Disrupted-in-schizophrenia 1 (DISC1) plays essential roles in mitochondria in collaboration with Mitofilin Young-Un Parka,b, Jaehoon Jeonga, Haeryun Leeb,JiYoungMunc, Joung-Hun Kima,JongSeoLeed,MinhDangNguyene, Sung Sik Hanc, Pann-Ghill Suhf, and Sang Ki Parka,1 aDivision of Molecular and Life Science, Department of Life Science, Biotechnology Research Center, Pohang University of Science and Technology, Pohang 790-784, Korea; bDivision of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea; cSchool of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea; dAbClon lnc, Seoul 152-779, Korea; eHotchkiss Brain Institute, Departments of Clinical Neurosciences, Cell Biology and Anatomy, and Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada T2N 4N1; and fSchool of Nano- Biotechnology and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-798, Korea Edited by Thomas C. Südhof, Stanford University School of Medicine, Palo Alto, CA, and approved August 31, 2010 (received for review March 31, 2010) Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophre- mitochondrial dysfunction has been found to be associated with nia-susceptibility gene affecting various neuronal functions. In this various psychiatric disorders, including schizophrenia (11). For study, we characterized Mitofilin, a mitochondrial inner membrane example, Maurer et al. (12) reported a deficit in the process of protein, as a mediator of the mitochondrial function of DISC1. A oxidative phosphorylation in the postmortem brains of patients fraction of DISC1 was localized to the inside of mitochondria and with schizophrenia. Deformations and reductions in the number directly interacts with Mitofilin. A reduction in DISC1 function in- of mitochondria have also been reported in postmortem studies duced mitochondrial dysfunction, evidenced by decreased mito- (13). Although these observations are critical insights into the link chondrial NADH dehydrogenase activities, reduced cellular ATP between schizophrenia and mitochondrial dysfunctions, the un- contents, and perturbed mitochondrial Ca2+ dynamics. In addition, derlying molecular mechanisms explaining this relationship have deficiencies in DISC1 and Mitofilin induced a reduction in mitochon- not been well defined. drial monoamine oxidase-A activity. The mitochondrial dysfunc- In this study, we characterized Mitofilin, also called inner- tions evoked by the deficiency of DISC1 were partially pheno- membrane protein, mitochondrial (IMMT) (14), as a mitochon- copied by an overexpression of truncated DISC1 that is associated drial interacting partner of DISC1, through which DISC1 plays with schizophrenia in human. DISC1 deficiencies induced the ubiq- essential roles in mitochondria. Mitofilin has been well-charac- uitination of Mitofilin, suggesting that DISC1 is critical for the sta- terized as an essential component for the functional integrity of bility of Mitofilin. Finally, the mitochondrial dysfunction induced by mitochondria (15, 16); thus, the data shown in this study may DISC1 deficiency was partially reversed by coexpression of Mitofi- provide a mechanistic link between DISC1 and mitochondrial lin, confirming a functional link between DISC1 and Mitofilin for the function, the deregulation of which might have implications in normal mitochondrial function. According to these results, we pro- the pathogenesis of schizophrenia. pose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin. Results Mitofilin as an Interacting Partner of DISC1. To elucidate DISC1 IMMT | mitochondrial dysfunctions | hyperdopaminergia | functions potentially relevant to schizophrenia, we attempted to calcium buffering | psychiatric disorders identify components of the DISC1–NDEL1-containing protein complex by using a yeast two-hybrid screen on a human fetal brain chizophrenia is a mental illness characterized by impairments library using NDEL1 as bait. Then, we tested whether any of the ’ positives from this screening could interact with DISC1. As a re- Sin an individual s perceptions of reality, which are commonly fi manifested in the form of hallucinations, paranoid or bizarre sult, we identi ed a cDNA clone encoding amino acid residues, 230–758, of Mitofilin, a mitochondrial inner membrane protein. delusions, and/or disorganized speech and thinking (1). The We noticed that the potential interaction between DISC1 and lifetime prevalence of schizophrenia is 0.55–1% of population, Mitofilin has been consistently recognized in the previous reports with up to 80% of genetic inheritance (2). Although the mo- describing various potential DISC1 interacting partners, mostly lecular mechanisms underlying the etiology and