Identification of Presinilin downstream targets in neuronal survival

阿尔茨海默病（AD）是痴呆和神经变性的最常见原因，但没有 疾病改善疗法是可用的。早老素（PSEN）基因含有约90%的与 家族性AD（FAD），强调其在AD发病机制中的重要性。这些FAD PSEN突变大多是 错义突变（>260）分散在整个编码序列中，与功能丧失一致 机制早老素（PS）是学习记忆、突触功能和神经元存活所必需的 在衰老过程中，并含有γ-分泌酶的活性位点。早老素条件性双敲除（PS cDKO） 在成年大脑皮层缺乏PS表达的小鼠概括了AD的关键特征，包括深刻的 年龄依赖性神经变性、神经胶质增生、炎症反应和tau蛋白过度磷酸化。研究 C.线虫、果蝇和培养的哺乳动物细胞表明，FAD突变损害PS功能， 分泌酶活性我们最近开发了两种表达FAD PSEN 1突变的敲入（KI）小鼠，L435 F和 C410 Y和纯合子KI/KI小鼠显示出与PS1-/-小鼠惊人的相似性，包括围产期致死率， γ-分泌酶活性消失，神经发生受损，Notch信号减少，表明FAD 突变类似于体内的PS1无效突变。PS维持神经元功能的分子机制 功能和存活率尚不清楚。PS下游靶标和γ-分泌酶底物的鉴定将 不仅阐明PS功能和功能障碍的分子机制， 新的治疗靶点。在目前的应用中，我们将利用苍蝇的力量， 遗传学，以确定参与神经元调控的PS下游靶点和γ-分泌酶底物， 生存和长寿。具体来说，我们将产生条件突变果蝇，其中早老素直系同源物（Psn） 或Nicastrin直系同源物（Nct）在成年神经元中被shRNA诱导敲低，然后使用这些突变体 果蝇筛选参与介导神经元存活和寿命的γ-分泌酶底物， 在苍蝇和小鼠模型中进行验证（目标1）。我们还将进行基于全基因组RNAi的遗传筛选， 在果蝇原代培养的神经元中鉴定能够纠正PS功能障碍的RNAi细胞系，然后验证 在果蝇模型中鉴定出的基因恢复PS功能障碍的能力（目的2）。完成 拟开展的研究将阐明PS在衰老过程中保护神经元存活的分子途径 并可能提供新的靶点，可进一步探索用于AD的疾病改善治疗。

Alzheimer's disease (AD) is the most common cause of dementia and neurodegeneration, but no disease-modifying therapy is available. The Presenilin (PSEN) genes harbor ~90% of the mutations linked to familial AD (FAD), highlighting its importance in AD pathogenesis. These FAD PSEN mutations are mostly missense mutations (>260) scattered throughout the coding sequence, consistent with a loss-of-function mechanism. Presenilin (PS) is essential for learning and memory, synaptic function and neuronal survival during aging, and contains the active site of γ-secretase. Presenilin conditional double knockout (PS cDKO) mice lacking PS expression in the adult cerebral cortex recapitulate key features of AD, including profound age-dependent neurodegeneration, gliosis, inflammatory responses and tau hyperphosphorylation. Studies in C. elegans, Drosophila and cultured mammalian cells showed that FAD mutations impair PS function and γ- secretase activity. We recently developed two knockin (KI) mice expressing FAD PSEN1 mutations, L435F and C410Y, and homozygous KI/KI mice show striking resemblance to PS1-/- mice, including perinatal lethality, abolished γ-secretase activity, impaired neurogenesis and decreased Notch signaling, demonstrating that FAD mutations resemble the PS1-null mutation in vivo. The molecular mechanism by which PS maintains neuronal function and survival is unclear. Identification of PS downstream targets and γ-secretase substrates will not only elucidate the molecular mechanism underlying PS function and dysfunction, but may provide novel therapeutic targets as well. In the current application, we will take advantage of the power of fly genetics to identify PS downstream targets and γ-secretase substrates involved in the regulation of neuronal survival and longevity. Specifically, we will generate conditional mutant flies, in which Presenilin ortholog (Psn) or Nicastrin ortholog (Nct) are inducibly knocked down by shRNA in adult neurons, and then use these mutant flies to screen for γ-secretase substrates involved in mediating neuronal survival and lifespan followed by validation in fly and mouse models (Aim 1). We will also perform whole-genome RNAi-based genetic screens in Drosophila primary cultured neurons to identify RNAi lines that can correct PS dysfunction, and then validate the identified genes in fly models for their abilities to restore PS dysfunction (Aim 2). Completion of the proposed studies will elucidate the molecular pathways by which PS protects neuronal survival during aging and may provide novel targets that can be further explored for disease-modifying therapy of AD.