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• W2046335251 abstract "It is indicated that there are important molecules interacting with brain nervous systems to regulate feeding and energy balance by influencing the signaling pathways of these systems, but relatively few of the critical players have been identified. In the present study, we provide the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a mediator of feeding behavior through interaction with serotonin receptor 2C (5-HT2CR), known for its critical role in feeding and appetite control. First, we demonstrated the co-localization and interaction between hypothalamic Ahi1 and 5-HT2CR. Ahi1 promoted the degradation of 5-HT2CR through the lysosomal pathway. Then, we investigated the effects of fasting on the expression of hypothalamic Ahi1 and 5-HT2CR. Fasting resulted in an increased Ahi1 expression and a concomitant decreased expression of 5-HT2CR. Knockdown of hypothalamic Ahi1 led to a concomitant increased expression of 5-HT2CR and a decrease of food intake and body weight. Last, we found that Ahi1 could regulate the expression of neuropeptide Y and proopiomelanocortin. Taken together, our results indicate that Ahi1 mediates feeding behavior by interacting with 5-HT2CR to modulate the serotonin signaling pathway. It is indicated that there are important molecules interacting with brain nervous systems to regulate feeding and energy balance by influencing the signaling pathways of these systems, but relatively few of the critical players have been identified. In the present study, we provide the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a mediator of feeding behavior through interaction with serotonin receptor 2C (5-HT2CR), known for its critical role in feeding and appetite control. First, we demonstrated the co-localization and interaction between hypothalamic Ahi1 and 5-HT2CR. Ahi1 promoted the degradation of 5-HT2CR through the lysosomal pathway. Then, we investigated the effects of fasting on the expression of hypothalamic Ahi1 and 5-HT2CR. Fasting resulted in an increased Ahi1 expression and a concomitant decreased expression of 5-HT2CR. Knockdown of hypothalamic Ahi1 led to a concomitant increased expression of 5-HT2CR and a decrease of food intake and body weight. Last, we found that Ahi1 could regulate the expression of neuropeptide Y and proopiomelanocortin. Taken together, our results indicate that Ahi1 mediates feeding behavior by interacting with 5-HT2CR to modulate the serotonin signaling pathway. Obesity and its associated ailments such as diabetes have become a worldwide epidemic carrying with them a heavy toll of morbidity and mortality. Over the past decades, it has become evident that neural circuits in the central nervous system play a direct and crucial role in controlling feeding and energy homeostasis (1Morton G.J. Cummings D.E. Baskin D.G. Barsh G.S. Schwartz M.W. Central nervous system control of food intake and body weight.Nature. 2006; 443: 289-295Crossref PubMed Scopus (1919) Google Scholar, 2Sandoval D. Cota D. Seeley R.J. The integrative role of CNS fuel-sensing mechanisms in energy balance and glucose regulation.Annu. Rev. Physiol. 2008; 70: 513-535Crossref PubMed Scopus (151) Google Scholar). Disruptions in the mechanisms of central nervous system energy sensing are able to alter the standard homeostatic responses and are factors that contribute to the pathophysiology of obesity and diabetes. The brain serotonin system has long been implicated in the neural regulation of food intake and energy metabolism, as highlighted by the clinical use of serotonin releasers and reuptake inhibitors as appetite suppressant and weight loss medication (3Jespersen S. Scheel-Krüger J. Evidence for a difference in mechanism of action between fenfluramine- and amphetamine-induced anorexia.J. Pharm. Pharmacol. 