Identification of Presinilin downstream targets in neuronal survival

阿尔茨海默病(AD)是痴呆症和神经退行性变的最常见原因，但没有 疾病修正疗法是可用的。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。这些流行的PSEN突变大多是 错义突变(&gt；260)散布在整个编码序列中，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-分泌酶的活性部位。早老素条件双击倒(PS CDKO) 在成年大脑皮层中缺乏PS表达的小鼠概括了AD的关键特征，包括深刻的 年龄依赖性神经退行性变、神经胶质增生、炎症反应和tau过度磷酸化。研究项目： 线虫、果蝇和培养的哺乳动物细胞显示FAD突变损害PS功能和γ- 分泌酶活性。我们最近培育了两只表达Fad PSEN1突变的敲门(KI)小鼠，L435F和 C410Y和纯合子Ki/Ki小鼠与PS1-/-小鼠表现出惊人的相似之处，包括围产期死亡率， 抑制γ-分泌酶活性，损害神经发生，减少Notch信号转导，表明FAD 突变类似于体内的PS1零突变。多糖核酸维持神经元功能的分子机制 功能和生存尚不清楚。鉴定PS下游靶标和γ分泌底物将 不仅阐明了PS功能和功能障碍的分子机制，而且可能提供 新的治疗靶点也是如此。在当前的应用中，我们将利用Fly的力量 识别PS下游靶点和参与神经元调节的γ分泌底物的遗传学 生存和长寿。具体地说，我们将产生条件突变果蝇，其中早老素直系同源基因(PSN) 或尼古丁同源异构体(NCT)在成年神经元中被shRNA诱导下调，然后使用这些突变体 苍蝇筛选参与调节神经元存活和寿命的γ分泌底物 苍蝇和老鼠模型的验证(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可以纠正PS功能障碍的RNAi系，然后验证 在果蝇模型中识别的基因恢复PS功能障碍的能力(目标2)。已完成的 拟议的研究将阐明PS在衰老过程中保护神经元存活的分子途径 并可能为AD的疾病调节治疗提供新的靶点。

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.