INTEGRINS: LINKING SENILE PLAQUES AND NEURITIC DYSTROPHY

我们打算建立老年斑和神经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，这两个病变之间的联系在海马神经元。 我们的工作建立在这些模型中，纤维状β-淀粉样蛋白（Abeta）添加到培养的神经元中导致神经毒性。将Abeta添加到成熟海马培养物中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亚型的选择性过度磷酸化。 因此，该提案的具体目的是为了获得对Abeta沉积，tau过度磷酸化和神经突变性之间的机制联系的进一步和新的见解。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联蛋白介导的促分裂原活化蛋白激酶（MAPK）信号转导通路的激活; 2）MAPK的这种激活有助于增强成年tau亚型的磷酸化，其在神经突变性的机制中起关键作用。 我们将确定：1）纤维状Abeta对MAPK的激活是否由整合素介导。 我们将尝试通过用已知抑制不同整合素功能的抗体预处理细胞来阻断由Abeta诱导的MAPK的活化。 我们将使用通过同源重组技术或反义寡核苷酸去除不同整合素的海马神经元重复这些实验。 2)MAPK参与A β诱导的成人tau蛋白亚型的选择性磷酸化 实验将使用从用可溶性或纤维状Abeta处理的野生型、tau敲除和人tau转基因小鼠制备的成熟海马培养物。 MAPK的活性可被特异性抑制剂抑制。 将使用tau抗体和通过磷酸化测定来确定Tau磷酸化。 将在光学和电子显微镜水平评估是否存在神经突变性体征。

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.