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&&产品价格:&1098.00元DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites
From the ?Department of Neuroscience, Physiology, and Pharmacology, University College London, Gower Street, London WC1E 6BT, United
the ?Neuroscience Research Unit, Pfizer, Cambridge, Massachusetts 02139, and
the §Department of Neuropathology, Heinrich Heine University, Moorenstrasse 5, 40225 Dusseldorf, Germany
8 To whom correspondence should be addressed. Tel.: 44-207-679-3218; Fax: 44-207-916-7968; E-mail: j.kittler{at}ucl.ac.uk.
6 Present address: UCL Cancer Inst., Paul O'Gorman Bldg., 72 Huntley St., London WC1E 6DD, United Kingdom.
The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism.
Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport
in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its
role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the
mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and
Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live
cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also
show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria,
where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to
endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria
contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of
the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator
of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles,
and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development.
Introduction
The disrupted in schizophrenia 1 (DISC1) protein is a promising candidate susceptibility factor for major mental illness (). Multiple genetic studies have shown an association between DISC1 and schizophrenia, bipolar disorder, major depression,
and autism (, ). DISC1 was first discovered due to a balanced chromosomal translocation in a family with a high incidence of schizophrenia
and other major mental illness (). This translocation results in the truncation of DISC1 after exon 8 and the fusion to another gene, Boymaw (also known as DISC1FP1 for DISC1 fusion partner 1), leading to the expression of a DISC1-Boymaw fusion protein (, ). DISC1 affects multiple cellular functions including neuronal proliferation, migration, and integration via its roles at
the centrosome in the anchoring of key proteins such as Bardet-Biedl syndrome (BBS) proteins BBS1 and BBS4 (). DISC1 also regulates intracellular signaling pathways such as the Wnt/β-catenin and PDE4 signaling pathways (, ) and regulates neurite outgrowth. Point mutations or truncation of DISC1 leads to decreased dendritic complexity, both in vivo and in dissociated culture (), highlighting the necessity for normal DISC1 function in neuronal development. However, the mechanisms by which DISC1 contributes
to altered neuronal development, function, and pathology remain poorly understood. Moreover, the cellular impact of expression
of the Boymaw fusion protein also remains unclear.
Mitochondria are highly dynamic organelles that undergo constant trafficking, fission, fusion, and turnover. In neurons, the
tight regulation of mitochondrial transport is critical to allow controlled delivery of these organelles to sites where they
are required for energy provision and calcium buffering (). Disruption of mitochondrial localization can lead to defects in synaptic function and plasticity in addition to affecting
neuronal morphology (, ). Detailed studies have revealed mitochondrial distribution and bidirectional trafficking to be regulated in a calcium-dependent
manner via the mitochondrial Rho GTPases Miro1 and Miro2 (, ). These outer mitochondrial membrane proteins possess two calcium-sensing EF-hand domains flanked by two GTPase domains on
their cytoplasmic face (, ). Miro1 interacts with kinesin and dynein motors and their TRAK adaptor proteins (). TRAK1 has been recently demonstrated to be axonally targeted, whereas TRAK2 favors a dendritic localization (, ). Knockdown of either the TRAK1 or TRAK2 adaptor significantly reduces the numbers of moving mitochondria in cultured hippocampal
axons and dendrites, respectively (, ). Currently, however, the molecular nature of other components of the Miro-TRAK machinery remain poorly understood.
Mitochondrial trafficking and morphology are tightly linked (). Mitochondrial morphology is dependent on the balance of fission and fusion. Fission is regulated by Drp1 (dynamin-related
protein 1), which is recruited to the mitochondria by anchors such as Fis1 (mitochondrial fission protein 1). Fusion is coordinated
by the GTPases Mitofusin1 and -2 at the outer mitochondrial membrane, which tether two mitochondria together, and OPA1 at
the inner membrane (). These fusion events are necessary for the exchange of mitochondrial contents, e.g. mitochondrial DNA and metabolites, maintaining mitochondrial function, and mitochondrial biogenesis (). Mitofusin2 also plays an important role in bridging mitochondria to the endoplasmic reticulum (ER) (). Mitochondria-ER contacts facilitate communication between these two organelles, including the transfer of calcium and lipids
(), and are known sites of autophagosome biogenesis (). Additionally, contacts between the ER and mitochondria are proposed to be involved in both fission-fusion and the trafficking
of mitochondria (); interestingly, the yeast homologue of Miro1, Gem1, is also known to be localized to these sites (). However the role of Miro in pathology at Mito-ER contacts is unclear.
DISC1 can be found localized to mitochondria (, ) and has been demonstrated previously to modulate the function and transport of mitochondria and other key cargo in neuronal
axons (, ), whereas disease-associated DISC1 point mutations lead to disrupted mitochondrial trafficking (, ). Although DISC1 appears to be important for mitochondrial trafficking in neuronal axons, whether DISC1 also impacts mitochondrial
trafficking in dendrites, a key locus for altered neuronal function in schizophrenia and other major mental illness, is unknown.
Moreover, the mechanisms by which DISC1 regulates mitochondrial trafficking and the impact of this regulation on neuronal
development remain unclear. Additionally, the effects of the schizophrenia-associated DISC1-Boymaw fusion protein on mitochondrial
dynamics and neuronal development are also poorly understood.
Here we explored further the role that DISC1 plays in mitochondrial dynamics, addressing the interactions of DISC1 with mitochondrial
trafficking complexes and fusion machinery. We investigated effects of DISC1 on mitochondrial trafficking in dendrites and
subsequent actions on dendritic development. Using biochemical assays together with live cell imaging experiments, we have
demonstrated that DISC1 forms protein complexes with the dendritic TRAK2/Miro trafficking complex and with the mitofusins.
We biochemically mapped the interaction among Miro, TRAK, and DISC1 to the DISC1 N terminus, demonstrating that overexpression
of the Miro N-terminal binding domain of DISC1 disrupts both mitochondrial dynamics and dendritic development in neurons.
Furthermore, we have shown that the DISC1-Boymaw fusion protein, resulting from the chromosomal translocation described in
a Scottish pedigree (), acts in a dominant negative fashion, significantly impairing mitochondrial dynamics, the mitochondria-ER interface, and
dendritic morphogenesis.
Materials and Methods
Antibodies and Constructs
Antibody against neurofascin (clone A12/18) was from NeuroMab (IC 1:100). Antibody against GFP was from Santa Cruz Biotechnology
(Western blot 1:500, sc-8334) or NeuroMab (clone N86/8). For Sholl analysis, anti-GFP antibody was from Nacalai Tesque, Inc.
