Alterations of ubiquitin related proteins in the pathology and development of schizophrenia: Evidence from human and animal studies.

Gene expression analyses in post-mortem schizophrenia brains suggest that a number of ubiquitin proteasome system (UPS) genes are associated with schizophrenia; however the status of UPS proteins in the schizophrenia brain is largely unknown. Ubiquitin related proteins are inherently involved in memory, neuronal survival and morphology, which are processes implicated in neurodevelopmental disorders such as schizophrenia. We examined levels of five UPS proteins (Protein Inhibitor of Activated STAT2 [PIAS2], F-Box and Leucine rich repeat protein 21 [FBXL21], Mouse Double Minute 2 homolog [MDM2], Ubiquitin Carboxyl-Terminal Hydrolase-L1 [UCHL1] and Ubiquitin Conjugating Enzyme E2D1 [UBE2D1]) involved in these neuronal processes, within the dorsolateral prefrontal cortex of post-mortem schizophrenia subjects and matched controls (n = 30/group), in addition to across neurodevelopmental time-points (juvenile, adolescent and adult stages of life), utilizing a well-established neurodevelopmental phencyclidine (PCP) animal model of schizophrenia. We observed significant reductions in PIAS2, FBXL21 and MDM2 in schizophrenia subjects compared to controls (p-values ranging from 0.002 to 0.004). In our developmental PCP model, MDM2 protein was significantly reduced in adult PCP-treated rats compared to controls (p = 0.034). Additionally, FBXL21 (p = 0.022) and UCHL1 (p = 0.022) were significantly decreased, whilst UBE2D1 was increased (p = 0.022), in juvenile phencyclidine-treated rats compared to controls. This is the first study reporting alterations of UPS proteins in post-mortem human schizophrenia subjects and in a neurodevelopmental model of schizophrenia. The findings from this study provide strong support for a role of these UPS proteins in the pathology and development of schizophrenia.

Shifting towards a model of mGluR5 dysregulation in schizophrenia: Consequences for future schizophrenia treatment.

Metabotropic glutamate receptor subtype 5 (mGluR5), encoded by the GRM5 gene, represents a compelling novel drug target for the treatment of schizophrenia. mGluR5 is a postsynaptic G-protein coupled glutamate receptor strongly linked with several critical cellular processes that are reported to be disrupted in schizophrenia. Accordingly, mGluR5 positive allosteric modulators show encouraging therapeutic potential in preclinical schizophrenia models, particularly for the treatment of cognitive dysfunctions against which currently available therapeutics are largely ineffective. More work is required to support the progression of mGluR5-targeting drugs into the clinic for schizophrenia treatment, although some obstacles may be overcome by comprehensively understanding how mGluR5 itself is involved in the neurobiology of the disorder. Several processes that are necessary for the regulation of mGluR5 activity have been identified, but not examined, in the context of schizophrenia. These processes include protein-protein interactions, dimerisation, subcellular trafficking, the impact of genetic variability or mutations on protein function, as well as epigenetic, post-transcriptional and post-translational processes. It is essential to understand these aspects of mGluR5 to determine whether they are affected in schizophrenia pathology, and to assess the consequences of mGluR5 dysfunction for the future use of mGluR5-based drugs. Here, we summarise the known processes that regulate mGluR5 and those that have already been studied in schizophrenia, and discuss the consequences of this dysregulation for current mGluR5 pharmacological strategies. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Molecular evidence of synaptic pathology in the CA1 region in schizophrenia.

