Natalie Matosin

Homer1/mGluR5 Activity Moderates Vulnerability to Chronic Social Stress

Stress-induced psychiatric disorders, such as depression, have recently been linked to changes in glutamate transmission in the central nervous system. Glutamate signaling is mediated by a range of receptors, including metabotropic glutamate receptors (mGluRs). In particular, mGluR subtype 5 (mGluR5) is highly implicated in stress-induced psychopathology. The major scaffold protein Homer1 critically interacts with mGluR5 and has also been linked to several psychopathologies. Yet, the specific role of Homer1 in this context remains poorly understood. We used chronic social defeat stress as an established animal model of depression and investigated changes in transcription of Homer1a and Homer1b/c isoforms and functional coupling of Homer1 to mGluR5. Next, we investigated the consequences of Homer1 deletion, overexpression of Homer1a, and chronic administration of the mGluR5 inverse agonist CTEP (2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine) on the effects of chronic stress. In mice exposed to chronic stress, Homer1b/c, but not Homer1a, mRNA was upregulated and, accordingly, Homer1/mGluR5 coupling was disrupted. We found a marked hyperactivity behavior as well as a dysregulated hypothalamic–pituitary–adrenal axis activity in chronically stressed Homer1 knockout (KO) mice. Chronic administration of the selective and orally bioavailable mGluR5 inverse agonist, CTEP, was able to recover behavioral alterations induced by chronic stress, whereas overexpression of Homer1a in the hippocampus led to an increased vulnerability to chronic stress, reflected in an increased physiological response to stress as well as enhanced depression-like behavior. Overall, our results implicate the glutamatergic system in the emergence of stress-induced psychiatric disorders, and support the Homer1/mGluR5 complex as a target for the development of novel antidepressant agents.

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.

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.

Neurodevelopmental Expression Profile of Dimeric and Monomeric Group 1 mGluRs: Relevance to Schizophrenia Pathogenesis and Treatment

Group 1 metabotropic glutamate receptors (mGluR1/mGluR5) play an integral role in neurodevelopment and are implicated in psychiatric disorders, such as schizophrenia. mGluR1 and mGluR5 are expressed as homodimers, which is important for their functionality and pharmacology. We examined the protein expression of dimeric and monomeric mGluR1α and mGluR5 in the prefrontal cortex (PFC) and hippocampus throughout development (juvenile/adolescence/adulthood) and in the perinatal phencyclidine (PCP) model of schizophrenia. Under control conditions, mGluR1α dimer expression increased between juvenile and adolescence (209–328%), while monomeric levels remained consistent. Dimeric mGluR5 was steadily expressed across all time points; monomeric mGluR5 was present in juveniles, dramatically declining at adolescence and adulthood (−97–99%). The mGluR regulators, Homer 1b/c and Norbin, significantly increased with age in the PFC and hippocampus. Perinatal PCP treatment significantly increased juvenile dimeric mGluR5 levels in the PFC and hippocampus (37–50%) but decreased hippocampal mGluR1α (−50–56%). Perinatal PCP treatment also reduced mGluR1α dimer levels in the PFC at adulthood (−31%). These results suggest that Group 1 mGluRs have distinct dimeric and monomeric neurodevelopmental patterns, which may impact their pharmacological profiles at specific ages. Perinatal PCP treatment disrupted the early expression of Group 1 mGluRs which may underlie neurodevelopmental alterations observed in this model.

Understanding the Molecular Mechanisms Underpinning Gene by Environment Interactions in Psychiatric Disorders: The FKBP5 Model

Epidemiologic and genetic studies suggest common environmental and genetic risk factors for a number of psychiatric disorders, including depression, bipolar disorder, and schizophrenia. Genetic and environmental factors, especially adverse life events, not only have main effects on disease development but also may interact to shape risk and resilience. Such gene by adversity interactions have been described for FKBP5, an endogenous regulator of the stress-neuroendocrine system, conferring risk for a number of psychiatric disorders. In this review, we present a molecular and cellular model of the consequences of FKBP5 by early adversity interactions. We illustrate how altered genetic and epigenetic regulation of FKBP5 may contribute to disease risk by covering evidence from clinical and preclinical studies of FKBP5 dysregulation, known cell-type and tissue-type expression patterns of FKBP5 in humans and animals, and the role of FKBP5 as a stress-responsive molecular hub modulating many cellular pathways. FKBP5 presents the possibility to better understand the molecular and cellular factors contributing to a disease-relevant gene by environment interaction, with implications for the development of biomarkers and interventions for psychiatric disorders.

