title: The contribution of MAPKs to microglial immune responses, and a pathogen type comparison study to probe maternal immune activation effects on the foetal brain
Creators: name: Kwon, Jaedeok
Publisher: name: University of Glasgow
Year: 2021

description: Schizophrenia is a psychiatric disorder, and despite a rapid increase in understanding of the disease both clinically and preclinically, the aetiology of the disease remains somewhat uncertain. Inflammation is increasingly correlated with schizophrenia, with evidence from patients' serum and post-mortem brain samples. Moreover, in the brain, microglia are the primary cells to respond to immune stimulation; thus, their cellular processes in neuroinflammation in terms of contribution of schizophrenia-related pathological changes should be investigated. Regarding microglial immune responses, this study looks specifically at the mitogen-activated protein kinase (MAPK) pathways, including c-jun N-terminal kinase (JNK), because the evidence from genome-wide genetic association studies suggests that some of the genetic risk factors for schizophrenia are related to this pathway. Furthermore, as suggested from the neurodevelopmental theory, maternal stress, or infection during pregnancy, is one risk factor in the development of schizophrenia in the offspring. Indeed, Maternal Immune Activation (MIA) animal studies have suggested that prenatal immune stress could negatively affect CNS development and offspring behaviours. This thesis hypothesises that maternal immune activation caused by environmental challenges affects foetal microglial immunity via the MAPK pathway, and that differing responses are observed depending on the nature of the immune challenge. In order to examine the statement, this thesis introduces research to study the neurobiological functions of MAPKs in neuronal (primary mouse cortical cultured neurons) and microglial (SIM-A9 mouse cell line) cells, with preclinical studies of the impact of MIA in mice on immunological changes following the administration of different inflammatory agents. Results in cultured primary mouse cortical neurons showed that two different TLR agonists, LPS (TLR4 agonist), and resiquimod (TLR7/8 agonist), did not cause any significant changes in the level of activated phospho-JNKs (pJNKs); however, poly I:C (TLR3 agonist) stimulation showed potential effects on JNK activation. Interestingly, chemokine stimulation, with CXCL10 in particular, affected the level of pJNKs. Continuing the in vitro studies, microglial immune responses were investigated. Resiquimod significantly increased the activation levels of MAPKs. LPS also increased the activation levels of MAPKs; however, it required a longer time than resiquimod. Interestingly, poly I:C did not significantly increase activation levels of any MAPKs. LPS and resiquimod initiated microglial immune responses (measured by RT-qPCR and ELISA); however, protein levels did not always reflect mRNA level changes. Besides, MAPK pathway contributions to the microglial immune reactions were suggested via MAPK inhibitor experiments, but in a stimulusdependent manner. Interestingly, investigation of microglial markers, Tspo, Aif-1, and Tmem119, following TLR-mediated stimulations in microglial cells (SIM-A9) showed changes that did not necessarily reflect levels of microglial activity. Alongside these in vitro studies, MIA models, induced by dsRNA (poly I:C) and ssRNA (resiquimod) virus mimetics, were investigated. Initial data suggested that the level of CXCL12 in placental tissues was increased by maternal exposure to poly I:C, but placental CXCL10 was not affected, even though poly I:C administration meaningfully induced maternal inflammation. The data from the main MIA study suggested that administration of a TLR7/8 (resiquimod), but not a TLR3 (poly I:C), agonist, induced the upregulation of cytokine and chemokine expression in embryo brains, even though the evidence of inflammation caused by both poly I:C and resiquimod was detected in maternal serum and placentae. These findings suggest that MIA has the potential to alter foetus brain immune status and that the response could be pathogen dependent, with single-stranded RNA (ssRNA) virus exposure potentially producing greater effects on the foetus. Corresponding with the in vitro microglia experiment, microglial markers in foetal brains were changed by MIA; these findings indicated these markers are changed by environmental conditions rather than remaining stable. These data provide compelling evidence of the roles of MAPKs in microglial involved neuroinflammation, although the precise action of these signalling molecules on microglia related pathology in schizophrenia remains unclear. In addition to previous works, a significant impact of maternal ssRNA virus (resiquimod) exposure on the developing foetal brain, but the doublestranded RNA (dsRNA) (poly I:C) virus did not. This means that resiquimod is potentially more effective than imiquimod or poly I:C in terms of producing an immune response; therefore, an MIA model using resiquimod may be a good model for the study of environmental contribution to psychiatric disease risk. Further work into the interaction between environmental and genetic factors in the MIA, and associated behaviour changes at later developmental ages, will provide insight into how maternal immune reactions and foetal CNS immune reactions are related to foetal brain development.