Noah M. Walton

Deficiency of Schnurri-2, an MHC Enhancer Binding Protein, Induces Mild Chronic Inflammation in the Brain and Confers Molecular, Neuronal, and Behavioral Phenotypes Related to Schizophrenia

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

Detection of an immature dentate gyrus feature in human schizophrenia/bipolar patients

Hippocampus-associated cognitive impairments are a common, highly conserved symptom of both schizophrenia (SCZ) and bipolar disorder (BPD). Although the hippocampus is likely an impacted region in SCZ/BPD patients, the molecular and cellular underpinnings of these impairments are difficult to identify. An emerging class of mouse models for these psychiatric diseases display similar cognitive impairments to those observed in human patients. The hippocampi of these mice possess a conserved pathophysiological alteration; we term the ‘immature dentate gyrus’ (iDG), characterized by increased numbers of calretinin-positive immature neuronal progenitors, a dearth of calbindin-positive mature neurons and (often) constitutively increased neurogenesis. Although these models provide a link between cellular dysfunction and behavioral alteration, limited translational validity exists linking the iDG to human pathophysiology. In this study, we report the initial identification of an iDG-like phenotype in the hippocampi of human SCZ/BPD patients. These findings suggest a new motif for the etiology of these diseases and link an emerging class of mouse models to the human disease condition.

Adult Neurogenesis Transiently Generates Oxidative Stress

An increasing body of evidence suggests that alterations in neurogenesis and oxidative stress are associated with a wide variety of CNS diseases, including Alzheimer’s disease, schizophrenia and Parkinson’s disease, as well as routine loss of function accompanying aging. Interestingly, the association between neurogenesis and the production of reactive oxidative species (ROS) remains largely unexamined. The adult CNS harbors two regions of persistent lifelong neurogenesis: the subventricular zone and the dentate gyrus (DG). These regions contain populations of quiescent neural stem cells (NSCs) that generate mature progeny via rapidly-dividing progenitor cells. We hypothesized that the energetic demands of highly proliferative progenitors generates localized oxidative stress that contributes to ROS-mediated damage within the neuropoietic microenvironment. In vivo examination of germinal niches in adult rodents revealed increases in oxidized DNA and lipid markers, particularly in the subgranular zone (SGZ) of the dentate gyrus. To further pinpoint the cell types responsible for oxidative stress, we employed an in vitro cell culture model allowing for the synchronous terminal differentiation of primary hippocampal NSCs. Inducing differentiation in primary NSCs resulted in an immediate increase in total mitochondria number and overall ROS production, suggesting oxidative stress is generated during a transient window of elevated neurogenesis accompanying normal neurogenesis. To confirm these findings in vivo, we identified a set of oxidation-responsive genes, which respond to antioxidant administration and are significantly elevated in genetic- and exercise-induced model of hyperactive hippocampal neurogenesis. While no direct evidence exists coupling neurogenesis-associated stress to CNS disease, our data suggest that oxidative stress is produced as a result of routine adult neurogenesis.

Immature dentate gyrus: an endophenotype of neuropsychiatric disorders.

Adequate maturation of neurons and their integration into the hippocampal circuit is crucial for normal cognitive function and emotional behavior, and disruption of this process could cause disturbances in mental health. Previous reports have shown that mice heterozygous for a null mutation in α-CaMKII, which encodes a key synaptic plasticity molecule, display abnormal behaviors related to schizophrenia and other psychiatric disorders. In these mutants, almost all neurons in the dentate gyrus are arrested at a pseudoimmature state at the molecular and electrophysiological levels, a phenomenon defined as “immature dentate gyrus (iDG).” To date, the iDG phenotype and shared behavioral abnormalities (including working memory deficit and hyperlocomotor activity) have been discovered in Schnurri-2 knockout, mutant SNAP-25 knock-in, and forebrain-specific calcineurin knockout mice. In addition, both chronic fluoxetine treatment and pilocarpine-induced seizures reverse the neuronal maturation, resulting in the iDG phenotype in wild-type mice. Importantly, an iDG-like phenomenon was observed in post-mortem analysis of brains from patients with schizophrenia/bipolar disorder. Based on these observations, we proposed that the iDG is a potential endophenotype shared by certain types of neuropsychiatric disorders. This review summarizes recent data describing this phenotype and discusses the datas potential implication in elucidating the pathophysiology of neuropsychiatric disorders.

The immature dentate gyrus represents a shared phenotype of mouse models of epilepsy and psychiatric disease.

