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• W3113241336 abstract "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. 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. ATP-binding cassette (ABC) proteins constitute a transporter superfamily that plays important physiological roles in all living organisms (1Ueda K. ABC proteins protect the human body and maintain optimal health.Biosci. Biotechnol. Biochem. 2011; 75: 401-409Crossref PubMed Scopus (62) Google Scholar, 2Schneider E. Hunke S. ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains.FEMS Microbiol. Rev. 1998; 22: 1-20Crossref PubMed Google Scholar). ABC proteins couple the energy of ATP binding and hydrolysis to many biological processes such as the translocation of various substrates including lipids, ions, peptides, and xenobiotics (3Davidson A.L. Dassa E. Orelle C. Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems.Microbiol. Mol. Biol. Rev. 2008; 72: 317-364Crossref PubMed Scopus (959) Google Scholar). Defects in the function and expression of ABC proteins are related to various diseases (1Ueda K. ABC proteins protect the human body and maintain optimal health.Biosci. Biotechnol. Biochem. 2011; 75: 401-409Crossref PubMed Scopus (62) Google Scholar). ATP-binding cassette subfamily A member 13 (ABCA13) is a transmembrane protein with the typical structure of ABC proteins: two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) characterized by Walker A, Walker B, and ABC signature motifs (4Prades C. Arnould I. Annilo T. Shulenin S. Chen Z.Q. Orosco L. Triunfol M. Devaud C. Maintoux-Larois C. Lafargue C. Lemoine C. Denèfle P. Rosier M. Dean M. The human ATP binding cassette gene ABCA13, located on chromosome 7p12.3, encodes a 5058 amino acid protein with an extracellular domain encoded in part by a 4.8-kb conserved exon.Cytogenet. Genome Res. 2002; 98: 160-168Crossref PubMed Scopus (29) Google Scholar). ABCA13 is predicted to be the largest member of the ABC protein family, and the human form includes 5058 amino acid residues and a long N-terminal region. In addition, alternative ABCA13 transcripts and protein lacking the N-terminal region were also reported (5Maeß M.B. Stolle K. Cullen P. Lorkowski S. Evidence for an alternative genomic structure, mRNA and protein sequence of human ABCA13.Gene. 2013; 515: 298-307Crossref PubMed Scopus (6) Google Scholar, 6Barros S.A. Tennant R.W. Cannon R.E. Molecular structure and characterization of a novel murine ABC transporter, Abca13.Gene. 2003; 307: 191-200Crossref PubMed Scopus (20) Google Scholar). The calculated molecular masses of full-size and shorter human ABCA13 are about 570 kDa and 260 kDa, respectively. ABCA13 is expressed in the human trachea, testis, bone marrow, brain, and other tissues (4Prades C. Arnould I. Annilo T. Shulenin S. Chen Z.Q. Orosco L. Triunfol M. Devaud C. Maintoux-Larois C. Lafargue C. Lemoine C. Denèfle P. Rosier M. Dean M. The human ATP binding cassette gene ABCA13, located on chromosome 7p12.3, encodes a 5058 amino acid protein with an extracellular domain encoded in part by a 4.8-kb conserved exon.Cytogenet. Genome Res. 2002; 98: 160-168Crossref PubMed Scopus (29) Google Scholar, 5Maeß M.B. Stolle K. Cullen P. Lorkowski S. Evidence for an alternative genomic structure, mRNA and protein sequence of human ABCA13.Gene. 2013; 515: 298-307Crossref PubMed Scopus (6) Google Scholar, 7Knight H.M. Pickard B.S. Maclean A. Malloy M.P. Soares D.C. McRae A.F. Condie A. White A. Hawkins W. McGhee K. van Beck M. MacIntyre D.J. Starr J.M. Deary I.J. Visscher P.M. et al.A cytogenetic abnormality and rare coding variants identify ABCA13 as a candidate gene in schizophrenia, bipolar disorder, and depression.Am. J. Hum. Genet. 2009; 85: 833-846Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). ABCA13 belongs to the ABCA subfamily (8Albrecht C. Viturro E. The ABCA subfamily--gene and protein structures, functions and associated hereditary diseases.Pflugers Arch. 2007; 453: 581-589Crossref PubMed Scopus (87) Google Scholar). Most proteins in this subfamily are reported to transport lipids (9Piehler A.P. Ozcürümez M. Kaminski W.E. A-subclass ATP-binding cassette proteins in brain lipid homeostasis and neurodegeneration.Front. Psychiatry. 