Alterations of postsynaptic density (PSD)95-complex proteins in schizophrenia ostensibly induce deficits in synaptic plasticity, the molecular process underlying cognitive functions. Although some PSD95-complex proteins have been previously examined in the hippocampus in schizophrenia, the status of other equally important molecules is unclear. This is especially true in the cornu ammonis (CA)1 hippocampal subfield, a region that is critically involved in the pathophysiology of the illness. We thus performed a quantitative immunoblot experiment to examine PSD95 and several of its associated proteins in the CA1 region, using post mortem brain samples derived from schizophrenia subjects with age-, sex-, and post mortem interval-matched controls (n=20/group). Our results indicate a substantial reduction in PSD95 protein expression (-61.8%). Further analysis showed additional alterations to the scaffold protein Homer1 (Homer1a: +42.9%, Homer1b/c: -24.6%), with a twofold reduction in the ratio of Homer1b/c:Homer1a isoforms (P=0.011). Metabotropic glutamate receptor 1 (mGluR1) protein levels were significantly reduced (-32.7%), and Preso, a protein that supports interactions between Homer1 or PSD95 with mGluR1, was elevated (+83.3%). Significant reduction in synaptophysin (-27.8%) was also detected, which is a validated marker of synaptic density. These findings support the presence of extensive molecular abnormalities to PSD95 and several of its associated proteins in the CA1 region in schizophrenia, offering a small but significant step toward understanding how proteins in the PSD are altered in the schizophrenia brain, and their relevance to overall hippocampal and cognitive dysfunction in the illness.

Possibility of a sex-specific role for a genetic variant in FRMPD4 in schizophrenia, but not cognitive function.

The neurotransmitter disturbances responsible for cognitive dysfunction in schizophrenia are hypothesized to originate with alterations in postsynaptic scaffold proteins. We have recently reported that protein levels of FRMPD4, a multiscaffolding protein that modulates both Homer1 and postsynaptic density protein 95 activity, is altered in the schizophrenia postmortem brain, in regions involved in cognition. Here, we set out to investigate whether genetic variation in FRMPD4 is associated with cognitive function in people with schizophrenia. We selected and examined a novel single nucleotide polymorphism, rs5979717 (positioned in the noncoding 3' untranslated region of FRMPD4 and potentially influencing protein expression), for its association with schizophrenia and nine measures of cognitive function, using age-matched and sex-matched samples from 268 schizophrenia cases and 268 healthy controls. Brain samples from 20 schizophrenia patients and 20 healthy controls were additionally genotyped to study the influence of this variant on protein expression of FRMPD4. Allelic distribution of rs5979717 was associated with schizophrenia in females (χ=4.52, P=0.030). No effects of rs5979717 were observed on cognitive performance, nor an influence of rs5979717 genotypes on the expression of FRMPD4 proteins in postmortem brain samples. These data provide initial support for a sex-specific role for common variation in rs5979717 in schizophrenia, which now warrants further investigation.

Genetic variants in Nogo receptor signaling pathways may be associated with early life adversity in schizophrenia susceptibility.

BACKGROUND: Schizophrenia is a severe neuropsychiatric disorder thought to result from abnormal brain development. Nogo, an oligodendrocyte bound molecule, signals by binding to the Nogo receptor (NgR) located on axonal membranes. The NgR co-receptors include p75 neurotrophin receptor or TNF receptor orphan Y (TROY). Nogo signaling is responsible for central nervous system myelin regulation and neurite outgrowth during neurodevelopment, and plasticity in the mature brain. METHODS: We examined single nucleotide polymorphisms (SNPs) in NgR, p75, and TROY receptor genes and downstream signaling partner With No Lysine (K) (WNK1) and Myelin transcription factor 1-like (Myt1l) genes in an Australian case-control schizophrenia cohort (n = 268/group). High-throughput SNP genotyping was performed using the MassARRAY® genotyping assay. RESULTS: Analysis revealed a significant association between the Myt1l SNP rs2304008 and female schizophrenia subjects. The WNK1 SNP rs1468326 and the Myt1l SNP rs3748988 showed significant associations with schizophrenia in subjects with a maternal mental history and in subjects who experienced childhood trauma respectively. Following Bonferroni correction, all significance was lost. CONCLUSIONS: Despite the lack of positive findings in our population after correction for multiple testing, previous gene expression and association studies in schizophrenia suggest the implication of NgR signaling pathway genes in the etiology of schizophrenia remains topical and timely. GENERAL SIGNIFICANCE: Further investigations will be necessary to fully assess the role of these genes in the pathophysiology of schizophrenia. However these genes may prove useful in further understanding the mechanism by which negative experiences early in life can affect myelin-related processes in the context of schizophrenia.

