Microglia are essential to the brain’s innate immune response and play a vital role in neuropathology related to tauopathies. Understanding how microglia change in response to differential expression of RNA binding protein T-cell intracellular antigen 1 (TIA1) will lend insight to microglial function in tauopathy. In preliminary studies our laboratory has shown that decreasing the expression of TIA1 has an inverse and dose dependent effect on activated microglial density in the dentate gyrus of the P301S mouse (PhD Candidate Chelsey LeBlang, 2018). Here, we utilized serial sectioning electron microscopy to define whether this relationship between TIA1 level and microglia remains consistent in the hilus and granule cell layer (GCL) of the dentate gyrus. Our analyses of microglial volume and microglial interactions within the neuropil have yielded four conclusions. First, the hilus, but not the GCL, exhibited a significant decrease in microglial volume per volume of tissue with the knock out and heterozygous expression of TIA1. Second, the number of appositions on microglia steadily increased on AT8+ and AT8- presynaptic and postsynaptic appositions in the hilus with decreasing TIA1 expression. Third, with the exception of one AT8- somatic apposition, the surface area of microglia apposing AT8+ somata is greater than any other structure and exhibits a dose dependent decrease with decreasing TIA1 expression. Fourth, in the GCL there is a larger fractional surface area of AT8- presynaptic and AT8+ postsynaptic structures when compared to their respective synaptic counterparts. Though not entirely consistent with previous data, this study has important implications for microglial function in tauopathy and related diseases.

Nicotinic acetylcholine receptor (nAChR) activation is neuroprotective and nicotine is a cognitive enhancer. Loss of nAChRs, deposition of tau neurofibrillary tangles, cleavage of amyloid precursor protein (APP) and inflammation are well documented in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β- and γ-secretase enzymes generates soluble Aβ peptides, with oligomeric forms of Aβ implicated in both the control of synaptic excitability and dysregulation of synaptic transmission and induction of neuronal death in AD. Aβ production is inhibited by calcium-dependent recruitment of α-secretase, as exemplified by activation of N-methyl-D-aspartate receptors (NMDAR). All neurodegenerative diseases are associated with inflammation, arising from altered homeostasis of the innate immune system, resulting in heightened activation of immune cells and induction of a pro-inflammatory environment. Stimulation of the α7 subtype of nAChR is anti-inflammatory and also enhances cognition and promotes neuronal survival. This work addressed the hypotheses that stimulation of highly calcium-permeable α7nAChR inhibits Aβ production by promoting α-secretase-mediated processing of APP and also modulates inflammatory cellular behaviour of microglia. Thus, this study assessed the role of α7nAChR at glutamatergic synapses, through probing effects on APP processing and phagocytosis in primary cortical neurons and microglia, respectively. Primary cortical neurons expressed functional α7nAChR and glutamate receptors, and through a number of experimental approaches, including immunoblotting and a cleavage reporter assay, results indicated α7nAChR activation with the α7nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596 had no effect on APP and Tau, in contrast to NMDAR activation that significantly modulated these proteins. Data suggest low expression of α7nAChR, coupled with distinct localisation of presynaptic α7nAChR and postsynaptic APP could explain the lack of effect. In addition, primary microglia were highly responsive to lipopolysaccharide and possessed functional α7nAChR that coupled to ERK phosphorylation. Microglial α7nAChR activation promoted neuroprotective phagocytic behaviour, in agreement with the ‘cholinergic anti-inflammatory pathway’. This study supports the hypothesis that α7nAChR are modulators of anti-inflammatory behaviour, thus α7nAChR-selective ligands are viable candidates for the treatment of AD and promoting cognitive enhancement.

