INTEGRINS: LINKING SENILE PLAQUES AND NEURITIC DYSTROPHY

我们打算建立老年斑和神经原纤维缠结之间的关系，这是阿尔茨海默病的两个标志性病变。最近，我们获得的数据表明，海马神经元的这两种病变之间存在联系。我们的工作建立在这些模型的基础上，在这些模型中，纤维状β -淀粉样蛋白（Abeta）添加到培养的神经元中会导致神经毒性。在成熟海马培养物中添加Abeta会导致神经元形态的严重改变，包括轴突和树突的进行性变性，以及成年tau亚型的选择性超磷酸化。因此，这一建议的具体目的是针对Abeta沉积，tau过度磷酸化和神经突变性之间的机制联系获得进一步和新颖的见解。具体目标将解决以下假设：1)Abeta的沉积导致成熟中枢神经元中整合素介导的丝裂原活化蛋白激酶（MAPK）信号转导通路的激活；2) MAPK的激活有助于增强成人tau亚型的磷酸化，这在神经突变性的机制中起关键作用。我们将确定：1)纤原β对MAPK的激活是否由整合素介导。我们将尝试通过用已知的抑制不同整合素功能的抗体预处理细胞来阻断由β诱导的MAPK的激活。我们将通过同源重组技术或反义寡核苷酸重复这些实验，使用缺乏不同整合素的海马神经元。2) MAPK参与了β诱导的成人tau亚型的选择性磷酸化。实验将使用野生型、tau基因敲除和人类tau基因转基因小鼠制备的成熟海马培养物，并用可溶性或纤维性β处理。MAPK的活性会被多种特异性抑制剂抑制。Tau蛋白磷酸化将通过Tau抗体和磷酸化测定来确定。神经突变性迹象的存在将在光镜和电子显微镜水平上进行评估。

We intend to establish the relationship between senile plaques and neurofibrillary tangles, the two hallmark lesions in Alzheimer's Disease. Recently, we have obtained data suggesting a link between these two lesions in hippocampal neurons. Our work builds on those models in which fibrillar beta-amyloid (Abeta) added to neurons in culture results in neurotoxicity. The addition of Abeta to mature hippocampal cultures resulted in severe alterations of neuronal morphology, including the progressive degeneration of axons and dendrites, and the selective hyperphosphorylation of adult tau isoforms. Therefore, the specific aims of this proposal are directed to obtain further and novel insights into the mechanistic link between Abeta deposition, tau hyperphosphorylation and neurite degeneration. The specific aims will address the following hypotheses: 1) the deposition of Abeta results in the integrin-mediated activation of the mitogen-activated protein kinase (MAPK) signal transduction pathway in mature central neurons; and 2) this activation of MAPK contributes to the enhanced phosphorylation of adult tau isoforms which in term, plays a key role in the mechanism underlying neurite degeneration. We shall determine: 1) whether the activation of MAPK by fibrillar Abeta is mediated by integrins. We will attempt to block the activation of MAPK induced by Abeta by pretreating the cells with antibodies known to inhibit the function of different integrins. We will repeat these experiments using hippocampal neurons depleted of different integrins by means of homologous recombination techniques or antisense oligonucleotides. 2) The participation of MAPK in the selective phosphorylation of adult human tau isoforms induced by Abeta. The experiments will be using mature hippocampal cultures prepared from wild type, tau knockout and human tau transgenic mice treated with soluble or fibrillar Abeta. The activity of MAPK will be suppressed by means specific inhibitors. Tau phosphorylation will be determined using tau antibodies and by phosphorylation assays. The presence of signs of neurite degeneration will be assessed at the light and electron microscopy levels.