A decade from discovery to therapy: Lingo-1, the dark horse in neurological and psychiatric disorders.

Leucine-rich repeat and immunoglobulin domain-containing protein (Lingo-1) is a potent negative regulator of neuron and oligodendrocyte survival, neurite extension, axon regeneration, oligodendrocyte differentiation, axonal myelination and functional recovery; all processes highly implicated in numerous brain-related functions. Although playing a major role in developmental brain functions, the potential application of Lingo-1 as a therapeutic target for the treatment of neurological disorders has so far been under-estimated. A number of preclinical studies have shown that various methods of antagonizing Lingo-1 results in neuronal and oligodendroglial survival, axonal growth and remyelination; however to date literature has only detailed applications of Lingo-1 targeted therapeutics with a focus primarily on myelination disorders such as multiple sclerosis and spinal cord injury; omitting important information regarding Lingo-1 signaling co-factors. Here, we provide for the first time a complete and thorough review of the implications of Lingo-1 signaling in a wide range of neurological and psychiatric disorders, and critically examine its potential as a novel therapeutic target for these disorders.

Metabotropic glutamate receptor 5, and its trafficking molecules Norbin and Tamalin, are increased in the CA1 hippocampal region of subjects with schizophrenia.

Metabotropic glutamate receptor 5 (mGluR5) is involved in hippocampal-dependent learning and memory, which are processes disrupted in schizophrenia. Recent evidence from human genetic and animal studies suggests that the regulation of mGluR5, including its interaction with trafficking molecules, may be altered in the disorder. However there have been no investigations of hippocampal mGluR5 or mGluR5 trafficking molecules in the postmortem schizophrenia brain to confirm this. In the present study, we investigated whether protein expression of mGluR5, as well as Norbin and Tamalin (modulators of mGluR5 signalling and trafficking), might be altered in the schizophrenia brain, using postmortem samples from the hippocampal CA1 region of schizophrenia subjects and matched controls (n=20/group). Protein levels of mGluR5 (total: 42%, p<0.001; monomer: 25%, p=0.011; dimer: 52%, p<0.001) and mGluR5 trafficking molecules (Norbin: 47%, p<0.001; Tamalin: 34%, p=0.009) were significantly higher in schizophrenia subjects compared to controls. To determine any influence of antipsychotic drug treatment, all proteins were also correlated with lifetime chlorpromazine equivalents in patients, and separately measured in the hippocampus of rats exposed to haloperidol or olanzapine treatment. mGluR5 was negatively correlated with lifetime antipsychotic drug exposure in schizophrenia patients, suggesting antipsychotic drugs could reduce mGluR5 protein in schizophrenia subjects. In contrast, mGluR5 and mGluR5 trafficking molecules were not altered in the hippocampus of antipsychotic drug treated rats. This investigation provides strong support for the hypothesis that mGluR5 is involved in the pathology of schizophrenia, and that alterations to mGluR5 trafficking might contribute to the hippocampal-dependent cognitive dysfunctions associated with this disorder.

Alterations of p75 neurotrophin receptor and Myelin transcription factor 1 in the hippocampus of perinatal phencyclidine treated rats.

