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<p><strong>Purpose:</strong> Microglia are the innate immune cells of the central nervous system. Activated microglia release nitric oxide, glutamate, and superoxide radicals, which are harmful to retinal ganglion cells (RGCs). They may also benefit surviving cells by removing toxic cellular debris or by secretion of neurotrophic factors. The paradoxical role of microglia remains controversial because the nature and time-course of the injury that determines whether microglia acquire a neuroprotective or pro-inflammatory phenotype is unknown. HAPI cells are an immortalized microglial cell line, whose phenotype can be manipulated <em>in vitro</em>. It is my HYPOTHESIS that pharmacological manipulation of microglia to acquire either a pro-inflammatory or pro-survival phenotype will exacerbate neuronal cell death or enhance neuronal survival after injury, respectively.</p> <p><strong>Method:</strong> Lipopolysaccharides (LPS) were used to hyper-stimulate the HAPI cells and minocycline to maintain the HAPI cells in a quiescent state. Prior to the experiments, the HAPI cells were labelled with Wheat Germ Agglutinin conjugated to Texas Red. The HAPI cells were cultured and exposed to minocycline (10 µg/mL for 1 hour) or LPS (1 µg/mL for 24 hours). Sprague-Dawley rats then recieved intraocular (30,000 cells) or tail vein (5 million cells) injections of either the minocycline treated HAPI cells or the LPS treated HAPI cells and an optic nerve crush. Retinas were examined at 4-14 days later and the number of surviving RGCs will be determined by Brn3a labelling of RGCs. BM88 antibody labelling was done to determine the severity of the injury and to determine molecular changes after neuroinflammation.</p> <p><strong>Results: </strong>Injection of untreated HAPI cells resulted in the greater loss of RGCs early after ONC when injected into the vitreous and later after ONC when injected into the tail vein. LPS activated HAPI cells injected into the vitreous resulted in greater RGC loss with and without injury. When injected into the tail vein with ONC there was no loss of RGCs 4 days after ONC but later there was greater loss of RGCs. Minocycline treated HAPI cells injected into the vitreous resulted in greater RGC survival than when untreated HAPI cells were injected. However, when injected into the tail vein with ONC there was greater loss of RGCs. There was also BM88 down regulation after injury and this was more pronounced after HAPI cell injection.</p> <p><strong>Conclusion:</strong> Neuroprotection or cytotoxicity of microglia depends on the type of activation, time course of the injury, and if the microglia act on the axon or cell body of the retinal ganglion cell.</p> / Doctor of Philosophy (PhD)

Major depressive disorder (MDD) is a highly heterogeneous disorder, with some patients displaying immune activation and altered intestinal microbiota composition when compared to healthy controls. In recent years, the transfer of fecal microbiota pooled from several MDD patients has been used to model depression in recipient rodents. However, we have previously observed the induction of donor-specific phenotypes in mice receiving microbiota from individual irritable bowel syndrome and generalized anxiety disorder patients. Therefore, we assessed the efficacy of fecal microbiota transplant (FMT) using individual versus pooled MDD patient microbiota to induce depressive-like behaviour in recipient rodents. We observed that pooling microbiota from several patients abrogated microbial features unique to individual donors. Mice that received pooled microbiota displayed different behavioural and immune phenotypes when compared to mice that received individual patient microbiota. Two individual MDD microbiota donors, patients MDD1 and MDD5, altered the behaviour of recipient mice when compared to controls. We identified several microbial species that may underlie the anxiety- and depressive-like behaviours observed in MDD1 and MDD5 mice. Additionally, altered expression of neural and immune genes was observed along the gut-brain axis of mice colonized with MDD1 microbiota. As microglia activation may play a role in our model, we developed a protocol for the isolation and phenotyping of adult mouse microglia that will facilitate future research efforts. Overall, our results demonstrate the heterogeneity of the microbial underpinnings of MDD and support the use of individual patient microbiota in future FMT experiments. / Thesis / Master of Science (MSc)

