PROTEASOME AND PARKIN AS DRUG TARGETS AGAINST METHAMPHETAMINE TOXICITY

描述（由申请人提供）：甲基苯丙胺（冰毒）是一种高度成瘾性的精神兴奋剂药物，长期或急性高剂量服用时具有神经毒性。甲基苯丙胺选择性损伤实验动物和人类纹状体多巴胺能终端。尽管多年来对甲基安非他明神经毒性的研究一直很活跃，但目前还没有开发出专门的药物来抵消甲基安非他明对大脑的破坏性影响。由于其广泛滥用，迫切需要有效的药物来保护和/或恢复大脑免受急性过量和慢性滥用冰毒的毒性作用。因此，确定分子药物靶点是开发新型药物的必要条件。我的长期目标是开发神经保护疗法来治疗使用冰毒的毒性作用。本研究的目的是为了更好地了解甲基安非他明暴露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连接酶帕金是否是潜在的药物靶点，可用于促进体内过量和慢性剂量甲基安非他明后多巴胺能末端的生存和恢复。暴饮暴食服用冰毒后，蛋白酶体和帕金森氏蛋白都在短时间内下降，这些缺陷也与帕金森氏病的病因有关。我们假设增加它们的功能将为长期和急性暴露于冰毒的大鼠提供神经保护。特异性目标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）来评估20S和26S蛋白酶体在甲基安非他明[多巴胺能末端存活及其从暴饮和慢性]中恢复]中的相对作用。TAT是人类免疫缺陷病毒1型的一个结构域，它能迅速穿过血脑屏障。这两种形式的蛋白酶体在暴露于甲基安非他明诱导的氧化应激时表现不同；因此，评估它们各自在甲基苯丙胺神经毒性中的作用是有必要的。特异性目标2将使用野生型、过度表达和敲除parkin的大鼠来评估parkin在冰毒（暴食和慢性冰毒的生存和恢复）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通过免疫组织化学和共聚焦显微镜进行研究。特异性Aim 3将确定甲基甲醚暴露的大鼠脑中蛋白酶体和帕金是否在功能上联系。对于临床干预而言，了解parkin水平的变化如何影响20S和26S功能是很重要的，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-蛋白酶体系统中的调节过程，这可能对多巴胺神经元的内源性生存和恢复机制很重要，因此具有临床重要性。拟议研究的结果可能会为冰毒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.