INTEGRINS: LINKING SENILE PLAQUES AND NEURITIC DYSTROPHY

描述(申请人提供)：最近，我们获得了老年斑和神经原纤维缠结的直接证据，这是阿尔茨海默病(AD)的两个标志性病变。我们培养了来自野生型、tau基因敲除和人tau转基因小鼠的海马神经元，并用Fifiillar AB处理它们。形态分析表明，在AB存在的情况下，表达小鼠或人tau蛋白的神经元退化。另一方面，在AB存在的情况下，tau耗竭的神经元没有表现出退化的迹象。这些结果表明，tau在AB诱导的神经退行性变中是必不可少的。然而，tau介导中枢神经元AA毒性效应的机制在很大程度上是未知的。该领域的大部分研究都集中在tau磷酸化在轴突变性中的作用。我们提出了一种替代机制，即tau可以介导AB诱导的轴突变性。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变性相关的神经细胞死亡。为了验证这一假设，我们建议：1)确定AB沉积是否诱导tau蛋白的裂解；2)确定在纤维AB存在的情况下，负责tau裂解的蛋白水解酶；3)确定tau裂解片段在多大程度上介导AB的神经毒性；以及4)确定能够阻止AA诱导的tau裂解的因素是否能够防止神经元变性和随后的细胞死亡。拟议的实验将使用成熟的海马神经元原代培养和AD动物模型进行。将使用包括蛋白质印迹分析、体外蛋白酶分析、定点突变和通过反义寡核苷酸或siRNA抑制蛋白质表达的技术组合。

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.