Preclinical and Clinical Evidence of DNA Methylation Changes in Response to Trauma and Chronic Stress

Exposure to chronic stress, either repeated severe acute or moderate sustained stress, is one of the strongest risk factors for the development of psychopathologies such as post-traumatic stress disorder and depression. Chronic stress is linked with several lasting biological consequences, particularly to the stress endocrine system but also affecting intermediate phenotypes such as brain structure and function, immune function, and behavior. Although genetic predisposition confers a proportion of the risk, the most relevant molecular mechanisms determining those susceptible and resilient to the effects of stress and trauma may be epigenetic. Epigenetics refers to the mechanisms that regulate genomic information by dynamically changing the patterns of transcription and translation of genes. Mounting evidence from preclinical rodent and clinical population studies strongly support that epigenetic modifications can occur in response to traumatic and chronic stress. Here, we discuss this literature examining stress-induced epigenetic changes in preclinical models and clinical cohorts of stress and trauma occurring early in life or in adulthood. We highlight that a complex relationship between the timing of environmental stressors and genetic predispositions likely mediate the response to chronic stress over time, and that a better understanding of epigenetic changes is needed by further investigations in longitudinal and postmortem brain clinical cohorts.

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 (&khgr;2=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.

Poster #S173 METABOTROPIC GLUTAMATE RECEPTOR 5 DYSREGULATION IN SCHIZOPHRENIA

(MBP), another myelinationand schizophrenia-related gene, is the bestcharacterized QKI mRNA ligand in mice (Larocque et al., 2002). Therefore, we tested this hypothesis using the human immature oligodendrocytic cell line, that levels of hnRNP C1 and C2 would influence the expression of these myelination-related genes. We also attempted to elucidate the functional differences between hnRNP C1 and C2. Methods: We constructed expression vectors for the two hnRNP C variants (hnRNP C1 and C2), and investigated, using the quantitative real-time RT-PCR, whether the overexpression of these proteins would change the expression of myelination-related genes, such as the QKI isoforms (QKI-5, -6, -7, and -7b) and MBP, in the human immature oligodendrocytic cell lime MO3.13 and during differentiation of this cell line by PMA treatment. Results: Endogenous expressions of both hnRNP C1 and C2 were found decreased during MO3.13 differentiation. In the same condition, the levels of expression of QKIs (QKI-5, -6, -7, and -7b) were significantly elevated. The up-regulation of QKIs were not influenced by overexpression of either the hnRNP C or C2. MBP was increased during differentiation of MO3.13, consistent with previous reports (Buntinx et al., 2003; Boscia et al., 2012). The up-regulations were not influenced by overexpression of either the hnRNP C1 or C2. However, intriguingly, MBP was up-regulated under the overexpression of hnRNP C2, but not hnRNP C1, in PMA-untreated cells. Discussion: We observed that the overexpression of hnRNP C1 and C2 do not affect the differential expression of QKIs and MBP during MO3.13 differentiation. But MBP seems to be regulated by hnRNP C2 in PMA-untreated cells. Similarly, we have reported previously that hnRNP C2 regulates MBP expression in a direct fashion, and that such regulation is not mediated by QKI in the human neuroblastoma cell line SK-N-SH (Iwata et al., 2011). Thus, we propose that the mechanism underlying the differential expression of hnRNP C1/2 in schizophrenia patients brains may involve changes of MBP expression. MBP has been found differentially expressed at gene and protein level (Martins-de-Souza et al. 2012; Matthews et al. 2012). Although no functional differences have been reported thus far for the two variants of hnRNP C protein, our findings indicate the existence of distinct functions between hnRNP C1 and C2. Further exploration of separate roles of hnRNP C1 and C2 associated to schizophrenia will lead to a better understanding the pathobiology of this disorder in a molecular level.

