PROTEASOME AND PARKIN AS DRUG TARGETS AGAINST METHAMPHETAMINE TOXICITY

描述（由申请人提供）：甲基苯丙胺（METH）是一种高度成瘾的精神兴奋剂药物，当长期或急性服用高剂量时具有神经毒性。METH选择性损伤实验动物和人类的纹状体多巴胺能末梢。尽管对METH神经毒性进行了多年的积极研究，但尚未开发出特定的药物来抵消METH对大脑的破坏作用。由于其广泛的滥用，迫切需要有效的药物，可以保护和/或恢复大脑免受急性METH过量和慢性METH滥用的毒性作用。因此，有必要确定分子药物靶标，以开发新的药物。我的长期目标是开发神经保护疗法来治疗METH使用的毒性作用。这项研究的目的是更好地了解在暴露于甲基苯丙胺的大鼠大脑中调节泛素-蛋白酶体系统的分子机制，并确定该系统的两个组成部分，蛋白酶体和E3连接酶帕金蛋白，是否是潜在的药物靶点，可用于促进毒性剂量的[狂欢和慢性]甲基苯丙胺后体内多巴胺能末梢的[存活和恢复]。蛋白酶体和parkin都在大量服用METH后不久下降，这些缺陷也参与了帕金森病的病因学。我们假设，增加它们的功能将提供长期和急性暴露于METH的大鼠神经保护。具体目标1将通过使用蛋白酶体抑制剂和一种调节蛋白酶体活性的新方法（即，全身注射干扰蛋白酶体组装的TAT标记肽），评价20 S和26 S蛋白酶体对[多巴胺能末梢的存活及其从狂欢和慢性中的恢复] METH的相对作用。达特是人类免疫缺陷病毒1型的一个结构域，可快速穿过血脑屏障。这两种形式的蛋白酶体在暴露于METH诱导的氧化应激时表现不同;因此，有必要评价它们各自在METH神经毒性中的作用。具体目标2将使用野生型、parkin过表达和parkin敲除大鼠评价parkin在METH [存活和从狂欢和慢性恢复]中的作用。帕金在黑质纹状体多巴胺神经元细胞内包涵体形成中的作用将使用免疫组织化学和共聚焦显微镜进行研究。具体目标3将确定蛋白酶体和parkin是否在暴露于MET的大鼠脑中功能上相关。为了临床干预的目的，重要的是要知道帕金水平的变化如何影响20 S和26 S功能，反之亦然。这些目标在概念上是联系在一起的，因为它们研究了泛素-蛋白酶体系统内的调节过程，这可能对多巴胺神经元的内源性存活和恢复机制很重要，因此具有临床重要性。这项研究的结果可能会为METH使用者带来新的治疗方法。

DESCRIPTION (provided by applicant): Methamphetamine (METH) is a highly addictive psychostimulant drug that is neurotoxic when taken at high doses chronically or acutely. METH selectively damages striatal dopaminergic terminals in experimental animals and humans. Despite years of active research on METH neurotoxicity, no specific medications have been developed to counteract the damaging effects that METH has on the brain. Due to its widespread abuse, there is a compelling need for effective pharmaceuticals that can protect and/or restore the brain from the toxic effects of acute METH overdose and chronic METH abuse. Thus, it is necessary to identify molecular drug targets in order to develop novel pharmaceuticals. My long-term goal is to develop neuroprotective therapies to treat the toxic effects of METH use. The goal of the proposed research is to better understand the molecular mechanisms regulating the ubiquitin-proteasome system in the METH-exposed rat brain and to determine whether two components of this system, proteasome and the E3 ligase parkin, are potential pharmaceutical targets that can be used to promote [survival and recovery] of dopaminergic terminals in vivo after toxic doses of [binge and chronic] METH. Both proteasome and parkin are decreased shortly after binge METH administration, and those deficits are also involved in the etiology of Parkinson's disease. We hypothesize that increasing their functions will provide neuroprotection in rats chronically and acutely exposed to METH. Specific Aim 1 will evaluate the relative roles of 20S and 26S proteasomes on the [survival of dopaminergic terminals and their recovery from binge and chronic] METH by using proteasomal inhibitors and a novel approach to regulate proteasomal activity, namely, systemic injections of TAT-tagged peptides that interfere with proteasomal assembly. TAT is a domain of the human immunodeficiency virus type 1 that rapidly crosses the blood brain barrier. These two forms of the proteasome behave differently upon exposure to METH- induced oxidative stress; thus, evaluation of their respective roles in METH neurotoxicity is warranted. Specific Aim 2 will evaluate the role of parkin in the [survival of and recovery from binge and chronic] METH using wild- type, parkin-overexpressing, and parkin knock-out rats. The role of parkin in the formation of intracellular inclusions in the nigrostriatal dopamine neurons will be investigated using immunohistochemistry and confocal microscopy. Specific Aim 3 will determine whether proteasomes and parkin are functionally linked in the METH-exposed rat brain. For clinical intervention purposes, it is important to know how variations in parkin levels influence 20S and 26S function and vice versa. These aims are conceptually linked as they investigate regulatory processes within the ubiquitin-proteasome system that may be important for [endogenous survival and recovery mechanisms] in dopamine neurons and, therefore, clinically important. The findings from the proposed research may lead to novel treatments for METH users.