NEUROFILAMENT KINASES AND ALZHEIMERS DISEASE TAU

阿尔茨海默病的两个主要病理标志是 神经纤维缠结和老年斑 老年斑包括 细胞外淀粉样纤维，由β-淀粉样肽组成， β-淀粉样前体蛋白的蛋白水解片段， 营养不良的神经突、活化的小胶质细胞和星形胶质细胞。 其他蛋白质 在AD斑块中也发现，包括α 1-抗胰凝乳蛋白酶和 载脂蛋白E 神经元细胞体中的神经纤维缠结是 由成对的螺旋丝（PHF）组成。 大量研究 现在显示PHF由微管相关蛋白tau组成， 其被异常磷酸化。最近的研究表明，lys-ser- tau蛋白上的pro（KSP）序列是异常磷酸化的序列之一 老年痴呆症 高分子量的神经丝蛋白，NF-H被磷酸化， 相似的KSP共有序列。 这个序列是目前超过50 在NF-H分子的尾部，这些位点中的大多数通常是 在体内磷酸化。 最近的研究表明，CDK 5，一种激酶， 与细胞周期依赖性激酶CDC 2相关，在脑中表达 并与神经丝和微管结合。 该激酶 磷酸化NF-H的一些但不是全部KSP位点， 使AD中一些异常磷酸化位点上的tau磷酸化。 这项建议的重点是这种激酶的具体功能， 神经系统以及它如何与tau蛋白的异常磷酸化有关 在AD中。 此外，我们将尝试分离其他激酶， 磷酸化剩余KSP位点上的NF-H，并且其也可以作用于 在老年痴呆症的tau蛋白上异常。 本提案的目的是：1.为了研究过度表达 cdk 5对NFH和tau蛋白磷酸化的影响 在转基因小鼠中克隆到神经元表达载体中的cDNA。 2.到 通过突变来确定在转基因小鼠中抑制CDK 5的效果 cdk 5cDNA克隆在其活性位点T产生失活激酶，其 将抑制内源性CKD 5。这种突变激酶将被引入 植入带有神经元特异性表达载体的转基因小鼠， 确定抑制NF-H和tau磷酸化的效果。 3.为了分离使剩余KSP上的NF-H磷酸化的其他激酶， 位点的蛋白质化学方法，以及酵母双杂交系统。

The two major pathological hallmarks of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Senile plaques consist of extracellular amyloid fibrils, composed of the beta-amyloid peptide, a proteolytic fragment of the beta-amyloid precursor protein surrounded by dystrophic neurites, activated microglia and astrocytes. Other proteins are also found in the AD plaques, including alpha1-antichymotrypsin and apolipoprotein E. The neurofibrillary tangles in neuronal cell bodies are composed of paired helical filaments (PHF). A large number of studies have now shown that PHFs are made up of the microtubule associated protein tau, which is abnormally phosphorylated. Recent studies have shown that lys-ser- pro (KSP) sequence on tau are among the sequences abnormally phosphorylated in Alzheimer's Disease. The high molecular weight neurofilament protein, NF-H is phosphorylated on similar KSP consensus sequences. This sequence is present more than fifty times in the tail of the NF-H molecule and most of these sites are normally phosphorylated in vivo. Recent studies have shown that cdk5, a kinase related to the cell cycle dependent kinase cdc2, is expressed in the brain and associates with neurofilaments, as well as microtubules. This kinase phosphorylates some, but not all of the KSP sites of NF-H and is also able to phosphorylate tau on some of the sites abnormally phosphorylated in AD. This proposal focuses on the specific function of this kinase in the nervous system and how it may relate to the abnormal phosphorylation of tau in AD. In addition, we will attempt to isolate other kinases which phosphorylate NF-H on the remaining KSP sites, and which may also act abnormally on tau in Alzheimer's Disease. The aims of this proposal are: 1. To study the effects of overexpression of cdk5 on the phosphorylation of NFH and tau in vivo by introducing its cDNAs cloned in a neuronal expression vector in transgenic mice. 2. To determine the effect of inhibition of cdk5 in transgenic mice by mutating the cdk5 cDNA clone in its active site t produce an inactive kinase, which will inhibit the endogenous ckd5. This mutant kinase will be introduced into transgenic mice with a neuron specific expression vector and we will determine the effect of the inhibition of phosphorylation of NF-H and tau. 3. To isolate other kinases which phosphorylate NF-H on the remaining KSP sites by protein chemical methods, as well as the yeast two-hybrid system.