NEUROFILAMENT KINASES AND ALZHEIMERS DISEASE TAU

阿尔茨海默病的两个主要病理特征是 神经原纤维缠结和老年斑。老年斑包括 细胞外淀粉样原纤维，由β-淀粉样多肽，a 被包围的β-淀粉样前体蛋白的蛋白分解片段 营养不良的神经突起，激活的小胶质细胞和星形胶质细胞。其他蛋白质 也在AD斑块中发现，包括α1-抗糜蛋白酶和 载脂蛋白E神经元胞体中的神经原纤维缠结是 由成对的螺旋丝(PHF)组成。大量的研究表明 现在证明PHF由微管相关蛋白tau组成， 它被异常的磷酸化了。最近的研究表明，Lys-Ser- Tau上的Pro(KSP)序列是异常磷酸化的序列之一 阿尔茨海默氏症。 高分子神经丝蛋白，核因子-H被磷酸化 相似的KSP共识序列。这个序列出现了50多个 在核因子-H分子尾部的时间，这些位置中的大多数通常是 在体内被磷酸化。最近的研究表明，一种名为CDK5的激酶 与细胞周期依赖激酶cdc2相关，在大脑中表达。 与神经丝和微管联系在一起。这种激活剂 使核因子-H的部分(但不是全部)KSP位点磷酸化，也能够 使AD中一些异常磷酸化的部位上的tau磷酸化。 本研究的重点是该激酶在细胞周期中的具体功能。 神经系统及其与tau异常磷酸化的关系 在公元后。此外，我们还将尝试分离出其他 在剩余的KSP位点上磷酸化NF-H，这也可能起作用 阿尔茨海默病患者对tau的异常反应。 这项建议的目的是：1.研究过度表达的影响 CDK5通过引入其在体内对NFH和tau磷酸化的影响 克隆在转基因小鼠神经元表达载体中的cDNA。2.至 通过突变检测CDK5在转基因小鼠中的抑制作用 在其活性部位t处克隆的cdk5会产生一种失活的激酶，它 会抑制内源性CKD5。这种突变的激酶将被引入 转化成具有神经元特异性表达载体的转基因小鼠，我们将 测定抑制核因子-H和tau蛋白磷酸化的作用。 3.分离其他使剩余KSP上的核因子-H磷酸化的激酶 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.