Aging is associated with a loss of cognitive function related to learning, memory, and executive function with varying severity. Although there is no age-related loss of neurons in healthy aging, myelin damage accumulates and is associated with cognitive decline. The brain’s resident macrophages, microglia, are responsible for clearing damaged myelin and promoting subsequent oligodendrocyte-mediated remyelination. To test the hypothesis that age-related dysfunction of microglial phagocytosis and oligodendrocyte remyelination capacity contributes to myelin pathology and cognitive impairment. To test this, rhesus monkeys from across the lifespan (7-30 years of age) were tested in three specific aims. 1) To characterize gene expression of myelin basic protein (MBP) in the brain and clearance of MBP to the cerebrospinal fluid (CSF) in relation to age-related myelin pathology. The density of myelinated axons visualized using label-free spectral confocal reflectance imaging did not correlate with age, but was significantly lower in aged animals with cognitive impairment. Next, MBP gene expression was measured using qPCR in the dorsal prefrontal cortex along with quantification of MBP protein levels in the CSF using ELISA. Age-dependent increases of MBP gene expression in the brain and MBP protein levels in the CSF were observed. Interestingly, MBP levels in the CSF were lower in animals with cognitive impairment. 2) To test the hypothesis that microglia would become increasingly primed for phagocytosis with age-related myelin pathology. The number of microglia immunostained with galectin-3, a marker for phagocytic activation, was quantified in the frontal white matter and increases in both aging and cognitive decline were detected. 3) To evaluate the hypothesis that lipofuscin, an age-related accumulation indicative of autophagic dysfunction, would accumulate and impair glial cells of the white matter in aged animals. Lipofuscin accumulation was increased with age in the frontal white matter and the size of lipofuscin clusters was associated with cognitive impairment. Lipofuscin was found primarily in microglia and oligodendrocytes, but not in astrocytes. These data suggest that lipofuscin burden in microglia and oligodendrocytes inhibits their homeostatic functions resulting in improper myelin clearance and turnover, leading to a devastating feed-forward cycle of myelin damage that contributes to age-related cognitive impairment.

A disfunção do sistema imune inato e a neuroinflamação tem sido cada vez mais reconhecidas como elementos importantes na fisiopatologia do transtorno bipolar (TB). Como componentes essenciais da imunidade inata, os macrófagos tem múltiplas funções tanto na inibição como na promoção da proliferação celular e na reparação tecidual, sendo a diversidade e a plasticidade características marcantes deste tipo celular. A polarização M1 clássica e a polarização alternativa M2 de macrófagos representam dois extremos de um estado dinâmico na mudança da ativação dos mesmos. Os macrófagos do tipo M1 sintetizam citocinas próinflamatórias que inibem a proliferação de células circundantes e danificam tecidos, enquanto os macrófagos do fenótipo M2 liberam citocinas antiinflamatórias que podem promover reparo tecidual. Um desequilíbrio da polarização M1-M2 dos macrófagos é frequentemente associado a várias doenças ou condições inflamatórias. O objetivo desta tese foi, além de revisar a importância da inflamação sistêmica na modulação da resposta inflamatória da microglia/macrófagos e consequentemente seu potencial envolvimento na fisiopatologia do TB, avaliar o perfil de polarização M1/M2 em cultura de macrófagos de sujeitos com TB comparados a indivíduos saudáveis. Monócitos foram isolados a partir de sangue periférico de dez sujeitos com TB e dez indivíduos saudáveis e diferenciados em macrófagos através da adição de fator estimulante de colônia de macrófagos (MCSF) ao meio de cultura. Para induzir a polarização M1 ou M2, as culturas foram incubadas com IFN-y e LPS ou IL-4 respectivamente. Após a incubação, recolheram-se os sobrenadantes e mediram-se as citocinas (IL-1β, IL-6, IL-10 e TNF-α) por ensaio multiplex. A secreção das citocinas IL-1β, TNF-α e IL-6 características do protótipo M1 e citocinas IL-10 do protótipo M2 foram semelhantes entre os pacientes e