1973; 25: 49-54Crossref PubMed Scopus (83) Google Scholar, 4Shor-Posner G. Grinker J.A. Marinescu C. Brown O. Leibowitz S.F. Hypothalamic serotonin in the control of meal patterns and macronutrient selection.Brain Res. Bull. 1986; 17: 663-671Crossref PubMed Scopus (173) Google Scholar, 5Vickers S.P. Benwell K.R. Porter R.H. Bickerdike M.J. Kennett G.A. Dourish C.T. Comparative effects of continuous infusion of mCPP, Ro 60-0175, and d-fenfluramine on food intake, water intake, body weight, and locomotor activity in rats.Br. J. Pharmacol. 2000; 130: 1305-1314Crossref PubMed Scopus (88) Google Scholar). Depletion of central serotonin using selective neurotoxins has been shown to result in hyperphagia and obesity, whereas the release of serotonin and the inhibition of reuptake by d-fenfluramine potently reduce feeding and body weight (6Pinder R.M. Brogden R.N. Sawyer P.R. Speight T.M. Avery G.S. Fenfluramine: a review of its pharmacological properties and therapeutic efficacy in obesity.Drugs. 1975; 10: 241-323Crossref PubMed Scopus (167) Google Scholar). More recently, several lines of evidence show that serotonin receptor agonists can significantly improve glucose tolerance and reduce plasma insulin in mouse models of obesity and type 2 diabetes (7Vezzosi D. Cartier D. Régnier C. Otal P. Bennet A. Parmentier F. Plantavid M. Lacroix A. Lefebvre H. Caron P. Familial adrenocorticotropin-independent macronodular adrenal hyperplasia with aberrant serotonin and vasopressin adrenal receptors.Eur. J. Endocrinol. 2007; 156: 21-31Crossref PubMed Scopus (77) Google Scholar, 8Kring S.I. Werge T. Holst C. Toubro S. Astrup A. Hansen T. Pedersen O. Sørensen T.I. Polymorphisms of serotonin receptor 2A and 2C genes and COMT in relation to obesity and type 2 diabetes.PLoS One. 2009; 4: e6696Crossref PubMed Scopus (62) Google Scholar, 9Zhou L. Sutton G.M. Rochford J.J. Semple R.K. Lam D.D. Oksanen L.J. Thornton-Jones Z.D. Clifton P.G. Yueh C.Y. Evans M.L. McCrimmon R.J. Elmquist J.K. Butler A.A. Heisler L.K. Serotonin 2C receptor agonists improve type 2 diabetes via melanocortin-4 receptor signaling pathways.Cell Metab. 2007; 6: 398-405Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Numerous serotonin receptor subtypes have been identified in which serotonin receptor 2C (5-HT2CR) 4The abbreviations used are: 5-HT2CRserotonin receptor 2CNPYneuropeptide YARCarcuate nucleusPOMCproopiomelanocortinmCPPmeta-chlorophenylpiperazineChlqchloroquineGFPgreen fluorescent proteinSH3Src homology 3dpidays post-injection. is recognized specifically as a mediator of serotonin-induced appetite and glucose regulation (10De Vry J. Schreiber R. Effects of selected serotonin 5-HT(1) and 5-HT(2) receptor agonists on feeding behavior: possible mechanisms of action.Neurosci. Biobehav. Rev. 2000; 24: 341-353Crossref PubMed Scopus (154) Google Scholar, 11Nonogaki K. Strack A.M. Dallman M.F. Tecott L.H. Leptin-independent hyperphagia and type 2 diabetes in mice with a mutated serotonin 5-HT2C receptor gene.Nat. Med. 1998; 4: 1152-1156Crossref PubMed Scopus (421) Google Scholar, 12Tecott L.H. Sun L.M. Akana S.F. Strack A.M. Lowenstein D.H. Dallman M.F. Julius D. Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors.Nature. 1995; 374: 542-546Crossref PubMed Scopus (1144) Google Scholar, 13Vickers S.P. Easton N. Webster L.J. Wyatt A. Bickerdike M.J. Dourish C.T. Kennett G.A. Oral adnmnistration of the 5-HT2C receptor agonist, mCPP, reduces body weight gain in rats over 28 days as a result of maintained hypophagia.Psychopharmacology (Berl.). 