(1:2000, G090R). The monoclonal antibodies 9E10 (recognizing Myc) and 12CA5 (recognizing HA) were obtained from respective
hybridomas (Western blot and immunofluorescence 1:100, mouse). Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence
1:100 mouse) (); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation),
and anti-TRAK1 was also from Atlas Antibodies (HPA005853). Mitofusin1 was from Abcam (Ab57602), and TOM20 was from Santa Cruz
Biotechnology (FL-145). Secondary antibodies for immunofluorescence were from Invitrogen and were used at 1:1000. Secondary
horseradish peroxidase-conjugated antibodies were from Rockland and used at 1:10,000. The cDNA construct encoding human mycDISC1-FL was a kind gift from N. Brandon (Cambridge, MA). Untagged human DISC1 was in a pRK5 expression vector (). Mitochondrially targeted monomeric DsRed fluorescent protein (MtDsRed2), synaptophysinGFP, GFPMiro1, and GFPMiro2 were described previously (, , , ). Endoplasmic reticulum-targeted DsRed fluorescent protein (ERDsRed) was from Clontech. GFPTRAK1 and GFPTRAK2 were cloned by insertion of the mouse TRAK sequences into the EGFP-C1 vector. HA-tagged DISC1 deletion constructs were
described previously (). MycTRAK constructs were a kind gift from F. A. Stephenson (University College London School of Pharmacy). TRAK Miro binding domain
(MBD) was described previously (). HABoymaw, a kind gift from M. Geyer (University of California, San Diego), was subcloned into the pRK5 expression vector (). The following constructs were from Addgene: mycMitofusin1 (plasmid 23212) and -2 (23213), Su9-EGFP-(23214) (), and mito-PAGFP-(23348) ().
Cell Culture and Transfection
COS7 and SH-SY5Y cells were maintained in 10-cm dishes containing 10 ml of enhanced DMEM supplemented with penicillin-streptomycin
and 10% FBS at 37 °C and 5% CO2, transfected by nucleofection using an Amaxa electroporator, and allowed 24–48 h for protein expression. For preparation
of the primary neuronal cultures, embryonic day 18 (E18) pups were removed from the dam under sterile conditions. Brains were
removed from the skulls, and hippocampal dissection was carried out in Hanks' balanced salt solution at 4 °C prior to incubation
in a 0.125% trypsin-EDTA solution for 15 min at 37 °C. Hippocampi were washed three times with 10 ml of Hanks' balanced salt
solution and triturated 10 times using a fire-polished Pasteur pipette in prewarmed attachment medium. Cells were plated at
350,000 cells in 5 ml of prewarmed attachment medium (minimum Eagle's medium plus 10% horse serum) in 6-cm dishes containing
washed glass coverslips precoated overnight in 500 μg/ml poly-l-lysine. After 5 h the medium was removed and replaced with prewarmed maintenance medium (Neurobasal medium supplemented with
2% B-27 (Gibco), 6% glucose, GlutaMAX, and penicillin-streptomycin). Calcium phosphate precipitation or lipofection methods
were used for transfection of hippocampal cultures at 7 days in vitro (DIV) for Sholl analysis or 8 DIV for live imaging. For calcium phosphate, 1–2 μg of DNA was prepared in 27 μl of Tris-EDTA,
3 μl of 2.5 m CaCl2, and 30 μl of 2× HEPES-buffered saline. Coverslips were treated in 1 ml of prewarmed unsupplemented Neurobasal medium with
the calcium phosphate preparation. The dishes were then returned to the 37 °C, 5% CO2 incubator for 30 min or until a fine precipitate was formed. Coverslips were washed twice, and samples were maintained in
the original conditioned medium for 24–48 h for live imaging or 72 h for Sholl analysis in the 37 °C, 5% CO2 incubator to allow expression of the transfected vectors. Lipofection was carried out according to the manufacturer's instructions
(Invitrogen) in unsupplemented Neurobasal medium with 6% glucose.
Biochemical Assays
Co- immunoprecipitation experiments were carried out in lysis buffer (50 mm HEPES, pH7.5, 0.5% Triton X-100, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, and 1 μg/ml antipain, pepstatin, and leupeptin) using GFP trap beads (Chromotek) or rabbit anti-Myc beads (Sigma).
For native co-immunoprecipitation experiments, the brains of transgenic rats expressing full-length, non-mutant, human DISC1
were used (). Co-immunoprecipitation (co-IP) was carried out in lysis buffer with 1.5% Triton X-100. The homogenate was incubated overnight
with antibodies in the above described buffer supplemented with 1% BSA. Protein A-beads (Sigma) were used, and IPs were washed
four times in incubation buffer and once in lysis buffer.
Western Blotting
SDS-PAGE and Western blotting samples were denatured at 94 °C for 5 min in 3× SDS sample buffer (150 mm Tris, pH 8, 6% SDS, 0.3 m DTT, 0.3% bromphenol blue, and 30% glycerol). Polyacrylamide gels were prepared using 10% running gels and 5% stacking gels
in Novex 1.5-mm cassettes and run using the Novex XCell SureLock Mini-Cell system. Gels were transferred onto Hybond-C nitrocellulose
membrane (GE Healthcare). Membranes were blocked in 4% milk for 1 h and incubated overnight at 4 °C with shaking in the appropriate
antibody. HRP-conjugated secondary antibodies were from Rockland (1:10,000). Bands were visualized using Crescendo chemiluminescent
substrate (Millipore) together with an ImageQuant LAS 4000 charge-coupled device camera system (CCD, GE Healthcare).
Immunocytochemistry
As shown in , fluorescent labeling was used during live imaging to determine the axonal compartment prior to mitochondrial imaging (). Thus, neurofascin antibody (1 μl) was incubated for 15 min on ice with the secondary fluorescently conjugated antibody
(0.3 μl). 100 μl of live imaging block solution was added (10% horse serum and 90% extracellular solution), the solution was
mixed, and a coverslip containing the cells for imaging was incubated in this mixture for 8 min at room temperature. Following
one rinse in 1× PBS, the coverslip was used for imaging as described below. Fixed cell imaging was carried out by fixation
with 4% paraformaldehyde for 10 min at room temperature followed by blocking for 10 min in 10% horse serum, 0.5% BSA, and
0.2% Triton X-100 in PBS. Coverslips were incubated in the relevant primary antibodies diluted in blocking solution for 1
h, washed five times in PBS, and incubated in secondary antibodies diluted in blocking solution. Coverslips were washed five
times in PBS, mounted onto slides using Prolong(R) Gold antifade reagent (Invitrogen), and later sealed with nail varnish. Proximity
ligation assays (Duolink) were carried out using anti-Rhot1 (HPA010687) and anti-DISC1 antibodies (14F2 both 1:200) or anti-DISC1
alone for control proximity ligation assays. Samples were fixed and blocked before primary antibodies against Miro1 (Rhot1)
and DISC1 raised in either mouse or rabbit were applied to the cells. Following primary antibody incubation, cells were washed
in PBS before incubation with secondary antibodies conjugated with oligonucleotides. Ligation and amplification reactions
were conducted at 37 °C, as described in the Duolink manual, before mounting and visualization using confocal microscopy (). Cell fusion assays were carried out as described previously (). Briefly, cells were nucleofected with MtDsRed2 or Su9-EGFP and plated together. 24 h later, the medium was replaced with
50% polyethylene glycol 1500 in unsupplemented DMEM for 45 s and washed three times every 10 min. Normal medium was replaced
and supplemented with 30 μg/ml cycloheximide. Cells were fixed and imaged 3 h later. Imaging was carried out using a Zeiss
LSM 700 upright confocal microscope using an Apochromat 63× oil immersion lens with a 1.4 numerical aperture. Images were
captured digitally using Zen 2010 software. For ER-mitochondria contact analysis, post-acquisition processing on stacks was
carried out in ImageJ using denoise and deconvolution plugins (, ) followed by a three-dimensional rendering with VolumeJ. Images of ER-mitochondria contacts were generated using the “image
calculator” function of ImageJ to generate images specifically of colocalized regions. Structured illumination microscopy
(SIM) was performed using a Zeiss Elyra PS.1 equipped with 405-, 488-, 555-, and 642-nm lasers. Images were acquired with
a 63 × 1.4 numerical aperture oil immersion objective using a pco.edge sCMOS camera and Zen 2012 image analysis software.