os controles. Utilizou-se a razão TNF-α / IL-10 do fenótipo M1 para refletir o estado inflamatório dos participantes. Não foi observada diferença entre os grupos (p=0,627). Duas hipóteses diferentes poderiam explicar esses resultados: todos os pacientes incluídos neste estudo representam um estágio inicial da doença como evidenciado pela pontuação FAST total inferior a 11. De acordo com o modelo de estadiamento em TB, as alterações biológicas (incluindo a inflamação) parecem estar relacionadas com os episódios de humor e progressão da doença. Juntamente com estudos anteriores, os nossos dados sugerem que os pacientes nos estágios iniciais ainda preservam a função do sistema imunológico sem apresentar um desequilíbrio a favor do perfil de macrófagos M1 como tem sido observado em pacientes no estágio tardio, destacando a relevância da intervenção precoce no TB. Ainda, estes pacientes estavam em tratamento com estabilizadores de humor e é plausível especular que esses fármacos exerçam efeitos sobre a polarização de macrófagos. Estudos futuros em pacientes drug-free são essenciais para avaliar esta questão. Em conclusão, nossos achados sugerem que os pacientes TB não apresentam desequilíbrio na polarização dos macrófagos em favor do fenótipo pró-inflamatório M1. O fato de todos estes pacientes estarem em estágios iniciais da doença reforça os efeitos protetores da intervenção precoce no TB na prevenção de alterações do sistema imune e, consequentemente, na progressão da doença. / Innate immune system dysfunction and neuroinflammation have been recognized as important elements in the pathophysiology of bipolar disorder (BD). As essential players of innate immunity, macrophages have multiple roles in inhibition and promotion of cell proliferation and tissue repair. The classical M1 polarization and the M2 alternative polarization of macrophages represent two extremes of a dynamic state in their change of activation. M1 macrophages synthesize proinflammatory cytokines that inhibit the proliferation of surrounding cells and damage tissues, whereas macrophages of the M2 phenotype release antiinflammatory cytokines that may promote tissue repair. An imbalance of the M1-M2 polarization of macrophages is often associated with various diseases or inflammatory conditions. The aim of this thesis was to review the importance of systemic inflammation in modulating the inflammatory response of microglia/ macrophages and consequently their potential involvement in the pathophysiology of BD, and also evaluate the M1/M2 polarization profile in macrophages of patients with BD compared to healthy individuals. Blood monocytes were obtained from ten BD patients and ten healthy controls. These cells were activated/polarized into the M1 (IFNγ + LPS) or M2(IL-4) phenotype. Supernatants were collected and the cytokines (IL-1β, IL-6, IL-10 and TNF-α) were measured by multiplex assay. Secretion of the IL- 1β, TNF-α, IL-6 and IL-10 were similar between patients and controls. The TNF-α/IL- 10 ratio of the M1 phenotype was used to reflect the inflammatory state of the participants. There was no difference between groups (p = 0.627). Two hypotheses could explain these results: all patients included in this study represent an early stage of disease as evidenced by the FAST score below 11. According to the BD staging model, biological changes (including inflammation) appear to be related to mood episodes and disease progression. Together with previous studies, our data suggest that patients in early stages of BD still preserve immune system function without presenting an imbalance in favor of M1 macrophages as has been observed in latestage patients, highlighting the relevance of early intervention. Moreover, these patients were under treatment with mood stabilizers and it is plausible to speculate that these drugs have effects on macrophage polarization. Future studies in drug-free patients are essential to assess this issue. In conclusion, our findings suggest that BD patients do not present imbalance in macrophage polarization in favor of the M1 proinflammatory phenotype. The fact that all these patients are in the early stages of the disease reinforces the protective effects of early intervention in BD to prevent changes in the immune system and, consequently, prevent the progression of the disease.

