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The Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1) is a protein complex containing the schizophrenia susceptibility factor dysbindin, which is encoded by the gene DTNBP1. However, mechanisms engaged by dysbindin defining schizophrenia susceptibility pathways have not been quantitatively elucidated. Here, we discovered prevalent and novel cellular roles of the BLOC-1 complex in neuronal cells by performing large-scale Stable Isotopic Labeling of Cells in Culture quantitative proteomics (SILAC) combined with genetic analyses in dysbindin-null mice (Mus musculus) and the genome of schizophrenia patients. We identified 24 proteins that associate with the BLOC-1 complex many of which were altered in content/distribution in cells or tissues deficient in BLOC-1. New findings include BLOC-1 interactions with the COG complex, a Golgi apparatus tether, and antioxidant enzymes peroxiredoxins 1-2. Importantly, loci encoding eight of the 24 proteins are affected by genomic copy number variation in schizophrenia patients. Thus, our quantitative proteomic studies expand the functional repertoire of the BLOC-1 complex and provide insight into putative molecular pathways of schizophrenia susceptibility.
Schizophrenia is a psychotic disorder where genetic factors account for 80% of disease susceptibility (Tandon et al., 2008). The identity of these genetic factors has been under scrutiny leading to the discovery of DTNBP1, the gene encoding dysbindin (Ross et al., 2006; Allen et al., 2008; Sun et al., 2008; Talbot et al., 2009; Ghiani and Dell’angelica, 2011; Mullin et al., 2011). Dysbindin expression is reduced in hippocampi and cortical areas of schizophrenia patients highlighting the relevance of DTNBP1 and dysbindin in molecular pathways leading to schizophrenia (Talbot et al., 2004; Tang et al., 2009a; Mullin et al., 2011; Talbot et al., 2011). Dysbindin is a subunit of the endosome-localized BLOC-1 complex, an octamer composed of dysbindin, pallidin, muted, snapin, cappuccino, and BLOS1-3 subunits (Li et al., 2004; Di Pietro and Dell’Angelica, 2005; Wei, 2006; Raposo and Marks, 2007; Dell’angelica, 2009; Lee et al., 2012). Buds and vesicles containing BLOC-1 and AP-3 participate in a cellular route that culminates with the delivery of cargo between early endosomes and late endosomal/lysosomal compartments (Dell’Angelica et al., 1998; Li et al., 2004; Di Pietro and Dell’Angelica, 2005; Borner et al., 2006; Di Pietro et al., 2006; Wei, 2006; Raposo and Marks, 2007; Setty et al., 2007; Dell’angelica, 2009; Salazar et al., 2009). In neurons, BLOC-1 and AP-3 also define a route that delivers membrane proteins from cell body endosomes to the synapse (Larimore et al., 2011). This route would explain in part why null alleles in BLOC-1 alter the composition of synaptic vesicles and the surface expression of neurotransmitter receptors (Talbot et al., 2006; Iizuka et al., 2007; Ji et al., 2009; Newell-Litwa et al., 2009; Tang et al., 2009b; Marley and von Zastrow, 2010; Newell-Litwa et al., 2010), which in turn trigger neurobehavioral phenotypes resembling those found in schizophrenia patients (Hattori et al., 2008; Bhardwaj et al., 2009; Cox et al., 2009; Dickman and Davis, 2009; Talbot, 2009; Cheli et al., 2010; Papaleo et al., 2010). This evidence points toward fundamental vesicle transport processes controlled by dysbindin-BLOC-1 in neurons delineating a schizophrenia susceptibility pathway. However, the expanse of mechanisms controlled by dysbindin and the BLOC-1 complex in neurons remain largely unexplored. Here we approached this question, revealing novel neuronal pathways that intersect with the BLOC-1 complex and their relationship to schizophrenia.
We quantitatively identified dysbindin-BLOC-1 complex interactors and pathways using mass spectrometry and stringent biochemical and genetic criteria. We identified the subunits of the BLOC-1 and AP-3 complexes as well as the Golgi tether, the COG complex (Smith and Lupashin, 2008), and the antioxidant enzymes peroxiredoxins 1-2 (Bell and Hardingham, 2011; Finkel, 2011). Importantly, genes encoding one third of the proteins identified as dysbindin-BLOC-1 interactors are included in genomic copy number variation (CNV) regions identified in schizophrenia individuals (Consortium, 2008; Karayiorgou et al., 2010). Our proteomic analysis expanded upon the functional repertoire of the BLOC-1 complex. Our findings suggest that endosome trafficking controlled by the BLOC-1 complex and associated factors constitute a cell-autonomous molecular pathway of schizophrenia susceptibility.
