FBXW7 regulates DISC1 stability via the ubiquitin-proteosome system.

Disrupted in schizophrenia 1 (DISC1) is a multi-functional scaffolding protein that has been associated with neuropsychiatric disease. The role of DISC1 is to assemble protein complexes that promote neural development and signaling, hence tight control of the concentration of cellular DISC1 in neurons is vital to brain function. Using structural and biochemical techniques, we show for we believe the first time that not only is DISC1 turnover elicited by the ubiquitin proteasome system (UPS) but that it is orchestrated by the F-Box protein, FBXW7. We present the structure of FBXW7 bound to the DISC1 phosphodegron motif and exploit this information to prove that disruption of the FBXW7-DISC1 complex results in a stabilization of DISC1. This action can counteract DISC1 deficiencies observed in neural progenitor cells derived from induced pluripotent stem cells from schizophrenia patients with a DISC1 frameshift mutation. Thus manipulation of DISC1 levels via the UPS may provide a novel method to explore DISC1 function.

Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1.

Exploring drug targets based on disease-associated molecular mechanisms during development is crucial for the generation of novel prevention and treatment strategies for neurodevelopmental psychiatric conditions. We report that prefrontal cortex (PFC)-specific postnatal knockdown of DISC1 via in utero electroporation combined with an inducible knockdown expression system drives deficits in synaptic GABAA function and dendritic development in pyramidal neurons, as well as abnormalities in sensorimotor gating, albeit without profound memory deficits. We show for the first time that DISC1 is specifically involved in regulating cell surface expression of α2 subunit-containing GABAA receptors in immature developing neurons, but not after full maturation. Notably, pharmacological intervention with α2/3 subtype-selective GABAA receptor positive allosteric modulators during the early postnatal period ameliorates dendritic deficits and behavioral abnormalities induced by knockdown of DISC1. These findings highlight a critical role of DISC1-mediated disruption of postnatal GABA signaling in aberrant PFC maturation and function.

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.

DISC1 regulates trafficking and processing of APP and Aß generation.

We report the novel regulation of proteolytic processing of amyloid precursor protein (APP) by DISC1, a major risk factor for psychiatric illnesses, such as depression and schizophrenia. RNAi knockdown of DISC1 in mature primary cortical neurons led to a significant increase in the levels of intracellular α-C-terminal fragment of APP (APP-CTFα) and the corresponding N-terminal-secreted ectodomain product sAPPα. DISC1 knockdown also elicited a significant decrease in the levels of amyloid beta (Aß)42 and Aß40. These aberrant proteolytic events were successfully rescued by co-expression of wild-type DISC1, but not by mutant DISC1 lacking the amino acids required for the interaction with APP, suggesting that APP-DISC1 protein interactions are crucial for the regulation of the C-terminal proteolysis. In a genetically engineered model in which a major full-length DISC1 isoform is depleted, consistent changes in APP processing were seen: an increase in APP-CTFα and decrease in Aß42 and Aß40 levels. Finally, we found that knockdown of DISC1 increased the expression of APP at the cell surface and decreased its internalization. The presented DISC1 mechanism of APP proteolytic processing and Aß peptide generation, which is central to Alzheimer's disease pathology, suggests a novel interface between neurological and psychiatric conditions.

Transcriptional regulation of neurodevelopmental and metabolic pathways by NPAS3.

The basic helix-loop-helix PAS (Per, Arnt, Sim) domain transcription factor gene NPAS3 is a replicated genetic risk factor for psychiatric disorders. A knockout (KO) mouse model exhibits behavioral and adult neurogenesis deficits consistent with human illness. To define the location and mechanism of NPAS3 etiopathology, we combined immunofluorescent, transcriptomic and metabonomic approaches. Intense Npas3 immunoreactivity was observed in the hippocampal subgranular zone-the site of adult neurogenesis--but was restricted to maturing, rather than proliferating, neuronal precursor cells. Microarray analysis of a HEK293 cell line over-expressing NPAS3 showed that transcriptional targets varied according to circadian rhythm context and C-terminal deletion. The most highly up-regulated NPAS3 target gene, VGF, encodes secretory peptides with established roles in neurogenesis, depression and schizophrenia. VGF was just one of many NPAS3 target genes also regulated by the SOX family of transcription factors, suggesting an overlap in neurodevelopmental function. The parallel repression of multiple glycolysis genes by NPAS3 reveals a second role in the regulation of glucose metabolism. Comparison of wild-type and Npas3 KO metabolite composition using high-resolution mass spectrometry confirmed these transcriptional findings. KO brain tissue contained significantly altered levels of NAD(+), glycolysis metabolites (such as dihydroxyacetone phosphate and fructose-1,6-bisphosphate), pentose phosphate pathway components and Kreb's cycle intermediates (succinate and α-ketoglutarate). The dual neurodevelopmental and metabolic aspects of NPAS3 activity described here increase our understanding of mental illness etiology, and may provide a mechanism for innate and medication-induced susceptibility to diabetes commonly reported in psychiatric patients.

