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Regulation of Brain-Derived Neurotrophic Factor Exocytosis and Gamma-Aminobutyric Acidergic Interneuron Synapse by the Schizophrenia Susceptibility Gene Dysbindin-1

  • Author Footnotes
    ⁎ Authors QY and FY contributed equally to this work.
    Qiang Yuan
    Footnotes
    ⁎ Authors QY and FY contributed equally to this work.
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
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  • Author Footnotes
    ⁎ Authors QY and FY contributed equally to this work.
    Feng Yang
    Footnotes
    ⁎ Authors QY and FY contributed equally to this work.
    Affiliations
    Lieber Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, Maryland
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  • Yixin Xiao
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
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  • Shawn Tan
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
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  • Nilofer Husain
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
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  • Ming Ren
    Affiliations
    Lieber Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, Maryland
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  • Zhonghua Hu
    Affiliations
    Lieber Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, Maryland
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  • Keri Martinowich
    Affiliations
    Lieber Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, Maryland
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  • Julia S. Ng
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
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  • Paul J. Kim
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
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  • Weiping Han
    Affiliations
    Singapore Bioimaging Consortium, Singapore, Singapore
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  • Koh-ichi Nagata
    Affiliations
    Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan
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  • Daniel R. Weinberger
    Affiliations
    Lieber Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, Maryland
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  • H. Shawn Je
    Correspondence
    Address correspondence to H. Shawn Je, Ph.D., Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore;
    Affiliations
    Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore

    Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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  • Author Footnotes
    ⁎ Authors QY and FY contributed equally to this work.
Open AccessPublished:August 27, 2015DOI:https://doi.org/10.1016/j.biopsych.2015.08.019

      Abstract

      Background

      Genetic variations in dystrobrevin binding protein 1 (DTNBP1 or dysbindin-1) have been implicated as risk factors in the pathogenesis of schizophrenia. The encoded protein dysbindin-1 functions in the regulation of synaptic activity and synapse development. Intriguingly, a loss of function mutation in Dtnbp1 in mice disrupted both glutamatergic and gamma-aminobutyric acidergic transmission in the cerebral cortex; pyramidal neurons displayed enhanced excitability due to reductions in inhibitory synaptic inputs. However, the mechanism by which reduced dysbindin-1 activity causes inhibitory synaptic deficits remains unknown.

      Methods

      We investigated the role of dysbindin-1 in the exocytosis of brain-derived neurotrophic factor (BDNF) from cortical excitatory neurons, organotypic brain slices, and acute slices from dysbindin-1 mutant mice and determined how this change in BDNF exocytosis transsynaptically affected the number of inhibitory synapses formed on excitatory neurons via whole-cell recordings, immunohistochemistry, and live-cell imaging using total internal reflection fluorescence microscopy.

      Results

      A decrease in dysbindin-1 reduces the exocytosis of BDNF from cortical excitatory neurons, and this reduction in BDNF exocytosis transsynaptically resulted in reduced inhibitory synapse numbers formed on excitatory neurons. Furthermore, application of exogenous BDNF rescued the inhibitory synaptic deficits caused by the reduced dysbindin-1 level in both cultured cortical neurons and slice cultures.

      Conclusions

      Taken together, our results demonstrate that these two genes linked to risk for schizophrenia (BDNF and dysbindin-1) function together to regulate interneuron development and cortical network activity. This evidence supports the investigation of the association between dysbindin-1 and BDNF in humans with schizophrenia.

      Keywords

      In the mammalian central nervous system, neurons receive inhibitory synaptic inputs from gamma-aminobutyric acid (GABA)ergic interneurons and excitatory inputs from glutamatergic neurons (
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      Dysbindin-1 in dorsolateral prefrontal cortex of schizophrenia cases is reduced in an isoform-specific manner unrelated to dysbindin-1 mRNA expression.
      ). In mice, a loss-of-function mutation in Dtnbp1 (dys-MT) disrupts both glutamatergic and GABAergic transmission in the prefrontal cortex, and this disruption is associated with working memory deficits (
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      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ). Most importantly, in dys-MT mice, cortical pyramidal neurons in the prefrontal cortex display enhanced excitability, presumably due to a reduction in GABAergic signaling (
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      Dysbindin-1 mutant mice implicate reduced fast-phasic inhibition as a final common disease mechanism in schizophrenia.
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      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ). The mechanism underlying the loss of inhibitory activity in dys-MT mice remains unclear because dysbindin-1 expression is largely restricted to excitatory neurons rather than interneurons (
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      Dysbindin-1 is reduced in intrinsic, glutamatergic terminals of the hippocampal formation in schizophrenia.
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      Dysbindin-1C is required for the survival of hilar mossy cells and the maturation of adult newborn neurons in dentate gyrus.
      ).
      In this study, we discovered an unexpected role of dysbindin-1 in the exocytosis of brain-derived neurotrophic factor (BDNF), an important trophic factor for the differentiation and survival of inhibitory neurons from excitatory neurons (
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      Brain-derived neurotrophic factor promotes the maturation of GABAergic mechanisms in cultured hippocampal neurons.
      ). We further determined that this reduction in BDNF secretion reduces the number of functional GABAergic synapses that are transneuronally formed on excitatory neurons (
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      Activity-dependent transfer of brain-derived neurotrophic factor to postsynaptic neurons.
      ). Given that interneurons cannot synthesize BDNF by themselves but require BDNF for the formation and maintenance of their synapses (
      • Woo N.H.
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      ), our results demonstrate that these two well-known schizophrenia susceptibility gene products (BDNF and dysbindin-1) function together to regulate interneuron synapse development. Furthermore, our results provide considerable insight into the cellular and molecular mechanisms that regulate the development of the neural circuitry in the brain and link these abnormalities in BDNF secretion to cognitive disease.

      Methods and Materials

      A detailed description of the experimental procedures can be found in the Supplement. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Duke-National University of Singapore Graduate Medical School and the Lieber Institute for Brain Development.

      Cell Culture

      Cortical neurons were prepared from embryonic day 17 rat embryos and cultured in Neurobasal medium with B27 supplement (Invitrogen, Carlsbad, California). At day-in-vitro (DIV) 7, cultures were transfected with a calcium phosphate transfection kit (Clontech, Madison, Wisconsin). Human embryonic kidney 293 cells and PC12 cells were maintained using standard protocols and were transfected with Lipofectamine 2000 (Invitrogen).