2003; 167: 274-280Crossref PubMed Scopus (88) Google Scholar). During the past few years, both pharmacological and genetic evidence has indicated that neuropeptide Y (NPY) and melanocortin systems are the necessary mechanisms by which 5-HT2CR agonists reduce appetite and improve diabetic conditions (14Brown C.M. Coscina D.V. Ineffectiveness of hypothalamic serotonin to block neuropeptide Y-induced feeding.Pharmacol. Biochem. Behav. 1995; 51: 641-646Crossref PubMed Scopus (16) Google Scholar, 15Heisler L.K. Cowley M.A. Tecott L.H. Fan W. Low M.J. Smart J.L. Rubinstein M. Tatro J.B. Marcus J.N. Holstege H. Lee C.E. Cone R.D. Elmquist J.K. Activation of central melanocortin pathways by fenfluramine.Science. 2002; 297: 609-611Crossref PubMed Scopus (409) Google Scholar, 16Rogers P. McKibbin P.E. Williams G. Acute fenfluramine administration reduces neuropeptide Y concentrations in specific hypothalamic regions of the rat: possible implications for the anorectic effect of fenfluramine.Peptides. 1991; 12: 251-255Crossref PubMed Scopus (68) Google Scholar). Although significant progress has been made in the study of serotonin-mediated regulation of energy metabolism, we are still far from understanding the whole picture. One of the more intriguing aspects of this area that has remained mysterious is the importance of cellular and molecular interactions that regulate energy homeostasis centrally. serotonin receptor 2C neuropeptide Y arcuate nucleus proopiomelanocortin meta-chlorophenylpiperazine chloroquine green fluorescent protein Src homology 3 days post-injection. Abelson helper integration site 1 (Ahi1) was initially identified as a common helper provirus integration site for murine leukemia and lymphomas (17Poirier Y. Kozak C. Jolicoeur P. Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA.J. Virol. 1988; 62: 3985-3992Crossref PubMed Google Scholar). A number of groups have identified Ahi1 mutations as a frequent cause of disease in patients with specific forms of Joubert syndrome, an autosomal recessive neurodevelopmental disorder, and its related disorders (JSRD) (18Dixon-Salazar T. Silhavy J.L. Marsh S.E. Louie C.M. Scott L.C. Gururaj A. Al-Gazali L. Al-Tawari A.A. Kayserili H. Sztriha L. Gleeson J.G. Mutations in the AHI1 gene, encoding jouberin, cause Joubert syndrome with cortical polymicrogyria.Am. J. Hum. Genet. 2004; 75: 979-987Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 19Utsch B. Sayer J.A. Attanasio M. Pereira R.R. Eccles M. Hennies H.C. Otto E.A. Hildebrandt F. Identification of the first AHI1 gene mutations in nephronophthisis-associated Joubert syndrome.Pediatr. Nephrol. 2006; 21: 32-35Crossref PubMed Scopus (76) Google Scholar, 20Valente E.M. Brancati F. Silhavy J.L. Castori M. Marsh S.E. Barrano G. Bertini E. Boltshauser E. Zaki M.S. Abdel-Aleem A. Abdel-Salam G.M. Bellacchio E. Battini R. Cruse R.P. Dobyns W.B. Krishnamoorthy K.S. Lagier-Tourenne C. Magee A. Pascual-Castroviejo I. Salpietro C.D. Sarco D. Dallapiccola B. Gleeson J.G. AHI1 gene mutations cause specific forms of Joubert syndrome-related disorders.Ann. Neurol. 2006; 59: 527-534Crossref PubMed Scopus (118) Google Scholar). The normal neural function of Ahi1, however, remains poorly defined. Both protein and mRNA studies have shown that Ahi1 is distributed in several brain areas implicated in feeding and metabolic regulation such as the hypothalamic paraventricular nucleus, the supraoptic nucleus, the arcuate nucleus (ARC), the lateral hypothalamic area, and the nucleus tractus solitarius in the brain stem (21Doering J.E. Kane K. Hsiao Y.C. Yao C. Shi B. Slowik A.D. Dhagat B. Scott D.D. Ault J.G. Page-McCaw P.S. Ferland R.J. Species differences in the expression of Ahi1, a protein implicated in the neurodevelopmental disorder Joubert syndrome, with preferential accumulation to stigmoid bodies.J. Comp. Neurol. 