BRAIN DAT/5-HTT DYSREGULATION IN HUMAN COCAINE USERS

描述：（申请人摘要） 多巴胺转运蛋白（DAT）上的结合位点显著增加， 人类可卡因使用者与匹配对照组的纹状体，这可能是由于 复杂的翻译后机制。 可卡因结合的改变 位点，以及伴随的潜在DAT功能的变化，可能 显著有助于可卡因引起临床现象 暴食，戒断和渴望症状，并建议DAT可能 代表多巴胺能细胞的重要调节焦点。 详细 DAT调节中涉及的分子变化的知识可能允许新的 药理学操纵其功能。 具体目标#1是 测试可卡因改变的人脑DAT证明了这一假设 对许多关键参数的灵敏度变化，包括缓冲液， 温度、pH、离子和不同的配体。 具体目标#2是 发现蛋白质-蛋白质相互作用，通过测定DAT 表观大小，或DAT mRNA剪接变体表达增加，或 单胺转运蛋白类似物mRNA种类，通过RNA酶检测保护 分析或PCR克隆，有助于发现在 人类可卡因吸食者 因为去甲肾上腺素转运蛋白（NET）仅 与DAT有微妙的不同，了解其监管可能有助于了解 其他单胺转运蛋白（MAT）的变形潜力。 具体目标#3是测试假设，即人脑NET是 在可卡因暴露的阻断反应中上调。 调控 自身受体和转运蛋白似乎是协调的过程，也许 涉及跨膜的直接相互作用。 因为自体感受器 调节可以补充转运蛋白调节，具体目标#4是测试 假设血清素和多巴胺的结合和mRNA水平 可卡因使用者与对照组相比，自身受体发生了改变。 具体目标#5 是为了验证多巴胺细胞在体内代谢过程中 因为可卡因的吸收受阻。 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的适应可能性， 他们感到不安。 人类死后的方法避免了复杂化 物种差异，并允许相关分析，难以建模 由于人类的体型， 大脑，对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（特别是表现出不同的神经元系统）， 人类神经解剖学与啮齿动物相比），人类独特的可能性 响应进一步增加。 由于这些原因，继续审查 现在人类样本中对可卡因暴露的脑单胺能适应 可用，沿着经验证的细胞模型系统的并行开发， 应该会很有价值和信息量

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.