Postnatal administration of phencyclidine (PCP) in rodents causes major disturbances to neurological processes resulting in severe modifications to normal behavioral traits into adulthood. It is routinely used to model psychiatric disorders such as schizophrenia, producing many of the dysfunctional processes in the brain that are present in this devastating disorder, including elevated levels of apoptosis during neurodevelopment and disruptions to myelin and plasticity processes. Lingo-1 (or Leucine-rich repeat and immunoglobulin domain-containing protein) is responsible for negatively regulating neurite outgrowth and the myelination of axons. Recent findings using a postmortem human brain cohort showed that Lingo-1 signaling partners in the Nogo receptor (NgR)/p75/TNF receptor orphan Y (TROY) signaling complex, and downstream signaling partners With No Lysine (K) (WNK1) and Myelin transcription factor 1 (Myt1), play a significant part in schizophrenia pathophysiology. Here we have examined the implication of Lingo-1 and its signaling partners in a neurodevelopmental model of schizophrenia using PCP to determine if these pathways are altered in the hippocampus throughout different stages of neurodevelopment. Male Sprague-Dawley rats were injected subcutaneously with PCP (10mg/kg) or saline solution on postnatal days (PN) 7, 9, and 11. Rats (n=6/group) were sacrificed at PN12, 5weeks, or 14weeks. Relative expression levels of Lingo-1 signaling proteins were examined in the hippocampus of the treated rats. p75 and Myt1 were decreased (0.001≤p≤0.011) in the PCP treated rats at PN12. There were no significant changes in any of the tested proteins at 5weeks (p>0.05). At 14weeks, p75, TROY, and Myt1 were increased in the PCP treated rats (0.014≤p≤0.022). This is the first report of an alteration in Lingo-1 signaling proteins in the rat hippocampus, both directly after PCP treatment in early development and in adulthood. Based on our results, we propose that components of the Lingo-1 signaling pathways may be involved in the acute neurotoxicity induced by perinatal administration of PCP in rats early in development and suggest that this may have implications for the hippocampal deficits seen in schizophrenia.

Alterations of mGluR5 and its endogenous regulators Norbin, Tamalin and Preso1 in schizophrenia: towards a model of mGluR5 dysregulation.

Knockout of genes encoding metabotropic glutamate receptor 5 (mGluR5) or its endogenous regulators, such as Norbin, induce a schizophrenia-like phenotype in rodents, suggesting dysregulation of mGluR5 in schizophrenia. Human genetic and pharmacological animal studies support this hypothesis, but no studies have explored mGluR5 dysfunction at the molecular level in the postmortem schizophrenia brain. We assessed mGluR5 mRNA and protein levels in the dorsolateral prefrontal cortex (DLPFC) using a large cohort of schizophrenia and control subjects (n = 37/group), and additionally measured protein levels of recently discovered mGluR5 endogenous regulators, Norbin (neurochondrin), Tamalin (GRASP-1), and Preso1 (FRMPD4), which regulate mGluR5 localization, internalization and signaling. While mGluR5 mRNA expression was unchanged, mGluR5 protein levels were significantly higher in schizophrenia subjects compared to controls (total: +22%; dimer: +54%; p < 0.001). Conversely, mGluR5 regulatory proteins were expressed at lower levels in schizophrenia subjects compared to controls (Norbin -37%, p < 0.001; Tamalin -30%, p = 0.084; Preso1 -29%, p = 0.001). mGluR5 protein was significantly associated with mGluR5 mRNA and mGluR5 endogenous regulators in control subjects, but these associations were lost in schizophrenia subjects. Lastly, there were no associations between protein measures and lifetime antipsychotic history in schizophrenia subjects. To confirm no antipsychotic influence, all proteins were measured in the prefrontal cortex of rats exposed to haloperidol or olanzapine; there were no effects of antipsychotic drug treatment on mGluR5, Norbin, Tamalin or Preso1. The results from our study provide compelling evidence that mGluR5 regulation is altered in schizophrenia, likely contributing to the altered glutamatergic signaling that is associated with the disorder.

Metabotropic glutamate receptor mGluR2/3 and mGluR5 binding in the anterior cingulate cortex in psychotic and nonpsychotic depression, bipolar disorder and schizophrenia: implications for novel mGluR-based therapeutics.

