Aggregated proteins in schizophrenia and other chronic mental diseases: DISC1opathies

Chronic mental diseases (CMD) like the schizophrenias are progressive diseases of heterogenous but poorly understood biological origin. An imbalance in proteostasis is a hallmark of dysfunctional neurons, leading to impaired clearance and abnormal deposition of protein aggregates. Thus, it can be hypothesized that unbalanced proteostasis in such neurons may also lead to protein aggregates in schizophrenia. These protein aggregates, however, would be more subtle then in the classical neurodegenerative diseases and as such have not yet been detected. The DISC1 (Disrupted-in-schizophrenia 1) gene is considered among the most promising candidate genes for CMD having been identified as linked to CMD in a Scottish pedigree and having since been found to associate to various phenotypes of CMD. We have recently demonstrated increased insoluble DISC1 protein in the cingular cortex in approximately 20% of cases of CMD within the widely used Stanley Medical Research Institute Consortium Collection. Surprisingly, in vitro, DISC1 aggregates were cell-invasive, i.e., purified aggresomes or recombinant DISC1 fragments where internalized at an efficiency comparable to that of α-synuclein. Intracellular DISC1 aggresomes acquired gain-of-function properties in recruiting otherwise soluble proteins such as the candidate schizophrenia protein dysbindin. Disease-associated DISC1 polymorphism S704C led to a higher oligomerization tendency of DISC1. These findings justify classification of DISC1-dependent brain disorders as protein conformational disorders which we have tentatively termed DISC1opathies. The notion of disturbed proteostasis and protein aggregation as a mechanism of mental diseases is thus emerging. The yet unidentified form of neuronal impairment in CMD is more subtle than in the classical neurodegenerative diseases without leading to massive cell death and as such present a different kind of neuronal dysfunctionality, eventually confined to highly selective CNS subpopulations.     

Generation,Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer''s disease (Aβ, tau), Parkinson''s Disease (α-synuclein), Huntington''s disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease^{1, 2}, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia³. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C⁴, and that DISC1 aggresomes generated in vitro were cell-invasive⁵, similar to what had been shown for Aβ⁶, tau^7-9, α-synuclein¹⁰, polyglutamine¹¹, or SOD1 aggregates¹². These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders. Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.     

DISC1 and the aggresome

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

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.     

NMDA receptor regulates migration of newly generated neurons in the adult hippocampus via Disrupted-In-Schizophrenia 1 (DISC1)

In the mammalian brain, new neurons are continuously generated throughout life in the dentate gyrus (DG) of the hippocampus. Previous studies have established that newborn neurons migrate a short distance to be integrated into a pre-existing neuronal circuit in the hippocampus. How the migration of newborn neurons is governed by extracellular signals, however, has not been fully understood. Here, we report that NMDA receptor (NMDA-R)-mediated signaling is essential for the proper migration and positioning of newborn neurons in the DG. An intraperitoneal injection of the NMDA-R antagonists, memantine, or 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) into adult male mice caused the aberrant positioning of newborn neurons, resulting in the overextension of their migration in the DG. Interestingly, we revealed that the administration of NMDA-R antagonists leads to a decrease in the expression of Disrupted-In-Schizophrenia 1 (DISC1), a candidate susceptibility gene for major psychiatric disorders such as schizophrenia, which is also known as a critical regulator of neuronal migration in the DG. Furthermore, the overextended migration of newborn neurons induced by the NMDA-R antagonists was significantly rescued by exogenous expression of DISC1. Collectively, these results suggest that the NMDA-R signaling pathway governs the migration of newborn neurons via the regulation of DISC1 expression in the DG.     

Interaction between FEZ1 and DISC1 in regulation of neuronal development and risk for schizophrenia

Disrupted-in Schizophrenia 1 (DISC1), a susceptibility gene for major mental disorders, encodes a scaffold protein that has a multifaceted impact on neuronal development. How DISC1 regulates different aspects of neuronal development is not well understood. Here, we show that Fasciculation and Elongation Protein Zeta-1 (FEZ1) interacts with DISC1 to synergistically regulate dendritic growth of newborn neurons in the adult mouse hippocampus, and that this pathway complements a parallel DISC1-NDEL1 interaction that regulates cell positioning and morphogenesis of newborn neurons. Furthermore, genetic association analysis of two independent cohorts of schizophrenia patients and healthy controls reveals an epistatic interaction between FEZ1 and DISC1, but not between FEZ1 and NDEL1, for risk of schizophrenia. Our findings support a model in which DISC1 regulates distinct aspects of neuronal development through its interaction with different intracellular partners and such epistasis may contribute to increased risk for schizophrenia.     