      Molecular Biology

      Dysbindin-1 and scrambled short hairpin RNAs (shRNAs) were generated using pLentilox 3.7 vector. Dysbindin-1 shRNA target sequences were conserved in both mouse and rat dysbindin-1A and C isoforms. pCAG-MCS2-myc-dysbindin-1 was generated as reported previously (
      • Ito H.
      • Morishita R.
      • Shinoda T.
      • Iwamoto I.
      • Sudo K.
      • Okamoto K.
      • Nagata K.
      Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation.
      ), and the shRNA-resistant mutant of dysbindin-1 was generated by polymerase chain reaction-based mutagenesis.

      Fluorescent Immunocytochemistry

      Cultured rat neurons and mouse brain slices (postnatal day [P] 28) were stained with various primary antibodies, followed by incubation with appropriate fluorophore-conjugated secondary antibodies and imaged. Detailed antibody information can be found in the Supplement.

      Imaging

      Images were acquired with an LSM 710 (Zeiss, Singapore) confocal microscope using either 40× or 63× oil objectives and total internal reflection fluorescence microscopy imaging was conducted as previously reported (
      • Mabb A.M.
      • Je H.S.
      • Wall M.J.
      • Robinson C.G.
      • Larsen R.S.
      • Qiang Y.
      • et al.
      Triad3A regulates synaptic strength by ubiquitination of Arc.
      ).

      Electrophysiology

      Cortical neurons were transfected with various constructs as previously described (
      • Mabb A.M.
      • Je H.S.
      • Wall M.J.
      • Robinson C.G.
      • Larsen R.S.
      • Qiang Y.
      • et al.
      Triad3A regulates synaptic strength by ubiquitination of Arc.
      ). Whole-cell voltage clamp recordings were performed on DIV14 to DIV15 neurons. For acute slice recording, wild-type and dys-MT male mice at P15 to P35 were used as previously described (
      • Papaleo F.
      • Yang F.
      • Garcia S.
      • Chen J.
      • Lu B.
      • Crawley J.N.
      • Weinberger D.R.
      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ).

      Statistical Analysis

      Unless otherwise stated, error bars represent the standard error of the mean. All statistical analyses are listed in the Supplement.

      Results

      Increased Excitatory Synaptic Transmission in Layer 2/3 Pyramidal Neurons in the Prefrontal Cortex of dys-MT Mice

      The prefrontal cortex (PFC) is a brain structure that is known to play a critical role in executive function and is prominently implicated in the pathophysiology of schizophrenia (
      • Papaleo F.
      • Yang F.
      • Garcia S.
      • Chen J.
      • Lu B.
      • Crawley J.N.
      • Weinberger D.R.
      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ). In the PFC, top-down information is transmitted via pathways from layer (L) 2/3 pyramidal neurons to pyramidal neurons in L5, which is a major corticofugal output layer of the PFC network. We characterized the synaptic properties of L2/3 pyramidal neurons within the prelimbic area (PrL), homologous to the dorsolateral prefrontal cortex in primates, by measuring spontaneous excitatory postsynaptic currents (sEPSCs) using whole-cell patch-clamp recordings (Figure 1A, B) (
      • Ji Y.
      • Yang F.
      • Papaleo F.
      • Wang H.X.
      • Gao W.J.
      • Weinberger D.R.
      • Lu B.
      Role of dysbindin in dopamine receptor trafficking and cortical GABA function.
      ). Intriguingly, we found that dys-MT mice exhibited a dramatic increase in the frequency of the sEPSCs that were recorded from L2/3 pyramidal cells, without changing the amplitude (WT: 2.69 ± .62 Hz, 10.50 ± .29 pA; dys-MT: 6.65 ± 1.50 Hz, 10.99 ± 1.25 pA; p < .05) (Figure 1C, D). These results indicate an increase in excitatory synaptic transmission in the PFC of dys-MT mice.
      Figure thumbnail gr1
      Figure 1Enhanced excitatory basal transmission of pyramidal neurons in the medial prefrontal cortex layer 2/3 in dysbindin-1 mutant (Dys-MT) mice. (A) Schematic diagram of medial prefrontal cortex. (B) Sample traces of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from the prelimbic (PrL) layer 2/3 (L2/3) pyramidal neurons in wild-type (WT) mice (left panel) and Dys-MT mice (right panel). (C, D) Cumulative plots and histograms of sEPSC frequency (C) and amplitude (D) recorded from the layer 2/3 pyramidal neurons in WT and Dys-MT mice (n = 9 and 11, respectively). (E) Sample traces of miniature inhibitory postsynaptic currents (mIPSCs) recorded from the layer 2/3 pyramidal neurons in WT and Dys-MT mice. (F, G) Cumulative plots and histograms of mIPSC frequency (F) and amplitude (G) recorded from the layer 2/3 pyramidal neurons in WT and Dys-MT mice (n = 26 and 19, respectively. *p < .05. Cg1, cingulate cortex; IL, intralimbic cortex.
      Previous reports suggested that the decreased excitability of interneurons and decreased inhibitory transmission onto pyramidal neurons partially account for the increased frequency of sEPSCs in the PFC of dys-MT mice (
      • Ji Y.
      • Yang F.
      • Papaleo F.
      • Wang H.X.
      • Gao W.J.
      • Weinberger D.R.
      • Lu B.
      Role of dysbindin in dopamine receptor trafficking and cortical GABA function.
      ). To test this further, we monitored spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in L2/3 pyramidal neurons within PrL. In the presence of glutamatergic antagonists and sodium channel blockers, we observed significant reduction in the frequency of mIPSCs (WT: 5.23 ± .30 Hz; dys-MT: 4.29 ± .27 Hz; p < .05) (Figure 1E, F). However, no change in mIPSC amplitude was detected in PrL of dys-MT mice (WT: 22.19 ± 1.98 pA; dys-MT: 18.31 ± 1.32 pA) (Figure 1E, G). Intriguingly, immunocytochemistry revealed that the density (WT: 1.52 ± .08 per 10 μm2; dys-MT: 1.24 ± .04 per 10 μm2; p < .01) and size (WT: 1.46 ± .05 μm2; dys-MT: 1.33 ± .04 μm2; p < .05) of vesicular GABA transporter (VGAT) (an inhibitory presynaptic marker)-expressing boutons, which represent GABAergic inhibitory synaptic terminals, in the L2/3 within the PrL of dys-MT mice were reduced significantly, indicating the reduced inhibitory inputs onto L2/3 pyramidal neurons (Supplemental Figure S1).

