Mechanisms of DJ-1 Protection against methamphetamine neurotoxicity.

描述(由申请人提供)：甲基苯丙胺诱导黑质纹状体系统中的多巴胺能轴突变性、氧化损伤和a-突触核蛋白(ASyn)聚集。类似的神经病理特征在帕金森氏病(PD)患者的大脑中也很明显。家族性和散发性帕金森病与DJ-1功能障碍有关，DJ-1是一种神经保护蛋白，通过激活各种有利于生存的机制来抑制氧化应激和aSyn聚集。总之，这些观察结果表明，DJ-1可能对甲基苯丙胺的神经毒性具有保护作用。这项拟议研究的长期目标是确定消除甲基苯丙胺引起的多巴胺能轴突变性的细胞反应，以此来刺激新疗法的发现。本申请中描述的研究重点是DJ-1在减轻甲基苯丙胺神经毒性方面的作用。这个问题的具体目的如下：(I)确定DJ-1是否抑制甲基苯丙胺诱导的轴突丢失；(Ii)评估DJ-1是否抑制甲基苯丙胺处理的细胞中的氧化应激和1-突触核蛋白聚集；以及(Iii)确定DJ-1介导的对甲基苯丙胺神经毒性的保护是否需要在胶质和/或神经元中表达该蛋白。原代表达人野生型DJ-1的中脑培养物将暴露在甲基苯丙胺中，无突起的多巴胺能神经元的百分比将通过免疫细胞化学来确定。这些细胞还将根据一系列DJ-1保护性反应进行表征。亚细胞定位的影响将通过测量DJ-1工程变异体的神经保护活性来研究，这些变异体优先针对胞浆、线粒体或细胞核。通过监测在神经元、星形胶质细胞或小胶质细胞中选择性表达DJ-1的培养物中的保护通路，将检验特定细胞类型DJ-1表达的作用。这个项目将促进我们对减轻甲基苯丙胺神经毒性的细胞反应的理解。最终，这项研究的见解将刺激制定策略，通过激活DJ-1调节的神经保护通路来治疗暴露于甲基苯丙胺的个人。 公共卫生相关性：该项目旨在评估DJ-1蛋白抑制甲基苯丙胺引发的神经元突起丧失的神经保护机制。来自这些研究的见解将刺激治疗策略的发展，以通过上调DJ-1保护反应来减轻甲基苯丙胺的神经毒性。

DESCRIPTION (provided by applicant): Methamphetamine induces dopaminergic neurite degeneration, oxidative damage, and a-synuclein (aSyn) aggregation in the nigrostriatal system. Similar neuropathological features are evident in the brains of individuals with Parkinson's disease (PD). Familial and sporadic PD involve dysfunction of DJ-1, a neuroprotective protein that suppresses oxidative stress and aSyn aggregation by activating various pro- survival mechanisms. Collectively, these observations suggest that DJ-1 may protect against methamphetamine neurotoxicity. The long-term objective of the proposed research is to identify cellular responses that abrogate dopaminergic neurite degeneration elicited by methamphetamine, as a way to stimulate the discovery of new therapies. The studies described in this application are focused on the role of DJ-1 in alleviating methamphetamine neurotoxicity. This question will be addressed with the following specific aims: (i) determine whether DJ-1 inhibits methamphetamine-induced neurite loss; (ii) assess whether DJ-1 suppresses oxidative stress and 1-synuclein aggregation in methamphetamine-treated cells; and (iii) determine whether DJ-1-mediated protection against methamphetamine neurotoxicity requires expression of the protein in glia and/or neurons. Primary midbrain cultures expressing human wild-type DJ-1 from an adenoviral construct will be exposed to methamphetamine, and the percentage of dopaminergic neurons without processes will be determined immunocytochemically. The cells will also be characterized in terms of a range of DJ-1 protective responses. The effect of subcellular localization will be investigated by measuring the neuroprotective activity of engineered DJ-1 variants preferentially targeted to the cytosol, mitochondria, or nucleus. The role of cell-type-specific DJ-1 expression will be examined by monitoring protective pathways in cultures that express DJ-1 selectively in neurons, astrocytes, or microglia. This project will advance our understanding of cellular responses that alleviate methamphetamine neurotoxicity. Ultimately, the insights from this study will stimulate the development of strategies to treat individuals exposed to methamphetamine via activation of neuroprotective pathways modulated by DJ-1. PUBLIC HEALTH RELEVANCE: This project is designed to assess neuroprotective mechanisms by which the protein DJ-1 suppresses the loss of neuronal processes triggered by methamphetamine. The insights from these studies will stimulate the development of therapeutic strategies to alleviate methamphetamine neurotoxicity via upregulation of DJ-1 protective responses.