Biochemical characterization of tau in tauopaty brains

(1) tau基因的生化特征通过分析日本家族tau基因（外显子10+11，+12,N279K, R5H, L266V）突变，外显子捕获法和体外微管组装法，研究了日本家族tau基因突变（外显子10+11，+12,N279K, R5H, L266V）的影响。在Exon10+12或N279患者的大脑中观察到过度磷酸化的4-重复（4R） tau沉积。R5H和L266V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4R亚型的tau沉积。Kii ALS-PDC患者的可溶性和不可溶性tau蛋白分析显示3R和4R tau蛋白过度磷酸化，可溶性tau蛋白正常。(2) AD脑Cdk5激活因子p25我们研究了calpain切割p35产生的Cdk5激活因子p25是否在AD脑中升高。对8例AD和9例对照病例的分析显示，AD脑组织中p25未见明显升高。此外，我们清楚地表明p35在死后的时间间隔内降解为p25。(3) α-synuclein的翻译后修饰我们利用生化和蛋白化学技术分析了路易体痴呆患者大脑中沉积的α-synuclein，发现不溶性α-synuclein在Ser129位点被过度磷酸化。我们还发现，部分磷酸化的不溶性α-突触核蛋白在α-突触核蛋白病变中泛素化，α-突触核蛋白的氨基酸残基31-109构成了丝状物的蛋白酶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