Injuries to the peripheral nervous system rank among the most common causes of chronic neuropathic pain. Afflicting millions of people for months or even years, symptoms of this condition have proven difficult to treat clinically. A thorough understanding of the pathophysiological changes induced by such nerve lesions is essential to the development of more efficient therapeutic options. Peripheral nerve injury induces a robust and tightly regulated innate immune response in the dorsal horn of the spinal cord. The precise molecular mechanisms regulating the spatiotemporal dynamics and functional impact of the response remain incompletely understood. Preclinical evidence suggests mitigating this immune response can have a significant therapeutic benefit in the treatment of neuropathic pain, however these findings have yet to be clinically validated. To elucidate the mechanisms regulating the spinal immune response, we used a mouse model of partial sciatic nerve injury exclusively in male adult (2-3-month-old) mice. The spared nerve injury (SNI) model employed throughout our studies induces robust, persistent neuropathic pain-like behavior. We established a time course for the spinal immune response to SNI and used mRNA extracted from the ipsilateral dorsal horn of lumbar spinal cord segments L4 and L5 to analyze changes in the transcriptome at the peak of the immune reaction 7 days after nerve lesion. We discovered upregulation of multiple elements of the triggering receptor expressed on myeloid cells 2 (Trem2) pathway. Trem2 is considered a regulator of toll-like receptor signaling in innate immune cells. It also promotes microglia-mediated phagocytosis in the central nervous system. Recent work from our lab has established neuronal apoptosis in the ipsilateral dorsal horn after SNI as an essential mechanism leading to the development of chronic neuropathic pain-like behavior. We used TUNEL staining of L4 spinal cord sections to compare the clearance of apoptotic cell profiles in Trem2-/- mice to wild-type littermates and discovered a key role for Trem2 in the clearance of apoptotic cells after SNI. We further used genetic deletion of Trem2 as well as administration of a Trem2 agonist in C57Bl/6 mice to assess the impact of Trem2 signaling on both the spinal immune response and neuropathic pain-like behavior after SNI. Neither removal nor augmentation of Trem2 signaling significantly affected the development of neuropathic pain-like behavior. Utilizing flow cytometry, we also evaluated the cellular composition of the spinal immune response. We found no evidence that monocytes from the peripheral circulation invade the spinal cord after SNI, as has been previously suggested. These findings were corroborated by immunohistochemical analysis of spinal cord sections from transgenic mice that express distinct fluorescent proteins in their monocyte and microglia cell populations. To better understand the different mechanisms modulating the spinal immune response, we further examined several transcriptionally regulated signaling pathways. We achieved the greatest reduction of mechanical allodynia in nerve-lesioned mice treated with a P2x4r antagonist. Surprisingly, the removal of fractalkine (Cx3cl1) signaling, another prominent chemokine signaling pathway in microglia, had no significant impact on either the spinal immune response or mechanical allodynia after SNI. Reducing the number of spinal microglia by blocking Csf1r activation did not prevent the development of mechanical allodynia after SNI either. Our findings reveal a more nuanced concept of microglial activation after nerve injury. The impact on neuropathic pain-like behavior and phagocytosis appear to be regulated by pathways that differ from those controlling immune cell recruitment and global activation. These findings provide a greater understanding of the complex mechanisms governing microglial function and offer new insight into molecular targets essential to the development of more efficient treatment options for neuropathic pain.

No description available.