There is accumulating evidence to suggest psychiatric disorders, such as bipolar disorder and schizophrenia, share common etiologies, pathophysiologies, genetics, and drug responses with many of the epilepsies. Here, we explored overlaps in cellular/molecular, electrophysiological, and behavioral phenotypes between putative mouse models of bipolar disorder/schizophrenia and epilepsy. We tested the hypothesis that an immature dentate gyrus (iDG), whose association with psychosis in patients has recently been reported, represents a common phenotype of both diseases.

SREB2/GPR85, a schizophrenia risk factor, negatively regulates hippocampal adult neurogenesis and neurogenesis‐dependent learning and memory

SREB2/GPR85, a member of the super‐conserved receptor expressed in brain (SREB) family, is the most conserved G‐protein‐coupled receptor in vertebrate evolution. Previous human and mouse genetic studies have indicated a possible link between SREB2 and schizophrenia. SREB2 is robustly expressed in the hippocampal formation, especially in the dentate gyrus, a structure with an established involvement in psychiatric disorders and cognition. However, the function of SREB2 in the hippocampus remains elusive. Here we show that SREB2 regulates hippocampal adult neurogenesis, which impacts on cognitive function. Bromodeoxyuridine incorporation and immunohistochemistry were conducted in SREB2 transgenic (Tg, over‐expression) and knockout (KO, null‐mutant) mice to quantitatively assay adult neurogenesis and newborn neuron dendritic morphology. Cognitive responses associated with adult neurogenesis alteration were evaluated in SREB2 mutant mice. In SREB2 Tg mice, both new cell proliferation and new neuron survival were decreased in the dentate gyrus, whereas an enhancement of new neuron survival occurred in SREB2 KO mouse dentate gyrus. Doublecortin staining revealed dendritic morphology deficits of newly generated neurons in SREB2 Tg mice. In a spatial pattern separation task, SREB2 Tg mice displayed a decreased ability to discriminate spatial relationships, whereas SREB2 KO mice had enhanced abilities in this task. Additionally, SREB2 Tg and KO mice had reciprocal phenotypes in a Y‐maze working memory task. Our results indicate that SREB2 is a negative regulator of adult neurogenesis and consequential cognitive functions. Inhibition of SREB2 function may be a novel approach to enhance hippocampal adult neurogenesis and cognitive abilities to ameliorate core symptoms of psychiatric patients.

Mouse Model of Chromosome 15q13.3 Microdeletion Syndrome Demonstrates Features Related to Autism Spectrum Disorder.

The chromosome 15q13.3 microdeletion is a pathogenic copy number variation conferring epilepsy, intellectual disability, schizophrenia, and autism spectrum disorder (ASD). We generated mice carrying a deletion of 1.2 Mb homologous to the 15q13.3 microdeletion in human patients. Here, we report that mice with a heterozygous deletion on a C57BL/6 background (D/+ mice) demonstrated phenotypes including enlarged/heavier brains (macrocephaly) with enlarged lateral ventricles, decreased social interactions, increased repetitive grooming behavior, reduced ultrasonic vocalizations, decreased auditory-evoked gamma band EEG, and reduced event-related potentials. D/+ mice had normal body weight, activity levels, sensory gating, and cognitive abilities and no signs of epilepsy/seizures. Our results demonstrate that D/+ mice represent ASD-related phenotypes associated with 15q13.3 microdeletion syndrome. Further investigations using this chromosome-engineered mouse model may uncover the common mechanism(s) underlying ASD and other neurodevelopmental/psychiatric disorders representing the 15q13.3 microdeletion syndrome, including epilepsy, intellectual disability, and schizophrenia. SIGNIFICANCE STATEMENT Recently discovered pathologic copy number variations (CNVs) from patients with neurodevelopmental/psychiatric disorders show very strong penetrance and thus are excellent candidates for mouse models of disease that can mirror the human genetic conditions with high fidelity. A 15q13.3 microdeletion in humans results in a range of neurodevelopmental/psychiatric disorders, including epilepsy, intellectual disability, schizophrenia, and autism spectrum disorder (ASD). The disorders conferred by a 15q13.3 microdeletion also have overlapping genetic architectures and comorbidity in other patient populations such as those with epilepsy and schizophrenia/psychosis, as well as schizophrenia and ASD. We generated mice carrying a deletion of 1.2 Mb homologous to the 15q13.3 microdeletion in human patients, which allowed us to investigate the potential causes of neurodevelopmental/psychiatric disorders associated with the CNV.