      Reduced Inhibitory Synapse Number in Cultured Cortical Excitatory Neurons Upon Dysbindin-1 Knockdown

      Our results indicated that decreased inhibitory synaptic transmission onto pyramidal neurons in the PrL of dys-MT mice might result in increased network activity. However, it remains unclear how the loss of dysbindin-1 results in these changes. First, we investigated whether the loss of endogenous dysbindin-1 in either pyramidal neurons or inhibitory neurons affects inhibitory synaptic input. To that end, we used dissociated cultured cortical neurons and generated a series of bicistronic lentiviral constructs expressing shRNAs together with enhanced green fluorescence protein (EGFP). The expression of a specific shRNA construct targeting rat and mouse dysbindin-1 (dys-shRNA) reduced the expression of recombinant dysbindin-1 to approximately 52% of control levels in human embryonic kidney cells and reduced the expression levels of endogenous dysbindin-1 in cultured cortical neurons to 48% (Supplemental Figure S2). Cultured cortical neurons were transfected with either scrambled (scr) shRNA or dys-shRNA and were subsequently immunostained against VGAT (Figure 2A). We restricted our analysis to excitatory neurons identified based on somatic morphology as well as a calcium/calmodulin-dependent protein kinase II reporter-driven, lentiviral probe that specifically labels glutamatergic excitatory neurons (Supplemental Figure S3) (
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      ). The knockdown of dysbindin-1 significantly reduced the inhibitory synapse density, as assessed using the number of VGAT-positive clusters compared with neurons transfected with scrambled shRNA (scr: .151 ± .018 boutons/μm; dys-shRNA: .081 ± .011 boutons/μm; p < .01) (Figure 2A, B). Moreover, we analyzed the number of inhibitory synapses using another inhibitory presynaptic marker, GAD65/67, and we found a similar reduction in synapses from dys-shRNA transfected neurons (scr: .145 ± .017 boutons/μm; dys-shRNA: .082 ± .016 boutons/μm; p < .01) (Supplemental Figure S4). To further confirm the specificity of the shRNA experiments, we performed a rescue experiment using an shRNA-resistant form of dysbindin-1 (dys-R) that harbors four silent mutations within the sequence targeted by the shRNA (Supplemental Figures S2A and S2B). Western blot analysis confirmed the resistance of dys-R to dys-shRNA in vitro (Supplemental Figures S2A and S2B). The effect of dysbindin-1 knockdown on the density of inhibitory synapses was rescued by the co-expression of dys-R (dys-shRNA + dys-R: .158 ± .015 boutons/μm) (Figure 2A, B). These data indicate that dysbindin-1 knockdown in excitatory neurons reduces the number of inhibitory synapses formed.
      Figure thumbnail gr2
      Figure 2Dysbindin-1 knockdown reduces the numbers of inhibitory synapses in rat cultured cortical neurons. (A) Representative images of cultured excitatory cortical neurons transfected with scrambled short hairpin RNA (shRNA) (scr), dysbindin-1 shRNA (dys-shRNA), or dysbindin-1 shRNA with rescue (dys-shRNA + dys-R). Green fluorescent protein (GFP)-positive neurons were analyzed at day-in-vitro (DIV) 14 by immunofluorescence using antibodies against vesicular gamma-aminobutyric acid transporter (VGAT). Scale bar = 5 μm. (B) Summary graph of the effects of dysbindin-1 knockdown on inhibitory synapse density (scr vs. dys-shRNA), quantified using VGAT boutons, and rescued by shRNA-resistant dysbindin-1 (dys-shRNA + dys-R) (n = 12, 16, and 16, respectively). (C–E) Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from DIV13 to DIV15 cortical neurons expressing scr, dys-shRNA, or dys-shRNA + dys-R (n = 15, 42, and 17, respectively). Shown are sample traces (C), cumulative plot and histograms of mIPSC frequency (D), and amplitude (E). All histogram data are shown as means ± SEM. Statistical significance was assessed using Student t test with the Bonferroni correction (**p < .01; ***p < .001).
      Given the reduction in the number of inhibitory synapses due to dysbindin-1 knockdown in the excitatory neurons, we recorded mIPSCs via whole-cell patch-clamp recordings of cortical pyramidal neurons that were transfected with either scr or dys-shRNA. The neurons expressing dys-shRNA displayed a significant reduction in the frequency (but not the amplitude) of the mIPSCs compared with the neurons expressing scr (scr: 1.38 ± .25 Hz, 42.6 ± 3.9 pA; dys-shRNA: .78 ± .07 Hz, 45.2 ± 2.1 pA; p < .01) (Figure 2C–E). This reduction in the mIPSC frequency was restored by co-expressing dys-R (dys-shRNA + dys-R: 1.41 ± .13 Hz, 47.1 ± 3.8 pA; p = .59) (Figure 2C–E). Taken together, our data strongly indicate that endogenously expressed dysbindin-1 in excitatory neurons regulates the number of inhibitory synaptic inputs.
      To unequivocally rule out any effect of the endogenously expressed dybsindin-1 in interneurons, we performed two additional sets of experiments. First, we used a bicistronic construct expressing only GFP in the interneurons using the GAD65 promoter and analyzed the complexity, length, and branch numbers of the dendrites. We found no significant difference between the groups (Supplemental Figures S5A and S5B). Additionally, the loss of dysbindin-1 expression from interneurons did not affect the density of their presynaptic GABA-containing synaptic vesicles as evaluated with GAD67 immunostaining (Supplemental Figure S5C and S5D). Together, these results indicate that the reduction in dysbindin-1 expression in interneurons did not change morphology or the number of GABA-containing vesicles.