Alzheimer's disease (AD) is defined as a progressive neurodegenerative disorder and is characterized by a devastating mental decline. There are three pathological hallmarks of the disease necessary for its diagnosis, these are extracellular amyloid plaques comprised of the beta-amyloid (Aβ) protein, intracellular neurofibrillary tangles comprised of hyperphosphorylated tau protein, and marked neuronal loss. Active immunization against Aβ1-42 or passive immunization with monoclonal anti-Aβ antibodies has been shown to reduce amyloid deposition and improve cognition in transgenic mouse models of AD, aged beagles, and nonhuman primates. Unfortunately, due to cerebrovascular adverse events, both active and passive immunization strategies targeting Aβ have failed in clinical trials. It is, therefore, necessary to identify novel amyloid-clearing therapeutics that do not induce cerebrovascular adverse events. We hypothesized that neuroinflammatory modulation could be a potential novel target. Triggering receptor expressed on myeloid cells-2 (TREM2) is a lipid and lipoprotein binding receptor expressed exclusively in the brain by microglia. Homozygous TREM2 loss of function mutations cause early-onset progressive presenile dementia while heterozygous, function-reducing point mutations triple the risk of sporadic, late-onset AD. Heterozygous TREM2 point mutations, which reduce either ligand binding or cell surface expression, are associated with a reduction in the number of microglia surrounding amyloid plaques, microglial inability to phagocytose compact Aβ deposits and form a barrier between plaques and neurons, an increase in the number of phospho- tau-positive dystrophic neurites and increased tau in the cerebrospinal fluid. Heterozygous mutations also double the rate of brain atrophy and decrease the age of AD onset by 3-6 years. Although human genetics supports the notion that loss of TREM2 function exacerbates neurodegeneration, it is unclear whether activation of TREM2 in a disease state is beneficial. The work we present here characterizes a TREM2 agonizing antibody as a potential therapeutic for amyloid reduction. We found that its administration results in immune modulation, recruitment of microglia to the site of amyloid plaques, reduced amyloid deposition and improvement in spatial learning and novel object recognition memory in the 5xFAD model of AD. More specifically, we show that intracranial injection of TREM2 agonizing antibodies into the frontal cortex and hippocampus of 5xFAD mice leads to clearance of diffuse and compact amyloid. We also show that systemic injection of TREM2 agonizing antibodies weekly over a period of 14 weeks results in clearance of diffuse and compact amyloid as well as elevated plasma concentrations of Aβ1-40 and Aβ1-42. Furthermore, systemic administration of these antibodies led to immune modulation and enhanced cognitive performance on radial arm water maze and novel object recognition tests. Importantly, we show the TREM2 agonizing antibody does not induce the adverse cerebrovascular events known to accompany amyloid modifying therapies. Though systemic administration of both TREM2 agonizing and anti-Abantibodies does not further enhance amyloid clearance or cognitive performance, co-administration mitigates the adverse cerebrovascular events associated with anti-Aβ antibodies. Collectively, these data indicate TREM2 activators may be an effective therapeutic target for the treatment of AD.

Alzheimers disease (AD) is pathologically characterized by amyloid plaques, neurofibrillary tangles, inflammation, and neurodegeneration. According to the amyloid hypothesis of AD, the central mediating event of the disease is the deposition of amyloid. The inflammation hypothesis of AD states that it is the inflammatory response to plaques and tangles, rather than the actual lesions, which causes the disease. Studies described here combine the two approaches into a single model. Four studies are presented using a basic protocol of intrahippocampal lipopolysaccharide (LPS) injection to stimulate inflammation in transgenic mice. The first study looked at alpha7 nicotinic receptors during the glial response to Abeta deposits and LPS. Reactive astrocytes which immunolabeled for alpha7 were co-localized with Congophilic deposits in APP and APP+PS1 mice, and increased after LPS injection. Unfortunately, LPS injection into alpha7 knock out mice revealed the alpha7 labeling to be nonspecific. The second study evaluated the time course of protein and gene expression after LPS injection into nontransgenic mice. This experiment identified both a transient and chronic microglial inflammatory response, with changes in cell morphology. The third study evaluated a similar time course in APP mice. Concurrent with the inflammatory response, transient reductions in Abeta burden were seen, though compact plaque load was unaffected. The fourth and final study used dexamethasone to inhibit LPS-induced inflammation in APP mice. LPS injection reduced Abeta burden, but was completely blocked by dexamethasone co-treatment. Though dexamethasone inhibited LPS-induced CD45 and complement receptor 3 levels (markers of general microglial activation), dexamethasone had no effect on scavenger receptor A or Fc gamma receptor II/III levels. An overall hypothesis regarding LPS mediated reductions in Abeta can be proposed: It is not the presence of the LPS molecule, nor the upregulation of receptors involved in phagocytosis, but rather general glial cell activation that mediates Abeta removal. Thus, a phagocytic cell must not only bind Abeta (by various receptors) but must also be capable of engulfing the material (via general cell activation). Taken together, these studies suggest that some level of inflammation in AD is beneficial and responsible for maintaining a balance between amyloid deposition and removal.