2008; 511: 238-256Crossref PubMed Scopus (38) Google Scholar, 22Ferland R.J. Eyaid W. Collura R.V. Tully L.D. Hill R.S. Al-Nouri D. Al-Rumayyan A. Topcu M. Gascon G. Bodell A. Shugart Y.Y. Ruvolo M. Walsh C.A. Abnormal cerebellar development and axonal decussation due to mutations in AHI1 in Joubert syndrome.Nat. Genet. 2004; 36: 1008-1013Crossref PubMed Scopus (326) Google Scholar, 23Sheng G. Xu X. Lin Y.F. Wang C.E. Rong J. Cheng D. Peng J. Jiang X. Li S.H. Li X.J. Huntingtin-associated protein 1 interacts with Ahi1 to regulate cerebellar and brainstem development in mice.J. Clin. Invest. 2008; 118: 2785-2795Crossref PubMed Scopus (69) Google Scholar). Genetic studies also indicate that Ahi1 may be related to energy metabolism. One group has reported a significant association between variants in the Ahi1 gene and type 2 diabetes in a Dutch population (24Salonen J.T. Uimari P. Aalto J.M. Pirskanen M. Kaikkonen J. Todorova B. Hyppönen J. Korhonen V.P. Asikainen J. Devine C. Tuomainen T.P. Luedemann J. Nauck M. Kerner W. Stephens R.H. New J.P. Ollier W.E. Gibson J.M. Payton A. Horan M.A. Pendleton N. Mahoney W. Meyre D. Delplanque J. Froguel P. Luzzatto O. Yakir B. Darvasi A. Type 2 diabetes whole-genome association study in four populations: the DiaGen consortium.Am. J. Hum. Genet. 2007; 81: 338-345Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In addition, single nucleotide polymorphism association studies identified two novel Ahi1 genetic variants linked with fasting blood glucose levels in Mexican American subjects (25Prior M.J. Foletta V.C. Jowett J.B. Segal D.H. Carless M.A. Curran J.E. Dyer T.D. Moses E.K. McAinch A.J. Konstantopoulos N. Bozaoglu K. Collier G.R. Cameron-Smith D. Blangero J. Walder K.R. The characterization of Abelson helper integration site-1 in skeletal muscle and its links to the metabolic syndrome.Metabolism. 2010; 59: 1057-1064Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Recent studies (26Han S.B. Choi B.I. Lee D. Kee S.H. Kim H.S. Sun W. Kim H. Regulation of AHI1 expression in adult rat brain: Implication in hypothalamic feeding control.Biochem. Biophys. Res. Commun. 2009; 390: 535-540Crossref PubMed Scopus (8) Google Scholar, 27Niu S.N. Huang Z.B. Wang H. Rao X.R. Kong H. Xu J. Li X.J. Yang C. Sheng G.Q. Brainstem Hap1-Ahi1 is involved in insulin-mediated feeding control.FEBS Lett. 2011; 585: 85-91Crossref PubMed Scopus (20) Google Scholar) also show that brain Ahi1 may play an important role in the regulation of feeding behavior. We noticed that Ahi1 and 5-HT2CR have similar distribution in the hypothalamus. This prompted us to investigate the relationship between Ahi1 and 5-HT2CR and their probable roles in feeding control. The findings in the present study provide evidence that Ahi1 interacts with 5-HT2CR to mediate feeding behavior. Our study reveals the normal neural function of Ahi1 in feeding control and offers insight into the understanding of how hypothalamic key molecules regulate the feeding behavior. Male C57BL/6J mice, 6–10 weeks old, were purchased from Southern Medical University, Guangdong Province, China. The animals were housed in a temperature- and humidity-controlled environment with a 12 h/12 h light/dark cycle with access to food and water ad libitum. Animals were acclimatized to laboratory conditions for a week before all tests. Animal care and all procedures for animal experiments conformed to the guidelines of the Animal Care and Use Committee of Guangzhou Biomedical and Health Institute, Chinese Academy of Sciences. The antibody against Ahi1 is described in our previous study (23Sheng G. Xu X. Lin Y.F. Wang C.E. Rong J. Cheng D. Peng J. Jiang X. Li S.H. Li X.J. Huntingtin-associated protein 1 interacts with Ahi1 to regulate cerebellar and brainstem development in mice.J. Clin. Invest. 