BACKGROUND: Metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5) are novel therapeutic targets for major depression (MD), bipolar disorder (BD) and schizophrenia. We aimed to determine whether mGluR2/3 and mGluR5 binding in the anterior cingulate cortex (ACC), a brain region essential for the regulation of mood, cognition and emotion, were differentially altered in these pathologies. METHODS: Using postmortem human brains derived from 2 cohorts, [(3)H]LY341495 binding to mGluR2/3 and [(3)H]MPEP binding to mGluR5 were measured by receptor autoradiography in the ACC. The first cohort comprised samples from individuals who had MD with psychosis (MDP), MD without psychosis (MDNP) and matched controls (n = 11-12 per group). The second cohort comprised samples from individuals who had MDNP, BD, schizophrenia and matched controls (n = 15 per group). RESULTS: No differences in mGluR2/3 or mGluR5 binding were observed in the MDP, MDNP, BD or schizophrenia groups compared with the control group (all p > 0.05). Importantly, there were also no differences in binding densities between the psychiatric disorders (p > 0.05). We did, however, observe age-related effects, with consistent negative associations between mGluR2/3 and age in the control group (r < -0.575, p < 0.025) and the psychotic disorder groups (MDP and schizophrenia: r = -0.765 to -0.515, p < 0.05), but not in the mood disorder groups (MDNP, BD). LIMITATIONS: Replication in larger independent cohorts and medication-naive individuals would strengthen these findings. CONCLUSION: Our findings suggest that mGluRs are unaltered in the ACC; however, the presence of altered receptor function cannot be discounted and requires further investigation. Taken together with previous studies, which report differential changes in mGluR2, 3 and 5 across these disorders, we suggest mGluRs may be affected in a brain region-specific manner.

Investigation of genetic variants in ubiquitin enzyme genes involved in the modulation of neurodevelopmental processes: a role in schizophrenia susceptibility?

Despite extensive research during the last few decades, the etiology of schizophrenia remains unclear. Evidence of both genetic and environmental influences in the developmental profile of schizophrenia has grown, and due to the complexity of this disorder, a polygenic aspect has been associated with this neuropsychiatric pathology. Unfortunately, no diagnostic strategies based on biological measurement or genetic testing is currently available for schizophrenia. Gene-expression profiling and recent protein studies have shown a decrease in the expression of ubiquitin pathway proteins in the prefrontal cortex of schizophrenia patients. We have examined single nucleotide polymorphisms (or SNPs) within three genes from the ubiquitin protein system: the ubiquitin conjugating enzyme E2D1 (UBE2D1) gene, the E3 SUMO-protein ligase protein inhibitor of activated STAT 2 (PIAS2) gene, and the E3 ubiquitin ligase F-box and leucine-rich repeat protein 21 (FBXL21) gene, in a Caucasian case-control population for schizophrenia. After Bonferroni correction for multiple testing was applied, no significant associations were reported for any of the tested SNPs. Additional genetic analyses will be necessary to fully explore the role of these three genes in schizophrenia. Regarding the rising interest in ubiquitin-related proteins as a therapeutic target in other pathologies such as cancer, further research into the role of ubiquitin pathways in schizophrenia seems topical and timely.

In vivo pharmacological evaluations of novel olanzapine analogues in rats: a potential new avenue for the treatment of schizophrenia.

