Alzheimer's disease (AD) remains a looming public health crisis, despite intensive research and pharmaceutical development efforts. No effective treatment option is currently available that can halt the disease process. The recent failures of high-profile clinical trials targeting the amyloid plaques and neurofibrillary tangles, the pathological hallmarks of the AD identified by Dr. Alois Alzheimer more than a century ago and the focus of extensive research and pharmaceutical development efforts, suggest that new directions in delineating the pathogenic mechanisms of AD are warranted before effective treatment of the disease can be achieved. Mitochondria are dynamic and complex organelles with essential roles in many aspects of biology, from energy production and intermediary metabolism to intracellular signaling and apoptosis. These broad functions position mitochondrion as a central player in human health. In neurons, mitochondria and synapses are intimately linked. In addition to the central role of mitochondria in bioenergetics, they are also critically important for maintaining cellular Ca2+ homeostasis. Ca2+ uptake by mitochondria helps buffer cytosolic Ca2+ transients arising from neuronal activation, protecting against the detrimental effects of bursts of Ca2+ influx. Under basal conditions, Ca2+ entry into mitochondria is needed for normal neuronal physiology. The ER- mitochondria contact site (ERMCS) are recognized as key cellular structures regulating mito-Ca2+ homeostasis. Moreover, there is an emerging recognition of ERMCS impairment in neurodegenerative diseases including AD. How ERMCS and mito-Ca2+ homeostasis are altered, and their contribution to disease phenotypes in in vivo settings, however, are not well understood. The goal of this proposal is to test the central hypothesis that an interplay between APP metabolism and ERMCS directs ER-mitochondrial Ca2+ signaling, and that defects in this process contributes to the etiology of AD. To test this hypothesis, we propose to achieve the following Specific Aims in this exploratory project: Aim 1. Examine defects in ERMCS formation in a Drosophila AD model and AD patient derived cells; Aim 2. Test the roles of ERMCS proteins that direct mito-Ca2+ homeostasis in mediating APP function in disease pathogenesis. By providing evidence for the involvement of ERMCS and mito-Ca2+ in APP function at the organellar, synaptic, and organismal levels, these studies will lay the foundation for future studies addressing the regulation and function of ERMCS in normal brain physiology, which will significantly advance our understanding of the fundamental roles of mitochondria and Ca2+ signaling in AD and ultimately offer novel therapeutic strategies.