      Reduced BDNF Secretion in Neurons Upon Dysbindin-1 Knockdown

      We observed that dysbindin-1 knockdown in excitatory neurons reduced the number of inhibitory synapses on the excitatory neurons. What mediates this transneuronal modulation? Recent studies have shown that dysbindin-1 interacts with and regulates the trafficking and exocytosis of secretory vesicles (
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      • Monteggia L.M.
      Brain-derived neurotrophic factor and neuropsychiatric disorders.
      ). First, gene ablation studies have shown that signaling mediated by BDNF and tropomyosin receptor kinase B (TrkB), a cognate receptor of BDNF, regulates inhibitory synapse formation and the maturation of inhibitory neurons in the cortex. Furthermore, BDNF knockdown in an individual excitatory neuron in the cerebral cortex results in a reduction in inhibitory synapses on that neuron (
      • Kohara K.
      • Yasuda H.
      • Huang Y.
      • Adachi N.
      • Sohya K.
      • Tsumoto T.
      A local reduction in cortical GABAergic synapses after a loss of endogenous brain-derived neurotrophic factor, as revealed by single-cell gene knock-out method.
      ). However, inhibitory neurons must rely on the BDNF that is secreted from excitatory neurons because inhibitory neurons do not express BDNF messenger RNA (
      • Rocamora N.
      • Pascual M.
      • Acsady L.
      • de Lecea L.
      • Freund T.F.
      • Soriano E.
      Expression of NGF and NT3 mRNAs in hippocampal interneurons innervated by the GABAergic septohippocampal pathway.
      ). Based on these findings, we sought to determine whether dysbindin-1 knockdown in cortical pyramidal neurons reduces BDNF secretion.
      First, we monitored the cellular localization of dysbindin-1 with respect to BDNF in neurons. In cortical neurons, GFP-tagged dysbindin-1 puncta co-localized with TdTomato-tagged BDNF signals, which were also overlapped with immunoreactivity of chromogranin B, a marker protein for large-dense core vesicles (LDCVs) (Supplemental Figure S6). We next examined the temporal and spatial relationship between dysbindin-1 and BDNF within neurons using confocal live-cell imaging. Live-cell imaging revealed that punctate spots of GFP-dysbindin-1 traveled together with TdTomato-BDNF-harboring vesicles along the dendrite (Movie S1) as well as an axon (Movie S2) in cortical neurons. These data suggested a potential role of dysbindin-1 in regulating the trafficking and exocytosis of BDNF secretory vesicles.
      Next, to directly visualize BDNF secretion from neurons, we took advantage of a pH-sensitive variant of EGFP (super-ecliptic pHluorin [SEP]), the fluorescence of which is quenched at low pH, and we fused this SEP to the C-terminal end of BDNF (BDNF-SEP) (Figure 3A) (
      • Miesenbock G.
      • De Angelis D.A.
      • Rothman J.E.
      Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.
      ). The distribution of BDNF-SEP, as revealed via co-immunostaining for EGFP, SMI312 (an axonal marker), and microtubule-associated protein 2 (a dendritic marker), was similar to that of endogenous BDNF stained with a BDNF-specific antibody, displaying punctate expression patterns in axons, dendrites, and cell bodies (Supplemental Figure S7). The neuronal processes that were not stained with the microtubule-associated protein 2 antibody were further confirmed to be axons; these processes were co-immunostained with an antibody against the axon-specific microtubule-associated protein SMI312 (Supplemental Figure S7). Reconstructed images in the axial plane of an axon, a dendrite, and a soma further supported the granular nature of the punctate fluorescent staining (Figure 3B), as is consistent with previous reports (Supplemental Figure S7).
      Figure thumbnail gr3
      Figure 3Dysbindin-1 knockdown reduces brain-derived neurotrophic factor (BDNF) secretion in neuronal cells and rat cultured cortical excitatory neurons. (A) Schematic illustration of BDNF-super-ecliptic pHluorin (SEP) fluorescence changing at different pH during BDNF secretion. (B) Total internal reflection fluorescence microscopic image of BDNF puncta in cultured cortical neurons transfected with BDNF-SEP (I, scale bar = 20 μm) and magnified views of BDNF puncta in boxed area of panel I at 1 second before and 10 seconds and 15 seconds after potassium chloride (KCl) stimulation, respectively (II, scale bar = 5 μm). Panel III, sample traces showing fluorescence change with time at boxed areas in panel (B), representing different events. (C) Histograms of total event numbers during 5 minutes after KCl stimulation in cultured cortical neurons transfected with scrambled short hairpin RNA (shRNA) (scr), dysbindin-1 shRNA (dys-shRNA) (n = 10 and 12, respectively). (D) Histograms and (E) cumulative plots of events number during 1 minute after KCl stimulation in PC12 cells transfected with scr or dys-shRNA (n = 6 and 5, respectively). All histogram data are shown as means ± SEM. Statistical significance was assessed using Student t test (*p < .05). K+, potassium ions; LDV, large dense core vesicle; PM, plasma membrane.
      To detect BDNF secretion via exocytosis, we performed time-lapse total internal reflection fluorescence imaging, which enables the selective excitation of fluorophores in the evanescent field above the cover glass (≤100 nm) (
      • Mabb A.M.
      • Je H.S.
      • Wall M.J.
      • Robinson C.G.
      • Larsen R.S.
      • Qiang Y.
      • et al.
      Triad3A regulates synaptic strength by ubiquitination of Arc.
      ,
      • Merrifield C.J.
      • Perrais D.
      • Zenisek D.
      Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells.
      ). When expressed in cultured cortical neurons, BDNF-SEP puncta were sparsely distributed in axons, dendrites, and cell bodies (Figure 3B). Bath application of ammonium chloride solution (50 mmol/L, pH 7.4) (
      • Kennedy M.J.
      • Davison I.G.
      • Robinson C.G.
      • Ehlers M.D.
      Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines.
      ), which deacidifies the vesicular lumen and thereby unquenches BDNF-SEP, resulted in a marked increase in the fluorescence intensity of each punctum and revealed many BDNF-SEP puncta in each neuron (Supplemental Figure S8), suggesting that BDNF-SEP was quenched in a majority of the vesicles.
      Next, we monitored the activity-induced exocytosis of BDNF-SEP-containing vesicles in cultured cortical neurons by applying a high concentration of potassium ions (K+) (60 mmol/L) to depolarize the neurons. Before high K+ stimulation, we detected discrete, randomly appearing puncta of BDNF-SEP fluorescence (Figure 3B). These randomly appearing BDNF-SEP puncta indicated spontaneous neuronal BDNF exocytosis (
      • Matsuda N.
      • Lu H.
      • Fukata Y.
      • Noritake J.
      • Gao H.
      • Mukherjee S.
      • et al.
      Differential activity-dependent secretion of brain-derived neurotrophic factor from axon and dendrite.
      ). After high K+ treatment, BDNF-SEP fluorescence was significantly increased in the control neurons (Figure 3B; Movie S3). Additionally, the high K+ treatment increased the number of BDNF exocytotic events, as quantified by the appearance of new fluorescent puncta at the cell surface (Figure 3C). In contrast, the increase in the number and intensity of BDNF-SEP puncta induced by high K+ treatment was dramatically reduced in neurons expressing dys-shRNA (Figure 3C). Additionally, we monitored the activity-dependent exocytosis of BDNF-SEP in PC12 cells and found that expressing dys-shRNA markedly reduced BDNF-SEP exocytosis (Figure 3D, E). Taken together, these data suggest that dysbindin-1 knockdown reduces activity-dependent BDNF secretion from neurons.