Typically, images were acquired with 34-μm grating and three rotations by exciting fluorophores with 1–3% laser intensity
and 120–150-ms exposure time. Post-acquisition, images were processed with Zen 2012 using the SIM reconstruction module with
drift corrections between the channels were performed with respect to 100-nm Tetraspec fluorescent microspheres
(Molecular Probes). For Sholl analysis an apochromat 40× oil immersion lens with a 1.3 numerical aperture was used. Neurites
were traced in NeuronStudio. The number of intersections was calculated using the neurite tracer plugin on ImageJ ().
Live Cell Imaging
For neuronal imaging of the mitochondria, E18 primary hippocampal neurons were transfected at 7–8 DIV, imaged at 9–10 DIV
under perfusion with imaging medium (125 mm NaCl, 10 mm HEPES, 10 mm glucose, 5 mm KCl, 2 mm CaCl2, and 1 mm MgCl2, pH 7.4) warmed to 37 °C, and flowed at a rate of 1–2 ml/min throughout the duration of each experiment (). For acquisition, fluorescence was captured using an Olympus microscope (BX60M) with a 60× Olympus objective coupled to
an EM-CCD camera (iXon, Andor Technology). Excitation was provided by a mercury arc lamp (Cairn Research) with the appropriate
filters (). Images were acquired at 1 frame/s for a period of 2 min. Axonal regions were acquired at a distance of 100–200 μm from
the cell body, and dendritic imaging was acquired at a distance of 50 μm from the cell body due to their reduced length. The
length of process assayed was ≈150 μm. To create kymographs, image sequences were opened within ImageJ. Curved processes were
straightened using the “straighten” macro, and kymographs were created using the “multiple kymograph” macro. The resulting
kymographs show the process along the x axis and time across the y axis. Mobility was assessed by counting the percentage of objects moving during an imaging period. Mitochondria and synaptophysinGFP positive vesicles were classed as moving if they moved more than 2 μm between the initial and final frames of acquisition
(). Photoactivation assays were carried out on a Zeiss LSM 700 upright confocal microscope using an apochromat 60× water immersion
lens with a 1.0 numerical aperture. Photoactivation was carried out at 405 nm after five frames, and spread of GFP signal
was measured in ImageJ over 95 frames at 1 frame/6 s.
Statistical Analysis
All data were obtained using cells from three different preparations unless otherwise stated. Individual differences were
assessed using individual Student's t tests at a 95% significance level. Statistical significance across groups was analyzed using one-way analysis of variance
and Tukey's post hoc test to compare all data groups. For Sholl analysis, we used two-way repeated measures analysis of variance
with a post hoc Bonferroni test for comparison of dendritic crossing and branch points. Data are shown as mean ± S.E. Both
the Pearson and Manders coefficients were calculated using the JACoP plugin within ImageJ.
DISC1 Couples to the TRAK-Miro Trafficking Complex to Affect Mitochondrial Trafficking in Axons and Dendrites
We and others have previously identified DISC1 as a regulator of mitochondrial trafficking in axons via knockdown and overexpression
studies (, ), and DISC1 was recently shown to interact with TRAK1, a predominantly axonal trafficking protein (). In contrast, whether DISC1 can interact with the dendritic mitochondrial trafficking adaptor TRAK2 to regulate dendritic
trafficking of mitochondria remains unclear. To address this question, we carried out co-IP experiments in COS7 cells co-expressing
the mitochondrial trafficking adaptors GFPTRAK1, GFPTRAK2, GFPMiro1 or GFPMiro2, and DISC1. Using GFP-TRAP beads we could readily pull down DISC1 with TRAK1 and TRAK2 and with Miro1 and Miro2 but
not with GFP alone (A). Conversely, TRAK2 could be robustly pulled down with immunoprecipitated DISC1 (data not shown). Thus, DISC1 interacts robustly
with TRAK2 in addition to Miro proteins.
View larger version:
DISC1 interacts with mitochondrial trafficking complex proteins to regulate transport in dendrites in addition to axons. A, GFP trap co-immunoprecipitation experiments from COS7 cells show robust interaction of DISC1 with GFPMiro1, GFPMiro2, GFPTRAK1, and GFPTRAK2. B, proximity ligation assay in SH-SY5Y cells with DISC1 antibody or a DISC1 and Miro1 antibody shows significantly increased
signal in the dual antibody condition over background, indicating that DISC1 and Miro1 interact within the cell (n = 3 individual preparations). Scale bar = 20 μm. IB, IN, input. C and D, co-immunoprecipitation experiments with rat brain homogenate showing DISC1 to be part of a native complex with Miro (C) and TRAK1 (n = 3) or TRAK2 (n = 3) (D). E, example of live labeling of the axon initial segment (AIS) to distinguish axons and dendrites within the same neuron. F, kymographs showing movement of mitochondria through the axons and dendrites over time. Moving mitochondria are indicated
by diagonal lines and stationary mitochondria by straight lines. G, dendritic compartments (gray bars) and axonal compartments (black bars) were assayed for mitochondrial movement, and the percentage of moving mitochondria was quantified with and without expression
of DISC1 (dendrites: n = 15 control neurons and n = 16 DISC1 neurons, *, p = 0.02; axons: n = 16 control neurons and n = 13 DISC1 neurons, *, p = 0.02). Scale bar = 5 μm. NS, not significant.
To confirm the interaction between endogenous DISC1 and Miro1 in situ, we also carried out proximity ligation assays in SH-SY5Y cells. This assay detects interactions between endogenous proteins
in fixed samples, giving a fluorescent readout after incubation with relevant primary antibodies, ligation, and amplification
steps (). The significant increase in puncta in the dual antibody condition indicated an intramolecular distance of &40 nm between
DISC1 and Miro1, suggesting that the interaction occurs with endogenous proteins under physiological conditions (B). To confirm with this finding, we performed co-IP experiments with rat brain homogenate. We demonstrated the interaction
of DISC1 and Miro/TRAK proteins in the brain tissue (, C and D). These experiments validate DISC1 as a part of the native mitochondrial trafficking complex and so able to mediate its effects
locally at the mitochondrion in neurons.
