Shinichi Kano, MD, PhD - US grants

DESCRIPTION (provided by applicant): Disturbance in brain development during adolescence may underlie schizophrenia (SZ). Although many reports showed that astrogliosis is not observed in SZ, accumulating evidence suggests that altered function of astrocytes and microglia as well as aberrant immune/inflammatory responses may also underlie SZ. Nonetheless, it is unclear whether or to what extent glial cells and inflammation are involved in altered brain development during adolescence. Recently, we have found that the expression of immune/stress related genes is altered in live neuronal cells derived from SZ patients. The most affected genes include glutathione S-transferase theta 2 (GSTT2) gene, which regulates cellular detoxification system and protects cells from reactive oxygen metabolites. Oxidative stress activates innate immune signaling and contributes to inflammation in various diseases such as diabetes, atherosclerosis, and neurodegenerative disorders. Indeed, we observed increased expression of proinflammatory cytokines by oxidative stress. In the proposed study, we will test the hypothesis that glial cell activation and inflammatory responses during adolescence contribute to altered development of glutamatergic synapses. We will perform in vivo knockdown of GSTT2 expression at specific developmental stages in mice as a model to induce glial cell activation and inflammation via increased oxidative stress. We will characterize the effects of knockdown on the activation of microglia and astrocytes as well as the production of proinflammatory cytokines. We will also assess the effects of knockdown on development of glutamatergic synapses and the expression of synaptic/dendritic immune molecules. Finally, we will address the role of innate immune signaling in microglia by using microglia-specific deletion of MyD88, a molecule that plays a central role in innate immune signaling. The training and research proposal will enable the candidate to develop into an independent investigator in neuropsychiatry research. The project will contribute to the understanding of the roles for glial cells and inflammation in altered brain development during adolescence relevant to SZ. PUBLIC HEALTH RELEVANCE: Although glial cell dysfunction and altered immune/inflammatory status have been suggested in patients with schizophrenia (SZ), it is not clear how they contribute to the pathology of SZ. In this study, we will characterize the role of glial cell activation and inflammatory response in altered brain maturation during adolescence. By utilizing our recent findings that SZ neurons have intrinsic susceptibility to oxidative stress, we will provide a model mechanism how SZ-associated neuronal defects lead to altered development of glutamatergic synapses via glia-mediated inflammatory responses. The study will contribute to the further understanding of altered development of glutamatergic synapses as well as identification of novel therapeutic target(s) that enables early intervention treatment for SZ.

ABSTRACT Adaptive immune cells in the periphery (T and B cells) and innate immune cells in the brain (microglia) have been implicated in the brain homeostasis in health and disease. Rodent studies using immunodeficient mice have revealed that the loss of adaptive immune cells (T and B cells) led to impaired learning and memory, anxiety-like behaviors, and impaired sociability. Nevertheless, it is not clear how adaptive immune cells communicate with microglia and affect brain development and function. Our long-term goal is to understand the molecular and cellular mechanisms underlying the communication between adaptive immune cells and brain cells during brain development and in adulthood. Our preliminary studies revealed that Rag1-/- and [Rag2- /-mice], lacking both T and B cells, exhibited impaired social behaviors. In Rag1-/- mice, increased c-Fos expression and altered microglial phenotypes in the medial prefrontal cortex (mPFC) were observed. This is consistent with previous reports that mPFC dysfunction is involved in social behaviors. [Notably, adoptive transfer of wild-type (WT) splenocytes (containing T and B cells) rescued Rag1-/- social behavioral deficits. Further, injection of WT serum exosomes rescued the same phenotype. The social behavioral deficits were also observed in Rag2-/- mice despite the fact that Rag2 is normally absent in the WT brain. Together, these findings suggest that T and B cells contribute to social behaviors via exosomes.] Indeed, we observed that exosomes from the sera of Rag1-/- mice lacked the expression of T and B cell markers and multiple microRNAs (miRNAs) presumably derived from T and B cells. The expression of predicted target gene(s) of these miRNAs, such as Ski, was enhanced in the PFC of Rag1-/- mice. In contrast, WT serum exosomes decreased Ski expression in microglia. Recent studies showed that microglia control neuronal synapses. Thus, our data suggest that deficient adaptive immune cell-microglia communication via exosomes impairs social behaviors by altering mPFC function. Hence, in this study, we will test our hypothesis that the lack of adaptive immune cell-derived exosomes and their miRNAs results in impaired social behaviors via altered microglial control of neuronal function in the medial PFC. We will first validate and extend our findings on serum exosomes and the mPFC neurons in Rag1-/- mice, and determine the causal role for the lack of adaptive immune cells by restoring them back into Rag1-/- mice with adoptive transfer technique (Aim 1). We will also examine the direct impact of impaired exosome release and miRNA production in adaptive immune cells on microglia and neurons in the mPFC and social behaviors by genetic approaches (Aim 2). [In addition, we will address the contribution of pyramidal neurons and microglia in the mPFC to impaired social behaviors (Aim 3).] This study will reveal novel mechanisms whereby adaptive immune cell-derived exosomes influence brain function and behavior and may eventually lead to novel therapeutic strategies in psychiatric disorders.

ABSTRACT First-episode psychosis (FEP) represents a critical stage of illness during which therapeutic interventions are believed to effectively influence long-term outcomes. Nonetheless, it is not clear what biological changes in the brain underlie FEP and subsequent disease progression. Moreover, there are no established molecular biomarkers that reflect psychosis and/or predict their longitudinal outcomes. Extracellular vesicles (EVs) are cell-derived microvesicles that contain various cellular components, such as nucleic acids, proteins, and metabolites, from the donor cells and, by fusing with other cells, transfer these components between cells in a paracrine and endocrine manner. An increasing body of evidence shows that EVs in the circulation may be useful in detecting various brain disorders at early stage and predicting their clinical outcomes. In this study, our goal is to evaluate the utility of EVs in the peripheral blood as molecular biomarkers to predict FEP and their subsequent progression. Our preliminary studies with a small cohort of FEP patients and controls suggested that microRNAs (miRNAs) were differentially expressed in plasma EVs derived from FEP patients. In parallel, our analysis of longitudinal changes in clinical/neurocognitive data of FEP patients identified potential subgroups. In the proposed study, we will test our hypothesis that peripheral blood EV-associated miRNAs (EV-miRNAs) will display changes reflecting clinical/neurocognitive data in FEP patients and their subgroups. We will first extend our preliminary findings on differential plasma EV-miRNA expression in FEP patients in a larger size of FEP patient and control samples, and then compare their profiles with those from CSF EV (Aim 1). We will also examine the utility of EV-miRNAs to distinguish FEP patient subgroups by combining unsupervised clustering of FEP patients based on longitudinal clinical/neurocognitive data changes and their plasma EV-miRNA data (Aim 2). This study will generate an unprecedented dataset of altered EV- miRNAs in the body fluids of FEP patients whose multimodal data are readily available. The findings will create a foundation for future studies to determine the utility of EV-miRNAs to predict long-term consequences of disease and potentially evaluate responsiveness to therapeutic interventions.