Molecular characterization of disrupted in schizophrenia-1 risk variant S704C reveals the formation of altered oligomeric assembly

DISC1 (Disrupted in schizophrenia-1) plays essential roles in neuronal proliferation, neuronal migration and axon guidance and has been implicated in schizophrenia and related psychiatric disorders. DISC1 forms a functional complex with nuclear distribution element-like protein-1 (NDEL1), a key component that regulates microtubule organization during cell division and neuronal migration. DISC1 polymorphisms at the binding interface of DISC1-NDEL1 complex have been implicated in schizophrenia. However, it is unknown how schizophrenia risk polymorphisms perturb its interaction with NDEL1 and how they change the inherent biochemical properties of DISC1. Here, we characterize the oligomerization and binding property of DISC1 and its natural schizophrenia risk variant, S704C. Our results show that DISC1 forms octamers via dimers as building blocks and directly interacts with tetramers of NDEL1. The schizophrenia risk variant S704C affects the formation of octamers of DISC1 and exhibits higher-order self-oligomerization. However, the observed formation of new oligomeric species did not influence its binding with NDEL1. These results suggest that the improper oligomeric assembly of DISC1-S704C may underlie the observed phenotypic variation due to the polymorphism.     

DISC1 and the aggresome: a disruption to cellular function?

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

DISC1 genetics, biology and psychiatric illness

Psychiatric disorders are highly heritable, and in many individuals likely arise from the combined effects of genes and the environment. A substantial body of evidence points toward DISC1 being one of the genes that influence risk of schizophrenia, bipolar disorder and depression, and functional studies of DISC1 consequently have the potential to reveal much about the pathways that lead to major mental illness. Here, we review the evidence that DISC1 influences disease risk through effects upon multiple critical pathways in the developing and adult brain.     

Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age‐related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP‐fused firefly luciferase (Fluc‐EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc‐EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc‐EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington’s disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc‐EGFP reporter mice enable new insights into proteostasis alterations in different diseases.     

DISC1, PDE4B, and NDE1 at the centrosome and synapse

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.     

Endoplasmic reticulum proteostasis impairment in aging

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one‐third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.     

Induction of heat shock proteins in differentiated human and rodent neurons by celastrol

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed protein misfolding disorders that are characterized by the neuronal accumulation of protein aggregates. Manipulation of the cellular stress-response involving induction of heat shock proteins (Hsps) in differentiated neurons offers a therapeutic strategy to counter conformational changes in neuronal proteins that trigger pathogenic cascades resulting in neurodegenerative diseases. Hsps are protein repair agents that provide a line of defense against misfolded, aggregation-prone proteins. These proteins are not induced in differentiated neurons by conventional heat shock. We have found that celastrol, a quinine methide triterpene, induced expression of a wider set of Hsps, including Hsp70B', in differentiated human neurons grown in tissue culture compared to cultured rodent neuronal cells. Hence the beneficial effect of celastrol against human neurodegenerative diseases may exceed its potential in rodent models of these diseases.     

DISC1 localizes to the centrosome by binding to kendrin

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation that segregated with major mental disorders in a Scottish family. Using the yeast two-hybrid system, we screened a human brain cDNA library for interactors of the DISC1 protein. One of the positive clones encoded kendrin/pericentrin-B, a giant protein known to localize specifically to the centrosome. The interaction between DISC1 and kendrin in mammalian cells was demonstrated by an immunoprecipitation assay. Residues 446-533 of DISC1 were essential for the interaction with kendrin. Immunocytochemical analysis revealed the colocalization of DISC1 and kendrin to the centrosome. These data indicate that DISC1 localizes to the centrosome by binding to kendrin. Kendrin has been reported to anchor the gamma-tubulin complex to the centrosome, providing microtubule nucleation sites. The present study suggests the possible involvement of DISC1 in the pathophysiology of mental disorders due to its putative effect on centrosomal function.     

A functional link between Disrupted-In-Schizophrenia 1 and the eukaryotic translation initiation factor 3

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a candidate gene for schizophrenia. DISC1 is disrupted by a balanced t(1;11)(q42.1;q14.3) translocation segregating with schizophrenia and related psychiatric illness in a large Scottish family. Here, we show that DISC1 interacts via its globular domain with the p40 subunit of the eukaryotic translation initiation factor 3. Furthermore, we found that overexpression of DISC1 in SH-SY5Y cells induces the assembly of eIF3- and TIA-1-positive stress granules (SGs), discrete cytoplasmic granules formed in response to environmental stresses. Our findings suggest that DISC1 may function as a translational regulator and may be involved in stress response.     

Quality control compartments coming of age

Maintenance of proteome integrity (proteostasis) is essential for cellular and organismal survival. Various cellular mechanisms work to preserve proteostasis by ensuring correct protein maturation and efficient degradation of misfolded and damaged proteins. Despite this cellular effort, under certain circumstances subsets of aggregation-prone proteins escape the quality control surveillance, accumulate within the cell and form insoluble aggregates that can lead to the development of disorders including late-onset neurodegenerative diseases. Cells respond to the appearance of insoluble aggregates by actively transporting them to designated deposition sites where they often undergo degradation. Although several protein aggregate deposition sites have been described and extensively studied, key questions regarding their biological roles and how they are affected by aging remained unanswered. Here we review the recent advances in the field, describe the different subtypes of these cellular compartments and outline the evidence that these structures change their properties over time. Finally, we propose models to explain the possible mechanistic links between aggregate deposition sites, neurodegenerative disorders and the aging process.     