2008; 118: 2785-2795Crossref PubMed Scopus (69) Google Scholar). For immunofluorescent staining, sections of brain tissue and coverslips plated with hypothalamic neurons or HEK293 cells were blocked with 5% bovine serum albumin (BSA) in 0.02 m PBS at room temperature for 1 h and followed by incubating with rabbit anti-Ahi1 antibody (1:300) and goat anti-5-HT2CR (1:50, Santa Cruz Biotechnology) at 4 °C overnight. Then the sections were incubated successively with Alexa Fluor 488-conjugated anti-goat secondary antibody and Alexa Fluor 594-conjugated anti-rabbit secondary antibody (both from Invitrogen) at room temperature for 30 min. Tissue sections and coverslips were mounted onto glass slides. Labeled samples were imaged using a ×100 objective Leica SP2 confocal microscope. Lysates from mouse hypothalami or from co-transfected HEK293 cells were extracted using lysis buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, and protease inhibitor mixture) and centrifuged at 12,000 × g at 4 °C for 15 min. 500 μl of supernatant (adjusted to 1 mg/ml) was first clarified by incubation with 40 μl of 50% protein A-Sepharose beads (Sigma) at 4 °C for 1 h to reduce nonspecific binding. After pelleting the beads, the supernatant was then incubated with antibody to mouse Ahi1 (1: 50), anti-hemagglutinin (HA) monoclonal antibody (Sigma, clone HA-7, diluted 1:4000), anti-GFP polyclonal antibody (Abcam, ab290, diluted 1:1000), or immunoglobulin G as control overnight at 4 °C followed by brief centrifugation. The immunoprecipitates were washed three times with low detergent buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, and 0.2% Tween 20) and subjected to Western blotting. A total of 15–20 mg of whole hypothalami from fasted mice or Ahi1 knockdown mice were adequately blended in 1 ml of pre-cold membrane purification buffer (MPB: 250 mm sucrose, 30 mm KCl, 20 mm Tris-Cl, pH 7.2, 0.2 mm DTT, 1 mm EDTA, 1 mm EGTA, 0.3% Triton X-114, and protease inhibitor mixture) on ice. The suspensions were ultracentrifuged at 59,000 × g at 4 °C for 15 min, and then the pellets (P1) were re-extracted as mentioned above in 1 ml of MPB with 0.8% Triton X-114. After a 30-min incubation on ice, the soluble materials were removed by ultracentrifugation (as above). The pellets (P2, purified membrane fraction) were washed twice with MPB and finally resuspended in MPB. Hypothalami were mechanically homogenized and sonicated in homogenization buffer on ice. 20 μg of tissue protein was size-fractionated using 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. Cell lysates (50 μg) from treated N18TG2 cells were also subjected to SDS-PAGE and blotted. After blotting with the antibodies, anti-mouse Ahi1 or anti-5-HT2CR and anti-γ-tubulin (Sigma), anti-β-actin (ACTB, GenScript), and anti-LAMP1 detection was performed using an enhanced chemiluminescent kit (Pierce) according to the manufacturer's instructions. Quantitations of gray density were performed using ImageQuant 5.2 software. Total RNA was isolated from tissue samples or cell samples using TRIzol reagent (Ambion). cDNA was synthesized by using Moloney murine leukemia virus (MMLV) reverse transcriptase (Fermentas). Primers for mouse Ahi1 (forward primer, 5′-GAC AGG AGA ACA AGT GGC AAT G-3′; reverse primer, 5′-ATC AGT GGT CAG CAC GAA CGA-3′), mouse NPY (forward primer, 5′-TAC TAC TCC GCT CTG CGA CAC-3′; reverse primer, 5′-CCA CAT GGA AGG GTC TTC AAG-3′), mouse proopiomelanocortin (POMC) (forward primer, 5′-CGA GCG GCC ATT AGG CTT-3′; reverse primer, 5′-CTT GTC CTT GGG CGG GTT-3′), and reference gene GAPDH (forward primer, 5′-CTG CAC CAC CAA CTG CTT AGC-3′; reverse primer, 5′-GGA AGG CCA TGC CAG TGA-3′) were optimized to an equal annealing temperature of 60 °C. Expression of Ahi1, NPY, POMC, and GAPDH was determined by real-time PCR using SYBR® Premix Ex Taq (Takara) on an MJ four-color real-time PCR system (Bio-Rad) according to the manufacturer's instructions. The expression ratio of target genes among the experimental groups was calculated and statistically analyzed as reported previously (28Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25871) Google Scholar, 29Yuan J.S. Reed A. Chen F. Stewart Jr., C.N. Statistical analysis of real-time PCR data.BMC Bioinformatics. 2006; 7: 85Crossref PubMed Scopus (1522) Google Scholar). For the knockdown of Ahi1, C57BL/6J mice (8-week-old males) were injected bilaterally with recombinant adenovirus vector encoding Ahi1-specific siRNA (Ad-siAhi1) or scramble-siRNA (Ad-scRNA) as control. A total of 1 × 1011 plaque-forming units in 1 μl of PBS were injected bilaterally intrahypothalamus at the stereotaxic positions (anteroposterior −1.1 mm, mediolateral −0.5 mm, dorsalventral −5.5 mm; anteroposterior −1.1 mm, mediolateral +0.5 mm, dorsalventral −5.5 mm). The adenoviral vector also independently expresses green fluorescent protein (GFP), which enabled us to trace the vector. Injection at the above coordinates allowed the adenovirus to mainly infect the ventromedial hypothalamus, ARC, dorsomedial hypothalamus, and other nuclei in the hypothalamus (Fig. 4A). The mice were single-housed in regular plastic cages before and after the surgical procedure. Food intake and body weight were recorded daily for 4 weeks. After a period of 3–5 days post-surgical recovery, the mice were tested for glucose tolerance tests and insulin tolerance tests performed as described previously (30Sutton G.M. Trevaskis J.L. Hulver M.W. McMillan R.P. Markward N.J. Babin M.J. Meyer E.A. Butler A.A. Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors.Endocrinology. 2006; 147: 2183-2196Crossref PubMed Scopus (118) Google Scholar). Glycemia was assessed using a blood glucose test meter. Co-localization of Ahi1 and 5-HT2CR was observed on the networks of cultured mice hypothalamic neurons. The details of culture preparations have been described previously (31Swandulla D. Misgeld U. Development and properties of synaptic mechanisms in a network of rat hypothalamic neurons grown in culture.J. Neurophysiol. 1990; 64: 715-726Crossref PubMed Scopus (36) Google Scholar). In brief, hypothalamic tissue was dissected from newborn mice. Hypothalamic neural cells were planted on poly-d-lysine-coated coverslips at a density of ∼2000 cells/cm2. Cultures were kept in Neurobasal medium supplemented with 2% B27 and 2 mm l-glutamine (all from Invitrogen) for 14 days in vitro. N18TG2 cells, the mouse neuroblastoma cell line with endogenous Ahi1 expression, were grown in high glucose Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (PAA Laboratories), 1 mm non-essential amino acids (Invitrogen), 2 mm l-glutamine (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). DNA constructs for expression of Ahi1 cDNA plasmids encoding full-length mouse Ahi1-(1–1047) were generated as in a previous study (32Jiang X. Hanna Z. Kaouass M. Girard L. Jolicoeur P. Ahi-1, a novel gene encoding a modular protein with WD40-repeat and SH3 domains, is targeted by the Ahi-1 and Mis-2 provirus integrations.J. Virol. 