Antibodies used in this study are all listed in Table I. SH-SY5Y and HEK293 (ATCC, Manassas, VA, USA) cells were cultured in DMEM media supplemented with 10% fetal bovine serum and 100 μg/mL penicillin and streptomycin (Hyclone, UT, USA) at 37°C in 10% CO2. SH-SY5Y and HEK cell lines were transfected with 3X FLAG Dysbindin (Cat No. EX-Mm12550-M12) or 3X-FLAG Muted (Cat No. EXT4795-M14) constructs obtained from Genecopoeia (Rockville, MD). Both constructs were in a pReceiver vector backbone and sequences were independently confirmed. These stably transfected cell lines were maintained in DMEM media supplemented with 10% FBS, 100 ug/ml penicillin and streptomycin and neomycin (0.2ug/ul) (Cat. No. SV30068, Hyclone, UT, USA) at 37°C in 10% CO2. For shRNA-mediated pallidin knockdowns, shRNA in a pLKO.1 vector for lentiviral infection were obtained from Open Biosystems (Clone ID: TRCN0000122781, Item No. RHS3979-98828366, Huntsville, AL). Control shRNA in pLKO.1 was obtained from Addgene (vector 1864; Cambridge, MA). SH-SY5Y cells were treated with lentiviral particles for 7 days to obtain efficient knockdown. After day 3 of infection cells were maintained DMEM media supplemented with 10% fetal bovine serum and puromycin (2ug/ml) (Invitrogen, CA, USA). In some cases, 12 hours before lysis, cells were treated with a 3mM sodium butyrate solution.
For SILAC labeling, cells were grown in DMEM media with either “light” unlabelled arginine and lysine amino acids (R0K0) or “heavy” 13C and 15N labeled arginine and 13C and 15N labeled lysine amino acids (R10K8) supplemented with 10% FBS and 100μg/ml penicillin and streptomycin and in some cases 0.2μg/μl neomycin. Cells were grown for a minimum of six passages ensuring maximum incorporation of the amino acids in the cellular proteins. All reagents for SILAC labeling were obtained from Dundee Cell Products (Scotland, UK). We confirmed the degree of incorporation of labeled amino acids in the total cellular pool as 97.5%.
To assess low-affinity interactions of BLOC-1 subunits, we performed cross-linking in intact cells with DSP-dithiobis(succinimidylpropionate) as previously described (Craige et al., 2008; Salazar et al., 2009; Zlatic et al., 2010). Briefly, untransfected HEK or SH-SY5Y cells or SH-SY5Y cells stably transfected either with FLAG-dysbindin or FLAG-muted were placed on ice, rinsed twice with PBS, and incubated either with 10 mM DSP (Pierce, Rockford, IL), or as a vehicle control DMSO, diluted in PBS for 2 h on ice. Tris, pH 7.4, was added to the cells for 15 min to quench the DSP reaction. The cells were then rinsed twice with PBS and lysed in buffer A (150 mM NaCl, 10 mM HEPES, 1 mM EGTA, and 0.1 mM MgCl2, pH 7.4) with 0.5% TritonX-100 and Complete anti-protease (Cat No. 11245200, Roche, IN, USA), followed by incubation for 30 min on ice. Cells were scraped from the dish, and cell homogenates were centrifuged at 16,100×g for 10 min. The clarified supernatant was recovered, and at least 500 μg of protein extract was applied to 30 microliters Dynal magnetic beads (Cat. No, 110. 31, Invitrogen, CA, USA) coated with antibody, and incubated for 2 hours at 4°C. In some cases, immunoprecipitations were done in the presence of the antigenic 3X FLAG peptide (340μM) (F4799, Sigma, MO, USA) as a control. The beads were then washed 4-6 times with buffer A with 0.1% TritonX-100. Proteins were eluted from the beads either with sample buffer or by 2 hours incubation with either buffer A alone as a control or 340 μM 3X-FLAG antigenic peptide on ice. Samples were resolved by SDS-PAGE and contents analyzed by immunoblot or silver stain. In the case of the large-scale proteomic analysis, proteins eluted from the beads were combined and concentrated by TCA precipitation. Samples were analyzed for SILAC protein identification by Dundee Cell Products (Scotland, U.K), MS Bioworks (MI, USA) and the Emory CND Proteomics Facility (GA, USA).
SILAC labeled samples were separated on a 4-12% Bis-Tris Novex mini-gel (Invitrogen, CA, USA) using the MOPS buffer system. The gel was stained with coomassie and the lane was excised into 20 equal segments using a grid. Gel pieces were processed using a robot (ProGest, DigiLab) with the following protocol. First, slices were washed with 25mM ammonium bicarbonate followed by acetonitrile. Reduced with 10mM dithiothreitol at 60°C followed by alkylation with 50mM iodoacetamide at RT. Samples were digested with trypsin (Promega) at 37°C for 4h. Quenched with formic acid and the supernatant was analyzed directly without further processing. Each gel digest was analyzed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher LTQ Orbitrap Velos. Peptides were loaded on a trapping column and eluted over a 75μm analytical column at 350nL/min; both columns were packed with Jupiter Proteo resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 FWHM resolution and MS/MS performed in the LTQ. The fifteen most abundant ions were selected for MS/MS. Data were processed through the MaxQuant software v184.108.40.206 (www.maxquant.org) which served data recalibration of MS, filtering of database search results at the 1% protein and peptide false discovery rate (FDR), and calculation of SILAC heavy:light ratios. Data were searched using a local copy of Mascot.
Mocha (STOCK gr +/+ Ap3d1mh/J, here referred to as Ap3d1mh/mh) and its control grizzled (STOCK gr +/+ Ap3d1+/J, here referred to as Ap3d+/+) and pallid (B6.Cg-Pldnpa/J, here referred to as Pldnpa/pa) breeding mouse pairs were obtained from Jackson Labs (Bar Harbor, Maine). Muted mice and their controls (B6C3 Aw-J/A-Mutedmu/J, Mutedmu/mu and CHMU+/mu) (Zhang et al., 2002) were obtained from Dr. Richard Swank (Roswell Park Cancer Institute, Buffalo, NY, USA) and bred in-house. Sandy (Dtnp1dntp1/dtnp1) mice in the C57/B6 background were obtained in breeding pairs from Dr. Konrad Talbott (University of Pennsylvania, PA, USA) as previously described (Cox et al., 2009). All mice were bred in-house following IUCAC approved protocols and used at six weeks of age. We used both male and female mice indistinctively.
