PROTEASOME AND PARKIN AS DRUG TARGETS AGAINST METHAMPHETAMINE TOXICITY

描述(申请人提供)：甲基苯丙胺(冰毒)是一种高度上瘾的精神刺激药物，长期或急性服用高剂量时会产生神经毒性。甲氨蝶呤选择性损害实验动物和人类的纹状体多巴胺能终末。尽管多年来对冰毒的神经毒性进行了积极的研究，但还没有开发出特定的药物来抵消冰毒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，使其免受急性冰毒过量和慢性冰毒滥用的毒性影响。因此，为了开发新的药物，有必要确定分子药物靶点。我的长期目标是开发神经保护性疗法来治疗冰毒的毒性效应。这项拟议的研究的目的是更好地了解在冰毒暴露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连接酶蛋白是否为潜在的药物靶点，可用于促进体内多巴胺能终末在中毒剂量[暴饮暴食和慢性]冰毒后的[存活和恢复]。在过量服用冰毒后不久，蛋白酶体和parkin都会减少，这些缺陷也与帕金森病的病因学有关。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。具体目的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标记多肽，来评估20S和26S蛋白酶体在[多巴胺能终末的存活及其从狂欢和慢性]方法中的恢复中的相对作用。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冰毒诱导的氧化应激下表现不同；因此，有必要评估它们在冰毒神经毒性中的各自作用。具体目标2将使用野生型、parkin过表达和parkin基因敲除的大鼠来评估parkin在[酗酒和慢性]冰毒存活和恢复中的作用。将使用免疫组织化学和共聚焦显微镜研究parkin在黑质纹状体多巴胺神经元细胞内包涵体形成中的作用。具体目标3将确定蛋白酶体和parkin在冰毒暴露的大鼠大脑中是否在功能上相关。为了临床干预的目的，重要的是要知道Parkin水平的变化如何影响20S和26S的功能，反之亦然。这些目标在概念上是相互联系的，因为它们研究泛素-蛋白酶体系统中的调控过程，该过程可能对多巴胺神经元的[内源性生存和恢复机制]很重要，因此在临床上也很重要。这项拟议研究的发现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.