[Abstract unavailable

CD8+ T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment1–4. Reactivating the cytotoxicity of CD8+ T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8+ T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme5, led to potentiated effector function and enhanced proliferation of CD8+ but not CD4+ T cells. This is due to the increase in the plasma membrane cholesterol level of CD8+ T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8+ T cells were better than wild-type CD8+ T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile6,7, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

Cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative diseases, including the abnormal accumulation of amyloid-β, one of the pathological hallmarks of Alzheimer disease (AD). Acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) are two enzymes that convert free cholesterol to cholesteryl esters. ACAT inhibitors have recently emerged as promising drug candidates for AD therapy. However, how ACAT inhibitors act in the brain has so far remained unclear. Here we show that ACAT1 is the major functional isoenzyme in the mouse brain. ACAT1 gene ablation (A1−) in triple transgenic (i.e., 3XTg-AD) mice leads to more than 60% reduction in full-length human APPswe as well as its proteolytic fragments, and ameliorates cognitive deficits. At 4 months of age, A1− causes a 32% content increase in 24-hydroxycholesterol (24SOH), the major oxysterol in the brain. It also causes a 65% protein content decrease in HMG-CoA reductase (HMGR) and a 28% decrease in sterol synthesis rate in AD mouse brains. In hippocampal neurons, A1− causes an increase in the 24SOH synthesis rate; treating hippocampal neuronal cells with 24SOH causes rapid declines in hAPP and in HMGR protein levels. A model is provided to explain our findings: in neurons, A1− causes increases in cholesterol and 24SOH contents in the endoplasmic reticulum, which cause reductions in hAPP and HMGR protein contents and lead to amelioration of amyloid pathology. Our study supports the potential of ACAT1 as a therapeutic target for treating certain forms of AD.Alzheimer disease | cholesterol esterification | lipid metabolism | oxysterols

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.

Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann–Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to 3 H-cholesteryl linoleate (CL) labeled-LDL, newly liberated 3 H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated 3 H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces ≥50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER.