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Microglia are a unique type of immune cell found within the brain, spinal cord and retina. In the healthy brain, their job is to support neurons, defend against infectious microbes, clear extracellular debris and remove dead or dying cells through phagocytosis. This diverse array of functions presents the possibility of unique subsets of microglia existing in the healthy brain, yet none have been described thus far. By utilizing cellular autofluorescence as a discriminating characteristic, we identified two novel subsets of microglia present in the healthy brains of mice and non-human primates. Approximately 70% of microglia displayed autofluorescence (AF+) while the remaining 30% did not (AF–). While the proportion of AF+ and AF– microglia remained constant throughout most of adult life, the autofluorescence intensity increased exclusively in the AF+ subset at an almost linear rate with age. This gain in autofluorescence correlated with equivalent increases in the size and complexity of storage bodies, as detected by transmission electron microscopy and increases in LAMP1 levels, a key component of the lysosomal compartment. As the brain ages, lysosomal storage material builds up inside AF+ microglia, further increasing the accumulation of autofluorescence as a result. The analysis of protein content in autofluorescent subsets revealed that AF+ microglia produced more proteins and enzymes involved in the storage and degradation of waste material, as well as more proteins involved in the regulation of mTOR, a key cellular pathway governing nutrient availability and energy production. Interestingly, the disruption of lysosomal function in microglia through genetic mutations accelerated the accumulation of storage material in AF+ cells, which led to impaired microglia physiology and increased cell death, mimicking the effects observed during advanced aging. Increasing evidence suggests that the accumulation of waste materials inside the brain contributes to diseases of aging and these data are suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

Microglia, the resident immune cells of the brain, play a critical role in brain homeostasis and neurological disease progression. In neurodegenerative diseases, microglia acquire a neurodegenerative phenotype (MGnD), the function of which is poorly understood. MicroRNA-155 (miR-155), a multifunctional microRNA enriched in cells of the immune system, and Apolipoprotein E (APOE), a lipoprotein which is significantly associated with Alzheimer’s disease (AD) risk, critically regulate MGnD. However, the role of these molecules in AD pathogenesis remains unclear. Here, we report that microglial deletion of miR-155 induces an early MGnD response activation state via interferon-ɣ (IFNɣ) signaling in mice. This phenotypic transition increases plaque-associated Apoe, enhances amyloid plaque compaction, reduces neuritic dystrophy and attenuates plaque-associated synaptic degradation, resulting in improved cognition. These findings provide a novel mechanism detailing the phenotypic switch from homeostatic microglia to MGnD, and highlight the beneficial role of IFNɣ responsive MGnD in restricting neurodegenerative pathology and preserving cognitive function in a mouse model of AD. In addition, we demonstrate that Apoe deficient microglia induce an MGnD signature comparable to controls, but enhance MGnD physiological phenotypes including enhanced cognitive behavioral performance and reduced plaque associated neuritic dystrophy. Furthermore, we highlight a potential mechanism by which Apolipoprotein C-1 may attenuate synaptic ß-amyloid accumulation in a mouse model of AD. These findings may serve as the basis novel immunomodulatory therapies targeting microglial miR-155 and APOE to treat AD.

Hypertension (HTN) is a critical public health issue estimated to contribute to 10% of deaths worldwide. Additionally, the salt sensitivity of blood pressure, an exaggerated pressor response to elevated dietary sodium intake, is estimated to be present in approximately 50% of the hypertensive population and 25% of the normotensive population. This is a critical problem as the average American consumes roughly three times the daily sodium intake recommended by the American Heart Association. Our laboratory has previously identified a critical role of Hypothalamic Paraventricular Nucleus (PVN) Gαi2 proteins in the maintenance of salt resistance and normotension in the rat. Salt resistant rats such as the Sprague-Dawley (SD) rat site- specifically upregulate these proteins in response to elevated dietary sodium intake to facilitate sympathoinhibition, natriuresis, and normotension. In contrast, in the Dahl Salt Sensitive (DSS) rat, and in salt resistant rats in which this protein is experimentally downregulated, our laboratory has identified the development of renal nerve-dependent sympathoexcitation and salt-sensitive hypertension (ssHTN). However, the neural mechanisms whereby PVN Gαi2 proteins facilitate salt resistance are unclear. In addition, there is a robust literature in other rat models of HTN suggesting that both neuroinflammation in the PVN as well as an imbalance between PVN inhibitory GABAergic and excitatory glutamatergic signaling contribute to elevations in sympathetic outflow to promote HTN. In this study, SD rats infused chronically with either targeted Gαi2 oligodeoxynucleotides (ODNs) or control scrambled (SCR) ODNs and challenged with either normal (0.6% NaCl) or high-salt (4% NaCl) diets were used to demonstrate that 1) PVN microglial activation and associated pro-inflammatory cytokine production contribute to the development of Gαi2 protein dependent ssHTN, 2) sex-dependent PVN microglial-mediated neuroinflammation precedes and likely drives the development of sympathoexcitation following high dietary sodium administration in male but not female Gαi2 protein dependent ssHTN, and 3) PVN GABAergic and glutamatergic signaling is disrupted and imbalanced, favoring excitation over inhibition, following elevated dietary sodium intake in Gαi2 protein dependent ssHTN. Together, these findings shed light on the pathological neural processes that occur in the absence of PVN Gαi2 protein upregulation and reveal potential mechanistic targets in the management of ssHTN.