      BDNF Application Rescues Dysbindin-1 shRNA-Mediated Inhibitory Synapse Defects

      The reduction in BDNF secretion from dysbindin-1 knockdown neurons suggested that BDNF could act as a crucial trophic factor in regulating the formation and/or maintenance of interneuron synapses. If this hypothesis were correct, the application of exogenous BDNF would rescue the reduced numbers of inhibitory synapses detected in cortical neurons after dysbindin-1 knockdown.
      To test this hypothesis, we exogenously applied recombinant BDNF (100 ng/mL) for 4 days to cortical neurons transfected with either scr or dys-shRNA. Dysbindin-1 knockdown in excitatory neurons reduced the inhibitory synapse density, as assessed using the number of VGAT-positive clusters (scr: .134 ± .024; dys-shRNA: .076 ± .009 boutons/μm) (Figure 4A, B). Intriguingly, the application of exogenous BDNF rescued this reduction in the inhibitory synapse density in dys-shRNA-expressing neurons (BDNF with dys-shRNA: .156 ± .015 boutons/μm; p < .001) (Figure 4A, B). We confirmed this result using GAD65 immunostaining as an alternative method to quantify the inhibitory synapse density and found a similar change (Supplemental Figure S9). To assess the specificity of this BDNF-mediated effect, we treated dys-shRNA-expressing neurons with recombinant nerve growth factor (100 ng/mL), which has multiple synaptic effects (Supplemental Figure S10). Treatment with nerve growth factor failed to rescue the inhibitory synapse reduction in dys-shRNA-expressing neurons, indicating that the rescue of the reduction in inhibitory synapse density in dysbindin-1 knockdown neurons was specific to BDNF.
      Figure thumbnail gr4
      Figure 4Brain-derived neurotrophic factor (BDNF) application rescues defects in inhibitory synapses induced by dysbindin-1 (dys) short hairpin RNA (shRNA) (dys-shRNA). (A) Representative images of rat cultured excitatory cortical neurons transfected with scrambled shRNA (scr) or dys-shRNA and treated with or without BDNF. Green fluorescent protein (GFP)-positive neurons were analyzed on day-in-vitro (DIV) 14 by immunofluorescence using antibodies against vesicular gamma-aminobutyric acid transporter (VGAT). Scale bar = 5 μm. (B) Summary graph of BDNF-mediated rescue of defects in inhibitory synapses caused by dysbindin-1 shRNA (scr vs. dys-shRNA vs. dys-shRNA + BDNF, n = 14, 23, and 15, respectively). (C–E) Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from DIV13 to DIV15 cortical neurons treated with or without BDNF. Shown are sample traces (C), cumulative plot and histograms of mIPSC frequency (D), and amplitude (E) (scr vs. dys-shRNA vs. dys-shRNA + BDNF, n = 20, 18, and 13, respectively). All histogram data are shown as means ± SEM. Statistical significance was assessed using Student t test with the Bonferroni correction (*p < .05; **p < .01).
      To further determine whether exogenous BDNF treatment rescues the deficits in inhibitory synaptic transmission in dys-shRNA-expressing neurons, we measured the mIPSCs from dys-shRNA-expressing neurons treated with either vehicle or BDNF (100 ng/mL). The application of BDNF (but not of the vehicle) rescued the reduction in frequency of mIPSCs recorded from dys-shRNA-expressing excitatory neurons (scr: 1.87 ± .30 Hz, 47.1 ± 3.5 pA; dys-shRNA: .73 ± .12 Hz, 54.24 ± 2.37 pA; dys-shRNA with BDNF: 1.63 ± .30 Hz, 56.2 ± 3.7 pA; p < .01) (Figure 4C–E). Taken together, we conclude that BDNF selectively rescues the reduction in the number of functional inhibitory synapses in cortical excitatory neurons induced by dysbindin-1 knockdown.

      Rescued Inhibitory Synaptic Transmission Upon BDNF Application in Ex Vivo Organotypic Hippocampal Slices

      To investigate the effects of the lack of endogenous dysbindin-1 expression on excitatory pyramidal neuron function ex vivo, we transfected our shRNA constructs into organotypic hippocampal slices via biolistic gene gun (
      • Kohara K.
      • Yasuda H.
      • Huang Y.
      • Adachi N.
      • Sohya K.
      • Tsumoto T.
      A local reduction in cortical GABAergic synapses after a loss of endogenous brain-derived neurotrophic factor, as revealed by single-cell gene knock-out method.
      ). The transfected neurons were marked with simultaneously introduced EGFP (Figure 5A). To induce an extremely sparse distribution of the transfected neurons, steel mesh was attached to the muzzle of the gene guns (
      • Lee S.J.
      • Escobedo-Lozoya Y.
      • Szatmari E.M.
      • Yasuda R.
      Activation of CaMKII in single dendritic spines during long-term potentiation.
      ). Typically, fewer than 10 EGFP-expressing neurons were detected among the vast number of neurons within each slice culture (Figure 5A).
      Figure thumbnail gr5
      Figure 5Dysbindin-1 knockdown reduces inhibitory synaptic transmission, which is restored by exogenous brain-derived neurotrophic factor (BDNF) in ex vivo rat hippocampal organotypic slices. (A) Representative image of a cultured organotypic slice transfected with dysbindin-1 (dys) short hairpin RNA (shRNA) (dys-shRNA). (B–D) Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from transfected neurons expressing scrambled shRNA (scr), dys-shRNA, or dys-shRNA + BDNF (n = 9, 12, and 12, respectively) in organotypic slices. Shown are sample traces (B), cumulative plot and histograms of mIPSC frequency (C), and amplitude (D). All histogram data are shown as means ± SEM. Statistical significance was assessed using Student t test with the Bonferroni correction (*p < .05). CA, cornu ammonis; DG, dentate gyrus.
      Next, we measured the inhibitory synaptic transmission in hippocampal pyramidal neurons expressing either scr or dys-shRNA. Analysis of the mIPSCs revealed marked decreases in the frequency of mIPSCs in dys-shRNA-expressing pyramidal neurons compared with scr-expressing pyramidal neurons (scr: 5.504 ± .476 Hz, 40.32 ± 1.70 pA; dys-shRNA: 3.6 ± .51 Hz, 34.6 ± 3.4 pA; p < .05) (Figure 5C, D). Intriguingly, these inhibitory synaptic deficits were rescued by BDNF application (100 ng/mL, 4 days) (dys-shRNA: 3.6 ± .51 Hz, 34.6 ± 3.4 pA; dys-shRNA with BDNF: 5.984 ± .561 Hz, 32.5 ± 2.12 pA; p < .01) (Figure 5C, D), as indicated by the significant shifts in the cumulative distribution curves for the mIPSC interevent interval.