The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function.

Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein-protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1-TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1-TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases.

Expression of D-serine and glycine transporters in the prefrontal cortex and cerebellum in schizophrenia.

The NMDA receptor co-agonists D-serine and glycine are thought to contribute to glutamatergic dysfunction in schizophrenia. They are removed from the synapse by specific neuronal and glial transporters, the status of which is clearly relevant to theories of D-serine and glycine function in the disorder. D-serine is primarily transported by Asc-1, and glycine by GlyT1 but maybe also by SNAT2. As a first step to addressing this issue, we studied Asc-1, GlyT1 and SNAT2 expression in dorsolateral prefrontal cortex and cerebellum of 18 subjects with schizophrenia and 20 controls, using immunoblotting and in situ hybridization. Asc-1 protein and SNAT2 mRNA were decreased in schizophrenia in both regions. GlyT1 mRNA and protein, and Asc-1 mRNA, were not altered. Antipsychotic administration for 14 days did not alter expression of the genes in rat brain. Unchanged GlyT1 suggests that glycine transport is not markedly affected in schizophrenia, and therefore that increased synaptic removal is not the basis for the putative deficit in glycine modulation of NMDA receptors in the disorder. Lowered Asc-1 in schizophrenia implies that D-serine reuptake is reduced, perhaps as a response to decreased synaptic D-serine availability. However, this interpretation remains speculative. Further investigations will be valuable in the evaluation of these transporters as potential therapeutic targets in psychosis.

The impact of Disrupted-in-Schizophrenia 1 (DISC1) on the dopaminergic system: a systematic review.

Disrupted-in-Schizophrenia 1 (DISC1) is a gene known as a risk factor for mental illnesses possibly associated with dopamine impairments. DISC1 is a scaffold protein interacting with proteins involved in the dopamine system. Here we summarise the impact of DISC1 disruption on the dopamine system in animal models, considering its effects on presynaptic dopaminergic function (tyrosine hydroxylase levels, dopamine transporter levels, dopamine levels at baseline and after amphetamine administration) and postsynaptic dopaminergic function (dopamine D1 and D2 receptor levels, dopamine receptor-binding potential and locomotor activity after amphetamine administration). Our findings show that many but not all DISC1 models display (1) increased locomotion after amphetamine administration, (2) increased dopamine levels after amphetamine administration in the nucleus accumbens, and (3) inconsistent basal dopamine levels, dopamine receptor levels and binding potentials. There is also limited evidence for decreased tyrosine hydroxylase levels in the frontal cortex and increased dopamine transporter levels in the striatum but not nucleus accumbens, but these conclusions warrant further replication. The main dopaminergic findings are seen across different DISC1 models, providing convergent evidence that DISC1 has a role in regulating dopaminergic function. These results implicate dopaminergic dysregulation as a mechanism underlying the increased rate of schizophrenia seen in DISC1 variant carriers, and provide insights into how DISC1, and potentially DISC1-interacting proteins such as AKT and GSK-3, could be used as novel therapeutic targets for schizophrenia.

Altered expression of histamine signaling genes in autism spectrum disorder.

The histaminergic system (HS) has a critical role in cognition, sleep and other behaviors. Although not well studied in autism spectrum disorder (ASD), the HS is implicated in many neurological disorders, some of which share comorbidity with ASD, including Tourette syndrome (TS). Preliminary studies suggest that antagonism of histamine receptors 1-3 reduces symptoms and specific behaviors in ASD patients and relevant animal models. In addition, the HS mediates neuroinflammation, which may be heightened in ASD. Together, this suggests that the HS may also be altered in ASD. Using RNA sequencing (RNA-seq), we investigated genome-wide expression, as well as a focused gene set analysis of key HS genes (HDC, HNMT, HRH1, HRH2, HRH3 and HRH4) in postmortem dorsolateral prefrontal cortex (DLPFC) initially in 13 subjects with ASD and 39 matched controls. At the genome level, eight transcripts were differentially expressed (false discovery rate <0.05), six of which were small nucleolar RNAs (snoRNAs). There was no significant diagnosis effect on any of the individual HS genes but expression of the gene set of HNMT, HRH1, HRH2 and HRH3 was significantly altered. Curated HS gene sets were also significantly differentially expressed. Differential expression analysis of these gene sets in an independent RNA-seq ASD data set from DLPFC of 47 additional subjects confirmed these findings. Understanding the physiological relevance of an altered HS may suggest new therapeutic options for the treatment of ASD.

Subunit-specific association of protein kinase C and the receptor for activated C kinase with GABA type A receptors.