Up-regulating DISC1 levels in neurons increases mitochondrial trafficking in axons (, ). The interaction between DISC1 and dendritic TRAK2 led us to explore whether DISC1 could also regulate mitochondrial motility
in dendrites. We compared DISC1-mediated trafficking effects on mitochondrial movement in axons versus dendrites within the same neurons. Neurons transfected at 8 DIV with MtDsRed2, a mitochondrially targeted red reporter construct,
with or without DISC1 were imaged at 9 DIV. DISC1 expression was confirmed by post hoc immunostaining. To unequivocally label
axons, the axon initial segment was live labeled with fluorescent anti-neurofascin antibody (), and the mitochondria were labeled by expressing MtDsRed2 (E) (). We found that in both axonal and dendritic compartments there was an equal enhancement in mitochondrial motility on co-expression
of DISC1 (, F and G, percentage of moving m 33.33 ± 2.16% for DISC1, n = 13 neurons, compared with 25.87 ± 0.82% for control, n = 16 in dendrites with DISC1, 32.55 ± 1.46% of mitochondria were moving, n = 16 neurons, compared with 23.84 ± 2.46% for control, n = 15 neurons). Thus, DISC1 interacts with the components of the dendritic transport machinery and can regulate the dendritic
trafficking of mitochondria.
Additionally, we investigated the subcellular localization of DISC1. DISC1 has a varied distribution within the cell and has
been demonstrated to adopt nuclear, cytosolic, and mitochondrial distributions (, , ). First, we investigated the subcellular localization of exogenous DISC1 in COS7 cells. Upon expression of DISC1 alone, ~20%
of the DISC1 protein showed mitochondrial localization as determined by colocalization with the mitochondrial marker MtDsRed2.
However, upon co-expression of the mitochondrial trafficking protein mycMiro1, this value increased ~2.5 fold (from 20.1 ± 1.84% to 48.4 ± 5.52%, , A and B, n = 13 control cells and n = 15 mycMiro1-expressing cells). The mitochondrial area was unchanged between the two conditions (data not shown). We also carried
out mitochondrial fractionation from COS7 cells to determine the levels of mitochondrial DISC1 upon overexpression of mycMiro1 or GFPTRAK1. In agreement with the immunofluorescence data, we observed an increase in the levels of DISC1 in the mitochondrial
fraction upon co-expression of mycMiro1 and GFPTRAK1, implying a recruitment of DISC1 to mitochondria and marking these trafficking complex proteins as acceptors for DISC1
on these organelles (C).
View larger version:
DISC1 is recruited to mitochondria by components of the mitochondrial trafficking complex. A, immunocytochemistry in COS7 cells showing localization of exogenous DISC1 with and without mycMiro1 overexpression. Mitochondria are labeled with MtDsRed2. Scale bar = 20 μm. B, percentage of DISC1 on mitochondria shown in A (n = 13–15 cells from three individual experiments, ***, p = 2.29 × 10-5). Overexpression of mycMiro1 recruits DISC1 to mitochondria. C, mitochondrial fractionation from COS7 cells shows an increase in DISC1 in this compartment with GFPTRAK1 and mycMiro overexpression (n = 3). IN, input.
The DISC1 N Terminus Is Critical for Interaction with Miro and TRAKs and for Mitochondrial Trafficking
The molecular determinants of the DISC1 interaction with TRAK1 and TRAK2 remain unclear. DISC1 has it
contains a globular N terminus followed by multiple protein-protein interaction domains (, ) (A). We sought to identify the region of DISC1 that interacts with Miro1 and TRAK2. Co-immunoprecipitation experiments with
mycMiro or GFPTRAK2 and HADISC1 deletion constructs encoding amino acids 1–301, 150–854, and 313–854 of DISC1 showed both the N-terminal 300 amino acids
of DISC1 and the longer 150–854-amino acid region as interacting with both Miro1 and TRAK2, whereas the C-terminal coiled-coil-containing
region (amino acids 313–854) did not co-IP with Miro or TRAK (, C and D). Thus, the interaction among DISC1, Miro, and TRAK requires the globular N-terminal domain, likely within amino acids 150–301,
with no role for the coiled-coil regions.
View larger version:
The DISC1 N terminus mediates the interaction with Miro and TRAKs. A, schematic of the DISC1 protein showing domains present in deletion constructs used. Coiled-coil domains are dark blue, and nuclear import or export signals are dark red. B, schematic of TRAK2 showing coiled-coil domains (dark blue). Cand D, mapping the region of DISC1 that interacts with mycMiro1 (C) and GFPTRAK2 (D). Co-IP experiments from COS7 cells show that the N-terminal 301 amino acids interact with Miro and TRAK2, whereas amino
acids 313–854 are not pulled down. Arrowhead, highlights full-length (FL) HADISC1 *, indicates nonspecific band. E and F, mapping the region of TRAK2 which interacts with DISC1. DISC1 interacts with full-length TRAK2 and TRAK2-(1–700) (E) but does not interact with the Miro binding domain (F). G, kymographs showing effect of overexpressing the DISC1-Miro binding domain on mitochondrial transport. Scale bar = 10 μm. H, percentage of moving mitochondria quantified in axons and dendrites. Overexpression of the DISC1-Miro binding domain prevents
mitochondrial transport (n = 23 ctrl and n = 21 DISC1-(1–301)-expressing neurons from three preparations, **, p = 0.002). I, quantification of percentage of moving mitochondria in axons and dendrites expressing MtDsRed2 (ctrl) or DISC1-(313–854),
the region that does not interact with Miro1 (n = 12 ctrl and 11 DISC1-(313–854)-expressing neurons from three preparations, p = 0.6). IB, IN, NS, not significant.
We also determined which region of TRAK2, another coiled-coil-rich protein, interacts with DISC1 (B). Co-IP experiments were carried out from COS7 cells expressing DISC1 and full-length mycTRAK2 or mycTRAK2-(1–700). TRAK2-(1–700) contains both the Miro binding domain and the N-terminal HAP1-like domain (the site of kinesin
binding (, )), as well as one of the binding sites for dynactin subunit p150glued (necessary for dynein motor function) (). In this case, we saw that both fragments interacted with DISC1 (E). Similar experiments with GFPTRAK2 or GFPTRAK2-(476–700), corresponding to the Miro1 binding domain of TRAK2 (), were also performed. In this case, we saw a marked decrease in the level of DISC1 pulled down with the Miro binding domain
of TRAK2-(476–700) in comparison with the full-length protein (F). Taken together, these data show that DISC1 binds at the N terminus of TRAK2 (residues 1–476) and not at the Miro1 binding
domain. Thus, DISC1 and Miro1 are unlikely to compete for the same interaction site on TRAK, allowing the formation of a functional
complex between these three proteins.
