Upregulation of MAM by C99 disrupts ACSL4 activity and phospholipid homeostasis in Alzheimer disease.
The structure and function of cellular and intracellular membranes are critically governed by the fatty acid (FA) composition of phospholipids (PLs), which is dynamically regulated by a network of enzymes that fine-tune lipid species according to cellular demands. In this study, we identify a mechanism through which the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) modulates the activity of the acyl-CoA synthetase long-chain family member 4 (ACSL4), an enzyme that channels polyunsaturated fatty acids (PUFAs) into phosphatidylcholine (PC) via the Lands cycle. Through integrated biochemical, proteomic, and lipidomic analyses in both cellular and animal models, we demonstrate that MAM formation enhances ACSL4 activity, promoting arachidonic acid (AA) activation and its preferential incorporation into PC in concert with the MAM-localized lysophospholipid acyltransferase 4 (LPCAT4). Our findings further uncover an unexpected link between this pathway and the pathogenesis of Alzheimers disease (AD). We show that elevated levels of C99--the {beta}-secretase cleavage product of amyloid precursor protein (APP)--induce MAM remodeling through cholesterol clustering, which in turn activates ACSL4 and alters PC composition. This effect is mirrored in AD models as well as in fibroblasts, neurons, and immune cells derived from both familial and sporadic AD patients, all of which exhibit chronically increased C99 levels, heightened ACSL4 activity, and enrichment of PUFA-containing PC species, leading to lipid imbalance and membrane dysfunction. Together, these results establish MAMs as dynamic lipid-regulatory hubs that coordinate ACSL4-dependent membrane remodeling and highlight the contribution of MAM dysregulation to lipid abnormalities observed in AD.