Hippocampal tissue was dissected from either wild type C57B6 mice or from dysbindin deficient Sandy mice and placed in cold PBS. 500 ul of lysis buffer (Buffer A, 0.5% Triton X-100 and Complete antiprotease (Cat No. 11245200, Roche, IN, USA) was added to each tissue followed by homogenization by sonication on ice. The sonicated tissue was allowed to rest on ice for 30 minutes followed by a high-speed spin at 16,100×g for 10 minutes at 4°C. The supernatant was then recovered, followed by protein measurements and then aliquoted for biochemical analysis.
Control or pallidin knockdown SH-SY5Y cells were rinsed twice with PBS and lysed in buffer A with 0.5% TritonX-100 supplemented with Complete antiprotease, followed by incubation for 30 min on ice. Cells were scraped from the dish, and cell homogenates were centrifuged at 16,100×g for 10 min. The clarified supernatant was recovered and measured for total protein content. Samples were then analyzed by immunoblot.
Cell lysates were resolved by sucrose sedimentation in 5-20% sucrose gradients as previously described (Salazar et al., 2009).
Control or pallidin knockdown SH-SY5Y cells were grown in selection media (DMEM+10% FBS+puromycin) for 7 days. Cells were then lifted by trypsin, pelleted and resuspended in PBS and the hydrogen peroxide sensitive probe DCFDA (4μl/ml) (D-399, Molecular Probes, CA, USA). As a negative control, cells were suspended only in PBS (non-stained control). The cells were then incubated at 37°C for 30 minutes. The cells were then pelleted at 800×g for 5 minutes and resuspended either in only PBS or PBS supplemented with 2 μM hydrogen peroxide as a positive control (added after resuspending in PBS). The samples were protected from light and analyzed by the LSR II FACScan analyzer (Becton Dickinson) using the 488nm excitation laser. Data was analyzed using the FlowJo software, version 8.2.2 (Tree Star Inc., OR, USA).
Brain slice sections were prepared from mice between 6 to 8 weeks of age. Animals were anesthetized with nembutal, then transcardially perfused with Ringer’s solution followed by perfusion of fixative (4% paraformaldehyde with 0.1% gluteraldehyde in PBS). Perfused brains were post-fixed with 4% paraformaldehyde, which was replaced with PBS within 12-18 hours. Brain tissue was cut into 60μm thick sections using a vibrating microtome and brain slice sections were stored in antifreeze (0.1M sodium phosphate monobasic, 0.1M sodium phosphate dibasic heptahydrate, 30% ethylene glycol, 30% glycerol) at −20°C until immunohistochemical preparation.
Brain sections containing the hippocampus formation were incubated for 20 minutes at room temperature in 1% sodium borohydride. Sections were rinsed with PBS, then, pre-incubated for 60 minutes at room temperature in a blocking solution (5% Normal Horse Serum and 1% BSA and 0.3% Triton X-100). Sections were incubated overnight at 4°C in primary antibody solutions (1% Normal Horse Serum and 1% BSA with the antibodies described in Table 1). The following day, sections were incubated for 60 minutes at room temperature in a secondary antibody solution (1% NHS and 1% BSA with 1:500 dilutions of Alexa-conjugated secondary antibodies: anti-mouse 488 or 568, anti-rabbit 488 or 568) (Invitrogen Molecular Probes, Carlsbad, CA, USA). Sections were rinsed and then incubated for 30 minutes at RT in cupric sulfate (3.854 W/V Ammonium Acetate, 1.596 W/V Cupric Sulfate in distilled water, pH 5). Sections were rinsed and mounted on glass coverslips with Vectashield mounting media (Vector Laboratories) for confocal microscopy analysis (Newell-Litwa et al., 2010). Confocal microscopy was performed with an Axiovert 100M microcope (Carl Zeiss) coupled to an Argon and HeNe1 lasers.
Images were acquired using Plan Apochromat 10×/0.5 dry, 20using Plan Apochromat 10/0.5 dry, and 40using Plan Apochromat 10/1.3 and 63using Plan Apochromat 10/1.4 DiC oil objectives. Emission filters used for fluorescence imaging were BP 505-530 and LP 560. Images were acquired with ZEN and LSM 510 software (Carl Zeiss). Fluorescence intensities were determined by Metamorph software (Newell-Litwa et al., 2010). At least four independent stainings from two animals were performed.
We searched for an overlap between the list of schizophrenia candidate genes identified in our study and all the CNVs reported by International Schizophrenia Consortium (3,391 patients with schizophrenia and 3,181 ancestrally matched controls)(Consortium, 2008) to determine if any one of our candidate genes was included. The complete list of all QC-passing CNVs (BED file format, hg17) can be accessed at http://pngu.mgh.harvard.edu/isc/cnv.html. We used UCSC Genome Browser’s “Batch Coordinate Conversion (liftOver)” utility to convert genome coordinate annotations from hg17 to hg19. This tool is publicly accessible at http://genome.ucsc.edu/index.html.
