BRAIN DAT/5-HTT DYSREGULATION IN HUMAN COCAINE USERS

描述：(申请人摘要) 多巴胺转运体(DAT)上的结合位点显著增加 人类可卡因吸毒者与匹配对照组的纹状体，这可能是由于 一种复杂的翻译后机制。可卡因结合的变化 站点以及基础DAT功能的相应更改可能 对可卡因引起的临床现象有显著影响，如 暴饮暴食、戒断和渴求症状，并表明DAT可能 代表着多巴胺能细胞的一个重要的调节焦点。一个详细的 对DAT调控中涉及的分子变化的了解可能会使新的 对其功能的药物治疗操作。具体的目标#1是 测试可卡因改变人脑DAT的假设表明 对许多关键参数的敏感度的变化，包括缓冲器， 温度、pH、离子，以及对不同配体的影响。具体的目标#2是 发现通过测定DAT检测到的蛋白质-蛋白质相互作用 表面大小，或DAT mRNA剪接变异体或表达增加 核糖核酸酶保护检测到的单胺转运体-类似的mRNA物种 分析或聚合酶链式反应克隆有助于在 人类可卡因吸毒者。因为去甲肾上腺素转运体(净值仅为 与DAT略有不同，了解其监管可能会有所启发 关于其他单胺转运体(MAT)的变质潜力。 具体的目标3是测试人类大脑网络是 由于暴露于可卡因而被封锁，因此表达上调。监管 自体受体和转运体似乎是相互协调的过程。 涉及跨膜的直接相互作用。因为自身受体 监管可能与运输商监管相辅相成，具体目标4是测试 5-羟色胺和多巴胺的结合和信使核糖核酸水平 可卡因使用者与对照组相比，自身受体发生了变化。特定目标#5 是为了验证这样一个假设，即多巴胺细胞在 因为可卡因的吸收被阻断，所以暴露在可卡因中。成功 针对多巴胺能功能的治疗方法可能需要采取 考虑到多巴胺神经元可获得的适应可能性 他们心烦意乱。人类尸检方法避免了复杂化 物种差异，并允许进行难以建模的相关分析 人类症状学和可能性，因为人类的大小 大脑，用于相互作用之间的大量相互关联分析 神经系统。当在多个神经元中发现改变时 系统(特别是5-羟色胺能神经元 人类神经解剖学与啮齿动物相比)，人类独一无二的可能性 回复进一步增加。出于这些原因，继续审查 人类标本对可卡因暴露的脑单胺能适应 随着经过验证的细胞模型系统的并行开发， 应该被证明是有价值的和信息丰富的。

DESCRIPTION: (Applicant's Abstract) Binding sites on the Dopamine Transporter (DAT) are markedly increased in striatum of human cocaine users versus matched controls, which is likely due to a complex post-translational mechanism. Alterations in cocaine binding sites, and accompanying changes in underlying DAT function, may significantly contribute to cocaine-induced clinical phenomenon such as binging, withdrawal, and craving symptoms and suggest that the DAT may represent an important regulatory focus for dopaminergic cells. A detailed knowledge of the molecular changes involved in DAT regulation may allow new pharmacotherapeutic manipulations of its function. SPECIFIC AIM #1 is to test the hypothesis that cocaine-altered DAT from human brain demonstrates changes in sensitivity to a number of critical parameters, including buffer, temperature, pH, ions, and to different ligands. SPECIFIC AIM #2 is to discover if either protein-protein interactions, detected by determining DAT apparent size, or increased expression of DAT mRNA splice-variants or monoamine transporter-analog mRNA species, detected by RNAase Protection assay or PCR cloning, contribute to the complex binding results found in human cocaine users. Because the norepinephrine transporter (NET is only subtly different from the DAT, understanding its regulation may shed light on the metamorphic potential of other monoamine transporters(MATs). SPECIFIC AIM #3 is to test the hypothesis that human brain NET is upregulated in response to blockade by cocaine exposure. The regulation of autoreceptors and transporters appear to be co-ordinated processes, perhaps involving direct interactions trans-membranally. Because autoreceptor regulation may compliment transporter regulation, SPECIFIC AIM #4 is to test the hypothesis that binding and mRNA levels for serotonin and dopamine autoreceptors are altered in cocaine users versus controls. SPECIFIC AIM #5 is to test the hypothesis that dopamine cells alter their metabolism during cocaine exposure because of cocaine's blockade of uptake. Successful therapeutic approaches targeted at dopaminergic function may need to take into account the adaptive possibilities available to dopamine neurons as they are perturbed. The human post mortem approach avoids complicating species differences, and allows correlative analyses with difficult to model human symptomatology and the possibility, because of the size of the human brain, for considerable inter-correlational analyses between interacting neuronal systems. As alterations are discovered in multiple neuronal systems (in particular serotonergic neurons which demonstrate a distinct human neuroanatomy compared to rodents), the likelihood of unique human responses further increases. For these reasons, continued examination of brain monoaminergic adaptations to cocaine exposure in human specimens now available, along with parallel development of validated cell-model systems, should prove valuable and informative.