2012; 3: 17Crossref PubMed Scopus (40) Google Scholar), including cholesterol and phosphatidylcholine by ABCA1 (10Tanaka A.R. Abe-Dohmae S. Ohnishi T. Aoki R. Morinaga G. Okuhira K. Ikeda Y. Kano F. Matsuo M. Kioka N. Amachi T. Murata M. Yokoyama S. Ueda K. Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.J. Biol. Chem. 2003; 278: 8815-8819Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 11Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar), surfactant lipids by ABCA3 (12Ban N. Matsumura Y. Sakai H. Takanezawa Y. Sasaki M. Arai H. Inagaki N. ABCA3 as a lipid transporter in pulmonary surfactant biogenesis.J. Biol. Chem. 2007; 282: 9628-9634Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Cheong N. Madesh M. Gonzales L.W. Zhao M. Yu K. Ballard P.L. Shuman H. Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome.J. Biol. Chem. 2006; 281: 9791-9800Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Weichert N. Kaltenborn E. Hector A. Woischnik M. Schams A. Holzinger A. Kern S. Griese M. Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells.Respir. Res. 2011; 12: 4Crossref PubMed Scopus (75) Google Scholar, 15Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells.FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (234) Google Scholar), N-retinylidene-phosphatidylethanolamine by ABCA4 (16Beharry S. Zhong M. Molday R.S. N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR).J. Biol. Chem. 2004; 279: 53972-53979Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), phosphatidylcholine and lysophosphatidylcholine by ABCA7 (17Tomioka M. Toda Y. Mañucat N.B. Akatsu H. Fukumoto M. Kono N. Arai H. Kioka N. Ueda K. Lysophosphatidylcholine export by human ABCA7.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862: 658-665Crossref PubMed Scopus (33) Google Scholar, 18Wang N. Lan D. Gerbod-Giannone M. Linsel-Nitschke P. Jehle A.W. Chen W. Martinez L.O. Tall A.R. ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux.J. Biol. Chem. 2003; 278: 42906-42912Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and glucosylceramide by ABCA12 (19Akiyama M. Sugiyama-Nakagiri Y. Sakai K. McMillan J.R. Goto M. Arita K. Tsuji-Abe Y. Tabata N. Matsuoka K. Sasaki R. Sawamura D. Shimizu H. Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J. Clin. Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (295) Google Scholar). These findings suggest that ABCA13 too transports lipids. However, the function of ABCA13 is unknown. Interestingly, rare genetic variants of human ABCA13 are related to susceptibility for schizophrenia, bipolar disorder, and major depression (7Knight H.M. Pickard B.S. Maclean A. Malloy M.P. Soares D.C. McRae A.F. Condie A. White A. Hawkins W. McGhee K. van Beck M. MacIntyre D.J. Starr J.M. Deary I.J. Visscher P.M. et al.A cytogenetic abnormality and rare coding variants identify ABCA13 as a candidate gene in schizophrenia, bipolar disorder, and depression.Am. J. Hum. Genet. 2009; 85: 833-846Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), but not without controversy. For example, one replication study on some of these rare variants failed to find this relation in an independent sample set (20Dwyer S. Williams H. Jones I. Jones L. Walters J. Craddock N. Owen M.J. O'Donovan M.C. Investigation of rare non-synonymous variants at ABCA13 in schizophrenia and bipolar disorder.Mol. Psychiatry. 2011; 16: 790-791Crossref PubMed Scopus (15) Google Scholar). A monkey carrying the heterozygous ABCA13 deletion displayed impaired social ability and restricted and repetitive behaviors that are most frequently associated with autism spectrum disorder (21Iritani S. Torii Y. Habuchi C. Sekiguchi H. Fujishiro H. Yoshida M. Go Y. Iriki A. Isoda M. Ozaki N. The neuropathological investigation of the brain in a monkey model of autism spectrum disorder with ABCA13 deletion.Int. J. Dev. Neurosci. 