PROJECT SUMMARY Lipid membranes compartmentalize and protect the different areas of the cell, and regulate several cellular functions. Thus, alterations in the maintenance of a membrane's lipid composition can result in cellular dysfunction and disease. In order to maintain the balance of the various lipid components required to maintain proper membrane integrity, the cell has evolved interconnected enzymatic reactions that regulate lipid homeostasis in specific areas of the cell, so that they can be co-regulated more easily. One such cellular area is located in a functional subregion of the endoplasmic reticulum (ER) in close apposition to mitochondria, called MAMs (mitochondria-associated ER membranes). Given that these MAMs are centers of lipid regulation, alteration of MAM regulation results in the loss of lipid homeostasis, and in changes in the composition of other membranes, which in turn can trigger cellular dysfunction. Neurons are highly dependent on the lipid composition of their membranes to carry out their functions in the brain. Therefore, it is not surprising that many neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with alterations in lipid homeostasis. The results presented in this application suggest that lipid deregulation in AD is triggered by MAM dysfunction. Notably, we have found that C99, the 99-aa C- terminal domain of the amyloid precursor protein (APP), a protein associated with familial AD, is involved in the regulation of MAM activity and in lipid homeostasis. C99 is generated when the full-length APP is cleaved by β- secretase. After that, C99 is then cleaved by γ-secretase in the ER, to generate the β-amyloid (Aβ) found in senile plaques. Our data show that γ-secretase dysfunction results in the accumulation of uncleaved C99 at the MAM, altering MAM's function in the regulation of lipid homeostasis. Furthermore, in cells from AD patients and in animal models of AD, C99 levels are increased in MAM, resulting in perturbed lipid homeostasis, triggering some of the features of AD, including mitochondrial dysfunction. Based on these findings, we propose that MAM is a key component in the regulation of neuronal functionality, predominantly via its effects on lipid homeostasis. Moreover, we also hypothesize that MAM- localized C99 is a component of MAM regulation, and that its accumulation in AD is central to the lipid abnormalities observed in the disease. We now propose studies aimed at understanding the mechanism(s) by which MAM participates in the regulation of cellular lipid homeostasis. Specifically, we will (1) determine the particular role of C99 in the control of lipid homeostasis via regulation of MAM; (2) analyze the impact of altered MAM function on the lipid composition and functionality of neuronal cultures; (3) extend our findings to AD models to understand the relevance of MAM-localized C99 in the pathogenesis of the disease We believe that this is novel a novel approach to ...