Chronic consumption of, and acute intoxication from, alcohol can have profound effects on the functional integrity of the central nervous system (CNS). The resident immunomodulatory cells of the CNS, microglia, provide signaling factors with both pro- and anti-inflammatory effects for protection. Microglial activation ranges through a multiplex of phases, of which have yet to be defined when induced by exposure to alcohol, and how the activation impacts surrounding cells. Exposure of alcohol has been revealed to induce an immune response in microglia, which can exhibit characteristics unique to a pro-inflammatory response depending on dose and time of alcohol exposure. To define the activation state produced by microglia in response to alcohol, ethanol-induced microglial protein and microRNA (miRNA) global profile expression changes were obtained in vitro, using the BV2 murine microglial cells, using mass spectrometry (MS)-based proteomics and microarray-based transcriptomic approaches, respectively, revealing potential regulatory miRNAs for inflammation mediation. The 2,277 protein groups identified through mass spectrometry and 3,195 miRNA genes identified using microarray analysis provided a strong foundation to determine miRNA-mRNA regulators and the pathways in which they are involved, that potentially play a role in microglial activation. The comparison of the miRNA expressed in microglia after lipopolysaccharide (LPS) and ethanol (EtOH) exposure, indicate that EtOH influenced miRNA does not signify having a pro-inflammatory activation phenotype, but the miRNA expressed under the influence of LPS does support this phenotype. The global pathway regulation evidence and defined proteins and miRNA-mRNA interactions upon microglial activation have the possibility to unite the pathways described in previous studies and further our understanding of EtOH-induced microglial activation, and their role in neuroinflammation and neurodegeneration. Further research to determine and validate the extent of gene regulation by miRNAs and subsequent impact on specific protein levels should be employed to define the miRNA transcriptome influence on pathways relevant to microglial function.

In both vertebrates and invertebrates, including leeches, microglia are rapidly activated by central nervous system (CNS) damage and migrate to the lesions. Adenosine triphosphate (ATP), nitric oxide (NO) and endocannabinoids have been implicated in controlling activation and migration, but details of the mechanisms are uncertain. This dissertation tests the hypothesis that endocannabinoids coordinate and influence the microglial response to nerve cord crushing. Chapter 1 reports that application of endocannabinoids to nerve cords at concentrations as low as 100 nM for arachidonylethanolamide (anandamide, or AEA) reduced the number of migrating microglia, but not when cords were pretreated with 10 µM of the CB2 cannabinoid receptor (CB2R) antagonist SR144528. In addition, immunoblots confirmed the expression of CB1-like and CB2-like receptors and immunohistochemistry showed that they were concentrated at lesions, where microglia accumulated. Benzoyl ATP (BzATP) also reduced microglia accumulation, an effect blocked by pretreatment of nerve cords with SR144528, whereas the G-protein coupled P2YR agonists uridine triphosphate (UTP) and methylthio-ATP (MeSATP) at 100 µM did not reduce accumulation. This result suggested that P2X7R activation elicited production and release of a CB2R agonist that influenced microglia movement. Chapter 2 reports that extracellular ATP levels were highest in the CNS within the first 30 min of injury and remained above unharmed controls for at least 2 hours. Application of 10 units (U) of the ATPase apyrase to nerve cords reduced accumulation of microglia at lesions, another indication that microglia require extracellular ATP to accumulate. Chapter 3 reports that AEA stopped ATP-induced movement of microglia, and that this effect was blocked by pretreatment of nerve cords with the CB1R antagonist SR141716A (10µM), the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L NAME, 2 mM) or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide (carboxy-PTIO or cPTIO, 1 mM). Thus the migration of microglia to lesions is regulated not only by ATP acting on P2YR and by NO acting on soluble guanylate cyclase (sGC), but also potentially by ATP binding to P2X7-like receptors to increase the production of cannabinoids. Cannabinoids, binding to the CB1R and CB2R cause production of NO, which suppresses microglia movement.