2018; 71: 130-139Crossref PubMed Scopus (13) Google Scholar, 22Yoshida K. Go Y. Kushima I. Toyoda A. Fujiyama A. Imai H. Saito N. Iriki A. Ozaki N. Isoda M. Single-neuron and genetic correlates of autistic behavior in macaque.Sci. Adv. 2016; 2e1600558Crossref PubMed Scopus (38) Google Scholar). However, the same monkey had a nonsense mutation in 5-hydroxytryptamine (serotonin) receptor 2C. 5-Hydroxytryptamine receptor 2C has been associated with neuropsychiatric disorders including autism spectrum disorder, leaving it unclear whether the heterozygous ABCA13 deletion caused the autism spectrum disorder-like phenotypes. Additionally, in a Drosophila model, knockdown of CG1718, which is homologous to human ABCA13, induced increased social space and abnormal circadian rhythm (23Ueoka I. Kawashima H. Konishi A. Aoki M. Tanaka R. Yoshida H. Maeda T. Ozaki M. Yamaguchi M. Novel Drosophila model for psychiatric disorders including autism spectrum disorder by targeting of ATP-binding cassette protein A.Exp. Neurol. 2018; 300: 51-59Crossref PubMed Scopus (22) Google Scholar). However, in reality, the amino acid sequence and protein size of CG1718 are more similar to that of ABCA3 than ABCA13. In this study, to elucidate the molecular functions of ABCA13, we examined the subcellular localization and function of ABCA13 in HEK293 cells transfected with mouse ABCA13. To study the physiological roles of ABCA13, we generated Abca13 KO mice using the clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) system and investigated the impact of ABCA13 deletion on behavioral phenotypes. In addition, we examined the intracellular cholesterol distribution and synaptic vesicle endocytosis in Abca13 KO cortical neuronal cultures. We performed ABCA13 mutant analysis to determine the effects of mutations associated with psychiatric disorders on ABCA13 function, finding that abnormal psychological behaviors correlated with impairments in the subcellular localization of and cholesterol trafficking by ABCA13. To determine the size of ABCA13 mainly expressed in mice, western blotting was performed using mouse kidney (Fig. 1A), where Abca13 is highly expressed. A major band was mainly detected at >460 kDa, which is consistent with the predicted molecular mass of full-size ABCA13 (570 kDa). This result indicates that ABCA13 is mainly expressed as a protein containing the long N-terminal region in mice. To study the biochemical activity of full-size ABCA13, we examined the protein's subcellular localization. HEK293 cells were transiently transfected with a plasmid expressing full-length mouse ABCA13 (5034 amino acids). Western blot analysis (Fig. 1B) showed a band of similar size to ABCA13 expressed in vivo. Immunostaining revealed that ABCA13 was localized in intracellular vesicles in HEK293 cells (Fig. 1C). Many members of the ABCA subfamily play important roles in lipid transport processes (8Albrecht C. Viturro E. The ABCA subfamily--gene and protein structures, functions and associated hereditary diseases.Pflugers Arch. 2007; 453: 581-589Crossref PubMed Scopus (87) Google Scholar, 10Tanaka A.R. Abe-Dohmae S. Ohnishi T. Aoki R. Morinaga G. Okuhira K. Ikeda Y. Kano F. Matsuo M. Kioka N. Amachi T. Murata M. Yokoyama S. Ueda K. Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.J. Biol. Chem. 2003; 278: 8815-8819Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 11Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 12Ban N. Matsumura Y. Sakai H. Takanezawa Y. Sasaki M. Arai H. Inagaki N. ABCA3 as a lipid transporter in pulmonary surfactant biogenesis.J. Biol. Chem. 2007; 282: 9628-9634Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 13Cheong N. Madesh M. Gonzales L.W. Zhao M. Yu K. Ballard P.L. Shuman H. Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome.J. Biol. Chem. 2006; 281: 9791-9800Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Weichert N. Kaltenborn E. Hector A. Woischnik M. Schams A. Holzinger A. Kern S. Griese M. Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells.Respir. Res. 2011; 12: 4Crossref PubMed Scopus (75) Google Scholar, 15Yamano G. Funahashi H. Kawanami O. Zhao L.X. Ban N. Uchida Y. Morohoshi T. Ogawa J. Shioda S. Inagaki N. ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells.FEBS Lett. 2001; 508: 221-225Crossref PubMed Scopus (234) Google Scholar, 16Beharry S. Zhong M. Molday R.S. N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR).J. Biol. Chem. 2004; 279: 53972-53979Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 17Tomioka M. Toda Y. Mañucat N.B. Akatsu H. Fukumoto M. Kono N. Arai H. Kioka N. Ueda K. Lysophosphatidylcholine export by human ABCA7.