2002; 76: 9046-9059Crossref PubMed Scopus (61) Google Scholar). We used PCR to generate C-terminally truncated mouse Ahi1-(1–820). These Ahi1 cDNAs were sequenced and cloned to the PRK vector that links the influenza HA epitope on the upstream of the Ahi1 coding region. For transfection of foreign DNAs into N18TG2, plasmid-polyethylenimine (Polysciences, Inc.) complexes incubated with cultures for 1 h. After 36 h of transfection, N18TG2 cells were stimulated under 5 μm meta-chlorophenylpiperazine (mCPP; Sigma) with or without 100 μm chloroquine (Chlq; Sigma) for the indicated time and then immediately collected for the following quantitative assays. For virus infection, the cells were infected by Ad-siAhi1 (1 × 107 plaque-forming units/ml medium) for 8 h and sampled at 24 h post-infection. HEK293 cells were cultured in DMEM containing 10% fetal bovine serum, 1 mm sodium pyruvate, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. When 50% confluence had been achieved, the HEK293 cells were co-transfected with constructs encoding DsRed-tagged Ahi1 and GFP-tagged 5-HT2CR as described above. N18TG2 cells, co-transfected with PRK-mAhi1-DsRed/PRK-DsRed and GFP-5-HT2CR, were washed once, fixed for 10 min at −20 °C in 50% (v/v) acetone/methanol, and air-dried. Cells were blocked by incubating them for 1 h in 5% BSA and then with anti-LAMP1 primary antibody (4 μg/ml, Abcam) at 4 °C for 24 h. Then coveslips were washed several times and incubated with Cy5-labeled anti-mouse IgG for 45 min at room temperature. After DAPI staining, four-color slides were visualized with a Leica SP2 confocal scanning microscope set up as follows: 403 nm laser (25% of power) window, 410–483 nm; 488 laser (25% of power) window, 493–538 nm; 543 nm laser (90% of power) window, 548–628 nm; and 633 nm laser (25% of power), window 638–700 nm. Images were collected using the microscope in sequential mode with a frame average and a format of 1024 × 1024 pixels and were analyzed using NIH ImageJ 1.40g software. For statistical analyses, images for all conditions were analyzed using identical acquisition parameters, and untreated and treated cells from the same culture preparation were always compared with one another. The images were also collected blind to experimental condition. The total thresholded area of fluorescently labeled overlay regions was automatically measured and divided by the total cell area, which was determined by setting a lower threshold level to measure the background fluorescence produced by the fixed cells. For each experiment, the fluorescence of all cells was normalized by dividing the average fluorescence of the untreated control cells. All cells that expressed 5-HT2CR with less than the average at 30% were excluded from analysis because of the variation in transfection efficiency. GraphPad Prism was utilized for data analysis. Data are presented as mean ± S.E. of at least three independent experiments. Statistical analyses were carried out by one-way analysis of variance followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant. First, we cultured hypothalamic neurons in vitro. In double-labeled immunofluorescence staining assays of cultured hypothalamic neurons, Ahi1 was observed to co-localize significantly with 5-HT2CR (Fig. 1A, top and middle rows). Staining of mouse brain sections also revealed an extensive overlap of the two proteins in hypothalamus nucleus such as ARC (Fig. 1A, bottom row). A co-immunoprecipitation study was performed in mice hypothalamus homogenates. As shown in Fig. 1B, 5-HT2CR selectively bound to endogenous Ahi1, whereas no signal was detected in immunoprecipitates from non-immune