Experimental conditions were compared with the non-parametric Wilcoxon-Mann-Whitney Rank Sum Test or One Way ANOVA, Dunnett’s Multiple Comparison using Synergy KaleidaGraph v4.03 (Reading, PA) or StatPlus Mac Built5.6.0pre/Universal (AnalystSoft, Vancouver, Canada). Data are presented as dot or boxplots. The latter display the four quartiles of the data, with the “box” comprising the two middle quartiles, separated by the median.
We used a highly sensitive quantitative proteomic approach to identify preponderant BLOC-1 interacting proteins to shed light on the broader functions of this complex. We took advantage of a well-established in-vivo cellular cross-linking protocol based on the cell permeant homobifunctional crosslinking agent DSP. DSP has an 11Å spacer arm and a disulphide bond allowing a reversal of chemical bridges between proteins by reducing agents (Lomant and Fairbanks, 1976; Zlatic et al., 2010). Upon DSP crosslinking, BLOC-1 proteins are presumably associated with other cellular proteins and therefore, predictably, we see a shift in the molecular sedimentation on a sucrose gradient of BLOC-1 component proteins, pallidin and dysbindin, compared to the BLOC-1 proteins that are not crosslinked (Fig 1A). We selectively enriched for BLOC-1 interacting proteins once molecular associations were chemically stabilized using immunomagnetic purification of protein complexes. We selected human neuroblastoma cell lines (SH-SY5Y) stably expressing either 3x-FLAG-tagged Dysbindin or 3x-FLAG-tagged Muted as a source of BLOC-1 complexes and interactors. We confirmed that tagged BLOC-1 subunits incorporated into the BLOC-1 complex in neuronal and non-neuronal cell lines (Fig. 4A-B, and unpublished data). Immunoprecipitation using the FLAG antibody from cross-linked FLAG-dysbindin expressing cells isolated dysbindin as well as other bands detected by silver stain (Fig. 1B, lane 4 asterisk marks FLAG-dysbindin). However, many of the bands precipitated were common with bands in control lanes (Fig. 1B, compare lane 4 with lane 2, 3 and 5). Therefore, to further purify and specifically enrich for polypeptides that would represent BLOC-1 interacting proteins with minimum background, we used the antigenic FLAG-peptide to selectively elute FLAG-dysbindin and its cross-linked interacting proteins from the immunomagnetic beads (Fig 1B, lane 7). We termed this method “immunoaffinity chromatography”. The immunoblot in Fig 1B, probed with the FLAG antibody, and the silver stain confirmed that FLAG-dysbindin and associated proteins were efficiently and specifically eluted using the antigenic FLAG peptide since only low levels of dysbindin and dysbindin-interacting polypeptides were detected in the magnetic beads after elution (Fig. 1B compare lanes 7 and 8). We further refined the selectivity of the FLAG immunoaffinity chromatography by coupling it to a two-tier protein identification strategy (Fig. 2A). First, protein identification was based on Stable Isotopic Labeling of Cells in Culture -SILAC-mass spectrometry followed by a curation of proteins against a database of polypeptides that non-selectively bind to FLAG antibody-decorated magnetic beads from untransfected, DSP cross-linked cell extracts (Ong et al., 2002; Mann, 2006; Trinkle-Mulcahy et al., 2008).
Cells expressing 3x-FLAG-tagged BLOC-1 subunits were stably labeled either with “light media” for controls (R0K0) or “heavy media” (R10K8, Fig 2A) to generate “light” and “heavy” cell extracts. We incubated “light” cell extracts with FLAG-decorated magnetic beads in the presence of an excess FLAG peptide. This approach completely prevented the binding of FLAG-dysbindin and associated polypeptides to beads making it a robust control to identify non-specific proteins bound to beads independent of the FLAG antibody (see Fig. 1B lane 2, 3 and 5). In contrast, “heavy” cell extracts were incubated only with FLAG-antibody decorated magnetic beads. We scaled-up the FLAG immunoaffinity chromatography for preparative purification from either FLAG-Muted or FLAG-Dysbindin expressing cell extracts (Fig 2B). Light and heavy-labeled eluted polypeptides were mixed 1:1 for MS/MS analysis. We identified a total of 105 eluted polypeptides associated with FLAG dysbindin (Fig. 2A). Subtraction of these 105 polypeptides with a non-specific polypeptide library generated from FLAG immunoaffinity chromatography eluates derived from untransfected cells reduced them to 43 specific polypeptides (Fig. 2A). We focused on 24 of these 43 proteins because their heavy/light labeling exceeded a ratio of 2 (see Fig 2A, 3A-B and Table II). The two-fold enrichment is a stringent cut-off criterion for SILAC experiments to reliably detect differences in the protein content between two samples (Ong et al., 2002; Ong and Mann, 2006; Trinkle-Mulcahy et al., 2008). The proteins were independently and reproducibly identified in multiple non-SILAC MS/MS experiments using cell lines transfected with two different subunits of the BLOC-1 complex (FLAG-Muted and FLAG-Dysbindin, Table II). Some of the proteins identified were known BLOC-1 binding partners validating the experimental approach. For example, we identified most subunits of the AP-3 adaptor protein complex and all eight subunits of the BLOC-1 complex (Di Pietro et al., 2006; Salazar et al., 2009). Importantly, we identified several novel putative BLOC-1 interacting proteins (listed in Table II and depicted in Fig 3). Those include two clathrin heavy chain isoforms, members of two tethering complexes, the COG and the exocyst complex; redox enzymes - peroxiredoxins I and II-, membrane proteins such as the SNARE Snap29 and the potassium channel KCNQ5, as well as proteins involved in axonal guidance and growth CRMP4 and α-N-catenin (Fig. 3C and Table II)(Schmidt and Strittmatter, 2007; Suzuki and Takeichi, 2008).