      Discussion

      Since the dysbindin-1 gene was identified, extensive molecular, cell biological, animal model, and human genotype-phenotype studies have been conducted to address the multiple functions and mechanisms of dysbindin-1 activity in the brain. Because dysbindin-1 is thought to mediate a range of intermediate phenotypes that underlie schizophrenia (
      • Papaleo F.
      • Yang F.
      • Garcia S.
      • Chen J.
      • Lu B.
      • Crawley J.N.
      • Weinberger D.R.
      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ,
      • Feng Y.Q.
      • Zhou Z.Y.
      • He X.
      • Wang H.
      • Guo X.L.
      • Hao C.J.
      • et al.
      Dysbindin deficiency in sandy mice causes reduction of snapin and displays behaviors related to schizophrenia.
      ,
      • Hattori S.
      • Murotani T.
      • Matsuzaki S.
      • Ishizuka T.
      • Kumamoto N.
      • Takeda M.
      • et al.
      Behavioral abnormalities and dopamine reductions in sdy mutant mice with a deletion in Dtnbp1, a susceptibility gene for schizophrenia.
      ), elucidating the precise biological functions of dysbindin-1 have received intensive study. Disruption of dysbindin-1 is thought to confer glutamatergic dysfunction (
      • Chen X.W.
      • Feng Y.Q.
      • Hao C.J.
      • Guo X.L.
      • He X.
      • Zhou Z.Y.
      • et al.
      DTNBP1, a schizophrenia susceptibility gene, affects kinetics of transmitter release.
      ). However, in mice, a loss-of-function mutation in dysbindin-1 (dys-MT) disrupts inhibitory neurotransmission in both the hippocampus and the prefrontal cortex (
      • Carlson G.C.
      • Talbot K.
      • Halene T.B.
      • Gandal M.J.
      • Kazi H.A.
      • Schlosser L.
      • et al.
      Dysbindin-1 mutant mice implicate reduced fast-phasic inhibition as a final common disease mechanism in schizophrenia.
      ,
      • Ji Y.
      • Yang F.
      • Papaleo F.
      • Wang H.X.
      • Gao W.J.
      • Weinberger D.R.
      • Lu B.
      Role of dysbindin in dopamine receptor trafficking and cortical GABA function.
      ,
      • Papaleo F.
      • Yang F.
      • Garcia S.
      • Chen J.
      • Lu B.
      • Crawley J.N.
      • Weinberger D.R.
      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ) via an unknown mechanism. In the present study, we discovered an unexpected role of dysbindin-1 for the exocytosis of BDNF from excitatory neurons that consequently transneuronally reduced the number of functional inhibitory synapses formed on the excitatory neurons.

