Jie Luo

Cholesterol must be precisely partitioned within cells, with the plasma membrane (PM) holding the highest levels. In contrast, the endoplasmic reticulum (ER)—the site of cholesterol synthesis—contains very little cholesterol. How newly synthesized cholesterol moves from the ER to the PM remains poorly defined. Here, we identify a COPII-independent trafficking route in which nascent cholesterol is exported via specialized cholesterol transport vesicles (CTVs). A genome-wide screen reveals that DEGS1-derived ceramide is essential for CTV formation at the ER. Biochemical purification shows that caveolins serve as the vesicle coat, and loss of caveolins eliminates CTV production. Silencing PACSINs or dynamins leads to intracellular accumulation of CTVs, suggesting impaired vesicle scission. CTV delivery to the PM depends on actin filaments and the motors MYO1C and MYO1E. In vitro reconstitution with giant unilamellar vesicles demonstrates that ceramide promotes inward budding, whereas CAV1 drives outward budding. This work identifies a previously unrecognized class of ER-derived, caveolin-coated vesicles that transport cholesterol to the PM.

Elevated blood low-density lipoprotein (LDL) levels are a major risk factor for cardiovascular disease. Increasing LDL receptor (LDLR) expression effectively reduces blood LDL and serves as a key therapeutic strategy for preventing and treating cardiovascular disease. However, it remains unclear how LDLR in different aortic cells contributes to atherosclerosis progression. In this study, we found that hepatocyte-specific deletion of Ldlr in combination with high-fat, high-cholesterol diet feeding induced hypercholesterolemia and atherosclerosis in mice. On this background, further deletion of Ldlr in endothelial cells or smooth muscle cells had no significant effects on atherosclerosis, whereas myeloid-selective ablation of Ldlr markedly attenuated atherosclerotic plaque formation. The decreased percentages of T cells and natural killer T cells in the aorta of mice lacking Ldlr in myeloid cells partially explained the reduced atherosclerotic burden, despite that bone marrow-derived macrophages from Ldlr knockout mice could still be induced to form foam cells in vitro. Therefore, LDLR is better to be elevated in a cell-specific manner for cardiovascular disease prevention and treatment.

Localization of lysosomes influences their properties, e.g., perinuclear lysosomes are more acidic but less mobile compared with the peripheral ones. Furthermore, the endoplasmic reticulum (ER) can actively regulate the dynamics and functions of lysosomes via membrane contact sites. In this study, we find that ER-resident apoptosis-resistant E3 ubiquitin protein ligase 1 (AREL1) establishes membrane contacts with lysosomes by directly interacting with the V _o a subunit of V-ATPase. AREL1 also catalyzes K33-linked polyubiquitylation of V-ATPase V ₁ B2 subunit, inducing its binding to UBAC2 localized in the perinuclear ER. Depletion of AREL1 or UBAC2 increases the number of peripheral lysosomes that possess partially assembled V-ATPase, elevated luminal pH, and attenuated degradative capacity. Knockdown of ZRANB1, the deubiquitylating enzyme that antagonizes AREL1-mediated V ₁ B2 ubiquitylation, promotes perinuclear clustering of lysosomes and increases lysosomal acidity and degradation. Mice lacking Arel1 exhibit age-dependent Purkinje cell loss, an ataxic phenotype, and motor impairment. Lipofuscin accumulation in the residual Purkinje cells of Arel1 ^−/− mice indicates lysosomal dysfunction. Orchestration of lysosomal positioning and function by the AREL1–UBAC2–V-ATPase axis underscores the physiological significance of ER-regulated perinuclear lysosomal positioning in neurons.

Cholesterol from low-density lipoprotein (LDL) can be transported to many organelle membranes by non-vesicular mechanisms involving sterol transfer proteins (STPs). Fatty acid–binding protein (FABP) 7 was identified in our previous study searching for new regulators of intracellular cholesterol trafficking. Whether FABP7 is a bona fide STP remains unknown. Here, we found that FABP7 deficiency resulted in the accumulation of LDL-derived cholesterol in lysosomes and reduced cholesterol levels on the plasma membrane. A crystal structure of human FABP7 protein in complex with cholesterol was resolved at 2.7 Å resolution. In vitro, FABP7 efficiently transported the cholesterol analog dehydroergosterol between the liposomes. Further, the silencing of FABP3 and 8, which belong to the same family as FABP7, caused robust cholesterol accumulation in lysosomes. These two FABP proteins could transport dehydroergosterol in vitro as well. Collectively, our results suggest that FABP3, 7, and 8 are a new class of STPs mediating cholesterol egress from lysosomes.

