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 (A?) 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 A? 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 A? 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.