INTEGRINS: LINKING SENILE PLAQUES AND NEURITIC DYSTROPHY

描述（由申请人提供）：最近，我们获得了老年斑和神经原纤维缠结这两种阿尔茨海默病（AD）标志性病变之间的直接证据。我们培养了从野生型、tau敲除和人类tau转基因小鼠中获得的海马神经元，并用纤维状AB处理它们。形态学分析表明，在AB存在的情况下，表达小鼠或人类tau蛋白的神经元都发生了变性。另一方面，tau缺失的神经元在AB存在的情况下没有变性的迹象。这些结果表明，tau对AB诱导的神经变性至关重要。然而，tau介导中枢神经元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都集中在tau磷酸化在神经突变性中的作用上。我们提出了另一种机制，通过tau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蛋白的分裂。截断的tau可能导致与中枢神经元神经退行性变相关的神经元细胞死亡。为了验证这一假设，我们提出：1)确定AB沉积是否诱导tau蛋白的切割；2)鉴定纤原性AB存在时负责tau蛋白切割的蛋白酶；3)确定tau裂解片段介导AB神经毒性的程度；4)确定能够阻止aa诱导的tau蛋白裂解的因子是否能够阻止神经元变性并导致细胞死亡。该实验将使用成熟的海马神经元原代培养物和AD动物模型进行。将使用包括Western blot分析、体外蛋白酶测定、定点诱变和通过反义寡核苷酸或siRNA抑制蛋白质表达在内的技术组合。

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.