impact our understanding of human development and disease by providing genetically distinct, functional sources of human cells. By completing the aims set forth in this proposal we expect to provide a detailed characterization of dopaminergic neurotransmission function in patients afflicted with addiction and provide insight into the pathophysiology of this complex disease as well as the contribution of genetic variants in monoamine transporter genes to addiction. We have established an interdisciplinary team that combines strengths in ethnographic study of drug addicts, neural differentiation and dopaminergic function analysis, as well as pluripotency and iPS cells to interrogate novel questions about the cellular and molecular dysfunction that contributes to addiction. We expect that results from our studies will have immediate relevance to the understanding and treatment of this human disease. PUBLIC HEALTH RELEVANCE: Addiction can be defined as a loss of control of substance use despite adverse consequences. The World Health Organization estimates that there are 2 billion alcohol users, 1.3 billion tobacco users and 185 million illicit drug users worldwide. People who are addicted cannot control their need for the substance of abuse, even in the face of negative health, social or legal consequences. As a result, addiction to legal and illegal substances destroys the lives of both patients and their families, exerting tremendous cost and burden on the society. Unfortunately, the cause of addiction is currently unknown. The molecular- and cellular-based mechanisms that contribute to the initiation and development of addition remain to be elucidated. Results of our studies will provide a detailed characterization of brain cell function in patients afflicted with addiction and will offer insight into the mechanisms that contribute to this complex, devastating disease.