We used biochemical and genetic approaches to further authenticate putative BLOC-1 interactors isolated by FLAG-dysbindin immunoaffinity chromatography, namely, co-immunoprecipitation and analyzing changes in the content or distribution of the candidate BLOC-1 interactors in the dentate gyrus of BLOC-1 null mouse brains (Fig. 4--5).5). This combined strategy has previously been used to characterize the interaction between the adaptor protein complex AP-3 and BLOC-1 (Fig. 4)(Di Pietro et al., 2006; Salazar et al., 2009; Newell-Litwa et al., 2010). Various subunits of the AP-3 complex co-immunoprecipitated with FLAG-dysbindin either from SH-SY5Y or HEK293 cell extracts (Fig 4A-B). Importantly, this biochemical interaction manifested itself as a decrease in the AP-3 immunoreactivity in the dentate gyrus of BLOC-1 deficient mice sandy (Dtnbp1sdy/sdy, Fig 4 F and F’) by quantitative confocal microscopy, a finding similar to our previous report in Mutedmu/mu null dentates (Newell-Litwa et al., 2010). The robustness of this BLOC-1 null phenotype is highlighted by the reciprocal decrease in pallidin immunoreactivity in the dentate gyrus of AP-3 deficient mice (mocha, Ap3d1mh/mh) (Fig. 4 H and H’).
To test the relevance of novel putative BLOC-1 interactors, we used, as a confidence criterion, either co-immunoprecipitation with tagged dysbindin and/or alteration in antigen distribution/content in the dentate gyrus of dysbindin-null mouse brains -Dtnbp1sdy/sdy. FLAG-dysbindin co-precipitated BLOC-1 (pallidin and Blos3) and AP-3 (AP-3 β3) subunits (Fig. 4A-B and and5A).5A). Importantly, putative novel interactors such as the exocyst subunits (sec6 and sec8), snap29, and peroxiredoxin I also co-precipitated selectively with FLAG-dysbindin. Importantly, abundant proteins such as cofilin and actin were not found in FLAG-dysbindin precipitates (Fig. 5A compare lanes 3 and 4). Other proteins identified by SILAC such as αN-catenin, CRMP4, and KCNQ5 could not be detected reliably in FLAG-dysbindin or FLAG-muted immunoprecipitations. Therefore, we resorted to distribution/content modifications of these antigens in the dentate gyrus of Dtnbp1sdy/sdy mouse brains using quantitative confocal microscopy. The distribution/content of KCNQ5, Sec8, CRMP4, and αN-catenin was specifically altered in the dentate gyrus of BLOC-1 deficient Dtnbp1sdy/sdy mice. While the immunoreactivity of KCNQ5 and αN-catenin was reduced in Dtnbp1sdy/sdy dentate gyri, CRMP4 immunoreactivity increased (Fig 5B-F). All these phenotypes were selective as evidenced by the co-labeling with VAMP2 or synaptophysin (Sphysin) antibodies, which detect two synaptic vesicle protein not affected by BLOC-1 deficiencies (Newell-Litwa et al., 2009). As an additional control for KCNQ5, we analyzed the distribution/content of another unrelated potassium channel, Kv1.2. Kv1.2 was not affected in Dtnbp1sdy/sdy brains (Fig. 5B and G). Overall, our results revealed that our unbiased and quantitative mass-spectrometry identification of BLOC-1 interactors generated a specific database of proteins whose expression can phenotypically distinguish wild-type from Dtnbp1sdy/sdy dentate gyri. Moreover, these results point to two previously unsuspected associations of the BLOC-1 complex: first, with the COG Golgi tethering complex, and second with peroxiredoxins, enzymes involved in redox metabolism.
We focused on the COG complex, a hetero-octameric protein complex that acts as a tether for vesicles fusing with the cis-Golgi that regulates the steady state content of Golgi proteins (Shestakova et al., 2006; Ungar et al., 2006; Smith and Lupashin, 2008). The COG complex is organized into two distinct lobes – lobe A (Cog1-4) and lobe B (Cog5-8) (Ungar et al., 2002; Lees et al., 2010). Our proteomic analysis directly identified 2 subunits from lobe B that co-isolate with the FLAG dysbindin -Cog 6 and Cog 8 (Fig 3 and Table II). However, upon further analysis we discovered that FLAG dysbindin co-precipitates subunits of both lobe A (Cog 3 and 4) and lobe B (Cog 5, 6, 7 and 8) (Fig 6A). Using a Cog5 antibody, we reciprocally co-immunoprecipitated FLAG-dysbindin from transfected cell lines (Fig 6B) as well as the endogenous BLOC-1 subunit pallidin from untransfected cell lines (Fig 6C). Notably, the chemical crosslinker DSP was not essential to reveal the interaction (see lane 6 in Fig 6B and lane 7 in Fig 6D) indicating that the COG-BLOC-1 interaction does not require the stringency of our precipitation assay and is maintained even in the absence of chemical stabilization. Importantly, and consistent with previously published data (Ungar et al., 2002), the COG complex isolated with Cog5 antibodies did not co-precipitate the exocyst complex as revealed by the absence of Sec8 in protein complexes precipitated with Cog5 antibodies (Fig 6C; IB: Sec 8). The BLOC-1-COG interaction is conserved irrespective of the cell type used (neuronal cell type in Fig 6B and 6C and epithelial cell line in Fig 6D) suggesting that a ubiquitous pathway between BLOC-1 and the COG tether may exist in all cells.
