Exploring FOXO Signaling in Promoting Neurodegeneration in AD

描述(申请人提供)：我们的长期目标是确定有效的治疗目标，以防止阿尔茨海默病(AD)的神经退化。在这个建议中，我们发现了转录因子FOXO1和FOXO3a作为新的细胞周期蛋白依赖性激酶-5(CDK5)底物，从而探讨了AD发病的新机制。Foxo1和FOXO3a在大脑中高度表达，特别是在阿尔茨海默病易患神经变性的区域。然而，FOXO在AD中的作用和调控尚不清楚。FOXO的激活及其翻译后修饰尚未在人类临床样本中进行分析。我们发现FOXO的激活早期发生在AD的临床组织中，并且在b-淀粉样蛋白介导的信号转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被激活：(I)CDK5介导的磷酸化(FOXO1在S249，FOXO3a在S173)；(Ii)钙调神经磷酸酶介导的FOXO在Akt位点的去磷酸化。已知AKT能使FOXO1和FOXO3a磷酸化，使它们失去活性。假设：CDK5和钙调神经磷酸酶协同促进FOXO的激活，导致神经毒性的b-淀粉样蛋白加工和细胞死亡，这是AD的两个特征。这项工作的影响是，它的成功完成将为AD的治疗提供追溯的生物标志物和潜在的新策略。这一假说将通过追求三个特定的目标来检验：目的1：剖析CDK5和钙调神经磷酸酶激活FOXO的分子机制。目的2：确定活性FOXO在促进大鼠和人胎神经元β-淀粉样蛋白加工和神经毒性方面的作用。目的：探讨FOXO在两种AD小鼠模型(APP/PS1和p25转基因小鼠)和人类临床组织中磷酸化的临床意义。虽然APP/PS1小鼠将确认抗体在FOXO信号转导中的全球作用，但p25-小鼠将专门展示CDK5‘S在FOXO激活中的作用及其与神经退行性变的关系。创新：该假说是基于使用高度创新的化学遗传学方法发现的新型CDK5底物FOXO1和FOXO3a而形成的。其次，本研究表明，直接抑制FOXO或靶向FOXO信号的上游调节因子将为预防AD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联系，胰岛素耗竭(AD的一个原因)，Akt抑制，FOXO的激活和AB1-42神经毒性中的神经退行性变。意义：我们认为激活Akt或抑制CaN和CDK5将 取消FOXO信号，在AD中起到神经保护作用。分析AD小鼠模型和人类临床样本中FOXO翻译后修饰与CDK5、CaN和Akt激活水平的关系，将有助于开发新的追溯分析工具，以更好地了解AD的发病机制。因此，我们预计，确定FOXO转录途径参与疾病发病的分子机制将是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.