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-淀粉样肽组成 β-淀粉样蛋白前体蛋白的蛋白水解片段被包围 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 AD 斑块中也发现了这些物质，包括 α1-抗胰凝乳蛋白酶和 载脂蛋白 E。神经元细胞体中的神经原纤维缠结是 由成对的螺旋丝（PHF）组成。 大量研究已经 现在表明 PHF 由微管相关蛋白 tau 组成， 这是异常磷酸化的。最近的研究表明，lys-ser- tau 上的 pro (KSP) 序列属于异常磷酸化的序列 在阿尔茨海默病中。 高分子量神经丝蛋白 NF-H 在 相似的 KSP 共有序列。 这个序列出现了五十多个 NF-H 分子尾部的时间，大多数这些位点通常是 在体内磷酸化。 最近的研究表明，cdk5，一种激酶 与细胞周期依赖性激酶 cdc2 相关，在大脑中表达 并与神经丝以及微管相关。 这种激酶 磷酸化 NF-H 的一些但不是全部 KSP 位点，并且还能够 使 AD 中某些异常磷酸化的位点上的 tau 蛋白磷酸化。 该提案重点关注该激酶在 神经系统及其与 tau 蛋白异常磷酸化的关系 在公元。 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，这也可能起作用 阿尔茨海默病中 tau 蛋白异常。 该提案的目的是： 1. 研究过度表达的影响 cdk5 对体内 NFH 和 tau 磷酸化的影响 cDNA 克隆到转基因小鼠的神经元表达载体中。 2. 至 通过突变确定转基因小鼠中cdk5的抑制作用 cdk5 cDNA 克隆在其活性位点产生非活性激酶， 会抑制内源性ckd5。这种突变激酶将被引入 进入带有神经元特异性表达载体的转基因小鼠中，我们将 确定抑制 NF-H 和 tau 磷酸化的效果。 3. 分离剩余 KSP 上磷酸化 NF-H 的其他激酶 通过蛋白质化学方法以及酵母双杂交系统进行位点。

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.