Proteomic characterization of aging cerebellum

描述（由申请人提供）：本研究的总体目标是详细描述大脑氧化敏感区域小脑蛋白质组中年龄依赖性变化的特征。该区域控制前庭功能、精细运动行为和一些高级认知过程，如情景记忆和嗅觉感知，所有这些在老年人中经常受损。此外，初步研究表明，老年大鼠小脑中氧化应激的标志物比其他任何脑区都多。在5、22和34个月时，F344/BNF1大鼠小脑中的蛋白表达将被量化，翻译后修饰将被确定和定位。分析将特别关注蛋白质磷酸化和蛋白质氧化修饰的积累，如氧化硫醇、3-硝基酪氨酸、3,4 -二羟基苯丙氨酸和4-羟基壬烯醛加合物。蛋白质组学分析将对参与神经传递和信号传导的三个小脑亚细胞部分进行：(i)小脑突触质膜，（ii）突触连接复合物，以及（iii）参与Ca+和一氧化氮（NO）依赖性细胞信号传导的多亚基谷氨酸受体，NMDA受体复合物。对这些组分的分析将对蛋白质复合物的组成和潜在的功能完整性产生具有代表性的结果。然而，这些亚细胞组分的选择允许有限的样本量（即要分析的蛋白质数量），允许通过鉴定蛋白质的最大序列覆盖来优化定量分析和确定翻译后修饰的定位。具体目标是1)进行定量蛋白质组学分析和小脑中可溶性蛋白和膜蛋白表达的年龄依赖性变化的差异显示，2)分析小脑蛋白的年龄依赖性磷酸化状态，3)鉴定和定位小脑蛋白的年龄依赖性氧化翻译后修饰，4)开发含dopa肽和蛋白的富集，表征和定量分析的新技术。这些蛋白质组学研究的结果将有助于建立新的数据库，为大脑功能随年龄下降的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.