DISC1-kendrin interaction is involved in centrosomal microtubule network formation

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation segregating with schizophrenia, bipolar disorder and other major mental illnesses in a Scottish family. We previously identified 446-533 amino acids of DISC1 as the kendrin-binding region by means of a directed yeast two-hybrid interaction assay and showed that the DISC1-kendrin interaction is indispensable for the centrosomal localization of DISC1. In this study, to confirm the DISC1-kendrin interaction, we examined the interaction between deletion mutants of DISC1 and kendrin. Then, we demonstrated that the carboxy-terminus of DISC1 is indispensable for the interaction with kendrin. Furthermore, the immunocytochemistry revealed that the carboxy-terminus of DISC1 is also required for the centrosomal targeting of DISC1. Overexpression of the DISC1-binding region of kendrin or the DISC1 deletion mutant lacking the kendrin-binding region impairs the microtubule organization. These findings suggest that the DISC1-kendrin interaction plays a key role in the microtubule dynamics.     

Disrupted‐in‐schizophrenia 1 functional polymorphisms and D2/D3 receptor availability: A [11C]‐(+)‐PHNO imaging study

The disrupted‐in‐schizophrenia 1 (DISC1) protein has been implicated in a range of biological mechanisms underlying chronic mental disorders such as schizophrenia. Schizophrenia is associated with abnormal striatal dopamine signalling, and all antipsychotic drugs block striatal dopamine 2/3 receptors (D_2/3Rs). Importantly, the DISC1 protein directly interacts and forms a protein complex with the dopamine D₂ receptor (D₂R) that inhibits agonist‐induced D₂R internalisation. Moreover, animal studies have found large striatal increases in the proportion of D₂R receptors in a high affinity state (D₂^highR) in DISC1 rodent models. Here, we investigated the relationship between the three most common polymorphisms altering the amino‐acid sequence of the DISC1 protein (Ser704Cys (rs821616), Leu607Phe (rs6675281) and Arg264Gln (rs3738401)) and striatal D_2/3R availability in 41 healthy human volunteers, using [¹¹C]‐(+)‐PHNO positron emission tomography. We found no association between DISC1 polymorphisms and D_2/3R availability in the striatum and D₂R availability in the caudate and putamen. Therefore, despite a direct interaction between DISC1 and the D₂R, none of its main functional polymorphisms impact striatal D_2/3R binding potential, suggesting DISC1 variants act through other mechanisms.     

Neurobiology of schizophrenia

With its hallucinations, delusions, thought disorder, and cognitive deficits, schizophrenia affects the most basic human processes of perception, emotion, and judgment. Evidence increasingly suggests that schizophrenia is a subtle disorder of brain development and plasticity. Genetic studies are beginning to identify proteins of candidate genetic risk factors for schizophrenia, including dysbindin, neuregulin 1, DAOA, COMT, and DISC1, and neurobiological studies of the normal and variant forms of these genes are now well justified. We suggest that DISC1 may offer especially valuable insights. Mechanistic studies of the properties of these candidate genes and their protein products should clarify the molecular, cellular, and systems-level pathogenesis of schizophrenia. This can help redefine the schizophrenia phenotype and shed light on the relationship between schizophrenia and other major psychiatric disorders. Understanding these basic pathologic processes may yield novel targets for the development of more effective treatments.     

No association of Disrupted-in-Schizophrenia-1 variation with prefrontal function in patients with schizophrenia and bipolar disorder

The Disrupted-in-Schizophrenia-1 (DISC1) gene has been implicated in both schizophrenia and bipolar disorder by linkage and genetic association studies. Altered prefrontal cortical function is a pathophysiological feature of both disorders, and we have recently shown that variation in DISC1 modulates prefrontal activation in healthy volunteers. Our goal was to examine the influence of the DISC1 polymorphism Cys704Ser on prefrontal function in schizophrenia and bipolar disorder. From 2004 to 2008, patients with schizophrenia (N = 44), patients with bipolar disorder (N = 35) and healthy volunteers (N = 53) were studied using functional magnetic resonance imaging while performing a verbal fluency task. The effect of Cys704Ser on cortical activation was compared between groups as Cys704 carriers vs. Ser704 homozygotes. In contrast to the significant effect on prefrontal activation we had previously found in healthy subjects, no significant effect of Cys704Ser was detected in this or any other region in either the schizophrenia or bipolar groups. When controls were compared with patients with schizophrenia, there was a diagnosis by genotype interaction in the left middle/superior frontal gyrus [family-wise error (FWE) P = 0.002]. In this region, Ser704/ser704 controls activated more than Cys704 carriers, and there was a trend in the opposite direction in schizophrenia patients. In contrast to its effect in healthy subjects, variation in DISC1 Cys704Ser704 genotype was not associated with altered prefrontal activation in patients with schizophrenia or bipolar disorder. The absence of an effect in patients may reflect interactions of the effects of DISC1 genotype with the effects of other genes associated with these disorders, and/or with the effects of the disorders on brain function.