Metabotropic Glutamate Receptor 5 as a Point of Convergence for Models of Obsessive-Compulsive Disorder and Autism Spectrum Disorder

Obsessive-compulsive disorder (OCD) is an anxiety-related condition that emerges either in prepuberty, which is more common in males, or in late adolescence, which is more common in females, and is distinguished by time-consuming and distressing patterns of repeated persistent thoughts, ideas, impulses, images, and behaviors. The prevalence of OCD in the United States is 2.3% and is slightly lower in pediatric populations (1). It has been estimated that up to 84% of youth diagnosed with OCD have comorbid disorders, such as major depressive disorder, alcohol dependence, and social phobia. Untreated OCD can lead to significant academic, social, and family dysfunction. The current pharmacological treatments for OCD include serotonin reuptake inhibitors and selective serotonin reuptake inhibitors. These drugs can cause unfavorable side effects, including gastrointestinal upset, insomnia, and headaches (2). In addition, these agents can require months to achieve their therapeutic effects, both delaying relief and reducing compliance. As an alternative to monoamine-based therapies, there has been a substantial effort to explore the role of various glutamate signaling pathway modulators as therapeutic targets for psychiatric conditions. Glutamate is the major excitatory neurotransmitter in the central nervous system, and drugs that modulate the glutamate system have emerged as potential treatments with reduced side effects for many neuropsychiatric disorders and as alternatives to dopamine, serotonin, and gamma-aminobutyric acid (GABA)–targeting drugs. With a few notable exceptions (e.g., amantadine, memantine, and ketamine), these drugs have not reached widespread clinical application. However, they may hold significant promise to positively impact and improve patient outcomes if linked to a basic mechanism of abnormal brain physiology underlying abnormal thought processes and behaviors. Building research from clinical and preclinical studies of behavior, physiology, genomics, and epidemiology over the past decade suggest that subsets of glutamate-related substrates may be appropriate targets for brain neurotransmitter and circuit manipulation. As one example of this approach, brain dysplasia in the cortico-striato-thalamic network has been reported in patients with OCD (3). The role of glutamate in the developing brain in general—and specifically in neuronal proliferation, migration, and differentiation—has brought about the hypothesis that abnormal glutamate homeostasis during development may contribute to the manifestation of OCD. Indeed, there have been trials of glutamate-modulating drugs to treat OCD, particularly those targeting the N-methyl-D-aspartate receptor

Perinatal administration of phencyclidine alters expression of Lingo-1 signaling pathway proteins in the prefrontal cortex of juvenile and adult rats

Abstract Postnatal administration of phencyclidine (PCP) in rodents causes major brain dysfunction leading to severe disturbances in behavior lasting into adulthood. This model is routinely employed to model psychiatric disorders such as schizophrenia, as it reflects schizophrenia-related brain disturbances including increased apoptosis, and disruptions to myelin and plasticity processes. Leucine-rich repeat and Immunoglobin-like domain-containing protein 1 (Lingo-1) is a potent negative regulator of both axonal myelination and neurite extension. The Nogo receptor (NgR)/tumor necrosis factor (TNF) receptor orphan Y (TROY) and/or p75 neurotrophin receptor (p75) complex, with no lysine (K) (WNK1) and myelin transcription factor 1 (Myt1) are co-receptors or cofactors in Lingo-1 signaling pathways in the brain. We have examined the developmental trajectory of these proteins in a neurodevelopmental model of schizophrenia using PCP to determine if Lingo-1 pathways are altered in the prefrontal cortex throughout different stages of life. Sprague–Dawley rats were injected with PCP (10 mg/kg) or saline on postnatal days (PN)7, 9, and 11 and killed at PN12, 5 or 14 weeks for measurement of Lingo-1 signaling proteins in the prefrontal cortex. Myt1 was decreased by PCP at PN12 (P=0.045), and at 14 weeks PCP increased Lingo-1 (P=0.037), TROY (P=0.017), and WNK1 (P=0.003) expression. This is the first study reporting an alteration in Lingo-1 signaling proteins in the rat prefrontal cortex both directly after PCP treatment in early development and in adulthood. We propose that Lingo-1 pathways may be negatively regulating myelination and neurite outgrowth following the administration of PCP, and that this may have implications for the cortical dysfunction observed in schizophrenia.