Targeting carbonic anhydrases in Alzheimer's disease

项目摘要 线粒体被认为是阿尔茨海默病(AD)的早期靶点之一。保存线粒体 功能是防止阿尔茨海默病大脑能量衰竭和抑制产生 活性氧(ROS)和caspase的激活，从而减少神经细胞、胶质细胞和血管细胞 功能障碍和死亡，并阻碍AD病理的进展。我们之前的研究和其他研究 研究表明，淀粉样β蛋白(Aβ)通过线粒体功能障碍和释放 细胞色素C(CytC)。我们的初步数据显示，甲唑胺(MTZ)和乙酰唑胺(ATZ)，两种 碳酸酐酶抑制剂(Cai)对线粒体碳酸酐酶(Ca)具有高度活性。 预防脑血管和神经细胞及动物模型中线粒体功能障碍和细胞色素C的释放 大脑淀粉样变性的发生，通过抑制线粒体过氧化氢的产生和线粒体 去极化。MTZ和ATZ等CAI目前被FDA批准用于青光眼、高原反应、 癫痫发作和其他临床指征。先前的研究报告了CA在衰老和衰老中的表达增加 AD脑，证实了CA作为AD治疗的一个新的和以前未探索的靶点的相关性。 我们假设CA抑制可阻止线粒体功能障碍和细胞死亡机制 A-β和Tau在体内外联合诱导改善神经血管病变和认知功能 AD模型中的损害。在目标1中，我们将在一个 同时表现为淀粉样变性和互动性病变的转基因小鼠模型。我们将喂养3xTg-AD转基因 有两个CAI的小鼠。我们将在小鼠发病前(从6个月大)开始治疗小鼠，并且 当病理已经存在时(从11个月龄开始)。线粒体功能、ROS产生、半胱氨酸天冬氨酸酶 激活、细胞死亡、淀粉样蛋白和tau沉积将在小鼠大脑中通过生化和 免疫组织化学。认知功能将从行为上进行评估。在目标2中，我们将在体外评估 CAIS预防tau介导的线粒体功能障碍的疗效 以及培养中的内皮细胞受到磷酸化(病理性)tau蛋白的挑战。在《目标3》中，我们将 验证线粒体碳酸酐酶作为AD病理和病理的重要靶点 易受淀粉样蛋白和tau蛋白的调节。我们将评估表达水平和特定的 CA-VA、-VB和-II在年龄匹配的阿尔茨海默病患者和健康人脑中的定位我们会让线粒体沉默 在血管内皮细胞、神经胶质细胞和神经细胞中检测CA沉默将阻止线粒体的假说 与CA抑制相似的A、β和tau病理性物种诱导的功能障碍通路和细胞死亡。 拟议的研究路线的目标是验证CA作为淀粉样变性和直肠肌萎缩症的靶点并将其翻译成 本研究旨在进行一项临床试验，以期在不久的将来将CAIS用于治疗AD和其他痴呆。

Project Summary Mitochondria are considered one of the early targets in Alzheimer’s disease (AD). Preserving mitochondrial function can be a key strategy to prevent energetic failure in the AD brain and to inhibit the production of reactive oxygen species (ROS) and activation of caspases, thereby reducing neuronal, glial and vascular cell dysfunction and death, and hindering the progression of AD pathology. Our previous studies and others demonstrated that amyloid beta (Aβ) causes brain cell death through mitochondrial dysfunction and release of cytochrome C (CytC). Our preliminary data revealed that methazolamide (MTZ) and acetazolamide (ATZ), two carbonic anhydrase inhibitors (CAIs) highly active on mitochondrial carbonic anhydrases (CAs) are able to prevent mitochondrial dysfunction and CytC release in brain vascular and neuronal cells and in animal models of cerebral amyloidosis, through the inhibition of mitochondrial H2O2 production and mitochondrial depolarization. CAIs such as MTZ and ATZ are currently FDA approved for glaucoma, high altitude sickness, seizures, and other clinical indications. Previous studies reported increases in CAs expression in the aging and AD brain, confirming the relevance of CAs as a novel and previously unexplored target for AD therapy. We hypothesize that CA inhibition prevents mitochondrial dysfunction and cell death mechanisms induced by both Aβ and tau in vitro and in vivo, ameliorating neurovascular pathology and cognitive impairment in AD models. In Aim 1, we will perform a pharmacological treatment with the CAIs in a transgenic mouse model presenting both amyloidosis and tauopathy. We will feed 3xTg-AD transgenic mice with the two CAIs. We will start treating mice prior to onset of pathology (from 6 months of age), and when pathology is already present (from 11 months of age). Mitochondrial function, ROS production, caspase activation, cell death, amyloid and tau deposition will be evaluated in the mouse brain biochemically and by immunohistochemistry. Cognitive function will be evaluated behaviorally. In Aim 2, we will assess in vitro the efficacy of CAIs for prevention of tau-mediated mitochondrial dysfunction, using human neuronal, glial and endothelial cells in culture challenged with phosphorylated (pathological) tau species. In Aim 3, we will validate mitochondrial carbonic anhydrases as important targets associated to AD pathology and susceptible to modulation by amyloid and tau challenge. We will assess expression levels and specific localization of CA-VA, -VB and -II in age-matched AD and healthy human brains. We will silence mitochondrial CAs in endothelial, glial and neuronal cells to test the hypothesis that CA silencing will prevent mitochondrial dysfunction pathways and cell death induced by Aβ and tau pathologic species similarly to CA inhibition. Goal of the proposed line of research is to validate CAs as a target for amyloidosis and tauopathy and translate this study to a clinical trial to repurpose CAIs for AD and other dementias in the close future.