Akira Sawa - US grants

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to characterize DISC-1 (Disrupted ln-Schizophrenia) and its interacting proteins and determine their roles in the pathogenesis of major mental illnesses including schizophrenia (SZ). In one large family with hereditary SZ, a balanced translocation in the DISC-1 gene segregates with major psychiatric illnesses with a LOD score of 7.1. The translocation leads to a truncation of the DISC-1 protein product. Thus, DISC-1 appears be the first causative gene mutation identified for SZ and related psychiatric conditions. Research in Alzheimer's and Parkinson's diseases has been greatly advanced by the understanding of rare familial cases, involving causative mutations in gene products such as presenilin, and alpha-synuclein. We propose to use a similar strategy for SZ, by focusing on DISC-l. While DISC-1 mutations may be very rare, the biology of DISC-1 and its protein interaction partners will help elucidate the pathogenesis of major mental disorders in the Scottish family and likely in some other familial, and possibly sporadic, cases as well. Our preliminary results suggest that DISC-1 and its interactors play a role in neurodevelopment and can be linked to cortical developmental disorders. Therefore our overall hypothesis is that cascades that include DISC-1 and its interactors may have important functions in cell structure and cell signaling during neural development. Aim 1 will screen DISC-1 protein interactors and characterize their binding by yeast two-hybrid (Y2H) assays. In Aim 2, the putative protein interactions will be confirmed and characterized by biochemical and molecular cell approaches. Aim 3 will provide functional analyses of DISC-1 and its interators in cell and animal models by modulating expression of these proteins by transfection of expression and RNAi constructs and examining the effects on cell structure and signaling, especially neurite outgrowth and neuronal migration. In Aim 4 autopsied brain samples from controls and patients with major mental illnesses such as SZ will be analyzed for changes in DISC-1 and its interactors. Together these studies will clarify the molecular and cellular functions of DISC-1 and its protein interaction partners. They will provide information to understand the pathogenesis of major mental illnesses in the Scottish family, and they may provide insight into some of the mechanisms that underlie SZ and major mental illnesses.

The Administration Core will provide the following four services to all projects, in coordination with other two cores, in this Schizophrenia Research Center at Johns Hopkins. Therefore, this core will 1) provide a centralized administrative facility for the entire Center. This service includes financial administration, organization of human and animal subject protocols, and coordination of research meetings inside this Center. 2) provide scientific leadership to the entire Center by centrally organizing scientific issues towards uniform conclusions inside the Center, coordinating collaborative research and material exchange with investigators outside the Center, and communicating with advisory committees and board. 3) provide the central data organization and make it available to the public. This includes construction of a database by using a web interface for all projects/cores, maintaining this database by assisting with data entry for each project/core centrally. In addition, by linking to this central database, this core will assist in publication and web page construction for the projects and cores. 4) provide statistical consultation for analysis of data as well as experimental design, by facilitating communications with the Biostatistical Consulting Center in the Department of Biostatistics in the Johns Hopkins Bloomberg School of Public Health. The mission of this Core is not only to assist each project/core towards overall productivity of this Schizophrenia Research Center but also to facilitate bi-directional exchange of scientific information between the Center and the broad scientific community.

DESCRIPTION (provided by applicant): Disrupted-In-Schizophrenia-1 (DISC1) is one of the most promising common susceptibility factors for major mental illnesses, such as schizophrenia and mood disorders. Our long-term goals are to clarify disease pathway(s) that commonly exist in major mental illnesses, especially schizophrenia. DISC1 appears to be a promising lead to advance analysis of the pathway(s). DISC1 protein is multifunctional with several distinct subcellular distributions, including the centrosome during neurodevelopment, the postsynaptic density, and the nucleus. Previous studies of autopsied brains and cell biology from our and other groups have suggested that DISC1 may have a regulatory role in gene transcription, which may be disturbed in major mental disorders. Thus, we hypothesize that study of a nuclear pathway of DISC1 may elucidate the pathophysiology of major mental illnesses. In Aim 1, we will determine biochemical mechanisms that regulate nuclear distribution of DISC1, focusing in particular on cis- elements and phosphorylation. In Aim 2, we plan to characterize protein interactions of DISC1 in the nucleus, focusing on ATF4/CREB2 (a transcription factor), N-CoR (a nuclear co-repressor), and PML (the main component of the nuclear body). We hypothesize that DISC1 functions as a scaffold at the nuclear body to recruit components of activator/repressor complex to CREB/ATF transcriptional machinery. Aim 3 is designed to elucidate mechanisms of how nuclear DISC1 regulates gene transcription, in particular CRE-mediated gene transcription. We hypothesize that possible recruitment of components of activator/repressor complex by DISC1 at the PML-nuclear body regulate CRE-mediated gene transcription at least via ATF4/CREB2. We hope that this mechanistic study will provide insight into understanding major mental illnesses and eventually lead to better therapeutic strategies for the diseases. PUBLIC HEALTH RELEVANCE: Disrupted-In-Schizophrenia-1 (DISC1) is one of the most promising common susceptibility factors for major mental illnesses, such as schizophrenia and mood disorders. Because several lines of evidence have suggested nuclear DISC1 may be involved in the pathophysiology of the diseases, we will study basic mechanism and regulation for nuclear DISC1. We will particularly focus on the key mechanisms that regulates nuclear functions of DISC1, including cis-elements for nuclear targeting, phosphorylation, SUMOylation, and protein interaction with other nuclear proteins, as well as its function in gene transcription.

Schizophrenia is a disorder of neuronal connectivity, at least in part, with developmental origin. Functional disturbance of the cerebral cortex, especially the prefrontal cortex, has been reproducibly reported. Recent genetic studies have suggested that some of the genetic susceptibility factors may have a role in formation of synapses. These factors include Disrupted-ln-Schizophrenia (DISC1), neuronal nitric oxide synthase (nNOS), CAPON, NDEL1, and ErbB4. DISC1 interacts with several intracellular proteins, including Kalirin and other susceptibility factors for the disease, as an adaptor protein in the postsynaptic density. Thus, we will study a role for molecular pathway(s) involving DISC1 in synaptic spine formation in the developing cerebral cortex. In the first half of this project (Aims 1 and 2), we will use primary cortical neurons to elucidate molecular pathways of DISC1/ Kalirin-7 and DISC1/nNOS/NDEL1 for proper spine formation in pyramidal neurons, possibly by influencing Rho-like small G-proteins, especially Rac1. We hypothesize that DISC1 regulates access of Kalirin-7 and NDEL1 to Rac1 and related proteins, in response to the activation of the NMDA glutamate receptor, and regulates synaptic spine formation in a proper manner. In the second half of this project (Aim 3), we will test how disturbance of DISC1 and its interaction with Kalirin-7 and other synaptic proteins lead to morphological changes of the spines in vivo and result in behavioral alterations, which will be studied in collaboration with Core B. Key mediators studied in this project, including Kalirin-7 and Rac1, will be evaluated by genetic analysis in Core C. Taken together, we hope to clarify how convergence of twto critical factors for schizophrenia, such as glutamate and DISC1, occurs at postsynaptic sites. We wish to elucidate mechanisms whereby this disease pathway, involving DISC1 and its interactors, leads to deficits in the synaptic spines in the pyramidal neurons of the frontal cortex and resultant behavioral abnormalities.

The overall goal of this Schizophrenia Research Center at Johns Hopkins is to conduct interdisciplinary studies towards understanding the molecular pathology of schizophrenia. One of the recent exciting developments in schizophrenia research is the identification of disease susceptibility genes from human genetic association studies. Functional studies of each susceptibility factor have suggested that, instead of functioning independently from each other, these factors act synergistically in several common "pathways" that may contribute to the disease pathology. Accumulating evidence also reinforces the emerging view that schizophrenia is a condition of neuronal development with an adult onset. How specific schizophrenia susceptibility factors, or pathways, regulate different aspects of neuronal development, however, is largely unknown. Our collaborative team has obtained compelling evidence from our preliminary studies that DISC1 interacts with other susceptibility factors as an adaptor and is involved in various phases of neurodevelopment. Thus, we hypothesize that DISC1 is a promising lead with which we can explore the disease "pathways" underlying the pathology of schizophrenia associated with neurodevelopment. On the basis of preliminary data that our group and others have collected, we hypothesize three aspects of neurodevelopment as an important foundation for studying and elucidating the pathology. These include: (1) synaptic formation in the developing cerebral cortex;(2) centrosomal organization in the developing cerebral cortex;and (3) neurogenesis in the hippocampus, which will be studied in each project (Projects 1-3), respectively. Core A will provide administration and overall scientific direction. Core B will characterize behavioral phenotypes/endophenotypes of mice under study as models for the molecular cellular biology in the three projects. Core C will conduct genetic studies exploring possible genetic variations in the targets originally from biological studies, which, in turn, will be studied in each project. The ultimate goal is to elucidate the disease "pathways" to have better understanding of the disease, build appropriate models for mechanistic studies and future therapeutic strategies, and eventually to identify novel ways to treat the disease.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-MH- 102 Schizophrenia Interactome. Schizophrenia is thought to be a complex neurodevelopmental disorder with many genetic factors contributing to its pathology, possibly in an interrelated or convergent manner. Two of the strongest susceptibility genes for Schizophrenia are neuregulin 1 (NRG1) and DISC1. Although they are broadly expressed in the developing brain and are thought to play important roles in neurodevelopmental processes, it remains unclear if these molecules are functionally related to each other and how this interaction affects cerebral cortical development. We hypothesize that changes in the functional interactions between these genetic factors and the resultant changes in the formation of neural circuitry in the cerebral cortex may lead to neurodevelopmental disorders such as schizophrenia. An understanding of the functional interactions between these important SZ susceptibility genetic factors in the developing cerebral cortex will be essential to delineate the pathophysiological processes that culminate in schizophrenia. To accomplish this, we will (1) determine the effects of NRG1 on DISC1 expression and the underlying signaling mechanisms in the developing cerebral cortex and (2) define the functional significance of NRG1-DISC1 interactions in the formation of cerebral cortex. This integrated analysis of how strong susceptibility genetic factors for schizophrenia interact during cortical development will help to decipher some of the key neurodevelopmental pathways whose disruption can lead to the development of schizophrenia. PUBLIC HEALTH RELEVANCE: Schizophrenia is a complex neurodevelopmental disorder with many genetic factors contributing to its pathology in an interrelated or convergent manner. Two of the strongest susceptibility genes for Schizophrenia are neuregulin 1 (NRG1) and DISC1. Changes in the functional interactions between these genetic factors and the resultant changes in the formation of neural circuitry in the cerebral cortex may lead to schizophrenia. Therefore, we aim to define how susceptibility genetic factors for schizophrenia interact during cortical development. This will help to decipher the key neurodevelopmental pathways whose disruption can lead to the development of schizophrenia.

DESCRIPTION (provided by applicant): Protein Interacting with C-Kinase (PICK1) is a multifunctional scaffold protein that interacts with many proteins in neurons and glial cells. We recently reported that PICK1 binds directly to the D-serine synthesizing enzyme, serine racemase (SR). Knockdown expression of PICK1 decreased levels of D-serine from SR in cell cultures. We published that decreased levels of D-serine were observed in neonatal forebrains in PICK1 knockout (PICK1 KO), consistent with the observation from cell biology. Several recent studies have suggested roles for D-serine in mental disturbances associated with cortical circuitry, including schizophrenia. To our knowledge, however, roles for PICK1 have largely been studied in the context of synaptic plasticity, mainly by using slices from the cerebellum and hippocampus. Therefore, in this proposed study, we will examine PICK1 knockout mice in behavioral assays and electrophysiological approaches and focus on the impact of the protein on D- serine disposition and brain functions, especially those associated with cortical circuitry. In our preliminary studies, we obtained (1) decreased levels of D-serine in the forebrain during the neonatal period, but not in the adulthood;(2) several behavioral deficits in adulthood, which includes those in spatial working memory and prepulse inhibition;and (3) decreased response of the cortical pyramidal neurons to NMDA in adulthood, in PICK1 KO mice. Based on preliminary data, our overall hypothesis is that neonatal deficits in D-serine, which is likely to associate with abnormal glutamate function, affect the maturation of prefrontal cortical circuits and result in deficits in synaptic and NMDA-dependent responses in pyramidal neurons and interneurons in adulthood, whereas at this time the levels of D-serine are normal. In Aim 1, we will examine levels of D-serine in the forebrain during development, especially in neonatal stages in PICK1 KO mice. In Aim 2, we will characterize adult PICK1 KO mice by a set of behavioral assays, in particular those for measuring cortical functions, and also by electrophysiological approaches. In Aim 3, we will normalize the levels of D-serine in PICK1 KO mice by administering D-serine, if necessary, combined with a DAAO inhibitor during the neonatal stage. We will then examine whether the D-serine treatment during neonatal periods influences possible abnormal behaviors and physiological phenotypes in adult PICK1 KO mice. Through these experiments, we will study a role for PICK1 in the neonatal forebrain in conjunction with D-serine, hoping that the information obtained from this study will be an important basis in understanding mental disorders and brain development. PUBLIC HEALTH RELEVANCE: PICK1 knockout mice Recent evidence has suggested that PICK1 may regulate glutamate neurotransmission via regulating D-serine metabolism in the neonatal forebrain. Formation of neuronal circuitry during neonatal stages is crucial for proper brain development and functions of adult brain. We will study role for PICK1 in the neonatal forebrain in conjunction with D- serine by characterizing PICK1 knockout mice, hoping that the information obtained from this study will be an important basis in understanding mental disorders and brain development.

This core will provide integrative services for administration, human resources, and data analysis. For administration, this core will have the following roles: 1) provision of a centralized administrative facility for the entire center, including financial administration, organization of human and animal subject protocols, and coordination of research meetings inside this center; 2) scientific leadership to the entire center by organizing scientific issues towards uniform goals, coordinating collaborative research and material exchange with investigators outside the center (resource/data sharing), and communicating with advisory committees and board; 3) training programs for young investigators and a summer undergraduate course; and 4) public outreach for both lay persons and scientific peers outside of the center. For human resources, this core will connect two established groups keeping world-class repositories of human genetic and tissue samples together with detailed clinical data. For data analyses, this core will provide consultation for experiments and data analyses, including microarray studies (gene expression profiling) and genetic sequencing. These data will be centrally analyzed together with currently available datasets in human tissue/genetic resources. This core will closely work with Core B for overall data analysis. Finally, this core will provide services for database production.

Schizophrenia (SZ) is believed to be a disorder of neural connectivity involving the prefrontal cortex. Several lines of evidence have suggested that abnormal synaptic reorganization, such as synaptic pruning, may underlie the pathology of SZ. We previously reported the localization of DISCI, a major risk factor of SZ, in the postsynaptic density in mature neurons. We then demonstrated that DISCI is required for proper maintenance of the synapse and spine by functionally linking to activation of NMDA-type glutamate receptor and conveying the signal to Kalirin-7 (Kal-7) and Rac1. Consistently, several groups, including ours, have obtained preliminary results that DISC1 mutant animal models show a decrease in the spine density in the cortex in adulthood. Considering a peak of DISC1 expression observed in early adolescence and importance of synaptic pruning possibly in the pathology of SZ, we now hypothesize that the DISC1-Kal-7- Rac1 cascade may, at least in part, play a role in the pathology of SZ, especially during the prodromal stage of the disorder in adolescence. We will have four aims as follows: (1) to examine synaptic disturbances in the cortex in several DISC1 mutant animal models in the developmental course from early adolescence to adulthood; (2) to identify whether the deficits of the pyramidal neurons, especially those associated with the spine, primarily lead to circuitry deficits of the frontal cortex and behavioral deficits relevant to SZ; (3) to test whether modulation of PAK1 (a key downstream mediator of Rac1) protects against DISC1-elicited synapse deterioration; and (4) to examine molecular profiles in the frontal cortex of DISC1 mutant animal models, especially those in the pyramidal neuron layers. Through these studies, we wish to characterize SZ-relevant synapse pathology in DISC1 animal models, identify mechanisms of how synaptic disturbance can lead to circuitry defects and behavioral abnormalities, pin down a way of intervention against the synapse pathology (towards therapeutic strategies), and establish changes in the molecular signature associated with the synaptic changes towards biomarker identification.

Adult brain function and behavior are influenced by neuronal network formation during development. Consequently, disturbances of brain development may underlie the pathology of adult mental disorders, such as schizophrenia (SZ) and mood disorders. Consistent with this notion, genetic susceptibility factors for these disorders that have been recently indentified, including Disrupted-in-Schizophrenia-1 (DISC1) and PCM1, have roles during neurodevelopment and are likely to cooperate, forming molecular pathways. Meanwhile, epidemiological studies have indicated that many environmental factors contribute to schizophrenia during neurodevelopment. P50 Schizophrenia Research Center at Johns Hopkins, therefore, is to address the key question of how defects of cortical development elicited by combinations of genetic and environmental risk factors lead to molecular, histological, and behavioral deficits associated with the frontal cortex in adulthood, which are relevant to SZ. Based on our preliminary studies, we hypothesize that DISC1 and its interactors are useful genetic probes for this study. Accordingly, the four major aims of this entire center are as follows: 1) to clarify the mechanisms whereby several different combinations of DISC1 and interactors (e.g., Karilin-7, PCM1, RPGRIP1L, CRMP2, nNOS, and NDEL1) mediate distinct processes during neurodevelopment, which in turn affect postnatal brain maturation and result in deficits of the frontal cortex and behavioral abnormalities relevant to SZ; 2) to determine how environmental factors relevant to SZ (prenatal immune activation, postnatal activation of complement cascade, and postnatal infection of Toxoplasma Gondii) influence genetic vulnerability associated with DISCI, which eventually contribute to the deficits of the frontal cortex and behavioral abnormalities relevant to SZ; 3) to identify molecular targets for possible biomarkers of SZ and SZ-associated endophenotypes by comparing altered expression profiles in preclinical models and human tissues; 4) to identify rare genetic variants associated with SZ and/or some endophenotypes associated with SZ by pinpointing novel candidates for genetic sequencing from biological studies. In this center, 6 projects and 2 cores will collaborate to achieve these scientific goals.

DESCRIPTION (provided by applicant): Schizophrenia (SZ) is a debilitating mental illness typically developing after puberty. Accumulating evidence suggests role for disturbances in postnatal brain maturation, which includes interneuron deficits. However, mechanistic understanding of SZ is not well developed. One major limitation that has blocked the progress, although mental disorders affect the brains, is the difficulty in accessing neuronal cells from patients. To overcome this dilemma, the program for which the PI serves as director, has systematically collected tissues and cells (lymphocytes, lymphoblasts, fibroblasts, induced pluripotent stem cells, and olfactory neurons via nasal biopsy) from patients with SZ as well as normal controls. Collection of blood cells is aimed to explore high throughput peripheral biomarkers. In our preliminary study, we observed excess levels of reactive oxygen species (ROS) in SZ lymphoblasts and olfactory neurons, compared with control cells. Interestingly, this difference between SZ and controls was accentuated following exposure of cells to increased glucose concentrations. Excess ROS in SZ was partially normalized by clozapine, which is utilized clinically in treatment of patients with SZ. Additional findings revealed redox imbalances in SZ cells. We conducted magnetic resonance spectroscopy of the individuals who provided tissue/cell samples and obtained preliminary data that suggest a decrease glutathione in the anterior cingulate cortex. On the basis of our preliminary data and previous studies by others, we hypothesize that cells derived from SZ patients may have intrinsic susceptibility that results in oxidative stress, which may be further represented under glucose overload. We hypothesize that this susceptibility is associated with SZ as a trait marker, being common in both neuronal and non-neuronal cells. In this proposal, we plan to address this cellular susceptibility in greater detail, by measuring the levels of ROS and protein oxidation as well as investigating possible cellular mechanisms underlying this susceptibility, such as the glutathione (GSH) cascade, NADPH oxidase, NAD/NADH, and mitochondrial functions. We will address whether neuroleptics may decrease excess ROS associated with this susceptibility. Then, in the final Aim, we will examine biochemical changes in brains of the same set of subjects from whom we obtain cells. We plan to examine the manner in which cellular changes and susceptibility observed in lymphoblasts and olfactory neurons are manifested in the brain. Evidence of oxidative stress in the pathology of SZ has been reported in the history of SZ research, which is now highlighted by reports that oxidative stress can elicit SZ-associated interneuron deficit, a key pathophysiology of this disorder. Thus, our study with human cells, especially neurons, may provide important information for SZ research. PUBLIC HEALTH RELEVANCE: Based on promising preliminary data, we study whether oxidative stress may play a role in cellular susceptibility associated with SZ by cell biology and brain imaging.