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862: 658-665Crossref PubMed Scopus (33) Google Scholar, 18Wang N. Lan D. Gerbod-Giannone M. Linsel-Nitschke P. Jehle A.W. Chen W. Martinez L.O. Tall A.R. ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux.J. Biol. Chem. 2003; 278: 42906-42912Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 19Akiyama M. Sugiyama-Nakagiri Y. Sakai K. McMillan J.R. Goto M. Arita K. Tsuji-Abe Y. Tabata N. Matsuoka K. Sasaki R. Sawamura D. Shimizu H. Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J. Clin. Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (295) Google Scholar). To determine whether ABCA13 is involved in lipid trafficking, we compared the intracellular cholesterol distribution in control and ABCA13-expressing HEK293 cells using the fluorescent cholesterol-binding probe filipin (Fig. 2A). ABCA13-positive vesicles were colocalized with the fluorescent signals in ABCA13-transfected cells. On the other hand, the intense signals of filipin in vesicles were much less observed in control cells. The fluorescent probe EGFP-D4 was reported to target cholesterol-rich membrane domains (24Liu S.L. Sheng R. Jung J.H. Wang L. Stec E. O'Connor M.J. Song S. Bikkavilli R.K. Winn R.A. Lee D. Baek K. Ueda K. Levitan I. Kim K.P. Cho W. Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.Nat. Chem. Biol. 2017; 13: 268-274Crossref PubMed Scopus (129) Google Scholar, 25Ishitsuka R. Saito T. Osada H. Ohno-Iwashita Y. Kobayashi T. Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution.J. Lipid Res. 2011; 52: 2084-2094Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Consistently, the vesicles where ABCA13 was detected were also strongly labeled with EGFP-D4 (Fig. 2B), but EGFP-D4 staining was hardly observed in control cells. Additionally, quantitative image analysis showed that EGFP-D4 staining was significantly higher in ABCA13-transfected cells compared with mock cells (Fig. 2C). These results suggest that transiently expressed ABCA13 causes cholesterol accumulation in vesicles. To examine whether human endogenous ABCA13 is localized in intracellular vesicles and affects intracellular cholesterol distribution, we used U2OS cells, a human osteosarcoma cell line that expresses endogenous ABCA13 (Fig. S1). As with our HEK293 experiments, in U2OS cells, intracellular vesicles where ABCA13 was detected were strongly stained with filipin (Fig. 2D). These results indicate that endogenously expressed human ABCA13 causes cholesterol accumulation in intracellular vesicles (ABCA13-vesicles) like mouse ABCA13. Lipids are transferred between the plasma membrane and subcellular organelles (26Ikonen E. Cellular cholesterol trafficking and compartmentalization.Nat. Rev. Mol. Cell Biol. 2008; 9: 125-138Crossref PubMed Scopus (996) Google Scholar, 27Jackson C.L. Walch L. Verbavatz J.M. Lipids and their trafficking: an integral part of cellular organization.Dev. Cell. 2016; 39: 139-153Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Since the plasma membrane contains 40–90% of total cellular cholesterol (28Litvinov D.Y. Savushkin E.V. Dergunov A.D. Intracellular and plasma membrane events in cholesterol transport and homeostasis.J. Lipids. 2018; 2018: 3965054Crossref PubMed Google Scholar), we hypothesized that the cholesterol accumulated in vesicles by ABCA13 was derived from the plasma membrane. To evaluate this possibility, we used EGFP-D4 to track plasma-membrane cholesterol in living cells. Incubation at 37 °C can internalize EGFP-D4, which binds to cholesterol of the exofacial leaflets of the plasma membrane, in living cells (29Maekawa M. Fairn G.D. Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.J. Cell Sci. 2015; 128: 1422-1433Crossref PubMed Scopus (144) Google Scholar). Accordingly, we found EGFP-D4 was internalized and colocalized in ABCA13-vesicles in HEK293 cells (Fig. 3A), but not in mock transfected cells under the same conditions. Pretreatment with MβCD, which removes cholesterol from the plasma membrane (30Zidovetzki R. Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies.Biochim. Biophys. Acta. 2007; 1768: 1311-1324Crossref PubMed Scopus (812) Google Scholar), resulted in no binding of EGFP-D4 to the cell even though ABCA13-vesicles were detected. These results suggest that ABCA13 accelerates the internalization of cholesterol from the plasma membrane. Because gangliosides are known to form microdomains containing cholesterol at the plasma membrane (31Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5152) Google Scholar, 32Fujita A. Cheng J. Hirakawa M. Furukawa K. Kusunoki S. Fujimoto T. Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling.Mol. Biol. Cell. 2007; 18: 2112-2122Crossref PubMed Scopus (204) Google Scholar, 33Komura N. Suzuki K.G. Ando H. Konishi M. Koikeda M. Imamura A. Chadda R. Fujiwara T.K. Tsuboi H. Sheng R. Cho W. Furukawa K. Yamauchi Y. Ishida H. Kusumi A. et al.Raft-based interactions of gangliosides with a GPI-anchored receptor.Nat. Chem. Biol. 2016; 12: 402-410Crossref PubMed Scopus (132) Google Scholar), we examined whether gangliosides such as GM1 and GM3 were also incorporated into ABCA13-vesicles. HEK293 cells transiently transfected with EGFP fused to ABCA13 at its C terminus or EGFP alone (control) were incubated at 37 °C with ATTO594-conjugated GM1 or GM3 (33Komura N. Suzuki K.G. Ando H. Konishi M. Koikeda M. Imamura A. Chadda R. Fujiwara T.K. Tsuboi H. Sheng R. Cho W. Furukawa K. Yamauchi Y. Ishida H. Kusumi A. et al.Raft-based interactions of gangliosides with a GPI-anchored receptor.Nat. Chem. Biol. 2016; 12: 402-410Crossref PubMed Scopus (132) Google Scholar), (Fig. 3B). ATTO594-conjugated GM1 and GM3 were substantially accumulated in ABCA13 vesicles, but their internalization was scarcely observed in control cells. Cholera toxin subunit B conjugated to Alexa Fluor 555, which binds to gangliosides (34Cho J.A. Chinnapen D.J. Aamar E. te Welscher Y.M. Lencer W.I. Massol R. Insights on the trafficking and retro-translocation of glycosphingolipid-binding bacterial toxins.Front. Cell Infect Microbiol. 2012; 2: 51Crossref PubMed Scopus (46) Google Scholar), was also delivered to ABCA13 vesicles (Fig. 3C). These results suggest ABCA13 also regulates the intracellular distribution of gangliosides. When cells were incubated with FM 4-64, a membrane-impermeant fluorescent lipid probe that is commonly used as a fluorescent reporter for endocytic vesicles and lipid trafficking (35Bandmann V. Mirsanaye A.S. Schäfer J. Thiel G. Holstein T. Mikosch-Wersching M. Membrane capacitance recordings resolve dynamics and complexity of receptor-mediated endocytosis in Wnt signalling.Sci. Rep. 2019; 9: 12999Crossref PubMed Scopus (7) Google Scholar, 36Wei Z. Su W. Lou H. Duan S. Chen G. Trafficking pathway between plasma membrane and mitochondria via clathrin-mediated endocytosis.J. Mol. Cell Biol. 2018; 10: 539-548Crossref PubMed Scopus (17) Google Scholar), we found FM 4-64 was localized in ABCA13 vesicles (Fig. 3D). The ratio of cholesterol to choline phospholipids in HEK293 cells was not altered by ABCA13 expression (Fig. S2), indicating that ABCA13 did not affect cellular cholesterol content. These results suggest that ABCA13 accelerates the internalization of plasma-membrane lipids by endocytic retrograde transport. Previous studies reported that ABCA13 is also expressed as a short version lacking the long N-terminal region, starting from methionine at 2892 (5Maeß M.B. Stolle K. Cullen P. Lorkowski S. Evidence for an alternative genomic structure, mRNA and protein sequence of human ABCA13.Gene. 2013; 515: 298-307Crossref PubMed Scopus (6) Google Scholar, 6Barros S.A. Tennant R.W. Cannon R.E. Molecular structure and characterization of a novel murine ABC transporter, Abca13.Gene. 2003; 307: 191-200Crossref PubMed Scopus (20) Google Scholar). Therefore, we assessed whether the long N-terminal region is required for ABCA13 function. ABCA13 mutant without the N-terminal region (ABCA13 ΔNter, Fig. 4A) was generated and transfected into HEK293 cells. We confirmed that ABCA13 ΔNter was expressed as a 240 kDa protein (Fig. 4B) and that it showed reticular localization (Fig. 4C), suggesting that the N-terminal region is required for the localization of ABCA13 to intracellular vesicles. ABC proteins couple the energy of ATP binding and hydrolysis to a variety of biological functions (3Davidson A.L. Dassa E. Orelle C. Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems.Microbiol. Mol. Biol. Rev. 2008; 72: 317-364Crossref PubMed Scopus (959) Google Scholar). We examined whether cholesterol internalization was dependent on ATP hydrolysis by ABCA13. We constructed three mutants: a mutant bearing one point mutation in NBD1 (ABCA13 K3849M), one in NBD2 (ABCA13 K4735M), and point mutations in both NBDs (ABCA13MM), in which conserved lysine residues in the Walker A motif crucial for ATP binding and hydrolysis were replaced by methionine (37Takahashi K. Kimura Y. Kioka N. Matsuo M. Ueda K. Purification and ATPase activity of human ABCA1.J. Biol. Chem. 2006; 281: 10760-10768Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 38Futamata R. Ogasawara F. Ichikawa T. Kodan A. Kimura Y. Kioka N. Ueda K. FRET analyses reveal a role of ATP hydrolysis-associated conformational changes in human P-glycoprotein.J. Biol. Chem. 2020; 295: 5002-5011Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) (Fig. 4A). These mutant proteins were expressed at similar levels (Fig. 4B) and localized to the intracellular vesicles like ABCA13 WT (Fig. 4C). However, EGFP-D4 staining was scarcely observed with the ATP hydrolysis-deficient mutants (Fig. 4C). These results suggest that cholesterol internalization is dependent on ATP hydrolysis by ABCA13. To study the physiological role of ABCA13, we examined the effects of ABCA13 deletion on phenotypes by generating Abca13 KO mice using the CRISPR/Cas system on the genetic background of C57BL/6N strain (39Mashiko D. Fujihara Y. Satouh Y. Miyata H. Isotani A. Ikawa M. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.Sci. Rep. 2013; 3: 3355Crossref PubMed Scopus (301) Google Scholar, 40Mashiko D. Young S.A. Muto M. Kato H. Nozawa K. Ogawa M. Noda T. Kim Y.J. Satouh Y. Fujihara Y. Ikawa M. Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes.Dev. Growth Differ. 2014; 56: 122-129Crossref PubMed Scopus (68) Google Scholar). To disrupt both full-size (5034 amino acids) and ΔNter (2143 amino acids) mouse Abca13, sgRNA was designed to target exon 21 of the Abca13 gene to induce frameshift mutations within the open reading frame (Fig. 5A). Plasmids expressing Cas9 and sgRNA against Abca13 were microinjected into zygotes of C57BL/6N mice, and Abca13 gene mutations were confirmed by DNA sequencing analysis. We created a mutant line harboring an 83 bp deletion in exon 21, leading to a premature stop codon in exon 22. The genotyping PCR products were represented in 576 bp for WT and 493 bp for KO (Fig. 5B). Western blotting was performed using the kidney, brain, and bone marrow to confirm the deletion of ABCA13 protein (Fig. 5C). A major band of ABCA13 was detected at >460 kDa in WT mice but not in KO mice. The bands detected around 240 kDa in both WT and Abca13 KO mice were considered to be nonspecific, because the frameshift mutation was introduced within the open reading frame of ΔNter ABCA13 (Fig. 5A) and the predicted molecular mass of ΔNter mouse ABCA13 is 240 kDa. These results indicate that the 83 bp deletion induced by the CRISPR/Cas system resulted in the deletion of ABCA13 protein. Abca13 KO mice were born normally and seemed to have normal appearance and life span. Because it was suggested that genetic variants of human ABCA13 are related to psychiatric disorders (7Knight H.M. Pickard B.S. Maclean A. Malloy M.P. Soares D.C. McRae A.F. Condie A. White A. Hawkins W. McGhee K. van Beck M. MacIntyre D.J. Starr J.M. Deary I.J. Visscher P.M. et al.A cytogenetic abnormality and rare coding variants identify ABCA13 as a candidate gene in schizophrenia, bipolar disorder, and depression.Am. J. Hum. Genet. 2009; 85: 833-846Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), we examined Abca13 KO mice using a battery of behavioral tests including body weight measurement, open field test, light and dark transition test, elevated plus maze test, hot pla" @default.
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