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. 2016 Nov;21(11):1561-1572.
doi: 10.1038/mp.2015.194. Epub 2016 Jan 12.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits

S V Trossbach  1 , V Bader  1 , L Hecher  1 , M E Pum  2 , S T Masoud  3 , I Prikulis  1 , S Schäble  2 , M A de Souza Silva  2 , P Su  4 , B Boulat  5 , C Chwiesko  5 , G Poschmann  6 , K Stühler  6 , K M Lohr  7 , K A Stout  7 , A Oskamp  8 , S F Godsave  9 , A Müller-Schiffmann  1 , T Bilzer  1 , H Steiner  10 , P J Peters  9 , A Bauer  8 , M Sauvage  5 , A J Ramsey  3 , G W Miller  7 , F Liu  4 , P Seeman  11 , N J Brandon  12 , J P Huston  2 , C Korth  1
Affiliations

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits

S V Trossbach et al. Mol Psychiatry. 2016 Nov.

Abstract

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

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Figures

Figure 1
Figure 1
Aggregated DISC1 detected by IHC in brains of the tgDISC1 rat. (a) Western blot comparing transgene expression and aggregation in four different tgDISC1 founder lines. Heterozygous rats of founder lines 1, 3, 5 and 7 displayed different levels of full-length human DISC1 transgene expression (homogenate) which were reflected in the insoluble fraction. No huDISC1 could be detected in the negative control animal. Beta-actin was used as loading control in the homogenate. Relative number of transgene copies in founder lines was determined by quantitative Real-Time-PCR of genomic DNA and revealed the least transgene copies in the gDNA of founder 3 (arbitrarily set to one for comparison between founder lines) and a Mendelian-like inheritance pattern upon breeding. Founder line 3 with the weakest DISC1 expression was chosen for further experiments to avoid artifacts of too strong transgene expression. (b) Confocal immunofluorescence of striatal (dStr, right panels) and frontal (left panels) cryosections of the transgenic DISC1 rat (middle and in higher magnification in lower panels) and a negative control rat (upper panels). Abundant punctuate, mainly perinuclear staining as evidence for the existence of DISC1 aggregates can be detected that are more frequent and bigger in the striatum. Green: DISC1; blue: DAPI; bar 40 μm (upper two panels), bar 10 μm (lower panels). (c) Densitometric analysis of biochemically purified insoluble fraction of the tgDISC1 rat mPFC and dorsal striatum. The tgDISC1 rat (n=6) had more aggregated DISC1 in the dStr than in the mPFC in accordance with the IHC. Wilcoxon *P=0.028. (d) NMR analysis of ventricle size. The tgDISC1 rat (n=8) had a larger total ventricle size than negative controls (n=10), namely 20.75±2.5 mm3 in tgDISC1 rats and 15.54±0.9 mm3 in negative controls. Unpaired t-test: P=0.052. All means±s.e.m. DISC1, Disrupted-in-Schizophrenia 1; gDNA, genomic DNA; NMR, nuclear magnetic resonance.
Figure 2
Figure 2
Behavioral phenotypes of the tgDISC1 rat. (a) Amphetamine hypersensitivity in the tgDISC1 rat shown by horizontal locomotion. Spontaneous locomotion before (saline; white bars) and after (gray bars) application of a single low dose of d-amphetamine (0.5 mg kg−1, i.p.) is presented. Whereas the d-amphetamine had no significant locomotor effect in the control animals (paired t-test: P=0.280, n=12), it led to increased locomotion in the tgDISC1 rat indicating hypersensitivity to d-amphetamine (paired t-test ***P<0.001, n=12). (b) Amphetamine hypersensitivity in the tgDISC1 rat shown by duration of rearing. Only tgDISC1 rats reacted with increased duration of rearing to d-amphetamine treatment (gray bars) compared with saline (white bars; paired t-test: negative controls (NC) P=0.146, TG ***P<0.001; TG and NC n=12). (c) The rotarod task as measure of motor learning ability and attention. Under constant speed of the wheel, the negative control animals showed a significant progressive improvement in