Loss-of-function of individual subunits of the BLOC-1 complex leads to downregulation of the other subunits of the BLOC-1 octamer suggesting that a loss of protein expression may be useful as an assay for structural or functional protein associations beyond those of the BLOC-1 complex proper (Zhang et al., 2002; Li et al., 2003; Starcevic and Dell’Angelica, 2004). Thus, we explored whether the down-regulation of the BLOC-1 subunit pallidin (Fig 6E) could alter the content of other BLOC-1 subunits and possibly components of the COG complex. SH-SY5Y cells treated with shRNA directed against pallidin down-regulated the BLOC-1 subunits muted and dysbindin, much like it is the case with murine null alleles of these BLOC-1 subunits (Fig 6E)(Zhang et al., 2002; Li et al., 2003; Starcevic and Dell’Angelica, 2004). Importantly, the content of the COG subunit Cog7 was also partially decreased in pallidin shRNA treated cells. This effect of BLOC-1 loss-of-function upon Cog7 content was recapitulated in hippocampi from BLOC-1 deficient Dtnbp1sdy/sdy mice (Fig 6F-G).
To further estimate the downstream effect of BLOC-1 loss of function upon COG function, we took advantage of the previously characterized “COG-sensitive” proteins. There are seven COG mutation-sensitive Golgi resident integral membrane proteins (GEARs) whose levels are down-regulated in the absence of the COG complex (Oka et al., 2004). Among those, we used CASP, an alternative splicing variant of the CUX1 gene, as a reporter for COG function (Gillingham et al., 2002; Oka et al., 2004). We hypothesized that down-regulation of COG complex subunits by BLOC-1 loss-of-function could in turn result in CASP down-regulation. Indeed, CASP levels in pallidin shRNA-treated cells were reduced to 77% of shRNA control-treated cells (Fig 6E, 77.2 ± 5.5% n=4 p=0.021 Wilcoxon-Mann-Whitney Rank Sum Test). Similarly, the content of CASP was reduced in the hippocampal formation of BLOC-1 deficient Dtnbp1sdy/sdy mice (Fig 6F-G). This evidence supports a biochemical and functional interaction between the BLOC-1 and the COG complex and suggests a novel pathway linking the endosome-localized BLOC-1 protein complex to the Golgi apparatus.
Our FLAG dysbindin proteomic analysis identified an additional novel association with peroxiredoxins I and II. These enzymes are involved in metabolism of peroxide and therefore the prediction is that BLOC-1 could modulate the redox state in cells (Bell and Hardingham, 2011). We tested the peroxiredoxin and BLOC-1 complex interaction analyzing the content/distribution of these enzymes in the dentate gyrus of BLOC-1 deficient Dtnbp1sdy/sdy mice as well as neuroblastoma cells rendered BLOC-1 deficient by shRNA down-regulation of another BLOC-1 subunit pallidin. Similar to other proteins identified by FLAG-dysbindin immunoaffinity chromatography, BLOC-1 deficiency either in the dentate gyrus of BLOC-1 deficient sandy Dtnbp1sdy/sdy mice or in pallidin shRNA-treated neuroblastoma cells demonstrated a significant decrease in peroxiredoxins immunoreactivity (Fig 7A-F). The Dtnbp1sdy/sdy dentate gyrus and pallidin shRNA induced peroxiredoxin phenotypes provide strong evidence of a functional consequence of the association between these redox enzymes and the BLOC-1 complex. Since peroxiredoxins scavenge hydrogen peroxide, we predicted that down-regulated peroxiredoxin I and II levels observed in BLOC-1 deficiencies would induce a steady state increase in cellular hydrogen peroxide levels. To test this hypothesis we employed a flow cytometry assay, utilizing neuroblastoma cells treated with control or pallidin targeting shRNA where cellular peroxide levels were measured in-vivo using the hydrogen peroxide specific fluoroprobe: 2′,7′-dichlorofluorescein (DCF) (Myhre et al., 2003; Cossarizza et al., 2009). Fluorescence intensity in control shRNA-treated neuroblastoma cells was increased 1.6 fold by the addition of a physiologically relevant concentration of 2 μM H2O2 (Fig 7G and I). Notably, shRNA down-regulation of one of the BLOC-1 subunit, pallidin, was sufficient to increase DCF fluorescence intensity robustly. The magnitude of this increase in BLOC-1 down-regulated cells was comparable to the increase seen with exogenously added H2O2 to control cells (Fig 7H and 7I). Thus, these results identify a novel function of the BLOC-1 complex regulating enzymes involved in redox metabolism.