GABA receptors (GABA(A)) are the major sites of fast synaptic inhibition in the brain and can be assembled from five subunit classes: alpha, beta, gamma, delta, and epsilon. Receptor function can be regulated by direct phosphorylation of beta and gamma2 subunits, but how kinases are targeted to GABA(A) receptors is unknown. Here we show that protein kinase C-betaII (PKC-betaII) is capable of directly binding to the intracellular domain of the receptor beta1 and beta3 subunits, but not to those of the alpha1 or gamma2 subunits. Moreover, associating PKC-betaII is capable of specifically phosphorylating serine 409 in beta1 subunit and serines 408/409 within the beta3 subunit, key residues for modulating GABA(A) receptor function. The receptor for activated C kinase (RACK-1) was found also to bind to the beta1 subunit intracellular domain, but PKC binding appeared to be independent of this protein. Using immunoprecipitation, the association of PKC isoforms and RACK-1 with neuronal GABA(A) receptors was seen. Furthermore, PKC isoforms associating with neuronal receptors were capable of phosphorylating the receptor beta3 subunit. Together, these observations suggest GABA(A) receptors are intimately associated with PKC isoforms via a direct interaction with receptor beta subunits. This interaction may serve to localize PKC activity to GABA(A) receptors in neurons allowing the rapid regulation of receptor activity by cell-signaling pathways that modify PKC activity.

Receptor cross talk: ligand-gated ion channels start to communicate.

Evidence is mounting for a direct mode of interaction between neurotransmitter receptors that modulates the activity of the interacting proteins. Brandon and Moss discuss two examples in which direct interactions between neurotransmitter receptors appear to regulate the receptors independently of the signal transduction cascades they activate. This type of interaction has important implications for rapid modulation of receptor function and the integration of multiple signals at a synapse.

Altered functional brain network connectivity and glutamate system function in transgenic mice expressing truncated Disrupted-in-Schizophrenia 1.

Considerable evidence implicates DISC1 as a susceptibility gene for multiple psychiatric diseases. DISC1 has been intensively studied at the molecular, cellular and behavioral level, but its role in regulating brain connectivity and brain network function remains unknown. Here, we utilize a set of complementary approaches to assess the functional brain network abnormalities present in mice expressing a truncated Disc1 gene (Disc1tr Hemi mice). Disc1tr Hemi mice exhibited hypometabolism in the prefrontal cortex (PFC) and reticular thalamus along with a reorganization of functional brain network connectivity that included compromised hippocampal-PFC connectivity. Altered hippocampal-PFC connectivity in Disc1tr Hemi mice was confirmed by electrophysiological analysis, with Disc1tr Hemi mice showing a reduced probability of presynaptic neurotransmitter release in the monosynaptic glutamatergic hippocampal CA1-PFC projection. Glutamate system dysfunction in Disc1tr Hemi mice was further supported by the attenuated cerebral metabolic response to the NMDA receptor (NMDAR) antagonist ketamine and decreased hippocampal expression of NMDAR subunits 2A and 2B in these animals. These data show that the Disc1 truncation in Disc1tr Hemi mice induces a range of translationally relevant endophenotypes underpinned by glutamate system dysfunction and altered brain connectivity.

Constitutive tyrosine phosphorylation of the GABA(A) receptor gamma 2 subunit in rat brain.

GABA(A) receptors are the major sites of fast synaptic inhibition in the brain, where they are predominantly composed of alpha, beta and gamma2 subunits. A role for direct tyrosine phosphorylation of residues 365 and 367 (Y365/367) within the intracellular domain of the gamma2 subunit has been suggested to be important in modulating GABA(A) receptor function, based on the study of recombinant receptors. To address the relevance of these observations for neuronal GABA(A) receptors we have studied the phosphorylation of the gamma2 subunit in the brain. In adult rat brain the gamma2 subunit is phosphorylated on tyrosine residues, including Y365/367 as defined using a phosphospecific antisera. In cultured cortical neurones, phosphorylation of Y365/367 is highly regulated and was only evident upon inhibition of tyrosine phosphatases. We also establish that the tyrosine kinase Src is capable of specifically interacting with the intracellular domains of receptor beta and gamma2 subunits. This may specifically localise tyrosine kinase activity to GABA(A) receptors, facilitating rapid receptor tyrosine phosphorylation upon kinase activation. Together our results suggests that tyrosine phosphorylation of the gamma2 subunit, possibly by closely associated Src, may be a dynamic mechanism for regulating GABA(A) receptor function in the brain.

Evidence for sustained elevation of IL-6 in the CNS as a key contributor of depressive-like phenotypes.

