Development of clinico-chemical and molecular-biological methods for diagnosis of Alzheimer's disease

(1)淀粉样蛋白β前体（APP）基因在阿尔茨海默病（AD）中的异常表达：我们发现，与对照组相比，AD大脑中含有和不含kunitz型蛋白酶抑制剂（KPI）结构域的APP mrna的比例平均升高1.5倍。研究还发现，随着年龄的增长，对照组大脑的这一比例也有所上升，不过比阿尔茨海默氏症患者晚20-25年。这一比例的变化在包括海马在内的大脑皮层灰质中最为明显。基于这些发现，我们提出APP mrna比值作为脑老化的分子指标。(2) AD中APP基因核苷酸序列无特异性变化：我们分析了AD和对照中APP基因转录物选择性剪接和产生具有或不具有KPI结构域的不同mrna的区域的核苷酸序列。在AD患者中没有发现特定的序列变化。AD患者脑脊液中KPI片段增加：应用新发明的胰蛋白酶- elisa法检测app衍生的KPI片段，发现AD患者脑脊液中KPI片段显著增加。(4) AD患者载脂蛋白E等位基因频率的偏差：我们证实，在迟发性AD患者中，载脂蛋白E （APOE）基因epsilon4等位基因频率高出2-3倍，在美国和日本病例中均有报道。我们进一步确定了epsilon4等位基因纯合性是迟发性AD的一个特别高的危险因素。肌萎缩性侧索硬化症（ALS）的新突变：我们在家族性ALS患者的超氧化物歧化酶-1基因中发现了一个迄今未知的突变，Leu^<106>*Val （CTC*GTC）。

(1) Aberrant expression of amyloid beta protein precursor (APP) gene in Alzheimer's disease (AD) : We found a 1.5-fold elevation, on the average, of the ratio of APP mRNAs with and without a Kunitz-type protease inhibitor (KPI) domain in AD brains compared with controls. The ratio was also found to be elevated in control brains with aging, though 20-25 years later than in AD.The change of the ratio was most apparent in the gray matter of cerebral cortex including hippocampus known to be affected in dementia. Based on these findings, we proposed the APP mRNAs ratio to serve as a molecular index of the brain aging.(2) No specific change in nucleotide sequences of APP gene in AD : We analyzed nucleotide sequences of the regions responsible for alternative splicing of APP gene transcript and producing distinct mRNAs with or without KPI domain in AD and control. There was no specific change found in the sequences among AD patients.Increase of KPI fragments in cerebrospinal fluid (CSF) of AD : Assay of APP-derived KPI fragments using a newly invented trypsin-ELISA method revealed a significant increse of the fragments in the CSF of AD patients.(4) Deviation of apolipoprotein E allele frequency in AD : We confirmed a 2-3-fold higher frequency of epsilon4 allele of apolipoprotein E (APOE) gene among late-onset AD patients, as reported for Americans, with Japanese cases. We further identified the epsilon4 allele homozygosity to be a particularly high risk factor for late-onset AD.New mutation for amyotrophic lateral sclerosis (ALS) : We found a hitherto unknown mutation, Leu^<106>*Val (CTC*GTC) in the superoxide dismutase-1 gene in a familial ALS patient.