Search for the candidate gene of a novel hereditary cerebellar degeneration : an approach using proteomics.

寻找新型遗传性小脑变性的候选基因：一种使用蛋白质组学的方法。

• 批准号: 15500231
• 负责人: TOYOSHIMA Yasuko
• 金额: $ 2.43万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15500231/
• 关键词: Cerebellar degeneration; 1 C2; polyglutamine disease; western blotting; two dimensional electrophoresis; アミノ酸配列; ポリグルタミン; 脊髄小脳変性症

We have found three novel polyglutamine (polyQ) disease families in last four years. In histological examination of the nervous system, each case has shown unique distribution of the polyQ-positive neuronal nuclei. We revealed one case was a homozygote of SCA 17,and reported the clinico-pathological findings.After informed consent, we analyzed the protein extracted from autopsied brain. We found a case had an extra band in western blotting pattern using antibody to polyQ stretches (1 C2).To profile the expression of proteins, we used 2D fluorescence difference gel electrophoresis (2D-DIGE) system (Amersham Bioscience). We chose a patient, who had been revealed as novel polyQ disease, and six controls. The protein samples were extracted from their cerebellum, and were labeled with CyDyes (patient : Cy5,control : Cy3,and internal standard : Cy2). The samples were separated over first and second dimensions according to their charge and size, respectively. Once the samples had been separated in the second dimension, gels were scanned for Cy2,Cy3 and Cy5 fluorescence using an appropriate scanner, Typhoon^<TM> 9400 imager (Amersham Bioscience). And image analysis was performed using DeCyder (Amersham Bioscience). As a result, we discovered a certain protein which was expressed massively in the patient's brain. Besides, from the result of 2D western blotting, the protein was the very thing that we have recognized as an extra band in the ID western blotting with 1 C2. We picked the protein spot, and sequenced the peptide fragments by MALDI-TOF mass spectrometry.

在过去四年中，我们发现了三个新的多聚谷氨酰胺 (polyQ) 疾病家族。在神经系统的组织学检查中，每个病例都显示出polyQ阳性神经元核的独特分布。我们发现一例是SCA 17纯合子，并报告了临床病理结果。在知情同意后，我们分析了从尸检大脑中提取的蛋白质。我们使用 PolyQ 延伸 (1 C2) 抗体发现一个病例在蛋白质印迹模式中存在额外条带。 为了分析蛋白质的表达，我们使用 2D 荧光差异凝胶电泳 (2D-DIGE) 系统 (Amersham Bioscience)。我们选择了一名已被证实患有新型 PolyQ 疾病的患者和六名对照者。从小脑中提取蛋白质样品，并用 CyDyes 标记（患者：Cy5，对照：Cy3，内标：Cy2）。样品根据其电荷和尺寸分别在第一和第二维度上分离。一旦在第二维中分离了样品，就使用合适的扫描仪Typhoon TM 9400成像仪(Amersham Bioscience)扫描凝胶的Cy2、Cy3和Cy5荧光。使用 DeCyder (Amersham Bioscience) 进行图像分析。结果，我们发现了一种在患者大脑中大量表达的蛋白质。此外，从2D蛋白质印迹结果来看，该蛋白质正是我们在1C2的ID蛋白质印迹中识别为额外条带的蛋白质。我们挑选了蛋白质点，并通过 MALDI-TOF 质谱法对肽片段进行了测序。

项目成果

Spinocerebellar ataxia type 17 repeat in patients with Huntington's disease-like and ataxia, reply

亨廷顿病样和共济失调患者中脊髓小脑共济失调 17 型重复，回复

• 发表时间: 2004
• 期刊: Annals of Neurology 56(1)
• 作者: Yasuko Toyoshima

Pathological involvement of the motor neuron system and hippocampal formation in motor neuron disease-inclusion dementia

运动神经元疾病-包涵性痴呆中运动神经元系统和海马结构的病理受累

• 发表时间: 2003
• 期刊: Acta Neuropathol 106
• 作者: Toyoshima Y;Yamada M;Onodera O;Shimohata M;Inenaga C;Fujita N;Morita M;Tsuji S;Takahashi H.;Yasuko Toyoshima;Yasuko Toyoshima
• 通讯作者: Yasuko Toyoshima

Spinocerebellar ataxia type 17 repeat in patients with Huntington's disease-like and ataxia, Reply.

亨廷顿病样和共济失调患者中脊髓小脑共济失调 17 型重复，回复。

• 发表时间: 2004
• 期刊: Ann Neurol 56
• 作者: Toyoshima Y;Yamada M;Onodera O;Shimohata M;Inenaga C;Fujita N;Morita M;Tsuji S;Takahashi H.
• 通讯作者: Takahashi H.

Pathological involvement of the motor neuron system and hippocampal formation in motor neuron disease-inclusion dementia.

运动神经元疾病包含性痴呆中运动神经元系统和海马结构的病理学参与。

• 发表时间: 2003
• 期刊: Acta Neuropathol 106
• 作者: Toyoshima Y;Piao YS;Tan OF;Morita M;Tanaka M;Oyanagi K;Okamoto K;Takahashi H.
• 通讯作者: Takahashi H.

Extracerebral ectopic mass with huge cysts in the anterior cranial cavity

前颅腔内有巨大囊肿的脑外异位肿块

• 发表时间: 2003
• 期刊: Neuropathology 23
• 作者: Toyoshima Y;Yamada M;Onodera O;Shimohata M;Inenaga C;Fujita N;Morita M;Tsuji S;Takahashi H.;Yasuko Toyoshima
• 通讯作者: Yasuko Toyoshima