There is compelling clinical literature implicating a role for cytokines in the pathophysiology of major depressive disorder (MDD). Interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) are pleiotropic inflammatory cytokines that have been reported to be elevated in patients with MDD. The present studies were undertaken to investigate the relationship between IL-6 and IL-1ß in animal models of depressive-like behavior. Analysis of brain tissue homogenates in the cortex of rats subjected to chronic stress paradigms revealed elevated levels of IL-6 protein in the absence of elevations in IL-1ß. Central administration of recombinant mouse IL-6 produced depressive-like phenotypes in mice, which were not accompanied by IL-1ß-induced increases in the brain tissue or IL-1ß-related sickness behavior typical of a general central nervous system inflammatory response. Systemic administration of fluoxetine in the presence of centrally administered IL-6 failed to produce the expected antidepressant-like response in mice relative to sham-infused controls. Further, administration of fluoxetine to mice with endogenous overexpression of brain IL-6 (MRL/MpJ-Fas(LPR/LPR) (LPR mice)) failed to produce the expected antidepressant-like effect relative to fluoxetine-treated control mice (MRL/MpJ(+/+)). Interestingly, blockade of IL-6 trans-signaling by coadministration of a gp130/Fc monomer or an anti-mouse IL-6 antibody with IL-6 prevented the IL-6-induced increases in immobility time as well as attenuated IL-6-induced increases of protein in the cortex. Taken together, these data indicate that elevations in IL-6 may have a pathophysiological role underlying depression and more specifically resistance to current classes of antidepressant medications and suggest that modulation of the IL-6 signaling pathway may have therapeutic potential for treatment-resistant depression.

Dissecting DISC1 function through protein-protein interactions.

Disrupted in schizophrenia 1 (DISC1) is emerging in the eyes of many as the most promising candidate of all the schizophrenia risk genes. This viewpoint is derived from the combination of genetic, clinical, imaging and rapidly advancing cell biology data around this gene. All of these areas have been reviewed extensively recently and this review will point you towards some of these excellent papers. My own personal view of the potential importance of DISC1 was echoed in a recent review which suggested that DISC1 may be a 'Rosetta Stone' for schizophrenia research [Ross, Margolis, Reading, Pletnikov and Coyle (2006) Neuron 52, 139-153]. Our own efforts to try to understand the function of DISC1 were through identification of its protein-binding partners. Through an extensive Y2H (yeast two-hybrid) and bioinformatics effort we generated the 'DISC1-Interactome', a comprehensive network of protein-protein interactions around DISC1. In two excellent industry-academia collaborations we focused on two main interacting partners: Ndel1 (nudE nuclear distribution gene E homologue-like 1), an enigmatic protein which may have diverse functions as both a cysteine protease and a key centrosomal structural protein; and PDE4B, a cAMP-specific phosphodiesterase. I will review the work around these two protein complexes in detail.

Disrupted in Schizophrenia 1 Interactome: evidence for the close connectivity of risk genes and a potential synaptic basis for schizophrenia.

Disrupted in Schizophrenia 1 (DISC1) is a schizophrenia risk gene associated with cognitive deficits in both schizophrenics and the normal ageing population. In this study, we have generated a network of protein-protein interactions (PPIs) around DISC1. This has been achieved by utilising iterative yeast-two hybrid (Y2H) screens, combined with detailed pathway and functional analysis. This so-called 'DISC1 interactome' contains many novel PPIs and provides a molecular framework to explore the function of DISC1. The network implicates DISC1 in processes of cytoskeletal stability and organisation, intracellular transport and cell-cycle/division. In particular, DISC1 looks to have a PPI profile consistent with that of an essential synaptic protein, which fits well with the underlying molecular pathology observed at the synaptic level and the cognitive deficits seen behaviourally in schizophrenics. Utilising a similar approach with dysbindin (DTNBP1), a second schizophrenia risk gene, we show that dysbindin and DISC1 share common PPIs suggesting they may affect common biological processes and that the function of schizophrenia risk genes may converge.

Behavioral and biochemical characterization of a mutant mouse strain lacking D-amino acid oxidase activity and its implications for schizophrenia.

D-amino acid oxidase (DAO) degrades D-serine, a co-agonist at the NMDA receptor (NMDAR). Hypofunction of the NMDAR has been suggested to contribute to the pathophysiology of schizophrenia. Intriguingly, DAO has been recently identified as a risk factor for schizophrenia through genetic association studies. A naturally occurring mouse strain (ddY/DAO-) has been identified which lacks DAO activity. We have characterized this strain both behaviorally and biochemically to evaluate DAO as a target for schizophrenia. We have confirmed that this strain lacks DAO activity and shown for the first time it has increased occupancy of the NMDAR glycine site due to elevated extracellular D-serine levels and has enhanced NMDAR function in vivo. Furthermore, the ddY/DAO- strain displays behaviors which suggest that it will be a useful tool for evaluation of the clinical benefit of DAO inhibition in schizophrenia.