Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity

Abstract

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer’s disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain unknown. Here, using biochemical fractionation techniques, we observe cholesterol accumulation at the MAM which leads to ACAT1/SOAT1 enrichment in this domain. MAM proteomics data suggests that ACAT1/SOAT1 inhibition strengthens the ER-mitochondria connection. Confocal and electron microscopy confirms that ACAT1/SOAT1 inhibition increases the number of ER-mitochondria contact sites and strengthens this connection by shortening the distance between these two organelles. This work demonstrates how directly manipulating local cholesterol levels at the MAM can alter inter-organellar contact sites and suggests that cholesterol buildup at the MAM is the impetus behind the therapeutic benefits of ACAT1/SOAT1 inhibition.

Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures

“…Regulation of the enzymatic activity through a differential localization would be a conceivable scenario. It is also possible that the translocation of ACAT into vesicles is associated with the strengthening of ERmitochondria connectivity in response to ACAT inhibition as recently reported [25].…”

Stealth Liposomes Encapsulating a Potent ACAT1/SOAT1 Inhibitor F12511: Pharmacokinetic, Biodistribution, and Toxicity Studies in Wild-Type Mice and Efficacy Studies in Triple Transgenic Alzheimer’s Disease Mice

“…Nanoparticles were prepared based on the procedure described in [ 48 ]. We previously found that when the high-affinity ACAT inhibitor such as F12511 or K604 are added to cells, the inhibition of the ACAT enzyme by F12511 or K604 remains unaffected by cell washing or the cell homogenization preparation process [ 24 , 48 ]. This finding implicates that once the inhibitor binds to the ACAT enzyme, it stays firmly bound for a few hours before dissociating from the enzyme.…”

“…In various cell types in the CNS, the gene expression levels of ACAT1 are much higher than those of ACAT2 (data retrieved from the public domain can be found in [ 20 ]). ACAT1 and ACAT2 are membrane proteins located in the endoplasmic reticulum (ER), and both enzymes are allosterically activated by sterols [ 21 , 22 , 23 , 24 ]. In addition, in microglia isolated from various neurodegenerative diseases and in vulnerable regions of human brains from LOAD, the ACAT1/SOAT1 gene is modestly induced (data retrieved from public domain can be found in [ 20 ]).…”

Towards a Unitary Hypothesis of Alzheimer’s Disease Pathogenesis

“…With respect to AD, C99 induces the formation of MAM via its ability to bind and “cluster” cholesterol, forming the raft, as described above. In turn, several proteins are recruited to the MAM so as to co-regulate multiple homeostatic pathways, some of which have been described above (e.g., cholesteryl ester synthesis via ACAT1 [ 219 ]). Alterations in the regulation of MAM formation, as occurs in AD, perturbs these pathways.…”