To investigate the consequences of disrupting the DISC1-Miro interaction, using live cell imaging we explored the impact of
expressing the DISC1-Miro-interacting domain (DISC1 residues 1–301) on mitochondrial transport dynamics. Hippocampal neurons
were transfected with MtDsRed2 or co-transfected with MtDsRed2 and HADISC1-(1–301). Assays were carried out as detailed under “Materials and Methods,” with HADISC1-(1–301) expression confirmed by immunocytochemistry following live imaging. Upon co-expression of the DISC1-Miro-interacting
domain, a significant decrease in moving mitochondria was detected (, G and H, ctrl = 14.7 ± 2.25%, DISC1-(1–301) = 5.07 ± 1.84%; n = 23 control and n = 21 DISC1-(1–301)-expressing neurons), as is also apparent by the decrease in diagonal lines in the kymographs. The effect
of DISC1-(313–854), which does not interact with Miro, was also investigated and showed no significant alteration in mitochondrial
trafficking (quantified data shown in I, ctrl = 14.6 ± 2.42%, DISC1-(313–854) = 12.6 ± 3.18%; n = 12 control neurons and 11 DISC1-(313–854)-expressing neurons); therefore the impairment in mitochondrial trafficking is
reliant on the DISC1-Miro-interacting domain.
The Schizophrenia-associated DISC1-Boymaw Fusion Protein Is Localized to Mitochondria and Impairs Mitochondrial Trafficking
The expression of the DISC1-Boymaw fusion protein results from the schizophrenia-associated chromosomal translocation, which
interrupts DISC1 in a Scottish pedigree (, ). As the fusion protein contains DISC1 amino acids 1–597 (which we show here to include the DISC1-Miro-interacting domain),
we investigated the localization of this protein and its effect on mitochondrial trafficking. In hippocampal neurons, the
DISC1-Boymaw fusion protein (labeled HABoymaw in –) adopts a mitochondrial distribution in neurons as shown by colocalization between MtDsRed2 and HABoymaw staining seen in the line scan of the zoomed process (, A and B). Additionally, Pearson colocalization analysis between MtDsRed2 and HABoymaw gives a coefficient of 0.65 ± 0.08, suggesting a preferential localization to mitochondria. Mitochondrial trafficking
assays in neurons transfected with HABoymaw and MtDsRed2 to label the mitochondria revealed expression of the DISC1-Boymaw fusion protein to significantly decrease
the percentage of moving mitochondria compared with control (, C and D, ctrl = 16.1 ± 2.20%, HABoymaw = 6.59 ± 1.4%; n = 32 control and n = 26 HABoymaw-expressing neurons). In contrast, HABoymaw expression did not significantly impact the trafficking of synaptophysinGFP positive vesicles (E, ctrl = 27.9 ± 2.7%, Boymaw = 28.5 ± 4.3%, n = 17–19 quantified in F), confirming that the Boymaw fusion protein is not responsible for an overall decrease in microtubule based transport but
specifically disrupts the trafficking of mitochondria. The impact of Boymaw is consistent with the dominant negative effect
of the DISC1-Miro-interacting domain on mitochondrial trafficking, suggesting that a disruption in DISC1-mediated mitochondrial
trafficking could be a pathological mechanism.
View larger version:
The DISC1-Boymaw fusion protein inhibits mitochondrial trafficking. A, immunocytochemistry in hippocampal neurons showing localization of the Boymaw protein. Scale bar = 20 μm, 5 μm on zoomed image (right). B, line scan of zoomed process showing that the DISC1-Boymaw fusion protein localizes to mitochondria in axons and dendrites.
(A.U. = arbitrary units.) C, kymographs showing mitochondrial transport in axons expressing MtDsRed2 and co-expressing HABoymaw. Scale bar = 10 μm. D, percentage of moving mitochondria in neurons was quantified with and without expression of HABoymaw. The presence of the HABoymaw fusion protein inhibits mitochondrial trafficking (n = 32 ctrl neurons and 26 HABoymaw-expressing neurons, ***, p = 0.001). E and F, Boymaw expression has no impact on synaptophysin trafficking (E), as quantified in F (n = 17 control and n = 19 HABoymaw-expressing neurons from three preparations, p = 0.9). NS, not significant. Scale bar = 10 μm.
DISC1 Couples to the Mitochondrial Fusion Machinery Proteins, Mitofusins
DISC1 had been shown previously to alter mitochondrial morphology (, ). Therefore, we measured the length of mitochondria in neurons upon expression of DISC1-(1–301) or the DISC1-Boymaw fusion
protein. We found a significant decrease in the length of mitochondria compared with control in each case (, A and B, ctrl = 2.1 ± 0.065 μm, DISC1-(1–301) = 1.8 ± 0.063 μm, n = 11 , C and D, ctrl = 1.81 ± 0.0858 μm, HABoymaw = 1.54 ± 0.0644 μm). This observed alteration in mitochondrial morphology led us to investigate the relationship between
DISC1 and the mitochondrial fusion machinery. We focused on mitofusins, crucial mediators of mitochondrial fusion and morphology
known to interact with Miro proteins (). Co-immunoprecipitation experiments from COS7 cells expressing mycMitofusin1 or -2 and human DISC1 revealed that DISC1 could be readily pulled down with both mycMitofusin1 and -2 (E). Moreover, mitochondrial fractionation from COS7 cells confirmed higher levels of mitochondrial DISC1 when either mycMitofusin1 or -2 was expressed (F). Importantly, we confirmed a biochemical interaction between DISC1 and Mitofusin1 from rat brain homogenate (G), showing no interaction with the outer mitochondrial membrane protein TOM20 (translocase of the outer membrane of 20 kDa),
a protein unrelated to mitochondrial trafficking (). This shows that DISC1 forms a specific, native complex with fusion proteins rather than interacting indiscriminately with
outer mitochondrial membrane proteins. Therefore, DISC1 may play a role in mitochondrial fission/fusion dynamics as well as
in trafficking.
View larger version:
DISC1 interacts with mitofusins. A and B, DISC1 1–301 decreases the length of mitochondria (A) as quantified in B (ctrl = 2. 1 μm ± 0.065, DISC1-(1–301) = 1.8 ± 0.063 μm, n = 11 axons, **, p = 0.004). C and D, the DISC1 Boymaw fusion protein decreases the length of mitochondria (ctrl = 1.81 ± 0.0858 μm, HABoymaw = 1.54 ± 0.0644 μm, n = 13 axons, *, p = 0.02). Scale bar = 10 μm. E, co-IP experiments from COS7 cells show that DISC1 interacts with MycMitofusin1 and -2. F, mitochondrial fractionation from COS7 cells shows that DISC1 is recruited to mitochondria upon overexpression of MycMitofusin1 and -2. G, co-IP experiments with rat brain homogenate showing DISC1 to be part of a native complex with Mitofusin1 but not with translocase
component TOM20 (n = 3). IB, IN, input.
