Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation.

Microglia, the resident phagocytes of the retina, are believed to influence the development of retinopathy, but their exact contributions to vascular integrity and neuronal loss are unknown. Therefore, utilizing two models of microglia depletion, we aimed to deplete and repopulate microglia to clarify the contribution of microglia to neuronal loss and vascular damage in the diabetic retina in an STZ-induced model of hyperglycemia. Here, we report that 2 weeks exposure to diphtheria toxin (DTx) in diabetic CX3CR1CreER:R26iDTR transgenic mice induced a 62% increase in Iba1+ microglia associated with an increase in TUJ1+ axonal density and prevention of NeuN+RBPMS+ neuronal loss. Conversely, diabetic PBS controls exhibited robust TUJ1+ axonal and NeuN+RBPMS+ neuronal loss compared to non-diabetic controls. A 2-week recovery period from DTx was associated with a 40% reduction in angiogenesis and an 85% reduction in fibrinogen deposition into the diabetic retina in comparison to diabetic PBS-treated controls. Analysis of microglia morphology and marker expression revealed that following a 2-week recovery period microglia displayed a P2RY12+Ly6C– phenotype and high transformation index (TI) values complimented by a ramified-surveillant morphology closely resembling non-diabetic controls. In contrast, diabetic PBS-treated control mice displayed P2RY12+Ly6C+ microglia, with a 50% reduction in TI values with an amoeboid morphology. To validate these observations were due to microglia depletion, we used PLX-5622 to assess vascular and neuronal damage in the retinas of diabetic mice. Confocal microscopy revealed that PLX-5622 also induced an increase in TUJ1+ axonal density and prevented fibrinogen extravasation into the diabetic retina. mRNAseq gene expression analysis in retinal isolates revealed that PLX-5622-induced microglia depletion and repopulation induced a downregulation in genes associated with microglial activation and phagocytosis, B2m, Cx3cr1, and Trem2, and complement-associated synaptic pruning, C1qa, C1qb, and C1qc. Although the levels of microglia depletion induced with DTx in the CX3CR1CreER:R26iDTR model and those induced with the CSF-1R antagonists are distinct, our results suggest that microglia depletion and replenishment is neuroprotective by inducing the proliferation of a homeostatic microglia pool that supports neuronal and vascular integrity.

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 limits the activation of pathogenic microglia and the human polymorphic CX3CR1 I249/M280 (hCX3CR1 I249/M280 ) variant increases disease progression in models of MS. However, the role of hCX3CR1 I249/M280 variant on microglial activation and central nervous system repair mechanisms remains unknown. Therefore, using transgenic mice expressing the hCX3CR1 I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to neuroinflammation and remyelination in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1 I249/M280 exhibit marked demyelination and microgliosis following acute cuprizone treatment. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1 I249/M280 but not CX3CR1-deficient mice up-regulated the cuprizoneinduced gene profile linked to inflammatory, oxidative stress, and phagocytic pathways. Although CX3CR1-deficient (CX3CR1-KO) and fractalkine-deficient (FKN-KO) mice displayed a comparable demyelination and microglial activation phenotype to hCX3CR1 I249/M280 mice, only CX3CR1-deficient and CX3CR1-WT mice showed significant myelin recovery 1 week from cuprizone withdrawal. Confocal microscopy | 431 Mendiola et al.

Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy (DR), but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Previous studies from diabetic mouse models showed accumulation of fibrinogen at vessel lesions surrounded by perivascular microglial clusters. The purpose of this study was to evaluate whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas, and to assess the effects of the defibrinogenating agent ancrod in retinal pathology and visual acuity in a two-hit inflammatory diabetic mouse model. Post-mortem human eyes were assessed for retinal and inflammatory gene expression by quantitative PCR. Immunohistochemical analyses in human and murine retinas were performed using markers of gliosis, vascular integrity, and fibrinogen deposition. An inflammatory microenvironment, with microgliosis and microaneurysms, was found in the diabetic human eye. Microglial activation, fibrinogen deposition, and axonal loss were also observed in the diabetic murine retina. Ancrod treatment correlated with reduced microgliosis, less fibrinogen deposition, and reduced pro-inflammatory cytokine levels in diseased retinal tissues. Together, these data suggest that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina. Since retinal microgliosis, vascular pathology, and vision deficits manifest in diabetic mice irrespective of CX3CR1 genotype, our results indicate that defibrinogenation can dampen systemic neuroinflammation and vascular insults, thereby improving vision at early stages of diabetes. Summary Statement Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.