walking on the rotarod over three subsequent trials, whereas the transgenics did not display such a learning curve (t-test of trial 3: *P=0.041; TG n=11, NC n=10). (d) Hyperexploration of tgDISC1 rats in the object recognition task (OR). Comparing duration of exploration of the new vs the old object in tgDISC1 rats and controls during OR test trial (TG n=12, NC: n=10) showed that tgDISC1 rats explored the new object more extensively than the negative controls (one-way ANOVA: #P=0.048), although both genotypes preferred the new over the old one (paired t-test: NC *P=0.045, TG ***P<0.001). (e) Hyperexploration of the tgDISC1 rats in the object place recognition task (OPR). Comparably, in the OPR task (TG n=12, NC n=8) the tgDISC1 rats explored the displaced object longer than control rats (one-way ANOVA: #P=0.019). Again, both genotypes favored the displaced over the stationary object (paired t-test; NC *P=0.021, TG **P=0.001). All means±s.e.m. DISC1, Disrupted-in-Schizophrenia 1; i.p., intraperitonial.
Figure 3
Figure 3
Dorsal striatum and dopamine homeostasis in the tgDISC1 rat. (a) Neurochemical quantification of post mortem dopamine (DA) in the dStr of tgDISC1 and negative control animals. TgDISC1 rats (n=12) had lower levels of DA as compared with negative controls (NCs) (n=12). TgDISC1 and negative control rats had 13 337±375 and 11 675±504 pg/mg DA. Unpaired t-test **P=0.005. (b) Total D2 receptor abundance in the dStr. Autoradiography was performed with the D2R specific radioligand [3H]raclopride. No difference in ligand binding and therefore total receptor density could be found in tgDISC1 rats (n=10) and controls (n=10) in the dorsal striatum. Mean receptor density was 870±26 fmol/mg protein for controls and 815±26 for tgDISC1 rats. Unpaired t-test P=0.156. (c) Elevated striatal D2High receptor portion in tgDISC1 rats. TgDISC1 rats (n=6) had an 81% increase in D2High receptor portions compared with negative controls (n=6) as measured by [3H]domperidone binding. Binding of the radioligand was challenged with either 1 nM or 100 nM DA, concentrations at which no significant occupation of D2Rs or D2High-specific binding occurs, respectively. Proportions of D2High receptors in relation to total D2 receptors were 33±3.9% in NCs and 59.7±6.5% in TGs. Unpaired t-test **P=0.005. (d) Peak DA release in the dStr of tgDISC1 rats. Fast-scan cyclic voltammetry measurement of DA in striatal slices revealed no difference in the peak release of DA in tgDISC1 rats (2.16±0.43 μm; n=4) and controls (1.88±0.58 μm; n=6). Mann–Whitney U-test P=0.762. (e) Clearance of extracellular DA in the dStr of tgDISC1 rats. TgDISC1 rats (tau=0.131±0.006; n=4) show increased extracellular DA clearance compared with negative controls (0.176±0.006; n=6) as measured by fast-scan cyclic voltammetry. Mann–Whitney U-test *P=0.036. (f) Striatal DAT levels in tgDISC1 rats. Preparation of the synaptic plasma membrane (SPM) and subsequent western blotting revealed a 19% increase in dopamine transporter levels in the dorsal striatum of tgDISC1 rats (NC, TG n=6). Densitometric analysis was performed by normalization of DAT to the Na/K-ATPase signal in the preparations. Unpaired t-test **P=0.004. All means±s.e.m.
Figure 4
Figure 4
Dopamine-induced DISC1 aggresome formation in cell models. (a) Confocal immunofluorescence light microscopy of SH-SY5Y human neuroblastoma cells induced for expression of full-length human DISC1 (S704, L607). DISC1 was diffusely expressed throughout the cytoplasm (left panel). Upon DA treatment (100 μm for 24 h; right panel) the previously diffusely distributed DISC1 built up aggresomes inside the cell. Bar 20 μm. (b) Cryoimmunogold electron microscopy for cells from a. Arrow marks the perinuclear, immunolabeled dopamine-induced DISC1 aggresome. Bar 100 nm. (c) Characterization of dopamine-induced DISC1 aggresomes as shown in a. Double-staining shows that DA-induced DISC1 aggresomes (red) are caged by vimentin (green). Bar 20 μm. (d) Co-immunoprecipitation of DISC1 by DAT in the tgDISC1 rat brain. In a plasma membrane preparation of the striatum the DAT antibody co-immunoprecipitated transgenic human DISC1. Upper two panels show DISC1 signal, lower two panels DAT signal at two different exposure times. DA, dopamine; DISC1, Disrupted-in-Schizophrenia 1; M, mitochondrion.
