Proteome of the cortex of old Pink1-SNCA A53T double mutant Parkinson mice

Parkinson's Disease (PD) is primarily caused by aggregates of alpha synuclein (SNCA) in dopaminergic neurons of the substantia nigra, but PD is a systemic disease and may lead to PD-associated dementia complex. PD-associated encephalopathy is a late manifestation in PD patients at risk for example owing to mutations of the lysosomal enzyme glucocerebrosidase. Defects of lysosomal waste removal and aggregation of mutant alpha synuclein (SNCA) impacts of the proteome. Here, we studied the proteome of the prefrontal cortex in Pink1-/-SNCA A53T double mutant mice in comparison with their wildtype controls. Pink1-/-SNCA A53T mice carry a loss of function knock-in mutation of PTEN induced kinase (Pink1), plus the human A53T mutation of alpha synuclein (SNCA-A53T) [1, 2]. Homozygous Pink1-/-SNCA A53T double mutant mice were generated by crossing Pink1-/- mice (background: 129/SvEv) with A53T-SNCA-overexpressing PrPmtA mice (background: FVB/N) and then, interbreeding the littermates. Wildtype (WT) control mice are hybrids from a crossbreeding of 129/SvEv and FVB/N mice, which were descended from littermates of the respective single mutant animals. Pink1-/-SNCA A53T mice develop spontaneous motor symptoms at advanced ages, with a progressive incidence above 15 months of age. The phenotype of Pink1-/-SNCA A53T and wildtype control mice was observed during aging. Mice were euthanized at an age of 1-1.5 years (matched with the controls). The cortices were rapidly removed and frozen in liquid nitrogen and processed for label free proteomic analyses.

帕金森病（Parkinson's Disease, PD）主要由黑质多巴胺能神经元内的α-突触核蛋白（alpha synuclein, SNCA）聚集引发，但PD实则为系统性疾病，还可诱发帕金森病相关痴呆综合征。由溶酶体酶葡糖脑苷脂酶突变等风险因素导致的帕金森病相关脑病，是PD患者的晚期表现。溶酶体废物清除缺陷与突变型α-突触核蛋白（alpha synuclein, SNCA）聚集会对蛋白质组造成影响。本研究针对Pink1基因敲除/SNCA A53T双突变小鼠的前额叶皮层蛋白质组展开研究，并与野生型对照小鼠进行对比。Pink1-/-SNCA A53T小鼠携带有PTEN诱导激酶（PTEN induced kinase, Pink1）的功能缺失敲入突变，以及人类α-突触核蛋白A53T突变（SNCA-A53T）[1, 2]。纯合子Pink1-/-SNCA A53T双突变小鼠的构建方式为：先将Pink1基因敲除小鼠（背景品系：129/SvEv）与过表达A53T-SNCA的PrPmtA小鼠（背景品系：FVB/N）杂交，随后对所得子代进行同胞兄妹交配。野生型（WT）对照小鼠为129/SvEv与FVB/N品系小鼠杂交所得的杂交一代，其亲本来自上述单突变小鼠的同胞后代。Pink1-/-SNCA A53T小鼠在老龄阶段会出现自发性运动症状，15月龄以上发病率逐渐升高。研究过程中观察了Pink1-/-SNCA A53T双突变小鼠与野生型对照小鼠在衰老过程中的表型变化。小鼠在1~1.5岁龄时实施安乐死（与对照组小鼠年龄匹配），快速分离皮层组织并置于液氮中速冻，随后进行无标记蛋白质组学分析。