Advanced stage tumors can acquire the ability to divide uncontrollably, invade the surrounding matrix, and circulate through the bloodstream or lymphatic system to distant organs in a process known as metastasis. The brain, which is shielded from the environment by the blood brain barrier, offers an immunocompetent lodging spot for the circulating cancer cells. Therefore, it is a "popular" destination for metastasized cancers which even surpass the incidents of primary brain tumors. It is hypothesized that the disseminated cancer cells engage with the host cells of the perivascular neural niche in a poorly understood crosstalk of molecular factors, that in turn augment the metastatic colonization of cancer cells. A better understanding of this crosstalk is indispensable to apprehending the complexity of the metastasis process, and to facilitating the discovery of biomarkers that predict metastatic potential and improve patient prognosis. The larger goal of this study was to adopt a mass spectrometry-based systems biology approach to investigate the molecular mechanisms and regulatory networks that underlie the complex phenomenon of breast cancer propagation at the brain metastatic site. To achieve this, the study was divided in three sub-projects designed around the following objectives, i.e., (a) to comprehensively characterize the protein landscape of the neural niche or the brain microenvironment comprised of astrocytes, microglia and endothelial cells, (b) to explore the immunological protein networks activated in microglia cells upon stimulation with anti-inflammatory cytokines released by tumor cells in the brain, and (c) to investigate the protein-level changes elicited in HER2+ breast cancer cells when grown under conditions that simulate the brain microenvironment in-vitro. Detailed characterization of the neural niche enabled us to propose molecular mechanisms that allow for the seeding and outgrowth of metastasized cancer cells in the brain. The study further provided novel insights into the signaling networks that regulate the immune functions of the microglia and their role during cancer development. Lastly, an in-depth investigation of breast cancer cells cultured in the presence of neural niche factors revealed potential novel mechanisms of cancer cell dormancy during metastasis. Altogether, large-scale proteomics data generated in this work will help clarify the mechanisms of metastatic cancer development, and will lay the groundwork for future studies that aim at the discovery of novel biomarkers and druggable targets for the treatment of brain metastatic cancers. / Doctor of Philosophy / In the US, the incidence of breast cancer ranks only second to lung cancer, and the primary cause for almost all cancer related deaths is the development of cancer metastasis in patients. The process of metastasis involves cancer cells leaving the primary tumor, entering the bloodstream or the lymphatic system, and spreading to other parts of the body during advanced stages of the disease. The brain is a common metastatic site for cancer cells to form a secondary tumor. Previous research has found that it is the brain cells which support the progression of secondary tumors in the central nervous system by releasing protein molecules that favor the survival and growth of disseminated cancer cells. Brain cells, including endothelial cells (that form the blood vessels), astrocytes (that regulate brain development), and microglia (that are the immune cells of the brain), are the first to respond to metastasized cancer cells. A better understanding of the behavior of these cells and of their signaling molecules which support the process of cancer metastasis can help us discover new drug targets to treat cancer patients. This study had three main objectives, i.e., (a) to profile the protein make-up of brain cells and identify specific proteins that support cancer development in the brain, (b) to investigate the changes in microglial proteins when these cells are exposed to conditions that simulate cancerous growth in the brain, and (c) to explore the proteins that become active in breast cancer cells when they are subjected to conditions that simulate the brain microenvironment. To accomplish this, we used a high-throughput technology called mass spectrometry, that allows for the identification of thousands of proteins in a sample at any given time. Overall, the study provided novel insights into the biological mechanisms of secondary tumor formation, and into the early response of breast cancer cells when they encounter unfamiliar conditions in the brain. The work further supports the discovery of specific proteins that can be targeted in anti-cancer therapies.

