INTEGRINS: LINKING SENILE PLAQUES AND NEURITIC DYSTROPHY

描述（由申请人提供）：最近，我们获得了直接证据，将老年斑和神经纤维缠结联系起来，这是阿尔茨海默病（AD）的两个标志性病变。我们培养了从野生型、tau基因敲除和人tau基因转基因小鼠获得的海马神经元，并用纤维状AB处理它们。形态学分析表明，表达小鼠或人tau蛋白的神经元在AB存在下退化。另一方面，tau耗尽的神经元在AB存在下没有显示出变性的迹象。这些结果表明，tau蛋白是必不可少的AB诱导的神经退行性变。然而，tau蛋白介导中枢神经元中Aa毒性作用的机制在很大程度上是未知的。该领域的大部分研究工作都集中在tau蛋白磷酸化在神经突变性中的作用上。我们提出了一种替代机制，tau蛋白可以介导AB诱导的神经突变性。我们假设纤维状AB的沉积导致tau蛋白的切割。截短的tau可能导致与中枢神经元中的神经变性相关的神经元细胞死亡。为了检验这一假设，我们提出：1）确定AB沉积是否诱导tau蛋白的切割; 2）鉴定在纤维状AB存在下负责tau切割的蛋白酶; 3）确定tau切割片段介导AB神经毒性的程度;和4）确定能够防止Aa诱导的tau切割的因子是否防止神经元变性，随后是细胞死亡。拟用海马神经元成熟原代培养物和AD动物模型进行实验。将使用包括蛋白质印迹分析、体外蛋白酶测定、定点诱变和通过反义寡核苷酸或siRNA抑制蛋白质表达的技术的组合。

DESCRIPTION (provided by applicant): Recently, we have obtained direct evidence linking senile plaques and neurofibrillary tangles, the two hallmark lesions in Alzheimer's disease (AD). We cultured hippocampal neurons obtained from wild type, tau knockout, and human tau transgenic mice, and treated them with fibrillar AB. Morphological analysis indicated that neurons expressing either mouse or human tau proteins degenerated in the presence of AB. On the other hand, tau-depleted neurons showed no signs of degeneration in the presence of AB. These results suggest that tau is essential to AB-induced neurodegeneration. However, the mechanism by which tau mediates Aa toxic effects in central neurons are largely unknown. Most of the research effort in the field has been focused on the role of tau phosphorylation in neurite degeneration. We propose an alternative mechanism by which tau could mediate AB-induced neurite degeneration. We hypothesize that the deposition of fibrillar AB results in the cleavage of tau proteins. Truncated tau may contribute to neuronal cell death associated with neurodegeneration in central neurons. To test this hypothesis we propose to: 1) determine whether AB deposition induces cleavage of tau proteins; 2) identify the proteases responsible for tau cleavage in the presence of fibrillar AB; 3) determine to what extent tau cleaved fragments mediate AB neurotoxicity; and 4) determine whether factors capable of preventing Aa-induced tau cleavage prevent neuronal degeneration followed by cell death. The proposed experiments will be carried out using mature primary cultures of hippocampal neurons and AD animal models. A combination of techniques including Western blot analysis, in vitro protease assays, site-directed mutagenesis, and the suppression of protein expression by means of antisense oligonucleotides or siRNA will be used.