We explored prevalent cellular roles of the BLOC-1 complex in neuronal cells by performing a large-scale SILAC proteomic analysis. We identified 105 proteins that after a stringent two-tier set of filters, zeroed in on 24 proteins that associate with the BLOC-1 subunit dysbindin – a schizophrenia susceptibility factor. Of these 24 proteins, 11 were novel BLOC-1 binding partners. Prominent among these 24 proteins are all the subunits of the BLOC-1 and AP-3 adaptor complexes. Each of these 24 proteins was independently confirmed to be a BLOC-1 interactor and many were altered in content/distribution in cells or tissues deficient in BLOC-1 complexes. Major new findings from our studies are BLOC-1 interactions with the COG complex Golgi tether, and biochemical and genetic evidence supporting interactions between BLOC-1 and the antioxidant enzymes peroxiredoxins I and II. Thus, our quantitative proteomic analysis expanded upon the functional repertoire of the BLOC-1 complex and provides insight into molecular pathways of schizophrenia susceptibility, which may now include endosome to TGN retrograde traffic (COG) as well as redox metabolism (peroxiredoxins).
A common way to elucidate the function of proteins or their complexes is to identify other proteins associated with it. However, there are pitfalls to the methods employed to define interactors, in particular those associated with BLOC-1 (Ghiani and Dell’angelica, 2011). To address several of the problems associated with studying the BLOC-1 complex, such as the low expression levels of BLOC-1 subunits, we employed a multifold approach. We quantitatively estimated peptides specifically enriched by immunoaffinity chromatography of FLAG-tagged BLOC-1 subunits purified from cells labeled with non-radioactive isotope tagged amino acids (Mann, 2006). This procedure was coupled with in vivo crosslinking with DSP to stabilize BLOC-1 interactions (Zlatic et al., 2010). Since DSP was used at a sub-stoichiometric level, we likely identified major BLOC-1 interacting proteins as attested by the enrichment of BLOC-1 and AP-3 subunits. The sub-stoichiometric use of DSP was intended to stabilize immediate or first order, interactors and to minimize formation of extended crosslinked higher order networks. For example, crosslinked complexes revealed that BLOC-1 co-isolated with either the COG complex or exocyst complex proteins and did not result in a large complex containing BLOC-1, COG and exocyst subunits altogether (Ungar et al., 2002). An additional layer of stringency in protein identification was the independent verification of protein interactions in BLOC-1 deficiencies, either using neuroblastoma cells rendered deficient by shRNA or hippocampal tissue from Dtnbp1sdy/sdy mice. We reasoned that if proteins interact with the BLOC-1 complex then they could reveal novel phenotypes in BLOC-1 deficiencies. One of these phenotypes is the co-down-regulation of BLOC-1 subunits in cellular lysates when one of the subunits is absent or reduced (Zhang et al., 2002; Li et al., 2003; Starcevic and Dell’Angelica, 2004). Such a phenotype was observed with peroxiredoxins I and II, as well as one of the COG subunits, Cog7. For another group of markers, we observed that their immunoreactivity was modified in the dentate gyrus of Dtnbp1sdy/sdy mice. Such is the case of AP-3 subunits, KCNQ5, peroxiredoxins I and II, αN-catenin, CRMP4, the exocyst subunit Sec8, and Snap29 (unpublished observations).
Dysbindin interactions and their function are frequently considered independent of the BLOC-1 complex. However, several lines of evidence summarized by Ghiani and Dell’Angelica emphasize dysbindin to be an integral component of the BLOC-1 complex (Ghiani and Dell’angelica, 2011; Mullin et al., 2011). Our data are in rapport with this paradigm. The BLOC-1 interactions reported here are reproducibly obtained with two independent BLOC-1 subunits – dysbindin and muted. Furthermore, phenotypes induced by down-regulation of a third BLOC-1 subunit, pallidin, are recapitulated in dysbindin null, Dtnbp1sdy/sdy mice. This is particularly evident in the down-regulation of Cog7 both in pallidin and dysbindin-deficient cells or tissues, respectively. What is the nature of the association between the COG and the BLOC-1 complex? One way we addressed this question was analyzing one of the COG-sensitive integral membrane Golgi proteins, collectively called GEAR proteins. GEAR proteins are reduced in COG-deficient cells and therefore provide a phenotypic readout for COG functions (Oka et al., 2004). CASP, the GEAR protein analyzed here, is an integral Golgi membrane protein (Gillingham et al., 2002). Our analysis demonstrated that when BLOC-1 was down-regulated, it led to a decreased level of COG complex proteins and in turn led to a reduction of CASP. These data indicate that the BLOC-1-dependent reduction of a COG complex subunit, although moderate, is sufficient to trigger a COG-dependent phenotype. These findings suggest that the BLOC-1 complex participates in an endosome route back to the Golgi complex delivering membrane proteins residen to or transiting through the Golgi complex (Smith et al., 2009). Since the CASP down-regulation observed in BLOC-1 deficiency is subtle, as expected from a moderate Cog7 reduction, we speculate that a subset of COG-dependent vesicles derived from endosomes and bound to the Golgi complex may be uniquely susceptible to BLOC-1 deficiency. These vesicles likely would use the Snap29 SNARE, another new associate of the BLOC-1 complex. In agreement with this model, we have recently identified Snap29 as a direct binding partner of Cog6 protein (V.L., unpublished data).