      Dysbindin-1, BDNF, and Their Roles in Inhibitory Synapse Development

      BDNF is a neurotrophic factor that regulates synapse development and plasticity in the mammalian brain (
      • Lu B.
      • Je H.S.
      Neurotrophic regulation of the development and function of the neuromuscular synapses.
      ,
      • Lu B.
      • Woo N.H.
      Trophic factors in synaptic plasticity and memory.
      ). Interestingly, BDNF regulates the maturation of interneurons, the dysfunction of which has been associated with schizophrenia (
      • Konopaske G.T.
      • Sweet R.A.
      • Wu Q.
      • Sampson A.
      • Lewis D.A.
      Regional specificity of chandelier neuron axon terminal alterations in schizophrenia.
      ). For example, in BDNF knockout mice, the number of interneurons is significantly reduced compared with that in wild-type mice (
      • Jones K.R.
      • Farinas I.
      • Backus C.
      • Reichardt L.F.
      Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development.
      ,
      • Altar C.A.
      • Boylan C.B.
      • Fritsche M.
      • Jackson C.
      • Hyman C.
      • Lindsay R.M.
      The neurotrophins NT-4/5 and BDNF augment serotonin, dopamine, and GABAergic systems during behaviorally effective infusions to the substantia nigra.
      ). Conversely, in transgenic mice overexpressing BDNF, the maturation of interneurons was significantly accelerated; this effect was accompanied by an increase in spontaneous network activity (
      • Huang Z.J.
      • Kirkwood A.
      • Pizzorusso T.
      • Porciatti V.
      • Morales B.
      • Bear M.F.
      • et al.
      BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex.
      ,
      • Aguado F.
      • Carmona M.A.
      • Pozas E.
      • Aguilo A.
      • Martinez-Guijarro F.J.
      • Alcantara S.
      • et al.
      BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl- co-transporter KCC2.
      ). Similarly, the application of exogenous BDNF to interneuron cultures resulted in accelerated formation of reciprocal connections via enhanced TrkB signaling (
      • Berghuis P.
      • Dobszay M.B.
      • Sousa K.M.
      • Schulte G.
      • Mager P.P.
      • Hartig W.
      • et al.
      Brain-derived neurotrophic factor controls functional differentiation and microcircuit formation of selectively isolated fast-spiking GABAergic interneurons.
      ).
      Intriguingly, GABAergic interneurons do not express BDNF messenger RNA and thus cannot produce BDNF themselves (
      • Ernfors P.
      • Wetmore C.
      • Olson L.
      • Persson H.
      Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family.
      ). Therefore, from whence do these neurons receive the BDNF that is necessary for their function? Kohara et al. (
      • Kohara K.
      • Kitamura A.
      • Adachi N.
      • Nishida M.
      • Itami C.
      • Nakamura S.
      • Tsumoto T.
      Inhibitory but not excitatory cortical neurons require presynaptic brain-derived neurotrophic factor for dendritic development, as revealed by chimera cell culture.
      ) reported that in co-cultures of BDNF-KO neurons with GFP-labeled wild-type neurons, interneurons receive BDNF released from excitatory neurons (
      • Kohara K.
      • Kitamura A.
      • Adachi N.
      • Nishida M.
      • Itami C.
      • Nakamura S.
      • Tsumoto T.
      Inhibitory but not excitatory cortical neurons require presynaptic brain-derived neurotrophic factor for dendritic development, as revealed by chimera cell culture.
      ). They later found that BDNF ablation in a single excitatory neuron induced a local reduction in the number of inhibitory synapses to that BDNF-knockout excitatory neuron (
      • Kohara K.
      • Yasuda H.
      • Huang Y.
      • Adachi N.
      • Sohya K.
      • Tsumoto T.
      A local reduction in cortical GABAergic synapses after a loss of endogenous brain-derived neurotrophic factor, as revealed by single-cell gene knock-out method.
      ). Likewise, enhanced BDNF exocytosis in synaptotagmin-IV knockout mice (
      • Dean C.
      • Liu H.
      • Staudt T.
      • Stahlberg M.A.
      • Vingill S.
      • Buckers J.
      • et al.
      Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity.
      ,
      • Dean C.
      • Liu H.
      • Dunning F.M.
      • Chang P.Y.
      • Jackson M.B.
      • Chapman E.R.
      Synaptotagmin-IV modulates synaptic function and long-term potentiation by regulating BDNF release.
      ) and reduced BDNF exocytosis in calcium-dependent activator protein for secretion 2 knockout mice have been shown to result in increased and decreased GABAergic synaptic transmission, respectively (
      • Shinoda Y.
      • Sadakata T.
      • Nakao K.
      • Katoh-Semba R.
      • Kinameri E.
      • Furuya A.
      • et al.
      Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network.
      ). These findings suggest that BDNF (presumably released from excitatory neurons) shapes and regulates local inhibitory synapse formation and/or maintenance. Therefore, it is conceivable that any defect in the cellular and molecular machinery that regulates BDNF trafficking and release from excitatory neurons could affect inhibitory synapse formation on these excitatory neurons.
      Here, we demonstrate that the loss of dysbindin-1 from cortical excitatory neurons leads to a reduction in functional inhibitory synapses, as demonstrated previously in the dys-MT mouse cortex and hippocampus (Figures 1 and 2) (
      • Ji Y.
      • Yang F.
      • Papaleo F.
      • Wang H.X.
      • Gao W.J.
      • Weinberger D.R.
      • Lu B.
      Role of dysbindin in dopamine receptor trafficking and cortical GABA function.
      ,
      • Papaleo F.
      • Yang F.
      • Garcia S.
      • Chen J.
      • Lu B.
      • Crawley J.N.
      • Weinberger D.R.
      Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways.
      ). However, the loss of dysbindin-1 from inhibitory neurons did not result in any defects in dendritic morphology or formation of axonal synaptic boutons (Supplemental Figure S4). This was potentially due to the lack of expression of dysbindin-1 in GABAergic neurons (
      • Wang H.
      • Yuan Y.
      • Zhang Z.
      • Yan H.
      • Feng Y.
      • Li W.
      Dysbindin-1C is required for the survival of hilar mossy cells and the maturation of adult newborn neurons in dentate gyrus.
      ). Additionally, we show that the impairment in activity-dependent BDNF release from dysbindin-1 knockdown cortical neurons and neuronal cells (Figure 3) and, intriguingly, the inhibitory synapse defects exhibited by dysbindin-1 knockdown neurons were rescued by BDNF application (Figure 4, Figure 5). Our data reveal novel transneuronal signaling between excitatory and inhibitory neurons and provide new insight into the cellular and molecular mechanisms that link these abnormalities in BDNF secretion to psychiatric disorders.

      Potential Mechanisms of Reduced BDNF Exocytosis in Dysbindin-1 Knockdown Neurons

      Dysbindin-1A is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) complex, and several studies have demonstrated that the loss of dysbindin-1 is accompanied by the loss of all BLOC-1 complex constituents (
      • Talbot K.
      • Ong W.Y.
      • Blake D.J.
      • Tang J.
      • Louneva N.
      • Carlson G.C.
      • et al.
      Dysbindin-1 and its protein family.
      ,
      • Wang H.
      • Yuan Y.
      • Zhang Z.
      • Yan H.
      • Feng Y.
      • Li W.
      Dysbindin-1C is required for the survival of hilar mossy cells and the maturation of adult newborn neurons in dentate gyrus.
      ,
      • Larimore J.
      • Zlatic S.A.
      • Gokhale A.
      • Tornieri K.
      • Singleton K.S.
      • Mullin A.P.
      • et al.
      Mutations in the BLOC-1 subunits dysbindin and muted generate divergent and dosage-dependent phenotypes.
      ,
      • Li W.
      • Zhang Q.
      • Oiso N.
      • Novak E.K.
      • Gautam R.
      • O’Brien E.P.
      • et al.
      Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1).
      ). Although our dysbindin-1 shRNA is designed to target conserved regions in both dysbindin-1A and dysbindin-1C isoforms, co-expression of the dysbindin-1A isoform rescued dysbindin-1 shRNA associated interneuron synaptic defects, indicating that the dysbindin-1A isoform might affect BDNF exocytosis. Further rescue experiments using dysbindin-1C isoform or isoform-specific shRNAs may elucidate which of the dysbindin-1 isoforms contribute to BDNF release and transneuronal interneuron synapse maintenance.
      In addition, dysbindin-1 and three other subunits of BLOC-1 (pallidin, snapin, and muted) interact with adaptor protein complex 3 (AP-3) and are also expressed in AP-3-containing vesicles in neurons (
      • Taneichi-Kuroda S.
      • Taya S.
      • Hikita T.
      • Fujino Y.
      • Kaibuchi K.
      Direct interaction of dysbindin with the AP-3 complex via its mu subunit.
      ). AP-3 has generally been considered to function in endocytic rather than biosynthetic pathways; however, a recent RNA interference-based screen identified that AP-3 functions in the formation, trafficking, and regulated exocytosis of LDCVs, which contain the BDNF that is secreted from neurons (
      • Asensio C.S.
      • Sirkis D.W.
      • Edwards R.H.
      RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway.
      ). Additionally, the loss of AP-3 affects the number, morphology, and density of LDCVs in neurons (
      • Asensio C.S.
      • Sirkis D.W.
      • Edwards R.H.
      RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway.
      ,
      • Kantheti P.
      • Qiao X.
      • Diaz M.E.
      • Peden A.A.
      • Meyer G.E.
      • Carskadon S.L.
      • et al.
      Mutation in AP-3 delta in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles.
      ). Furthermore, mice lacking the neuronal isoform of AP-3 (ap-3μ knockout mice) exhibited the reduced inhibitory neurotransmission and abnormal propagation of neuronal excitability that were detected in dys-MT mice (
      • Nakatsu F.
      • Okada M.
      • Mori F.
      • Kumazawa N.
      • Iwasa H.
      • Zhu G.
      • et al.
      Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor.
      ). More importantly, the BDNF content was reduced in the hippocampus of BLOC-1-deficient mice (
      • Larimore J.
      • Ryder P.V.
      • Kim K.Y.
      • Ambrose L.A.
      • Chapleau C.
      • Calfa G.
      • et al.
      MeCP2 regulates the synaptic expression of a Dysbindin-BLOC-1 network component in mouse brain and human induced pluripotent stem cell-derived neurons.
      ). Taken together, we speculate that defects in the dysbindin-1 interactome may impair BDNF release via AP-3-mediated LDCV function, but this possibility also requires further investigation.

