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。保持线粒体 功能可以是防止AD脑中能量衰竭和抑制 活性氧（ROS）和半胱天冬酶的活化，从而减少神经元，神经胶质和血管细胞 功能障碍和死亡，并阻碍AD病理学的进展。我们之前的研究和其他研究 表明淀粉样蛋白β（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，醋甲唑胺（MTZ）和乙酰唑胺（ATZ），两个 对线粒体碳酸酐酶（CA）具有高度活性的碳酸酐酶抑制剂（CAIs）能够 预防脑血管和神经元细胞以及动物模型中的线粒体功能障碍和CytC释放 脑淀粉样变性，通过抑制线粒体H2 O2的产生和线粒体 去极化CAIs如MTZ和ATZ目前被FDA批准用于青光眼，高原病， 癫痫发作和其他临床适应症。以前的研究报道了CA表达在衰老和衰老过程中的增加， AD脑，确认CA作为AD治疗的新的和以前未探索的靶点的相关性。 我们假设CA抑制防止线粒体功能障碍和细胞死亡机制 在体外和体内由Aβ和tau诱导，改善神经血管病理和认知功能 AD模型中的损伤。在目标1中，我们将使用CAIs进行药物治疗， 转基因小鼠模型呈现淀粉样变性和tau蛋白病。我们将饲养3xTg-AD转基因 两种CAIs的小鼠。我们将在病理学发作之前（从6月龄开始）开始治疗小鼠，并且 当病理已经存在时（从11个月大开始）。线粒体功能，活性氧产生，半胱天冬酶 将在小鼠脑中以生物化学方式和通过以下方式评估活化、细胞死亡、淀粉样蛋白和tau沉积： 免疫组化认知功能将从行为上进行评价。在目标2中，我们将在体外评估 使用人神经元、神经胶质细胞和神经胶质细胞的CAIs预防tau介导的线粒体功能障碍的功效 和用磷酸化（病理性）tau种类攻击的培养物中的内皮细胞。在目标3中，我们 验证线粒体碳酸酐酶作为与AD病理学相关的重要靶标， 易受淀粉样蛋白和tau蛋白刺激的调节。我们将评估表达水平和特异性 CA-VA、-VB和-II在年龄匹配的AD和健康人脑中的定位。我们将沉默线粒体 在内皮细胞、神经胶质细胞和神经元细胞中的CA，以检验CA沉默将阻止线粒体 与CA抑制类似，Aβ和tau病理种类诱导的功能障碍途径和细胞死亡。 拟议的研究路线的目标是验证CA作为淀粉样变性和tau蛋白病的靶点，并将其转化为 这项研究将在不久的将来成为一项临床试验，以重新利用CAIs治疗AD和其他痴呆症。

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.