DESCRIPTION (provided by applicant): The diagnostic boundaries defined by current diagnostic systems (e.g., DSM) are now being challenged by recent advances in the genetic architecture underlying psychiatric disorders. Meanwhile, NIMH has launched the Research Domain Criteria project (RDoC) to develop, for research purposes, new ways of classifying psychopathology based on dimensions of observable behavior and neurobiological measures beyond the current diagnostic systems. The DISC1 gene was originally discovered as the sole disrupted transcript with an open reading frame, at the breakpoint of an inherited chromosomal translocation in a Scottish pedigree: it segregates with a variety of major mental illnesses, including schizophrenia (SZ), bipolar disorder (BP), and major depression. Nonetheless, genome wide association studies have failed to detect associations between DISC1 locus and SZ (or other DSM-categorized diseases thus far). In contrast, the data from association studies of DISC1 with anatomical, physiological, and behavioral traits, which commonly underlie the pathology of major mental illnesses, have been promising. Thus, we hypothesize that DISC1 is a promising target to address mechanisms underlying mental illnesses across diagnostic categories in the RDoC framework: DISC1 may be a good probe that mediates translation from the discoveries in basic genetics, neuroscience, and behavioral science into clinical application. Based on our preliminary data, we further hypothesize that a decrease in the level of phosphorylation at serine-713 of human DISC1 (pS713-DISC1) underlies delayed neural differentiation, which disturbs neural circuitry formation in the brain development and, in turn, interferes with the acquisition of working memory. We will study relatively stable outpatients with SZ and BP, as well as well-matched healthy controls. Within the RDoC framework, our construct of interest is working memory (cognitive domain), whereas the independent variable is pS713-DISC1 (molecule). Our dependent variables include neuronal fate (cells), cortical surface area, thickness, and volume (circuit), and working memory (behavior). Johns Hopkins Schizophrenia Center has established an infrastructure of translational research in which we conduct clinical/neuropsychological assessment, brain imaging, and multiple tissue biopsies for molecular and cellular study simultaneously from each study participant. This infrastructure allows us to perform experiments in which molecular, anatomical, and behavioral data will be obtained from the same individuals. By utilizing this potential strength, we will address how behavior and neuroanatomical abnormalities relevant to psychotic disorders (SZ and BP currently categorized by DSM) are quantitatively associated with a specific molecular signature (phosphorylation of DISC1 in this study).

? DESCRIPTION (provided by applicant): There is a high degree of co-morbidity between drug addiction and other mental illnesses. Adolescence is a critical period for the onset of substance use disorders as well as that of mood disorders, schizophrenia, and anxiety disorders. Insults that occur during adolescent brain maturation, such as stress exposure, may enhance vulnerability to drug experimentation and on-going use and the subsequent development of addiction; similar relationships with stress exposure have been found for other mental illnesses. Persons with dual diagnoses often exhibit symptoms that are more severe, persistent, and resistant to treatment, in comparison to patients who have either disorder alone. Nonetheless, effective medications that address both conditions for these dual diagnosis (or co-morbid) populations have not been well established. There have been a limited number of preclinical models for co-morbid conditions. Given that preclinical models are useful to better understand neurobiological mechanisms of, and explore novel therapeutic strategies for brain disorders, further studies are warranted. We have recently reported a model that displays depression-associated behavioral alterations as well as molecular changes in dopaminergic pathways (Niwa et al, Science 2013): we observed mesocortical projection-specific epigenetic changes in the dopaminergic neurons in a glucocorticoid signaling-related manner. We have further expanded our preliminary study with additional data on abnormal responses to cocaine. On the basis of these two independent observations, we hypothesize that this disease model may be useful for studying the neurobiology of co-morbid cocaine addiction and depression, by making additional protocol changes in cocaine exposure and further optimization. First, we plan to establish a model that displays co-morbid phenotypes of aberrant response to cocaine and depression-relevant behaviors, and plan to use it to develop neurobiological mechanisms of co-morbid drug addiction and mental illness. Second, we will identify a way of intervening with the co-morbid conditions in the disease model with cocaine exposure. Using techniques already established in our group, we propose to study projection-specific effects of the hypothalamic-pituitary-adrenal axis on dopaminergic neurons and behavior in the model of co-morbidity. A successful completion of these studies will broaden our understanding of co-morbid conditions. Once we establish a model that displays the co-morbid phenotypes, the model will be useful for studying pathological mechanisms underlying such co-morbid conditions. The model will also provide a good template not only for screening compounds with better efficacy and fewer side effects, but also for prophylactic environmental readjustment, which is crucially important in clinical psychiatry.