no / Cryptolepine, an indoloquinoline alkaloid in Cryptolepis sanguinolenta, has anti-inflammatory property. In this study, we aimed to evaluate the effects of cryptolepine on lipopolysaccharide (LPS)- induced neuroinflammation in rat microglia and its potential mechanisms. Microglial activation was induced by stimulation with LPS, and the effects of cryptolepine pretreatment on microglial activation and production of proinflammatory mediators, PGE2/COX-2, microsomal prostaglandin E2 synthase and nitric oxide/iNOS were investigated. We further elucidated the role of Nuclear Factor-kappa B (NF-κB) and the mitogen-activated protein kinases in the antiinflammatory actions of cryptolepine in LPS-stimulated microglia. Our results showed that cryptolepine significantly inhibited LPS-induced production of tumour necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-1beta (IL-1β), nitric oxide, and PGE2. Protein and mRNA levels of COX-2 and iNOS were also attenuated by cryptolepine. Further experiments on intracellular signalling mechanisms show that IκB-independent inhibition of NF-κB nuclear translocation contributes to the anti-neuroinflammatory actions of cryptolepine. Results also show that cryptolepine inhibited LPS-induced p38 and MAPKAPK2 phosphorylation in the microglia. Cell viability experiments revealed that cryptolepine (2.5 and 5 μM) did not produce cytotoxicity in microglia. Taken together, our results suggest that cryptolepine inhibits LPS-induced microglial inflammation by partial targeting of NF-κB signalling and attenuation of p38/MAPKAPK2.

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Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage cancer for the majority of patients. The work presented in this dissertation addresses the multifaceted role of glutamate in cancer cell-induced pain signalling and provides several potential therapeutic directions. Several cell types, including breast cancer cells and microglia, release glutamate via the system xC- antiporter. To limit the excitotoxic tendency of breast cancer cells to release glutamate in excess, we first indirectly inhibited xCT, the active subunit of system xC-, with the TrkA inhibitor AG879. We demonstrated that the system xC- antiporter is functionally influenced by the actions of nerve growth factor on its cognate receptor, TrkA, and that inhibiting this complex reduced CIP via downstream actions on xCT. Co-culture studies then demonstrated the direct effect of glutamate released by wildtype MDA-MB-231 carcinoma cells on microglial activation, as well as functional system xC- activity, while knockdown of xCT in MDA-MB-231 cells mitigated microglial activation and cystine uptake. Blockade of system xC- with sulfasalazine attenuated nociception in an immunocompetent murine model of CIP and inhibited tumour-induced microglial activation in the dorsal horn of the spinal cord. Finally, tumour-induced nociceptive behaviours appeared to progress in parallel with microglial activation in the hippocampus, and ablating microglia delayed the onset and severity of tumour-induced nociceptive behaviours, confirming that microglia are implicated in CIP and regional microglia are influenced by this pain. This is the first experimental evidence to demonstrate the effects of peripheral tumour on hippocampal microglial activation in relation to cancer-related nociception. These data collectively demonstrate that the system xC- antiporter is functionally implicated in CIP and may be particularly relevant to pain progression through spinal microglia. Upregulated xCT in chronically activated microglia may be one pathway to central glutamate cytotoxicity. Therefore, microglial xCT may therefore be a valuable target for mitigating CIP. / Thesis / Doctor of Philosophy (Medical Science) / Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage metastatic cancer. Clinically, achieving analgesia often comes at the expense of patients’ quality of life, as current therapeutics fail to adequately manage this pain and induce dose-dependent side effects. Cancer cells secrete excess amounts of glutamate, a signalling molecule involved in CIP, which can activate immune cells called microglia within the spinal cord. Mice that demonstrate tumour-induced pain exhibit an amplified immune response that manifests through the activation pattern and quantity of microglia within the spinal cord, as well as brain regions implicated in pain and distress. Pharmacologically blocking glutamate release from cancer cells limits this pain response, in addition to several physiological indicators of pain, including microglial activation in the central nervous system. Changes in microglia-related glutamate signalling may reflect the emotional problems reported by patients with CIP. Better understanding the mechanisms of CIP will help generate more comprehensive treatment approaches.