Lipidome changes due to accumulation of cholesterol via APP‐C99 alters neuronal permeability

Abstract

γ-secretase activity is enriched at mitochondria-associated ER membranes (MAMs),a lipid-raft subdomain of the ER, where APP fragment C99 is delivered for its cleavage. Moreover, γ-secretase activity deficiency linked to AD mutations causes C99 accumulation at MAM, resulting in the upregulation of MAM activities such as sphingomyelin turnover and cholesterol esterification. We now explore whether the interaction of C99 with cholesterol could explain the link between C99 accumulation at MAM and the lipid abnormalities found in AD. Using cellular models of C99 accumulation versus a cholesterol-binding deficient C99, we assessed cholesterol uptake dynamics and sub-cellular distribution along with MAM-regulated functionalities. Also, we used a photo click activable cholesterol analog to study the proteins interacting with cholesterol at MAM in both conditions. Finally, we compare membrane permeability dynamics in neuronal models of Alzheimer's disease. C99 accumulation at MAM caused an increase in cholesterol uptake, cholesterol esterification, phospholipid synthesis and sphingomyelinase activity accompanied by a sub cellular redistribution of cholesterol to endolysosomes and ER, while C99-defective in cholesterol binding showed no changes in these activities. Moreover, when the cholesterol-interacting proteome of both conditions was compared, C99 caused a huge change/recruitment at MAM, especially of enzymes involved in lipid metabolism. ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4) activation was detected as an important player in the lipidome changes associated with C99 accumulation that led to membrane permeability alterations CONCLUSION: We report that the lipid alterations caused by pathogenic C99 accumulation are a consequence of an exacerbated uptake of extracellular cholesterol and mobilization towards MAM. The increase content of cholesterol at MAM driven by C99 might be responsible of the persistent activation of MAM and a subsequent alteration of the lipid metabolism since a cholesterol-binding deficient C99 fails to increase MAM activities and cholesterol trafficking. We also point to ACSL4 activation as a possible molecular switch for the permeability changes found in cellular models of AD.