Abstract (Project 3) Recent clinical studies that examine prodromal subjects and recent-onset schizophrenia (SZ) have indicated that stress-associated pathways are activated prior to and at the onset of the disease, in contrast to milder changes of the pathways in the chronic stages. In addition, human postmortem studies have demonstrated changes in dendritic spines of pyramidal neurons and parvalbumin (PV)-positive interneurons. These are key neural substrates for the excitatory-inhibitory (E-I) imbalance in prefrontal cortical (PFC) neuronal networks underlying cognitive deficits in SZ. Our preliminary data show changes in stress-associated molecules and interneurons in adolescence and young adulthood in mouse models that display altered adult behaviors relevant to SZ. These models carry genetic perturbations of microtubule-associated molecules and show mild deficits in early neurodevelopment. Based on this background, we propose the following two Aims: Aim 1 will determine and characterize the critical periods for changes in stress-associated cascades and E-I imbalance in several genetic mouse models with mild brain deficits in early development elicited by microtubule-associated genes; and Aim 2 will study the mechanisms of neurocircuitry-based behavioral changes associated with medial PFC (mPFC) and orbitofrontal cortex (OFC), such as working memory deficits and behavioral inflexibility. We will also investigate whether adolescent social isolation exacerbates the pathological signatures. Finally, we will intervene with the stress pathways in a molecule, cell type and brain region-specific manner during adolescence to try to rescue adult phenotypes (physiology, behavior). We believe the proposed study is innovative and will lead to the development of new tools for early diagnosis and intervention in cognitive deficits relevant to SZ and related mental disorders.

ABSTRACT For the past decade, several lines of evidence have shown a direct role for oxidative stress in the pathology of schizophrenia (SZ). Although peripheral changes associated with oxidative stress may be useful to establish biomarkers for the disease, connecting such peripheral changes to brain dysfunction has not yet been fully established. Furthermore, there is a need to develop high throughput assays for measuring such peripheral changes. We recently found that oxidative stress-associated endogenous autofluorescence (AF) is aberrantly augmented in SZ cells. AF is regulated by the GAPDH stress cascade, and the extent of AF is negatively correlated with cognitive flexibility evaluated by the Wisconsin Card Sorting Test. Meanwhile, we have recently found that the selectively activated GAPDH stress cascade in microglia in the prefrontal cortex is likely to mediate cognitive inflexibility in an oxidative stress-associated mouse model. We have observed that expression of Cd11b (a key factor for microglia to target to synapse) is regulated by the GAPDH stress cascade in this mouse model. Based on these promising preliminary data, we hypothesize that activation of the GAPDH stress cascade and associated altered AF triggers pathological changes in microglia, which in turn affects synaptic connectivity in the prefrontal cortex that underlies cognitive flexibility. To address this hypothesis, we propose the following three aims: 1) to establish a high throughput assay that measures cellular AF from human blood samples; 2) to identify specific cognitive domain(s) that is correlated with and predicted by augmented AF in blood cells; and 3) to identify a molecular mechanism by which the GAPDH stress cascade mediates cognitive inflexibility in an oxidative stress-associated animal model. Through these three Aims, we seek the translational potential of intervening in the GAPDH stress cascade to ameliorate cognitive deficits by using AF in blood cells as an objective high throughput marker.