Niemann-Pick C1-like 1 (NPC1L1) is essential for intestinal cholesterol absorption. Together with the cholesterol-rich and Flotillin-positive membrane microdomain, NPC1L1 is internalized via clathrin-mediated endocytosis and transported to endocytic recycling compartment (ERC). When ERC cholesterol level decreases, NPC1L1 interacts with LIMA1 and moves back to plasma membrane. However, how cholesterol leaves ERC is unknown. Here, we find that, in male mice, intracellular bile acids facilitate cholesterol transport to other organelles, such as endoplasmic reticulum, in a non-micellar fashion. When cholesterol level in ERC is decreased by bile acids, the NPC1L1 carboxyl terminus that previously interacts with the cholesterol-rich membranes via the A₁₂₇₂LAL residues dissociates from membrane, exposing the Q₁₂₇₇KR motif for LIMA1 recruitment. Then NPC1L1 moves back to plasma membrane. This study demonstrates an intracellular cholesterol transport function of bile acids and explains how the substantial amount of cholesterol in NPC1L1-positive compartments is unloaded in enterocytes during cholesterol absorption.

The low-density lipoprotein (LDL) is a major cholesterol carrier in circulation and is internalized into cells through LDL receptor (LDLR)-mediated endocytosis. The LDLR protein is highly expressed in the steroidogenic organs and LDL cholesterol is an important source for steroidogenesis. Cholesterol must be transported into the mitochondria, where steroid hormone biosynthesis initiates. However, how LDL cholesterol is conveyed to the mitochondria is poorly defined. Here, through genome-wide small hairpin RNA screening, we find that the outer mitochondrial membrane protein phospholipase D6 (PLD6), which hydrolyses cardiolipin to phosphatidic acid, accelerates LDLR degradation. PLD6 promotes the entrance of LDL and LDLR into the mitochondria, where LDLR is degraded by mitochondrial proteases and LDL-carried cholesterol is used for steroid hormone biosynthesis. Mechanistically, the outer mitochondrial membrane protein CISD2 binds to the cytosolic tail of LDLR and tethers LDLR⁺ vesicles to the mitochondria. The fusogenic lipid phosphatidic acid generated by PLD6 facilitates the membrane fusion of LDLR⁺ vesicles with the mitochondria. This intracellular transport pathway of LDL–LDLR bypasses the lysosomes and delivers cholesterol to the mitochondria for steroidogenesis.

Peroxisomal disorders (PDs) are a heterogenous group of diseases caused by defects in peroxisome biogenesis or functions. X-linked adrenoleukodystrophy is the most prevalent form of PDs and results from mutations in the ABCD1 gene, which encodes a transporter mediating the uptake of very long-chain fatty acids (VLCFAs). The curative approaches for PDs are very limited. Here, we investigated whether cholesterol accumulation in the lysosomes is a biochemical feature shared by a broad spectrum of PDs. We individually knocked down fifteen PD-associated genes in cultured cells and found ten induced cholesterol accumulation in the lysosome. 2-Hydroxypropyl-β-cyclodextrin (HPCD) effectively alleviated the cholesterol accumulation phenotype in PD-mimicking cells through reducing intracellular cholesterol content as well as promoting cholesterol redistribution to other cellular membranes. In ABCD1 knockdown cells, HPCD treatment lowered reactive oxygen species and VLCFA to normal levels. In Abcd1 knockout mice, HPCD injections reduced cholesterol and VLCFA sequestration in the brain and adrenal cortex. The plasma levels of adrenocortical hormones were increased and the behavioral abnormalities were greatly ameliorated upon HPCD administration. Together, our results suggest that defective cholesterol transport underlies most, if not all, PDs, and that HPCD can serve as a novel and effective strategy for the treatment of PDs.

High cholesterol is a major risk factor for cardiovascular disease¹. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease². ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins³. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces⁴, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.

Low-density lipoprotein (LDL) is the main carrier of cholesterol and cholesteryl ester in circulation. High plasma levels of LDL cholesterol (LDL-C) are a major risk factor of atherosclerotic cardiovascular disease (ASCVD). LDL-C lowering is recommended by many guidelines for the prevention and treatment of ASCVD. Statins, ezetimibe, and proprotein convertase subtilisin/kexin type 9 inhibitors are the mainstay of LDL-C-lowering therapy. Novel therapies are also emerging for patients who are intolerant to statins or respond poorly to standard treatments. Here, we review the most recent advances on LDL-C-lowering drugs, focusing on the mechanisms by which they act to reduce LDL-C levels. The article starts with the cornerstone therapies applicable to most patients at risk for ASCVD. Special treatments for those with little or no LDL receptor function then follow. The inhibitors of ATP-citrate lyase and cholesteryl ester transfer protein, which are recently approved and still under investigation for LDL-C lowering, respectively, are also included. Strategies targeting the stability of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol catabolism can be novel regimens to reduce LDL-C levels and cardiovascular risk.