Another interesting family of proteins identified in our SILAC proteomic analysis is peroxiredoxins. Peroxiredoxins I and II are ubiquitously expressed enzymes that remove low level peroxides generated as a result of steady state cellular metabolism (Bell and Hardingham, 2011). These two enzymes are down-regulated in pallidin shRNA-treated cells and its immunoreactivity is decreased in the dentate gyrus of the hippocampal formation from BLOC-1 deficient Dtnbp1sdy/sdy mice. This decrease in the peroxiredoxins, in turn, resulted in a significant increase in the steady levels of hydrogen peroxide in the range of low micromolar level. Apart from being a byproduct of oxidative metabolism, hydrogen peroxide also participates in cell signaling (Finkel, 2011). Consequently, peroxiredoxins play essential roles in mediating signaling cascades targeted by hydrogen peroxide (Neumann et al., 2009; Finkel, 2011). BLOC-1 may regulate the activity or subcellular location of peroxiredoxins, for example in signal transduction by tyrosine kinase receptors on endosomes. Alternatively, peroxiredoxins may modulate BLOC-1 function by a redox mechanism, such as by regulating the oxidation status of cysteines either in BLOC-1 subunits (all human BLOC-1 isoforms contain cysteine residues), or in membrane proteins in close proximity of BLOC-1. It is of interest that the levels of peroxiredoxin I are reduced in the frontal cortex of schizophrenia patients (Focking et al., 2011; Martins-de-Souza et al., 2011). Such a reduction is consistent with the low expression of dysbindin and other BLOC-1 subunits in cortical areas of patients with schizophrenia (Talbot et al., 2004; Tang et al., 2009a; Mullin et al., 2011; Talbot et al., 2011). This raises the possibility that schizophrenia pathogenesis hypotheses centered on redox alterations and those linked to dysbindin may converge on a common molecular mechanism.
Part of our interest in BLOC-1 and dysbindin biology stems from its correlation with schizophrenia risk (Talbot et al., 2009; Mullin et al., 2011). We hypothesized that if dysbindin is part of a molecular pathway contributing to or affected by schizophrenia, then genes encoding dysbindin interactors should be significantly represented among those genes with structural variants associated with schizophrenia risk, such as SNAP29 (Malhotra et al., 2011). To test this prediction we analyzed The International Schizophrenia Consortium database of cases carrying rare chromosomal deletions and duplications that increase risk of schizophrenia (Consortium, 2008). This database contains 3,391 schizophrenia cases and 3,181 controls. Our prediction is strongly supported by CNVs encompassing genes encoding dysbindin interactors (Table III). Eight of the 24 proteins identified as dysbindin-BLOC-1 interactors are represented among genes within CNVs exclusively found in schizophrenia patients. Among those we found COG and AP-3 complex subunits. None of these eight loci are affected in two genome wide analyses of unaffected individuals totaling 3,853 subjects (Consortium, 2008; Buizer-Voskamp et al., 2011). Strikingly, the dysbindin interactors Snap29 and the clathrin heavy chain isoform CLTCL1 (CHC22) were among those proteins whose genes are most frequently affected in schizophrenia individuals (Table III).) These two genes are located within the chromosome 22q11.2 region. Individuals with haploinsufficiency of this region have 22q11.2 deletion syndrome and develop schizophrenia at rate of ~30%. These deletions account for as many as ~2% of de novo schizophrenia cases in the general population (Karayiorgou et al., 2010). The 22q11.2 deletion syndrome also encompasses SEPT5, the gene encoding septin 5, a protein that binds septin 8 and AP-3 complexes both found in our dysbindin proteome (Baust et al., 2008; Nakahira et al., 2010). Thus, 22q11.2 deletion syndrome combines up to three haploinsufficiencies that converge on a pathway defined by the schizophrenia susceptibility factor, dysbindin. The molecular associations between a clathrin heavy chain isoform (CHC22), Snap29, and septin 5 suggest that the 22q11.2 deletion syndrome may have a pronounced deficiency of this pathway. These findings support the concept that quantitative proteomes of a schizophrenia susceptibility factor, such as dysbindin, can define putative schizophrenia susceptibility pathways by revealing unsuspected connections between the disease associated genomic loci. This hypothesis is supported by the dysbindin interactor Snap29 and the clathrin heavy chain isoform CLTCL1 (CHC22), which are among those proteins whose genes are frequently affected in schizophrenia individuals carrying CNVs associated to disease (Table III).
Pathogenic hypotheses for schizophrenia have tended to emphasize individual genes of “interest” rather than cell-autonomous pathways defined by the molecular interactions of a schizophrenia susceptibility factor (Ross et al., 2006; Tandon et al., 2008). One of those putative pathways is an endosomal hub defined by dysbindin and its protein interactors many of which remained unknown or were not prioritized based on their abundance in dysbindin isolates (Mead et al., 2010). Based on the work presented here we propose that defective endosome sorting mechanisms controlled by the BLOC-1 complex may contribute to the pathogenesis of schizophrenia and systemic disorders that characterize the 22q11.2 deletion syndrome.
This work was supported by grants from the National Institutes of Health to V.F. (NS42599, GM077569) and V.L. (GM083144). AG and JL were supported by National Institutes of Health FIRST program Fellowship K12 GM000680. We are indebted to the Faundez lab members and Dr. Frances Brodsky for their comments. Supported in part by the Neuronal Imaging Core of the Emory Neuroscience NINDS Core Facilities Grant P30NS055077 and by the Flow Cytometry Core Facility of the Emory University School of Medicine.