Svenja V. Trossbach

The functional role of the second NPXY motif of the LRP1 beta-chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors.

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-d-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.

Oligomer Assembly of the C-Terminal DISC1 Domain (640−854) Is Controlled by Self-Association Motifs and Disease-Associated Polymorphism S704C

Genetic studies have established a role of disrupted-in-schizophrenia-1 (DISC1) in chronic mental diseases (CMD). Limited experimental data are available on the domain structure of the DISC1 protein although multiple interaction partners are known including a self-association domain within the middle part of DISC1 (residues 403-504). The DISC1 C-terminal domain is deleted in the original Scottish pedigree where DISC1 harbors two coiled-coil domains and disease-associated polymorphisms at 607 and 704, as well as the important nuclear distribution element-like 1 (NDEL1) binding site at residues 802-839. Here, we performed mutagenesis studies of the C-terminal domain of the DISC1 protein (residues 640-854) and analyzed the expressed constructs by biochemical and biophysical methods. We identified novel DISC1 self-association motifs and the necessity of their concerted action for orderly assembly: the region 765-854 comprising a coiled-coil domain is a dimerization domain and the region 668-747 an oligomerization domain; dimerization was found to be a prerequisite for orderly assembly of oligomers. Consistent with this, disease-associated polymorphism C704 displayed a slightly higher oligomerization propensity. The heterogeneity of DISC1 multimers in vitro was confirmed with a monoclonal antibody binding exclusively to HMW multimers. We also identified C-terminal DISC1 fragments in human brains, suggesting that C-terminal fragments could carry out DISC1-dependent functions. When the DISC1 C-terminal domain was transiently expressed in cells, it assembled into a range of soluble and insoluble multimers with distinct fractions selectively binding NDEL1, indicating functionality. Our results suggest that assembly of the C-terminal domain is controlled by distinct domains including the disease-associated polymorphism 704 and is functional in vivo.

Convergence of Two Independent Mental Disease Genes on the Protein Level: Recruitment of Dysbindin to Cell-Invasive Disrupted-In-Schizophrenia 1 Aggresomes

BACKGROUND Both disrupted-in-schizophrenia 1 (DISC1) and dysbindin have been identified as schizophrenia candidate genes in independent genetic linkage studies. The proteins have been assigned distinct subcellular locations and functions. We investigated whether both proteins converge into a common pathway specific for schizophrenia or mental diseases. METHODS DISC1 and dysbindin were expressed as recombinant proteins with or without a fluorescent protein-tag in human or mouse neuroblastoma cells and as recombinant proteins in E. coli. Postmortem brains of patients with mental diseases from the Stanley Research Medical Institutes Consortium Collection were used to demonstrate molecular interactions in biochemically purified protein fractions. RESULTS First, upon overexpression in neuroblastoma cells, DISC1 formed aggresomes that recruited homologous soluble C-terminal DISC1 fragment or heterologous dysbindin. Domains involved in binding could be mapped to DISC1 (316-597) and dysbindin (82-173), indicating a specific interaction. In addition, recruitment was demonstrated when externally added, purified DISC1 aggresomes penetrated recipient cells after coincubation. Second, a direct interaction between soluble DISC1 protein and dysbindin was demonstrated in a cell free system using E. coli-expressed proteins. Third, co-aggregation of DISC1 and dysbindin was demonstrated in postmortem brains for a subgroup of cases with chronic mental disease but not healthy control subjects. CONCLUSIONS A direct interaction of soluble and insoluble DISC1 protein with dysbindin protein demonstrates convergence of so far considered independent mental disease genes by direct molecular interaction. Our findings highlight protein aggregation and recruitment as a biological mechanism in mental disease.

DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites

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.

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

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.

Intranasal dopamine treatment reinstates object-place memory in aged rats

Following oral or IV administration, dopamine (DA) cannot cross the blood-brain barrier to a significant extent, but can enter the brain when administered via the nasal passages. Intranasal administration of DA was shown to increase extracellular DA in the striatum, to have antidepressant action and to improve attention and working memory in rats. Here we show that aged (22-24 months old) rats are deficient in an object-place learning task, but that this learning/memory is intact and comparable with that of adult rats upon pre-trial administration of 0.3 mg/kg DA gel into the nasal passages. This result raises the possibility of the therapeutic application of intranasal DA treatment for age-related cognitive disorders.

Peripheral DISC1 protein levels as a trait marker for schizophrenia and modulating effects of nicotine.

The Disrupted-in-Schizophrenia 1 (DISC1) protein plays a key role in behavioral control and vulnerability for mental illnesses, including schizophrenia. In this study we asked whether peripheral DISC1 protein levels in lymphocytes of patients diagnosed with schizophrenia can serve as a trait marker for the disease. Since a prominent comorbidity of schizophrenia patients is nicotine abuse or addiction, we also examined modulation of lymphocyte DISC1 protein levels in smokers, as well as the relationship between nicotine and DISC1 solubility status. We show decreased DISC1 levels in patients diagnosed with schizophrenia independent of smoking, indicating its potential use as a trait marker of this disease. In addition, lymphocytic DISC1 protein levels were decreased in smoking, mentally healthy individuals but not to the degree of overriding the trait level. Since DISC1 protein has been reported to exist in different solubility states in the brain, we also investigated DISC1 protein solubility in brains of rats treated with nicotine. Sub-chronic treatment with progressively increasing doses of nicotine from 0.25mg/kg to 1mg/kg for 15 days led to a decrease of insoluble DISC1 in the medial prefrontal cortex. Our results demonstrate that DISC1 protein levels in human lymphocytes are correlated with the diagnosis of schizophrenia independent of smoking and thus present a potential biomarker. Reduced DISC1 protein levels in lymphocytes of healthy individuals exposed to nicotine suggest that peripheral DISC1 could have potential for monitoring the effects of psychoactive substances.

Simultaneous effects on parvalbumin-positive interneuron and dopaminergic system development in a transgenic rat model for sporadic schizophrenia.

To date, unequivocal neuroanatomical features have been demonstrated neither for sporadic nor for familial schizophrenia. Here, we investigated the neuroanatomical changes in a transgenic rat model for a subset of sporadic chronic mental illness (CMI), which modestly overexpresses human full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1), and for which aberrant dopamine homeostasis consistent with some schizophrenia phenotypes has previously been reported. Neuroanatomical analysis revealed a reduced density of dopaminergic neurons in the substantia nigra and reduced dopaminergic fibres in the striatum. Parvalbumin-positive interneuron occurrence in the somatosensory cortex was shifted from layers II/III to V/VI, and the number of calbindin-positive interneurons was slightly decreased. Reduced corpus callosum thickness confirmed trend-level observations from in vivo MRI and voxel-wise tensor based morphometry. These neuroanatomical changes help explain functional phenotypes of this animal model, some of which resemble changes observed in human schizophrenia post mortem brain tissues. Our findings also demonstrate how a single molecular factor, DISC1 overexpression or misassembly, can account for a variety of seemingly unrelated morphological phenotypes and thus provides a possible unifying explanation for similar findings observed in sporadic schizophrenia patients. Our anatomical investigation of a defined model for sporadic mental illness enables a clearer definition of neuroanatomical changes associated with subsets of human sporadic schizophrenia.

Neuropeptide precursor VGF is genetically associated with social anhedonia and underrepresented in the brain of major mental illness: its downregulation by DISC1

In a large Scottish pedigree, disruption of the gene coding for DISC1 clearly segregates with major depression, schizophrenia and related mental conditions. Thus, study of DISC1 may provide a clue to understand the biology of major mental illness. A neuropeptide precursor VGF has potent antidepressant effects and has been reportedly associated with bipolar disorder. Here we show that DISC1 knockdown leads to a reduction of VGF, in neurons. VGF is also downregulated in the cortices from sporadic cases with major mental disease. A positive correlation of VGF single-nucleotide polymorphisms (SNPs) with social anhedonia was also observed. We now propose that VGF participates in a common pathophysiology of major mental disease.

Intra-nasal dopamine alleviates cognitive deficits in tgDISC1 rats which overexpress the human DISC1 gene.

HIGHLIGHTSRats with overexpressed human DISC1 gene show object attention deficits.Rats with overexpressed human DISC1 gene show deficits in long‐term object memory.Intranasal dopamine can recover these cognitive deficits.DISC1 overexpression causes changes in monoamines and acetylcholine systems. ABSTRACT The Disrupted‐in‐Schizophrenia 1 (DISC1) gene has been associated with mental illnesses such as major depression and schizophrenia. The transgenic DISC1 (tgDISC1) rat, which overexpresses the human DISC1 gene, is known to exhibit deficient dopamine (DA) homeostasis. To ascertain whether the DISC1 gene also impacts cognitive functions, 14–15 months old male tgDISC1 rats and wild‐type controls were subjected to the novel object preference (NOP) test and the object‐based attention test (OBAT) in order to assess short‐term memory (1h), long‐term memory (24h), and attention. Results: The tgDISC1 group exhibited intact short‐term memory, but deficient long‐term‐memory in the NOP test and deficient attention‐related behavior in the OBAT. In a different group of tgDISC1 rats, 3mg/kg intranasally applied dopamine (IN‐DA) or its vehicle was applied prior to the NOP or the OBAT test. IN‐DA reversed cognitive deficits in both the NOP and OBAT tests. In a further cohort of tgDISC1 rats, post‐mortem levels of DA, noradrenaline, serotonin and acetylcholine were determined in a variety of brain regions. The tgDISC1 group had less DA in the neostriatum, hippocampus and amygdala, less acetylcholine in neostriatum, nucleus accumbens, hippocampus, and amygdala, more serotonin in the nucleus accumbens, and less serotonin and noradrenaline in the amygdala. Conclusions: Our findings show that DISC1 overexpression and misassembly is associated with deficits in long‐term memory and attention‐related behavior. Since behavioral impairments in tgDISC1 rats were reversed by IN‐DA, DA deficiency may be a major cause for the behavioral deficits expressed in this model.