Olanzapine (Olz) is one of the most effective antipsychotic drugs commonly used for treating schizophrenia. Unfortunately, Olz administration is associated with severe weight gain and metabolic disturbances. Both patients and clinicians are highly interested in the development of new antipsychotics which are as effective as atypical antipsychotics but which have a lower propensity to induce metabolic side effects. In the present study, we examined two new derivatives of Olz; OlzEt (2-ethyl-4-(4'-methylpiperazin-1'-yl)-10Hbenzo[b]thieno[2,3-e][1,4]diazepine), and OlzHomo (2-ethyl-4-(4'-methyl-1',4'-diazepan-1'-yl)-10H-benzo[b]thieno[2,3-e] [1,4]diazepine), for their tendency to induce weight gain in rats. Weight gain and metabolic changes were measured in female Sprague Dawley rats. Animals were treated orally with Olz, OlzEt, OlzHomo (3 or 6 mg/kg/day), or vehicle (n = 8), three times daily at eight-hour intervals for 5 weeks. Furthermore, a phencyclidine (PCP)-treated rat model was used to examine the prevention of PCP-induced hyperlocomotor activity relevant for schizophrenia therapy. Male Sprague Dawley rats were pre-treated with a single dose (3 mg/kg/day) of Olz, OlzEt, OlzHomo, or vehicle (n = 12), for 2 weeks. Locomotor activity was recorded following a subcutaneous injection with either saline or PCP (10 mg/kg). Olz was found to induce weight gain, hyperphagia, visceral fat accumulation, and metabolic changes associated with reduced histamatergic H1 receptor density in the hypothalamus of treated rats. In contrast, OlzEt and OlzHomo presented promising antipsychotic effects, which did not induce weight gain or fat deposition in the treated animals. Behavioural analysis showed OlzEt to attenuate PCP-induced hyperactivity to a level similar to that of Olz; however, OlzHomo showed a lower propensity to inhibit these stereotyped behaviours. Our data suggest that the therapeutic effectiveness of OlzHomo may be delivered at a higher dose than that of Olz and OlzEt. Overall, OlzEt and OlzHomo may offer a better pharmacological profile than Olz for treating patients with schizophrenia. Clinical trials are needed to test this hypothesis.

Novel olanzapine analogues presenting a reduced H1 receptor affinity and retained 5HT2A/D2 binding affinity ratio.

BACKGROUND: Olanzapine is an atypical antipsychotic drug with high clinical efficacy, but which can cause severe weight gain and metabolic disorders in treated patients. Blockade of the histamine 1 (H1) receptors is believed to play a crucial role in olanzapine induced weight gain, whereas the therapeutic effects of this drug are mainly attributed to its favourable serotoninergic 2A and dopamine 2 (5HT2A/D2) receptor binding affinity ratios. RESULTS: We have synthesized novel olanzapine analogues 8a and 8b together with the already known derivative 8c and we have examined their respective in vitro affinities for the 5HT2A, D2, and H1 receptors. CONCLUSIONS: We suggest that thienobenzodiazepines 8b and 8c with lower binding affinity for the H1 receptors, but similar 5HT2A/D2 receptor binding affinity ratios to those of olanzapine. These compounds may offer a better pharmacological profile than olanzapine for treating patients with schizophrenia.

Structural contributions of antipsychotic drugs to their therapeutic profiles and metabolic side effects.

Antipsychotic drugs have various neuropharmacological properties as a result of their structural diversity. Despite their therapeutic benefits, most of the prescribed atypical antipsychotics can induce severe side effects, including weight gain, type II diabetes mellitus, and cardiovascular diseases. Among the developed atypical antipsychotic agents, tetracyclic dibenzodiazepine and thienobenzodiazepine compounds, particularly clozapine and olanzapine, are associated with the greatest weight gain and metabolic disturbances. However, the unique chemical structure of these compounds causes the low risk of side effects reported for typical antipsychotics (e.g. extrapyramidal symptoms and tardive dyskinesia). This report reviews the recent discovery of the potential role of the chemical structure of antipsychotics in their therapeutic properties and metabolic disturbances. By developing structure-activity relationship studies for atypical antipsychotics, we will improve our understanding of the structural modifications of these chemical classes that lead to reduced weight gain, which will be an invaluable step toward the discovery of the next generation of atypical antipsychotics. In this review, we suggest that a novel dibenzodiazepine or thienobenzodiazepine antipsychotic drug with lower affinity for H(1) receptors may significantly advance schizophrenia therapy.