Proteomic characterization of aging cerebellum

描述(由申请人提供)：这项研究的总体目标是详细描述大脑氧化敏感区域小脑的蛋白质组随年龄的变化。这一区域控制前庭功能、精细运动行为以及一些高级认知过程，如情景记忆和嗅觉，所有这些在老年人中都经常受到影响。此外，初步研究表明，老年大鼠小脑的氧化应激标志物比其他任何大脑区域都多。在5个月、22个月和34个月的F344/BNF1大鼠的小脑中，蛋白质表达将被量化，翻译后修饰被识别和定位。分析将特别集中在蛋白质磷酸化和蛋白质氧化修饰的积累上，如氧化的硫醇、3-硝基酪氨酸、3，4-二羟基苯丙氨酸和4-羟基壬醛加合物。蛋白质组学分析将涉及神经传递和信号传递的三个小脑亚细胞部分：(I)小脑突触质膜，(Ii)突触连接复合体，以及(Iii)参与钙和一氧化氮(NO)依赖的细胞信号传递的多亚单位谷氨酸受体，NMDA受体复合体。对这些组分的分析将在蛋白质复合体的组成和潜在的功能完整性方面产生代表性的结果。然而，选择这些亚细胞组分允许有限的样本量(即要分析的蛋白质的数量)，从而允许通过最大限度地覆盖已识别蛋白质的序列来优化定量分析和最终定位翻译后修饰。其具体目的是：1)对小脑中随年龄变化的可溶性蛋白和膜蛋白的表达进行定量的蛋白质组学分析和差异显示；2)分析小脑蛋白随年龄变化的磷酸化状态；3)识别和定位小脑蛋白随年龄变化的氧化修饰；4)发展新的技术来丰富、表征和定量分析含有DOPA的多肽和蛋白质。这些蛋白质组学研究的结果将有助于新出现的数据库，为揭示随着年龄增长而导致功能衰退的大脑机制提供关键的见解。

DESCRIPTION (provided by applicant): The overall goal of this research is a detailed characterization of age-dependent changes in the proteome of an oxidation-sensitive area of the brain, the cerebellum. This region controls vestibular function, fine motor behaviors, and some higher cognitive processes such as episodic memory and olfactory perception, all of which are frequently compromised in aged individuals. Furthermore, preliminary studies showed more markers of oxidative stress in aged rat cerebellum than any other brain region. Protein expression will quantitated and post-translational modifications identified and localized in cerebella from F344/BNF1 rats at 5, 22, and 34 months. Analysis will focus specifically on protein phosphorylation and accumulation of protein oxidative modifications such as oxidized thiols, 3-nitrotyrosine, 3, 4-dihydroxyphenylalanine, and 4-hydroxynonenal adducts. Proteomic analysis will be carried out with three cerebellar subcellular fractions involved in neurotransmission and signaling: (i) cerebellar synaptic plasma membranes, (ii) synaptic junctional complexes, and (iii) a multi-subunit glutamate receptor involved in Ca+ and nitric oxide (NO)-dependent cell signaling, the NMDA receptor complex. Analysis of these fractions will yield representative results on composition and potential functional integrity of protein complexes. Yet, selection of these subcellular fractions allows for limited sample sizes (i.e., number of proteins to be analyzed) permitting optimization of quantitative analysis and definitive localization of post-translational modifications through maximum sequence coverage of identified proteins. The Specific Aims are to 1) conduct quantitative proteomic analysis and differential display of age-dependent changes in expression of soluble and membrane proteins in cerebellum, 2) analyze age-dependent phosphorylation status of cerebellar proteins, 3) identify and localize age-dependent oxidative post-translational modifications on cerebellar proteins, and 4) develop novel technologies for enrichment, characterization, and quantitative analysis of DOPA-containing peptides and proteins. Results of these proteomic studies will contribute to emerging databases providing critical insights into brain mechanisms underlying functional decline with age.