Exploring FOXO Signaling in Promoting Neurodegeneration in AD

描述（由申请人提供）：我们的长期目标是确定预防阿尔茨海默病（AD）神经变性的有效治疗靶点。在这个建议中，我们研究了一个新的机制，AD发病机制后，我们发现转录因子FOXO 1和FOXO 3a作为新的细胞周期蛋白依赖性激酶5（Cdk 5）的底物。FOXO 1和FOXO 3a在大脑中高度表达，特别是在AD中对神经变性敏感的区域。然而，FOXO在AD中的作用和调控尚不清楚。尚未在人类临床样品中分析FOXO的活化及其翻译后修饰。我们发现FOXO的激活发生在AD临床组织的早期，并且在b-淀粉样蛋白介导的信号传导中具有高度的神经毒性。此外，我们的数据表明FOXO通过两种独立的机制激活：（i）Cdk 5介导的磷酸化（S249处的FOXO 1，S173处的FOXO 3a）;（ii）Akt位点处的钙调神经磷酸酶介导的FOXO去磷酸化。已知Akt磷酸化FOXO 1和FOXO 3a，使其失活。假设：Cdk 5和钙调神经磷酸酶协同促进FOXO的活化，这导致神经毒性B-淀粉样蛋白加工和细胞死亡，这是AD的两个标志。这项工作的影响是，它的成功完成将为AD治疗提供回顾性生物标志物和潜在的新策略。这一假设将通过追求三个具体的目标进行测试：目标1：解剖的分子机制，其中FOXO被激活的Cdk 5和钙调神经磷酸酶在大鼠和人类胎儿神经元。目的2：确定活性FOXO在促进大鼠和人胎儿神经元中的β-淀粉样蛋白加工和神经毒性中的作用。目标3：研究两种AD小鼠模型（APP/PS1和p25转基因小鼠）和人类临床组织中FOXO磷酸化的临床相关性。虽然APP/PS1小鼠将证实Ab在FOXO信号传导中的全局作用，但p25-小鼠将具体证明Cdk 5在激活FOXO中的作用及其与神经变性的相关性。创新：该假设是基于新的Cdk 5底物FOXO 1和FOXO 3a，使用高度创新的化学遗传方法发现的。其次，本研究表明，直接抑制FOXO或靶向FOXO信号传导的上游调节因子将为预防AD中的神经变性提供新的治疗干预点。第三，我们的研究提供了一个新的分子之间的联系胰岛素耗竭（AD的原因），Akt抑制，激活FOXO和神经变性Ab 1 -42神经毒性。意义：我们认为激活Akt或抑制钙调神经磷酸酶和Cdk 5， 消除FOXO信号传导，在AD中引起神经保护。在AD小鼠模型和人类临床样本中分析FOXO翻译后修饰与Cdk 5、CaN和Akt激活水平将有助于开发用于回顾性分析的新工具，以更好地了解AD发病机制。因此，我们期望确定FOXO转录途径有助于疾病发病机制的分子机制将是AD预防和治疗的重要一步。

DESCRIPTION (provided by applicant): Our long-term goal is to identify effective therapeutic targets for preventing neurodegeneration in Alzheimer's disease (AD). In this proposal, we investigate a novel mechanism of AD pathogenesis following our discovery of transcription factors FOXO1 and FOXO3a as novel Cyclin Dependent Kinase-5 (Cdk5) substrates. FOXO1 and FOXO3a are highly expressed in the brain, specifically in areas susceptible to neurodegeneration in AD. However, the roles and regulation of FOXOs in AD are unclear. The activation of FOXOs and their post- translational modifications have not been analyzed in human clinical samples. We show that the activation of FOXOs occurs early in AD clinical tissues and is highly neurotoxic in b-amyloid-mediated signaling. Further, our data suggest that FOXOs are activated by two independent mechanisms: (i) Cdk5-mediated phosphorylation (FOXO1 at S249, FOXO3a at S173); (ii) calcineurin-mediated dephosphorylation of FOXOs at Akt sites. Akt is known to phosphorylate FOXO1 and FOXO3a, rendering them inactive. Hypothesis: Cdk5 and calcineurin synergistically promote FOXOs' activation, which leads to neurotoxic b- amyloid processing and cell death, two hallmarks of AD. The impact of this work is that its successful completion will provide retrospective biomarkers and potential new strategies for AD treatment. This hypothesis will be tested by pursuing three specific aims: Aim 1: Dissect the molecular mechanism by which FOXOs are activated by Cdk5 and calcineurin in rat and human fetal neurons. Aim 2: Determine the roles of active FOXOs in promoting b-amyloid processing and neurotoxicity in rat and human fetal neurons. Aim 3: Investigate the clinical relevance of FOXOs' phosphorylation in two AD mouse models (APP/PS1 and p25-transgenic mice) and human clinical tissues. While APP/PS1 mice will confirm a global role of Ab in FOXO signaling, p25-mice will specifically demonstrate Cdk5's role in activating FOXOs and their correlation with neurodegeneration. Innovation: The hypothesis is formulated based on novel Cdk5 substrates FOXO1 and FOXO3a, discovered using a highly innovative chemical genetic approach. Second, this study suggests that inhibiting FOXOs directly or targeting the upstream regulators of FOXO signaling will provide novel therapeutic intervention points for preventing neurodegeneration in AD. Third, our study provides a novel molecular link between insulin depletion (a cause for AD), Akt inhibition, activation of FOXOs and neurodegeneration in Ab1-42 neurotoxicity. Significance: We propose that activating Akt or inhibiting calcineurin and Cdk5 will abrogate FOXO signaling, causing neuroprotection in AD. Analysis of FOXO post-translational modifications coupled with Cdk5, CaN and Akt activation levels in AD mouse models and human clinical samples will aid in the development of novel tools for retrospective analysis to better understand AD pathogenesis. Thus, we expect that determining the molecular mechanisms by which FOXO transcriptional pathway contributes to disease pathogenesis will be an important step forward in AD prevention and treatment.