      Targeting Interneuron and BDNF/TrkB Signaling for Treatment of Schizophrenia

      Dysbindin-1 protein expression is widely distributed throughout the brain, but it is concentrated in synapses in brain regions that are commonly affected in schizophrenia, such as the hippocampus, the striatum, and the prefrontal cortex (
      • Talbot K.
      • Ong W.Y.
      • Blake D.J.
      • Tang J.
      • Louneva N.
      • Carlson G.C.
      • et al.
      Dysbindin-1 and its protein family.
      ). Interestingly, decreases in one or more dysbindin-1 isoforms have been detected in the cortex and the hippocampus of schizophrenic patients (
      • Talbot K.
      • Ong W.Y.
      • Blake D.J.
      • Tang J.
      • Louneva N.
      • Carlson G.C.
      • et al.
      Dysbindin-1 and its protein family.
      ,
      • Tang J.
      • LeGros R.P.
      • Louneva N.
      • Yeh L.
      • Cohen J.W.
      • Hahn C.G.
      • et al.
      Dysbindin-1 in dorsolateral prefrontal cortex of schizophrenia cases is reduced in an isoform-specific manner unrelated to dysbindin-1 mRNA expression.
      ,
      • Talbot K.
      • Louneva N.
      • Cohen J.W.
      • Kazi H.
      • Blake D.J.
      • Arnold S.E.
      Synaptic dysbindin-1 reductions in schizophrenia occur in an isoform-specific manner indicating their subsynaptic location.
      ). Furthermore, dys-MT mice exhibited behavioral deficits resembling schizophrenia intermediate phenotypes; thus, dys-MT mice represent a good animal model of schizophrenia.
      Impaired synaptic connectivity is a predominant hypothesis of the pathology of schizophrenia and has been suggested by the results of human postmortem studies (
      • Lewis D.A.
      • Gonzalez-Burgos G.
      Neuroplasticity of neocortical circuits in schizophrenia.
      ). However, there is insufficient information about the cellular and molecular mechanisms underlying this potentially impaired synaptic connectivity in schizophrenia. It is widely believed that the impaired synaptic connectivity between GABAergic and glutamatergic neurons may cause network imbalance and the eventual impairment of brain function that is commonly observed in schizophrenic patients (
      • Lewis D.A.
      • Hashimoto T.
      • Volk D.W.
      Cortical inhibitory neurons and schizophrenia.
      ,
      • Lewis D.A.
      • Gonzalez-Burgos G.
      Neuroplasticity of neocortical circuits in schizophrenia.
      ,
      • Goldman-Rakic P.S.
      Cellular basis of working memory.
      ,
      • Le Magueresse C.
      • Monyer H.
      GABAergic interneurons shape the functional maturation of the cortex.
      ). Here, we provide cellular and molecular evidence that interneuron dysfunction may be the primary cause of the overall synapse deficits associated with dysfunction of two genes implicated in the pathophysiology of this illness (
      • Weickert C.S.
      • Ligons D.L.
      • Romanczyk T.
      • Ungaro G.
      • Hyde T.M.
      • Herman M.M.
      • et al.
      Reductions in neurotrophin receptor mRNAs in the prefrontal cortex of patients with schizophrenia.
      ). If these suppositions are correct, it may be possible to ameliorate the impaired GABAergic synapse formation and the subsequent synaptic imbalance between excitatory and inhibitory signaling within local cortical circuits by enhancing or restoring the levels of BDNF signaling. Therefore, it will be exciting to evaluate the ability of BDNF mimetic peptides and small molecules that enhance BDNF translation (
      • Longo F.M.
      • Yang T.
      • Knowles J.K.
      • Xie Y.
      • Moore L.A.
      • Massa S.M.
      Small molecule neurotrophin receptor ligands: Novel strategies for targeting Alzheimer’s disease mechanisms.
      ,
      • Price R.D.
      • Milne S.A.
      • Sharkey J.
      • Matsuoka N.
      Advances in small molecules promoting neurotrophic function.
      ) in an attempt to restore inhibitory synapse function in dys-MT mice (
      • Nestler E.J.
      • Hyman S.E.
      Animal models of neuropsychiatric disorders.
      ).

      Acknowledgments and Disclosures

      This work was supported by the Singapore Ministry of Education Academic Research Fund (MOE2012-T2-1-021) and Duke-National University of Singapore Signature Research Program Block Grant (to HSJ).
      QY, FY, and HSJ designed the research; QY, FY, YX, ST, NH, MR, ZH, KM, JSN, PJK, and HSJ performed the research; WH, KN, and DRW provided reagents, materials, and analysis tools and edited the manuscript; QY, FY, YX, ST, and HSJ analyzed the data; and HSJ wrote the paper.
      We thank Dr. Marina Ceccarini at Centro Nazionale Malattie Rare for providing dysbindin-1 antibody and we also thank Dr. Zheng Li at the National Institute for Mental Health for providing dysbindin-1 mutant mice. We thank Hasini Ganegala for her excellent technical assistance. We thank Drs. Shirish Shenolikar and Zeng Li for their critical comments on the manuscript.
      All authors report no biomedical financial interests or potential conflicts of interest.

      Appendix A. Supplementary Materials

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