ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.

ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.

C-terminal of ABCA1 separately regulates cholesterol floppase activity and cholesterol efflux activity.

ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for cholesterol homeostasis. We recently reported that ABCA1 not only exports excess cholesterol in an apoA-I dependent manner, but that it also flops cholesterol from the inner to the outer leaflet of the plasma membrane. However, the relationship between these two activities of ABCA1 is still unclear. In this study, we analyzed the subcellular localization of ABCA1 by using a newly generated monoclonal antibody against its extracellular domain and the functions of eleven chimera proteins, in which the C-terminal domain of ABCA1 was replaced with those of the other ABCA subfamily members. We identified two motifs important for the functions of ABCA1. Three periodically repeated leucine residues were necessary for the cholesterol floppase activity but not the cholesterol efflux activity, while a VFVNFA motif was essential for both activities of ABCA1. These results suggest that the C-terminal of ABCA1 separately regulates the cholesterol floppase activity and the cholesterol efflux activity.

Lysophosphatidylcholine export by human ABCA7.

The ATP-binding cassette transporter A7 (ABCA7), which is highly expressed in the brain, is associated with the pathogenesis of Alzheimer's disease (AD). However, the physiological function of ABCA7 and its transport substrates remain unclear. Immunohistochemical analyses of human brain sections from AD and non-AD subjects revealed that ABCA7 is expressed in neuron and microglia cells in the cerebral cortex. The transport substrates and acceptors were identified in BHK/ABCA7 cells and compared with those of ABCA1. Like ABCA1, ABCA7 exported choline phospholipids in the presence of apoA-I and apoE; however, unlike ABCA1, cholesterol efflux was marginal. Lipid efflux by ABCA7 was saturated by 5µg/ml apoA-I and was not dependent on apoE isoforms, whereas efflux by ABCA1 was dependent on apoA-I up to 20µg/ml and apoE isoforms. Liquid chromatography-tandem mass spectrometry analyses revealed that the two proteins had different preferences for phospholipid export: ABCA7 preferred phosphatidylcholine (PC)≥lysoPC>sphingomyelin (SM)=phosphatidylethanolamine (PE), whereas ABCA1 preferred PC>>SM>PE=lysoPC. The major difference in the pattern of lipid peaks between ABCA7 and ABCA1 was the high lysoPC/PC ratio of ABCA7. These results suggest that lysoPC is one of the major transport substrates for ABCA7 and that lysoPC export may be a physiologically important function of ABCA7 in the brain.

A chemical probe that labels human pluripotent stem cells.

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

The effects of neurological disorder-related codon variations of ABCA13 on the function of the ABC protein.

Rare coding variants of ATP-binding cassette protein A13 (ABCA13) contribute to the risk of neurological disorders, but little is known about the physiological function of ABCA13 and how single nucleotide polymorphisms (SNPs) affect it. Here, we examined the effects of neurological disorder-related SNPs ABCA13, T4031A and R4843C in the context of ABCA1, and found that the former SNP (T1088A in ABCA1) severely impaired the ABCA1 functions of apolipoprotein A-I (apoA-I) binding and cholesterol efflux. The antibody against mouse ABCA13 reacted with neurons in the cerebral cortex, hippocampus, and cerebellum. These results suggest that the T4031A replacement affects the function of ABCA13 in the brain.

Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex.

The loading of antigenic peptides onto major histocompatibility complex class I (MHC I) molecules is an essential step in the adaptive immune response against virally or malignantly transformed cells. The ER-resident peptide-loading complex (PLC) consists of the transporter associated with antigen processing (TAP1 and TAP2), assembled with the auxiliary factors tapasin and MHC I. Here, we demonstrated that the N-terminal extension of each TAP subunit represents an autonomous domain, named TMD(0), which is correctly targeted to and inserted into the ER membrane. In the absence of coreTAP, each TMD(0) recruits tapasin in a 1:1 stoichiometry. Although the TMD(0)s lack known ER retention/retrieval signals, they are localized to the ER membrane even in tapasin-deficient cells. We conclude that the TMD(0)s of TAP form autonomous interaction hubs linking antigen translocation into the ER with peptide loading onto MHC I, hence ensuring a major function in the integrity of the antigen-processing machinery.

Function and regulation of ABCA1--membrane meso-domain organization and reorganization.

The ATP-binding cassette protein A1 (ABCA1) mediates the secretion of cellular-free cholesterol and phospholipids to an extracellular acceptor, apolipoprotein A-I, to form high-density lipoprotein. Because ABCA1 is a key factor in cholesterol homeostasis, elaborate transcriptional and post-transcriptional regulations of ABCA1 have evolved to maintain cholesterol homeostasis. Recent studies suggest that ABCA1 moves lipids not only between membranes but also within membranes to organize and reorganize membrane meso-domains to modulate cell proliferation and immunity.