The DISC1-Boymaw Fusion Protein Decreases Mitochondrial Fusion
Given the interaction between DISC1 and the mitofusins, as well as the effect of the DISC1-Boymaw fusion protein on mitochondrial
morphology, we also investigated the impact of Boymaw expression on mitochondrial fusion in primary hippocampal neurons. We
used a mitochondrially targeted, photoactivatable GFP (, ) as the control condition with co-expression of HABoymaw and MtDsRed2 expression to visualize the neurons prior to photoactivation. We carried out photoactivation in the neuronal
soma, a location of high mitochondrial density and therefore fusion events, to minimize the contribution of trafficking defects
to any mitochondrial fusion alteration (). A decrease in the spread of GFP signal post-photoactivation is seen in HABoymaw-expressing neurons, showing a decreased mitochondrial fusion rate (, A and B) (n = 17 control and 15 HABoymaw neurons, final normalized area ctrl = 1.44, HABoymaw = 1.20 arbitrary units).
View larger version:
The DISC1-Boymaw fusion protein inhibits mitochondrial fusion. A, HABoymaw inhibits mitochondrial fusion in neurons. Neurons were transfected with MtDsRed2 and mitochondrially targeted photoactivatable
GFP (ctrl) or coexpressing HABoymaw. Scale bar = 20 μm. B, the change in area of GFP signal after photoactivation is reduced in HABoymaw-expressing neurons (n = 17 control and n = 15 Boymaw-expressing neurons, p & 0.05 at 12–90 s post-photoactivation, **, p & 0.01 at 96–570 s post-photoactivation). C, schematic showing mitochondrial fusion after polyethylene glycol-mediated plasma membrane fusion. D and E, HABoymaw decreases mitochondrial fusion in COS7 cells (D) as quantified by colocalization analysis in E (n = 15 post-fusion cells from three individual preparations, ***, p = 0.0009). Scale bar = 20 μm.
Although this photoactivation assay gives an indication of the fusion rate independent from mitochondrial trafficking, there
is the potential for contribution of the reported trafficking defect to the decreased spread of GFP signal. To confirm our
results by another method, we used a previously described assay () involving transfection and co-culture of two populations of cells (in this case COS7 cells) with different mitochondrial
markers (in this case fluorophores Su9GFP and MtDsRed2 or with HABoymaw co-expressed with MtDsRed2) followed by PEG 1500 treatment to fuse the plasma membranes after 24 h. paraformaldehyde
fixation and immunostaining was carried out 3 h later (see schematic in C). This assay has the advantage of mitochondrial HABoymaw expression in just half of the cells, and therefore the trafficking of Su9GFP positive mitochondria is unimpeded and fusion can be investigated with a lesser contribution of Boymaw-dependent mitochondrial
trafficking deficits. Representative post-fusion cells are shown (D), and colocalization of Su9GFP and MtDsRed2 positive mitochondria, indicating fusion events, is quantified (E, n = 15 fused cells, ctrl = 27% ± 5.2, HABoymaw = 6.4 ± 1.9%). The noted decrease in colocalization indicates a Boymaw-dependent impairment in mitochondrial fusion
in addition to trafficking. This is consistent with Boymaw acting in a dominant negative manner for fusion as well as trafficking,
as suggested by the decrease in mitochondrial length caused by both the DISC1-Miro-interacting domain and HABoymaw expression. Taken together, these data support a role for DISC1 in mitochondrial fusion as well as trafficking.
The DISC1-Boymaw Fusion Protein Decreases the ER-Mitochondria Contact Area
The DISC1 interaction with Miro and Mitofusin 2 (Mfn2), known components of ER-mitochondria contact sites in yeast and mammalian
cells, respectively (, ), prompted us to investigate effects of DISC1 and the DISC1-Boymaw fusion protein on the ER-mitochondria interface. We used
COS7 cells because of their extensive ER network and expressed Su9GFP and ERdsRed to label the mitochondria and ER, respectively, along with HADISC1 or HABoymaw. Representative volume renderings are shown in A. Colocalization analysis between the ERdsRed and Su9GFP signals by Manders coefficient indicates the area of ER-mitochondria contacts, revealing a significant decrease in this area
in the presence of HABoymaw (C, M ctrl = 0.19 ± 0.04, HADISC1 = 0.18 ± 0.03, HABoymaw = 0.15 ± 0.05, ctrl versus HABoymaw, p = 0.03, ctrl versus DISC1 and DISC1 versus HABoymaw, not significant (NS), n = 15 cells from three experiments). Additionally, we used these colocalized regions, showing ER-mitochondria contacts for
colocalization studies with HADISC1 or HABoymaw (B) by generating images of the ER-mitochondria contacts. Despite the reduced ER-mitochondria interface, we found a greater
fraction of the HABoymaw signal present at contact sites compared with HADISC1 (D, M HADISC1 = 0.07 ± 0.01, HABoymaw = 0.14 ± 0.03, p = 0.03). This alteration in ER-mitochondria contact sites suggests potential roles for DISC1 in ER-mitochondria cross-talk
and mitophagosome biogenesis (). To further investigate the potential that DISC1 might be resident at ER-mitochondria contacts, we carried out SIM imaging.
This technique gives images with resolution approaching 120–130 nm and thus is of great value in imaging these microdomains
(). SIM imaging was performed in SH-SY5Y cells transfected with Su9GFP and ERdsRed and stained for endogenous DISC1. E shows SIM reconstructed images of the mitochondria and ER, and from these images ER-mitochondria contact images were determined
as described previously. On the ER-mitochondria image, the DISC1 image was overlaid and represented in F. A line scan showing an overlap of signal intensity in DISC1 and ER-mitochondria contact is shown in G. Interestingly, we observed that endogenous DISC1 adopts a punctate distribution, as suggested previously (), and it can be seen that these puncta colocalize in part with the contact sites between the ER and the mitochondria.
View larger version:
The DISC1-Boymaw fusion protein decreases the area of ER-mitochondria contacts. A, three-dimensional renderings of mitochondrial network (Su9GFP) and ER (ERdsred) in COS7 cells upon co expression of HADISC1 or HABoymaw. Colocalization shows regions of ER-mitochondria interface. Scale bar = 20 μm, 5 μm on zoomed images (right hand panel). B, colocalization of HADISC1 or HABoymaw with ER-mitochondria contacts. The images of the contact sites were generated from colocalization of images shown in
A. C, Manders colocalization coefficient of images in A shows a decrease in area of ER-mitochondria contacts upon HABoymaw expression (n = 15 cells from three experiments, ctrl versus Boymaw p & 0.05). NS, not significant. D, Manders coefficient for colocalization of HADISC1 or HABoymaw with ER-mitochondria contact sites. HABoymaw shows higher colocalization than HADISC1 (*, p = 0.03). E, structured illumination microscopy showing ER and mitochondrial network in SH-SY5Y cells. F, structured illumination microscopy shows partial colocalization between endogenous DISC1 and ER-mitochondria contacts in
SH-SY5Y cells. Scale bar = 10 μm (zoom 1 μm). G, line scan showing signal intensities of DISC1 and ER-mitochondria contacts. A.U.= arbitrary units.