rabbit serum control. To confirm this physical interaction, we subsequently expressed them in transfected HEK293 cells; GFP-5-HT2CR showed an extensive distribution throughout the plasma membrane and cytoplasm of the cell, whereas Ahi1 showed a unique distribution pattern characterized as dot-like structures. Co-expression of 5-HT2CR and Ahi1 displayed apparent co-localization in punctate aggregates in the cytoplasm of HEK293 cells (supplemental Fig. 1A). Their interaction was further demonstrated by co-immunoprecipitation using anti-Ahi1 antibody (supplemental Fig. 1B). Then, we constructed the C terminal-truncated Ahi1. In the immunofluorescence experiments, we found that the co-localization of Ahi1 and 5-HT2CR was lost in the cells transfected with C terminal-truncated Ahi1 (Fig. 1D). Similarly the combination of Ahi1 and 5-HT2CR was abolished in the C terminal-truncated Ahi1 (Fig. 1E). These data indicate that the C terminus of Ahi1 is the region where it interacts with 5-HT2CR. Given the WD40 repeat domains and the SH3 domain found in the Ahi1 protein (32Jiang X. Hanna Z. Kaouass M. Girard L. Jolicoeur P. Ahi-1, a novel gene encoding a modular protein with WD40-repeat and SH3 domains, is targeted by the Ahi-1 and Mis-2 provirus integrations.J. Virol. 2002; 76: 9046-9059Crossref PubMed Scopus (61) Google Scholar), we hypothesized that Ahi1 may play a role in neurotransmitter receptor trafficking as an adaptor between cytoskeleton and membrane protein. It has been reported that Ahi1 participates in the process of intracellular vesicle trafficking and is co-localized with microtubules and the microtubule-organizing center (33Keller L.C. Geimer S. Romijn E. Yates 3rd, J. Zamora I. Marshall W.F. Molecular architecture of the centriole proteome: the conserved WD40 domain protein POC1 is required for centriole duplication and length control.Mol. Biol. Cell. 2009; 20: 1150-1166Crossref PubMed Scopus (102) Google Scholar, 34Louie C.M. Caridi G. Lopes V.S. Brancati F. Kispert A. Lancaster M.A. Schlossman A.M. Otto E.A. Leitges M. Gröne H.J. Lopez I. Gudiseva H.V. O'Toole J.F. Vallespin E. Ayyagari R. Ayuso C. Cremers F.P. den Hollander A.I. Koenekoop R.K. Dallapiccola B. Ghiggeri G.M. Hildebrandt F. Valente E.M. Williams D.S. Gleeson J.G. AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis.Nat. Genet. 2010; 42: 175-180Crossref PubMed Scopus (153) Google Scholar). We postulated that Ahi1 could participate in 5-HT2CR vesicles sorting to degradation after endocytosis. To test this hypothesis, we treated cells with mCPP, an agonist of 5-HT2CR (13Vickers S.P. Easton N. Webster L.J. Wyatt A. Bickerdike M.J. Dourish C.T. Kennett G.A. Oral adnmnistration of the 5-HT2C receptor agonist, mCPP, reduces body weight gain in rats over 28 days as a result of maintained hypophagia.Psychopharmacology (Berl.). 2003; 167: 274-280Crossref PubMed Scopus (88) Google Scholar). As shown in Fig. 2A, the 5-HT2CR decreased in a time-dependent way in the cells transfected with full-length Ahi1, whereas in the cells transfected with truncated Ahi1 this decrease was inhibited. Then we addressed the pathway of 5-HT2CR degradation. We first investigated the possible involvement of the lysosomal pathway by treatment with Chlq, a lysosomal enzyme inhibitor. Analysis by Western blot revealed that treatment with Chlq increased the level of 5-HT2CR in mCPP treated N18TG2 cells (Fig. 2B, lane 3 versus lane 2), thereby indicating that the lysosomal pathway mediates internalized 5-HT2CR degradation. To address the possible function of Ahi1 in lysosomal sorting, we investigated whether the expression of" @default.
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