Figure 5
Figure 5
Dopamine-induced DISC1 high-molecular-weight (HMW) bands in cell and animal models. (a) Appearance of DISC1 HMW bands upon DA-treatment of cells. Western blot of the lysate (upper panel) or insoluble fraction (lower panel) of NLF human neuroblastoma cells transiently transfected with full-length DISC1 (S704, L607) and treated with DA at indicated concentrations. HMW DISC1-immunoreactive bands appear (arrow) with increasing concentrations of DA. (b) Analysis of the purified insoluble fraction of the mPFC of three transgenic mouse lines either overexpressing DAT (DAT-OE), with decreased VMAT2 expression (VMAT2-DE) and double transgenics (DAT-OE:VMAT2-DE). In the western blot (upper panel) a HMW Disc1 signal is visible in all transgenic mouse lines, but not in wild-type mice. As a loading control of the input material for the insoluble fraction preparation homogenates were blotted and incubated with beta-actin on a separate blot. Densitometric quantitation (lower panel) of insoluble HMW Disc1 in the mPFC of transgenic mice shows that while VMAT-DE animals exhibit a trend towards increased Disc1 aggregation in mPFC, in DAT-OE mice Disc1 aggregates were significantly elevated. DAT-OE:VMAT2-DE mice displayed nearly double the amount of Disc1 aggregates in mPFC compared with WT animals (Kruskal-Wallis with one-tailed Dunn's post hoc test; DAT-OE: *P=0.029; DAT-OE:VMAT2-DE: *P=0.014; n=4 each). (c) Western blots of the purified insoluble fraction showing the HMW band as a common signature of DA-induced aggregation of human, rat, and mouse DISC1. Depicted are the insoluble fractions of primary rat cortical neurons derived from tgDISC1 rats with (+) and without (−) incubation with 50 μm dopamine and of endogenous Disc1 from DAT-OE mouse brain, stained with the huDISC1 specific mAB 14F2 (green) and rodent Disc1 specific polyclonal C-term Ab (red), detecting human transgenic and endogenous mouse DISC1, respectively. Primary neuron samples show a dopamine induction-dependent, HMW immunoreactive band above 200 kDa (upper arrow) for endogenous rat as well as transgenic human DISC1. Also endogenous mouse Disc1 shows HMW bands in the insoluble fraction. The actin control demonstrates equal protein content for the lysate input of primary neurons. All means±s.e.m.
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References

    1. Millar JK, Wilson-Annan JC, Anderson S, Christie S, Taylor MS, Semple CA et al. Disruption of two novel genes by a translocation co-segregating with schizophrenia. Hum Mol Genet 2000; 9: 1415–1423. - PubMed
    1. Blackwood DH, Fordyce A, Walker MT St, Clair DM, Porteous DJ, Muir WJ. Schizophrenia and affective disorders—cosegregation with a translocation at chromosome 1q42 that directly disrupts brain-expressed genes: clinical and P300 findings in a family. Am J Hum Genet 2001; 69: 428–433. - PMC - PubMed
    1. St Clair D, Blackwood D, Muir W, Carothers A, Walker M, Spowart G et al. Association within a family of a balanced autosomal translocation with major mental illness. Lancet 1990; 336: 13–16. - PubMed
    1. Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. The DISC locus in psychiatric illness. Mol Psychiatry 2008; 13: 36–64. - PubMed
    1. Bradshaw NJ, Porteous DJ. DISC1-binding proteins in neural development, signalling and schizophrenia. Neuropharmacology 2012; 62: 1230–1241. - PMC - PubMed

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