Gastrin-Releasing Peptide Contributes to the Regulation of Adult Hippocampal Neurogenesis and Neuronal Development

In the postnatal hippocampus, newly generated neurons contribute to learning and memory. Disruptions in neurogenesis and neuronal development have been linked to cognitive impairment and are implicated in a broad variety of neurological and psychiatric disorders. To identify putative factors involved in this process, we examined hippocampal gene expression alterations in mice possessing a heterozygous knockout of the calcium/calmodulin‐dependent protein kinase II alpha heterozygous knockout gene (CaMK2α‐hKO), an established model of cognitive impairment that also displays altered neurogenesis and neuronal development. Using this approach, we identified gastrin‐releasing peptide (GRP) as the most dysregulated gene. In wild‐type mice, GRP labels NeuN‐positive neurons, the lone exception being GRP‐positive, NeuN‐negative cells in the subgranular zone, suggesting GRP expression may be relevant to neurogenesis and/or neuronal development. Using a model of in vitro hippocampal neurogenesis, we determined that GRP signaling is essential for the continued survival and development of newborn neurons, both of which are blocked by transient knockdown of GRPs cognate receptor (GRPR). Furthermore, GRP appears to negatively regulate neurogenesis‐associated proliferation in neural stem cells both in vitro and in vivo. Intracerebroventricular infusion of GRP resulted in a decrease in immature neuronal markers, increased cAMP response element‐binding protein (CREB) phosphorylation, and decreased neurogenesis. Despite increased levels of GRP mRNA, CaMK2α‐hKO mutant mice expressed reduced levels of GRP peptide. This lack of GRP may contribute to the elevated neurogenesis and impaired neuronal development, which are reversed following exogenous GRP infusion. Based on these findings, we hypothesize that GRP modulates neurogenesis and neuronal development and may contribute to hippocampus‐associated cognitive impairment. Stem Cells 2014;32:2454–2466

The impact of genetics on future drug discovery in schizophrenia

ABSTRACT Introduction: Failures of investigational new drugs (INDs) for schizophrenia have left huge unmet medical needs for patients. Given the recent lackluster results, it is imperative that new drug discovery approaches (and resultant drug candidates) target pathophysiological alterations that are shared in specific, stratified patient populations that are selected based on pre-identified biological signatures. One path to implementing this paradigm is achievable by leveraging recent advances in genetic information and technologies. Genome-wide exome sequencing and meta-analysis of single nucleotide polymorphism (SNP)-based association studies have already revealed rare deleterious variants and SNPs in patient populations. Areas covered: Herein, the authors review the impact that genetics have on the future of schizophrenia drug discovery. The high polygenicity of schizophrenia strongly indicates that this disease is biologically heterogeneous so the identification of unique subgroups (by patient stratification) is becoming increasingly necessary for future investigational new drugs. Expert opinion: The authors propose a pathophysiology-based stratification of genetically-defined subgroups that share deficits in particular biological pathways. Existing tools, including lower-cost genomic sequencing and advanced gene-editing technology render this strategy ever more feasible. Genetically complex psychiatric disorders such as schizophrenia may also benefit from synergistic research with simpler monogenic disorders that share perturbations in similar biological pathways.

Derivation of neural stem cells from an animal model of psychiatric disease.

Several psychiatric and neurological diseases are associated with altered hippocampal neurogenesis, suggesting differing neural stem cell (NSC) function may play a critical role in these diseases. To investigate the role of resident NSCs in a murine model of psychiatric disease, we sought to isolate and characterize NSCs from alpha-calcium-/calmodulin-dependent protein kinase II heterozygous knockout (CaMK2α-hKO) mice, a model of schizophrenia/bipolar disorder. These mice display altered neurogenesis, impaired neuronal development and are part of a larger family possessing phenotypic and behavioral correlates of schizophrenia/bipolar disorder and a shared pathology referred to as the immature dentate gyrus (iDG). The extent to which NSCs contribute to iDG pathophysiology remains unclear. To address this, we established heterogeneous cultures of NSCs isolated from the hippocampal neuropoietic niche. When induced to differentiate, CaMK2α-hKO-derived NSCs recapitulate organotypic hippocampal neurogenesis, but generate larger numbers of immature neurons than wild-type (WT) littermates. Furthermore, mutant neurons fail to assume mature phenotypes (including morphology and MAP2/calbindin expression) at the same rate observed in WT counterparts. The increased production of immature neurons which fail to mature indicates that this reductionist model retains key animal- and iDG-specific maturational deficits observed in animal models and human patients. This is doubly significant, as these stem cells lack several developmental inputs present in vivo. Interestingly, NSCs were isolated from animals prior to the emergence of overt iDG pathophysiology, suggesting mutant NSCs may possess lasting intrinsic alterations and that altered NSC function may contribute to iDG pathophysiology in adult animals.