pathophysiology from yeast two-hybrid screens (17, 18), although it has not been of schizophrenia have yet to be clearly defined, increased do- further analyzed or characterized functionally. paminergic activities in the mesolimbic pathway have been The interactions were confirmed by interaction-dependent consistently associated with several of the positive symptoms of β-galactosidase activity and growth on selective media (Fig. 1A). schizophrenia (3). Disrupted-in-schizophrenia In the subsequent interaction analyses using yeast two-hybrid 1 (DISC1) has emerged as a con- assays, regions of DISC1, amino acid residues 600–854 and 1–347, – vincing candidate gene that may explain many aspects of showed interaction-dependent signals with Mitofilin371 590 (Fig. – schizophrenia and related mood disorders. Familial mutations in S1A). The DISC1600 854 region showed strong interaction-de- – the DISC1 gene, including t(1;11)(q42.1;q14.3) translocation, pendent signals with Mitofilin400 590 (Fig. S1B). In addition, – cosegregate with symptoms related to schizophrenia, bipolar DISC1200 400 did not interact with Mitofilin371-590 (Fig. S1A) but – disorders, and recurrent major depression (4). DISC1 plays a showed signal with Mitofilin230 758 (Fig. S1C), suggesting that significant role in the processes of neurodevelopment, including neurite outgrowth, neuronal migration, neurogenesis (5–8), intra- cellular cAMP signaling (9), and many other neuronal processes Author contributions: Y.-U.P. and S.K.P. designed research; Y.-U.P., J.J., H.L., J.Y.M., and in collaboration with other cellular factors such as nudE nuclear S.K.P. performed research; Y.-U.P., J.-H.K., J.S.L., M.D.N., P.-G.S., and S.K.P. contributed distribution gene E homolog (A. nidulans)-like 1 (NDEL1) (6). new reagents/analytic tools; Y.-U.P., J.J., H.L., J.Y.M., S.S.H., and S.K.P. analyzed data; and Additionally, genetic evidence supporting a functional link be- Y.-U.P., J.J., H.L., S.S.H., and S.K.P. wrote the paper. tween DISC1 and NDEL1 in schizophrenia pathogenesis has The authors declare no conflict of interest. been reported (10). This article is a PNAS Direct Submission. Mitochondria play critical roles in fundamental cellular pro- 1To whom correspondence should be addressed. E-mail: [email protected]. cesses, encompassing energy production, programmed cell death, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NEUROSCIENCE 2+ Ca homeostasis, and monoamine metabolism. Intriguingly, 1073/pnas.1004361107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1004361107 PNAS | October 12, 2010 | vol. 107 | no. 41 | 17785–17790 Downloaded by guest on October 1, 2021 600-854 600-854 600-854 230-758 230-758 230-758 230-758 A B 598-854 598-854 -DISC1 -NDEL1 T T-NDEL1 fi pPC97-hDISC1 pPC86-Mitofilin pPC97-NDEL1 pPC86-hDISC1 pPC86-Mitofilin pPC86-hDISC1 pPC86-hDISC1 SDS-PAGE; GST GST GS GST GST-DISC1GS Fig. 1. Interactions among DISC1, NDEL1, and Mito lin. (A) + pPC86 + pPC97 + pPC86 + pPC97-NDEL1 + pPC97-NDEL1 + pPC97-Mitofilin + pPC97-Mitofilin Interactions of DISC1, NDEL1, and Mitofilin in the yeast two- b-galactosidase activity 72 hybrid assay. (Upper) Interaction-dependent β-galactosidase − 55 expression. (Lower)GrowthonHIS selective media contain- growth- on His /3-AT media ing 20 mM 3-amino-1,2,4-triazol (3-AT). hDISC1, human 43 DISC1. (B) Interaction of Mitofilin with DISC1 and NDEL1 in 34 vitro analyzed by a blot overlay assay. The PVDF membrane C 200-400 358-597 598-854 598-726 726-854 26 with the purified hDISC1 and NDEL1 protein bands trans- coomassie staining of overlay; GST-Mitofilin123-758 fi transfered proteins WB; anti-Mitofilin ferred from an SDS/PAGE was rst stained with Coomassie Input (2%) GST GST-hDISC1GST-hDISC1GST-hDISC1GST-hDISC1GST-hDISC1GST-NDEL1 Blue R250 (Left) and subjected to GST–Mitofilin blot overlay anti-Mitofilin Mitofilin followed by anti-Mitofilin Western blot analyses (Right). WB 72 fi 72 D Dashed lines are the distribution of puri ed recombinant 55 Input (2%) anti-myc IP proteins with degradation products. WB, Western blotting. flag-hDISC1 -+- + - --+- + - - fi 43 (C) Associations of Mito linwithDISC1andNDEL1de- flag-hDISC-tr - - - + +- - - - ++- Coommassie Mitofilin-myc -++-- + -++-- + staining 34 termined by GST pull-down assays. Lysates were prepared 26 flag-hDISC1 from SH-SY5Y cells that were differentiated for 36 h. (D) anti-flag 95 fi WB Coimmunoprecipitation of Mito lin with full-length and 72 72 truncated DISC1. HEK293 cells were transfected with flag- 55 flag-hDISC1-tr hDISC1 or flag-hDISC1-tr (amino acid residues 1–598), and anti-GST 43 fi WB anti-myc 95 Mito lin-myc constructs as indicated. IP, immunoprecipi- 34 WB Mitofilin-myc