DISC1-mediated Mitochondrial Trafficking Is Necessary for Normal Dendritic Arborization
DISC1 plays a key role in the regulation of neurite outgrowth both in vitro and in vivo (, ), and growing evidence suggests a link between mitochondrial dynamics and dendrite development and complexity (, , ). The interaction between DISC1 and dendritically targeted TRAK2 prompted us to investigate the effect of disrupting DISC1-mediated
mitochondrial dynamics on dendritic development. GFP expression was used to delineate neuronal morphology, and two markers
of comp dendritic length and dendritic branching. Neurons expressing the DISC1-Miro-interacting domain
(DISC1-(1–301)) to disrupt mitochondrial trafficking showed a decreased dendritic complexity (A). The total dendritic length per cell was decreased 31% compared with control (B, ctrl = 1669.4 ± 99.0 μm, DISC1-(1–301) = 1148.6 ± 88.0 μm, n = 16 cells from four preparations, p = 0.001). Next we carried out a Sholl analysis to study whether dendrite arbor complexity differs as a function of distance
from the soma. This showed the decrease in the number of intersections to be pronounced at 80 and 100 μm from the soma (C). A similar analysis was then carried out with the number of dendritic branch points per neuron. As with dendritic length,
the number of branch points per cell decreased by 31% upon expression of the DISC1-Miro-interacting domain compared with control
(, D and E, ctrl = 16 ± 1.9 branch points, DISC1-(1–301) = 11.5 ± 1.2 branch points), with the effect most noticeable at 90 μm from
View larger version:
DISC1-mediated mitochondrial trafficking is necessary for normal dendritic development. A, representative images showing control 10 DIV neurons and those expressing the DISC1-Miro binding domain (residues 1–301).
GFP was used to visualize neuronal morphology. Scale bar = 10 μm. B, total dendritic length/cell (***, p = 0.001). C, Sholl analysis reveals a significant decrease in intersections at 80 and 100 μm from the soma (*, p & 0.05). D, average number of branch points/cell is decreased with DISC1-Miro binding domain expression (*, p = 0.04). E, Sholl analysis of branch points (*, p & 0.05 at 90 μm from the soma) (n = 16 neurons from four preparations). F, representative images showing control 10 DIV neurons expressing GFP in the presence or absence of HABoymaw. Scale bar = 10 μm. G, total dendritic length is decreased when the DISC1-Boymaw fusion protein is expressed (**, p = 0.004). H, Sholl analysis showing the number of intersections at 10-μm intervals. A significant decrease in intersections in Boymaw-expressing
neurons is seen proximal to the soma at a distance of 50–80 μm away from the soma (*, p & 0.05 in each case). I, analysis of branch points reveals expression of the DISC1-Boymaw fusion protein to decrease in the number of branch points/cell
(*, p = 0.03). J, Sholl analysis reveals a significant decrease 50 μm from the soma (*, p & 0.05, n = 15–16 neurons from four preparations).
Repeating this analysis with neurons expressing the DISC1-Boymaw fusion protein showed the same effect (F). The calculation of total dendritic length reveals a decrease of 35% compared with control (G, ctrl = 1590.4 ± 142.7 μm, Boymaw = 1033.3 ± 101.0 μm, n = 15–16 neurons from four individual preparations, p = 0.004). A significant decrease in the number of dendritic intersections was noted at 50–80 μm from the soma (H). As with dendritic length, the total number of branch points was decreased 33% upon expression of the fusion protein (I, ctrl = 16 ± 1.7 branch points, Boymaw = 10.7 ± 1.5 branch points). Concurrently, a Sholl analysis revealed this decrease
to be most obvious at 50 μm from the soma (J). Taken together, these data demonstrate that the expression of the DISC1-Boymaw fusion protein has a severe negative impact
on dendritic development, showing this effect to be linked to impairment in the mitochondrial dynamics. These findings provide
further evidence for the importance of correct mitochondrial distribution in the development and maintenance of dendritic
arbors. Furthermore, our findings support a key role for DISC1 in this process and further dissect the pathways through which
this occurs.
Discussion
Here we demonstrate that the schizophrenia-associated protein, DISC1, interacts with Miro1 and Miro2 as well as TRAK1 and
TRAK2 to affect axonal and dendritic transport of mitochondria. We report the interaction of DISC1 with the mitofusins and
confirm DISC1 as part of a native complex with trafficking and fusion proteins in brain tissue. We also demonstrate that the
schizophrenia-associated DISC1-Boymaw fusion protein acts in a dominant negative fashion to disrupt mitochondrial trafficking
and fusion, as well as decreasing the area of ER-mitochondria contacts. Finally, we demonstrate the necessity of DISC1-mediated
mitochondrial dynamics for correct neuronal development and dendritic arborization.
We found Miro1 to be a major mitochondrial acceptor for the DISC1 protein, similar to its effect on the TRAK adaptor and the
E3 ubiquitin ligase Parkin, which is crucial for mitophagy (, ). Via interaction mapping and subsequent trafficking experiments in neurons, we have demonstrated the necessity of the DISC1-Miro-TRAK
interaction for normal mitochondrial transport. Our mapping experiments support the interaction between Miro and DISC1 as
occurring within amino acids 150–301 of DISC1. Furthermore, the same region of DISC1 interacting with both Miro1 and TRAK
suggests that the interaction occurs with one of these proteins via the other, e.g. DISC1 interacts with Miro1 via TRAK. Interestingly, the DISC1-TRAK1 interaction was shown to be increased by ~30% upon expression
of the R37W pathological DISC1 mutant (), consistent with our identification of the TRAK-interacting domain localizing to the DISC1 N terminus. Because our mapping
experiments support the idea that the binding site is within amino acids 150–301, this raises the possibility that the R37W
mutation may indirectly impact on DISC1 binding to TRAK1 via a conformational change in the DISC1 N terminus.
It is yet to be determined how DISC1 mediates its effects on trafficking. Although emerging evidence suggests differences
between the axonal and dendritic regulation of mitochondrial transport (, , , ), it would seem that DISC1 is a factor that is common to trafficking in both compartments. This is supported by our data
demonstrating interactions with both TRAK1 and TRAK2 and comparable up-regulation of mitochondrial motility in axons and dendrites.
In a recent article investigating DISC1 and mitochondrial trafficking in axons, DISC1 is reported to show a preference for
kinesin-mediated anterograde transport (). DISC1 interacts with kinesin motors (, ), raising the possibility of local regulati this is in agreement with our mitochondrial fractionation
assays. Indeed, DISC1 has been reported previously to inhibit GSK3β (), which in turn phosphorylates kinesin light chains, causing uncoupling of motors from cargo (reviewed in Ref. ). Moreover, DISC1 is known to interact with and inhibit PDE4 (phosphodiesterase 4), both directly and via transcriptional
down-regulation in a complex with ATF4 (activating transcription factor 4) (). A subsequent increase in cAMP could activate PKA, thus inhibiting GSK3β and rescuing a decrease in moving mitochondria
as has been reported in studies with a cAMP analogue and the PKA activator forskolin (). The contributions of these pathways, as well as others, will need to be addressed in the future to fully understand the
mechanism by which DISC1 regulates mitochondrial trafficking.
