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）蛋白质组的年龄依赖性变化。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到损害。此外，初步研究表明，老年大鼠小脑中的氧化应激标志物比任何其他大脑区域都多。在5、22和34个月时，对F344/BNF 1大鼠小脑中的蛋白质表达进行定量，并鉴定和定位翻译后修饰。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。将对参与神经传递和信号传导的三种小脑亚细胞组分进行蛋白质组学分析：（i）小脑突触质膜，（ii）突触连接复合物，和（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。这些馏分的分析将产生蛋白质复合物的组成和潜在功能完整性的代表性结果。然而，这些亚细胞级分的选择允许有限的样品大小（即，待分析的蛋白质的数量），从而允许通过所鉴定的蛋白质的最大序列覆盖来优化定量分析和翻译后修饰的确定性定位。具体目的是：1）对小脑中可溶性和膜蛋白表达的年龄依赖性变化进行定量蛋白质组学分析和差异显示，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，以及4）开发用于富集、表征、以及含DOPA的肽和蛋白质的定量分析。这些蛋白质组学研究的结果将有助于新兴的数据库提供关键的洞察大脑机制的功能下降与年龄。

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.