Neurodevelopmental disorders, such as autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder, are a heterogeneous set of developmental disorders affecting the central nervous system. Studies into their etiology remain challenging, as neurodevelopmental disorders frequently present with a wide range of biological, behavioral, and comorbid symptomologies. Increasing epidemiological reports of antibiotic use during pregnancy as a significant correlate of subsequent mental disorder diagnosis in children suggest a mechanism of influence via the maternal gut-fetal brain axis. Importantly, antibiotics cause dysbiosis of the gut microbiome and disrupt the delicate composition of the microbial inoculum transferred from mother to child, which is critical for development of the immune system and holds implications for long-term health outcomes. The research objective of this dissertation is to reveal a causal mechanism of maternal microbial influence on neurodevelopment by examining the brain's resident immune cells, microglia, and corresponding behavioral outcomes in a mouse model of antibiotics-driven maternal microbiome dysbiosis (MMD). We identify early gross motor deficits and social behavior impairments in offspring born to MMD dams, which paralleled hyperactivated microglia in brain regions specific to cognition and social reward. The MMD microglia also exhibited altered transcriptomic signatures reflective of premature cellular senescence that support evidence of impaired synaptic modeling found in MMD brains. We report that these deficits are rescued in the absence of Cx3cr1, a chemokine receptor expressed ubiquitously on microglia, to highlight a pathway in which maternal microbiota may signal to neonatal microglia to undergo appropriate neurodevelopmental actions. Finally, we characterize Lactobacillus murinus HU-1, a novel strain of an important gut bacterium found in native rodent microbiota, and demonstrate its use as a probiotic to restore microglial and behavioral dysfunction in MMD offspring. / Ph. D. / Population studies on neurodevelopmental disorders, such as autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder, highlight antibiotic use during pregnancy as a major correlate of subsequent diagnoses in children. These findings support a growing body of evidence from animal and human studies that the microbial ecosystems (“microbiome”) found in and on our bodies play significant roles in mental health, including mood, cognition, and brain function. Importantly, antibiotics during pregnancy create an imbalance of the gut microbiome (“dysbiosis”) and disrupt the microbial inoculum transferred from mother to child, which is critical for maturation of the infant immune system and holds implications for long-term health outcomes. Thus, the research objective of this dissertation is to identify a mechanism of influence from the mother’s gut to the neonate’s brain by examining the brain’s resident immune cells (“microglia”) in a mouse model of antibiotics-driven maternal microbiome dysbiosis (MMD). We uncover autism-like behavioral deficits and dysfunctional microglia in MMD offspring, and characterize signaling cues specific to microglia by which improper neurodevelopment may be taking place. We also reveal that the detrimental effects of MMD are reversed in mice born to mothers pretreated with a probiotic candidate, Lactobacillus murinus HU-1, to suggest maternally-derived Lactobacillus may help to mediate proper neurodevelopment.

The placenta plays a vital role in a healthy pregnancy by supporting the intricacies of fetal development. Over 10% of pregnancies experience impaired placental function, resulting in the loss of critical neuroactive steroids the fetal brain cannot yet make, thus leaving them vulnerable to perinatal brain injury and abnormal neurodevelopment. However, this vulnerability is not always equal. Many neurodevelopmental disorders exhibit a sex bias in incidence and severity. I hypothesize that loss of placental support during pregnancy results in sex differences in both behavioral presentation as well as on the cellular and transcriptomic levels. Utilizing the akr1c14cyp19aKO (plKO) mouse model, which features placenta-specific allopregnanolone (ALLO) knockdown, I investigated the sex specific impact of placental hormones on cerebellar development. Here I show that placental ALLO is essential for cerebellar white matter development and inflammatory regulation via microglial function. Male mice without placental ALLO exhibit signs of placental inflammation, accelerated postnatal myelination, and defects in microglial phagocytosis of excess myelin. Alternatively, females seem to be more resilient with a progressive anti-inflammatory profile across development and reduced myelination. Additionally male plKO show autism-like behaviors such as deficits in social behavior and increased stereotyped behavior. The females do not exhibit this phenotype. My main goals were threefold; to investigate how male and female inflammatory profiles differ and where this difference originates, to investigate how this inflammation impacts microglia and thereby oligodendrocytes, and how I can alter microglial function in a way to improve plKO outcomes. Mechanistically, these changes appear to be in part due to baseline sex differences in response to inflammatory stimuli which prime microglia to differentially support the surrounding white matter. Together, this work supports a novel link between placental ALLO loss, microglial function, and sex specific presentation of neurodevelopmental disorders.