We have shown that the schizophrenia-associated DISC1-Boymaw fusion protein, which targets to mitochondria (), localizes to mitochondria in MAP2 positive dendritic processes. DISC1-Boymaw expression results in a decrease in the length
of mitochondria and disrupts mitochondrial trafficking without impairing the trafficking of other cargoes. Although a role
for DISC1 in the trafficking of other cargoes (e.g. synaptic vesicles) has been demonstrated (), a specificity for mitochondria is not unexpected given the mitochondrial localization of the Boymaw fusion protein. Moreover,
this is consistent with the effect of DISC1 knockdown on mitochondrial trafficking () and interruption of the DISC1-Miro complex by expression of the DISC1-Miro-interacting domain. This may provide a disease
mechanism for the t(1;11) chromosomal translocation (). Multiple mutations in DISC1 have been reported previously to disrupt mitochondrial trafficking (, ), which can also be disrupted upon DISC1 aggresome formation (). The interruption of mitochondrial trafficking prevents localization of mitochondria at sites of high energy and calcium
buffering demand, e.g. synapses (), and a decrease in mitochondria at synapses has been reported previously in a schizophrenic cohort ().
We also have reported here that the mitofusins interact with DISC1 and have effects on the localization of DISC1 similar to
those of Miro1 and the TRAKs, consistent with these proteins being components of the mitochondrial trafficking complex (). Additionally, expression of both the DISC1-Miro-interacting domain and the DISC1-Boymaw fusion protein leads to alterations
in mitochondrial morphology, prompting investigation into mitochondrial fusion events. We found the DISC1-Boymaw fusion protein
to display dominant negative activity on mitochondrial fusion in COS7 cells and primary neurons. These data extend our findings,
demonstrating DISC1 as a regulator of mitochondrial fission/fusion dynamics in addition to mitochondrial trafficking. The
reported effect on fusion will impact the mitochondrial network, as fusion is crucial for the exchange of contents and mitochondrial
biogenesis (). Intriguingly, a role for DISC1 in the mitochondrial fusion pathway suggests its involvement in maintaining the function
of the mitochondrial population.
Here, we also investigated the localization of DISC1 and the DISC1-Boymaw fusion protein at sites of ER-mitochondria contact
by confocal microscopy and studied the effects of the DISC1-Boymaw fusion protein on these sites. We found that a greater
fraction of the DISC1-Boymaw fusion protein is present at these sites in comparison with wild type DISC1. Notably, we also
have demonstrated presence of endogenous DISC1 at these sites by super-resolution microscopy. Further, we have demonstrated
that Boymaw expression decreases the area of these contacts. This could reflect inhibition of Mitofusin2 tethering activity,
in agreement with our mitochondrial fusion assays and consistent with previous reports showing that Mitofusin2 knock-out mouse
embryonic fibroblasts (MEFs) have a significantly decreased area of ER-mitochondria colocalization (). In addition, the role of ER-mitochondria contacts as sites of trafficking and fission/fusion regulation remains an exciting
area for future study, and the effects of schizophrenia-associated DISC1 mutants on these contacts could account for alterations
in mitochondrial dynamics and turnover downstream of its effects on autophagosome biogenesis (). Indeed, alterations in the sites of ER-mitochondria contact have been noted in models of neurodegenerative disease such
as amyotrophic lateral sclerosis and Parkinson disease (, ).
Finally, we have reported evidence of interplay between DISC1 and mitochondrial trafficking, fusion, and ER-mitochondria contacts
in dendritic morphogenesis. Both DISC1 and the proteins mediating mitochondrial dynamics have been shown to affect neurite
development. The TRAK proteins have been recently reported to regulate neuronal morphology, with knockdown of TRAK2 decreasing
dendritic complexity (). Mitofusin2 is necessary for normal dendritic development in the cerebellum (), and studies of Mitofusin1 overexpression also show an alteration in dendritic arborization, suggesting that mitochondrial
distribution and fission/fusion play a critical role in dendrite development (). We have demonstrated a decrease in dendritic complexity upon expression of the DISC1-Miro-interacting domain and the Boymaw
fusion protein. Variations in dendritic morphology have been reported previously in models of neuropsychiatric illness, and
alteration of DISC1 function, either by truncation or point mutation, is known to decrease neuronal complexity (, , , ). This impairment in dendritic development could lead to disruptions in network connectivity and neurotransmission, leading
to the development of schizophrenic symptoms. Indeed, mice expressing Boymaw exhibit behavioral abnormalities such as increased
startle and anhedonia, consistent with schizophrenia and depression (). Collectively, our data support a mechanism whereby impaired DISC1 function leads to aberrant mitochondrial dynamics and
dendritic morphogenesis, a causative factor in schizophrenia and other major mental illness.
Author Contributions
R. N., S. M., T. A. A., W. D. H., and J. T. K. designed the research. R. N., S. M., T. A. A., N. B., D. I., and M. P. performed
and analyzed the research. S. V. T. and C. K. generated the tgDISC1 rat and C. K. provided DISC1 antibody. R. N., T. A. A.,
and J. T. K. wrote the paper.
Acknowledgments
We thank the members of the Kittler laboratory for constructive discussion. We thank the University College London Super Resolution
Imaging Facility for access to SIM.
1 An MRC CASE Award Ph.D. student sponsored by Pfizer.
2 Recipient of an EMBO Long-term Fellowship and Marie Curie International Incoming Fellowship (IIF) award.
3 A University College London (UCL) Impact Award Ph.D. student.
4 A member of the UCL MRC-DTA Ph.D. Program in Biomedicine. Present address: Dept. of Psychiatry, N. Y. State Psychiatric Inst.,
1051 Riverside Dr., No. 28, New York, NY 10032.
5 A Brain Research Trust Ph.D. student in the UCL Clinical Neuroscience Program.
7 Supported by a grant from the Brain Behavior and Research Foundation (NARSAD Independent Investigator Award 20350) and EU-FP7
(MC-ITN “IN-SENS” 607616).
* This work was supported by Starting Grant 282430 from the European Research Council (ERC), a Research Prize from the Lister
Institute of Preventive Medicine, the Wellcome Trust, and a Medical Research Council (MRC) senior non-clinical fellowship
(to J. T. K.).The authors declare that they have no conflicts of interest with the contents of this article.
9 The abbreviations used are:
endoplasmic reticulum
days in vitro
immunoprecipitation or immunoprecipitate
structured illumination microscopy
Received October 20, 2015.
Author's Choice—Final version free via .
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