Biochemical characterization of tau in tauopaty brains

（1）tau蛋白病脑组织中tau蛋白的生物化学特征我们通过分析患者可溶性和不溶性tau蛋白、外显子捕获试验和体外微管组装试验，研究了在日本家系中发现的tau基因突变（外显子10 +11，+12，N279 K，R5 H，L266 V）的影响。在外显子10 +12或N279患者的脑中观察到过度磷酸化的4-重复（4 R）tau沉积。R5 H和L266 V突变都影响了促进微管组装的能力，并且在这些大脑中检测到3R和4 R亚型的tau沉积。来自Kii ALS-PDC患者的可溶性和不溶性tau的分析揭示了过度磷酸化的3R和4 R tau积累和可溶性tau的正常模式。（2）AD脑内Cdk 5激活因子p25的表达我们研究了钙蛋白酶（calpain）裂解p35后产生的Cdk 5激活因子p25在AD脑内是否升高。对8例AD和9例对照病例的分析表明，AD脑中未检测到p25的显著增加。此外，我们清楚地表明，p35降解为p25在死后的时间间隔。（3）α-突触核蛋白的翻译后修饰我们利用生物化学和蛋白质化学技术分析了路易体痴呆患者脑中沉积的α-突触核蛋白，发现不溶性α-突触核蛋白在Ser 129处高度磷酸化。我们还发现一部分磷酸化的不溶性α-突触核蛋白在α-突触核蛋白病病变中被泛素化，并且α-突触核蛋白的氨基酸残基31-109构成了细丝的蛋白酶K抗性核心

(1)Biochemical characterization of tau from tauopathy brainsWe investigated effects of tau gene mutations (Exon10+11, +12, N279K, R5H, L266V) which were identified in Japanese families by analyzing soluble and insoluble tau from the patients, exon trapping assay and in vitro microtubule assembly assay. Hyperphosphorylated 4-repeat (4R) tau depositions were observed in brains of patients with either Exon10+12 or N279. Both R5H and L266V mutations affected the ability to promote microtubule assembly, and tau deposition of both 3R and 4R isoforms were detected in those brains. Analysis of soluble and insoluble tau from patients with Kii ALS-PDC revealed hyperphosphorylated 3R and 4R tau accumulations and normal pattern of soluble tau.(2)Cdk5 activator p25 in AD brainsWe investigated whether Cdk5 activator p25 that is produced by calpain cleavage of p35 is elevated in AD brain. Analysis of 8 AD and 9 control cases have shown that no significant increase of p25 was detected in AD brain. Furthermore, we clearly showed that p35 degraded to p25 during postmortem intervals.(3)Posttranslational modification on α-synucleinWe analyzed α-synuclein deposited in brains with dementia with Lewy body patients using biochemical and proteinchemical techniques, and identified that the insoluble α-synuclein is hyperphosphorylated at Ser129. We also found that a fraction of phosphorylated insoluble α-synuclein is ubiquitinated in α-synucleinopathy lesions, and that amino acid residues 31-109 of α-synuclein constitute Proteinase K resistant core of the filaments