Pharmacological depletion of microglia alleviates neuronal and vascular damage in the diabetic CX3CR1-WT retina but not in CX3CR1-KO or hCX3CR1I249/M280-expressing retina.

Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. There is no cure for DR, and medical interventions marginally slow the progression of disease. Microglia-mediated inflammation in the diabetic retina is regulated via CX3CR1-FKN signaling, where FKN serves as a calming signal for microglial activation in several neuroinflammatory models. Polymorphic variants of CX3CR1, hCX3CR1I249/M280 , found in 25% of the human population, result in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in CX3CR1-KO and FKN-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of hCX3CR1I249/M280 -expression in microglia-mediated inflammation in the diseased retina are relevant to identify mechanisms by which microglia contribute to disease progression. Our results show that hCX3CR1I249/M280 mice are significantly more susceptible to microgliosis and production of Cxcl10 and TNFα under acute inflammatory conditions. Inflammation is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to CX3CR1-WT mice. Therefore, to further investigate the role of hCX3CR1I249/M280 -expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1+ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic CX3CR1-WT prevented TUJ1+ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate TUJ1+ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in CX3CR1-KO mice but not in hCX3CR1I249/M280 mice, suggesting that hCX3CR1I249/M280 expressing microglia influences vascular pathology differently compared to CX3CR1-KO microglia. Currently CX3CR1-KO mice are the most commonly used strain to investigate CX3CR1-FKN signaling effects on microglia-mediated inflammation and the results in this study indicate that hCX3CR1I249/M280 receptor variants may serve as a complementary model to study dysregulated CX3CR1-FKN signaling. In summary, the protective effects of microglia depletion is CX3CR1-dependent as microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate retinal degeneration nor microglial morphological activation as observed in CX3CR1-WT mice.

Models of microglia depletion and replenishment elicit protective effects to alleviate vascular and neuronal damage in the diabetic murine retina.

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.

Noradrenaline in Alzheimer's Disease: A New Potential Therapeutic Target.

A growing body of evidence demonstrates the important role of the noradrenergic system in the pathogenesis of many neurodegenerative processes, especially Alzheimer's disease, due to its ability to control glial activation and chemokine production resulting in anti-inflammatory and neuroprotective effects. Noradrenaline involvement in this disease was first proposed after finding deficits of noradrenergic neurons in the locus coeruleus from Alzheimer's disease patients. Based on this, it has been hypothesized that the early loss of noradrenergic projections and the subsequent reduction of noradrenaline brain levels contribute to cognitive dysfunctions and the progression of neurodegeneration. Several studies have focused on analyzing the role of noradrenaline in the development and progression of Alzheimer's disease. In this review we summarize some of the most relevant data describing the alterations of the noradrenergic system normally occurring in Alzheimer's disease as well as experimental studies in which noradrenaline concentration was modified in order to further analyze how these alterations affect the behavior and viability of different nervous cells. The combination of the different studies here presented suggests that the maintenance of adequate noradrenaline levels in the central nervous system constitutes a key factor of the endogenous defense systems that help prevent or delay the development of Alzheimer's disease. For this reason, the use of noradrenaline modulating drugs is proposed as an interesting alternative therapeutic option for Alzheimer's disease.

Sphk2 deletion is involved in structural abnormalities and Th17 response but does not aggravate colon inflammation induced by sub-chronic stress.

The chronic inflammatory process that characterizes inflammatory bowel diseases (IBD) is mainly driven by T-cell response to microbial and environmental antigens. Psychological stress is a potential trigger of clinical flares of IBD, and sphingosine-1-phosphate (S1P) is involved in T-cell recruitment. Hence, stress impact and the absence of sphingosine kinase 2 (Sphk2), an enzyme of S1P metabolism, were evaluated in the colon of mice after sub-chronic stress exposure. Here, we show that sub-chronic stress increased S1P in the mouse colon, possibly due to a decrease in its degradation enzymes and Sphk2. S1P accumulation could lead to inflammation and immune dysregulation reflected by upregulation of toll-like receptor 4 (TLR4) pathway, inhibition of anti-inflammatory mechanisms, cytokine-expression profile towards a T-helper lymphocyte 17 (Th17) polarization, plasmacytosis, decrease in IgA+ lymphoid lineage cells (CD45+)/B cells/plasmablasts, and increase in IgM+ B cells. Stress also enhanced intestinal permeability. Sphk2 knockout mice presented a cytokine-expression profile towards a boosted Th17 response, lower expression of claudin 3,4,7,8, and structural abnormalities in the colon. Intestinal pathophysiology should consider stress and S1P as modulators of the immune response. S1P-based drugs, including Sphk2 potentiation, represent a promising approach to treat IBD.

CCL2 Inhibition of Pro-Resolving Mediators Potentiates Neuroinflammation in Astrocytes.

The chemokine CCL2 participates in multiple neuroinflammatory processes, mainly through the recruitment of glial cells. However, CCL2 has also been proven to exert different types of actions on these cells, including the modification of their response to inflammatory stimuli. In the present study we analyzed the effect of CCL2 on the resolution of inflammation in astrocytes. We observed that genetic removal of CCL2 increases the expression of the enzymes responsible for the synthesis of specialized pro-resolving mediators arachidonate 15-lipoxygenase and arachidonate 5-lipoxygenase in the brain cortex of 5xFAD mice. The expression of FPR2 receptor, known to mediate the activity of pro-resolving mediators was also increased in mice lacking CCL2.The downregulation of these proteins by CCL2 was also observed in cultured astrocytes. This suggests that CCL2 inhibition of the resolution of inflammation could facilitate the progression of neuroinflammatory processes. The production of the pro-inflammatory cytokine IL-1beta by astrocytes was analyzed, and allowed us to confirm that CCL2 potentiates the activation of astrocytes trough the inhibition of pro-resolving pathways mediated by Resolvin D1. In addition, the analysis of the expression of TNFalpha, MIP1alpha and NOS2 further confirmed CCL2 inhibition of inflammation resolution in astrocytes.

Microglial CX3CR1 production increases in Alzheimer's disease and is regulated by noradrenaline.

The loss of noradrenergic neurons and subsequent reduction of brain noradrenaline (NA) levels are associated with the progression of Alzheimer's disease (AD). This seems to be due mainly to the ability of NA to reduce the activation of microglial cells. We previously observed that NA induces the production of the chemokine Fractalkine/CX3CL1 in neurons. The activation of microglial CX3CR1, sole receptor for CX3CL1, reduces the activation of microglia, which is known to largely contribute to the neuronal damage characteristic of AD. Therefore, alterations of CX3CR1 production in microglia could translate into the enhancement or inhibition of CX3CL1 anti-inflammatory effects. In order to determine if microglial CX3CR1 production is altered in AD and if NA can control it, CX3CR1 expression and synthesis were analyzed in 5xFAD mice and human AD brain samples. In addition, the effects of NA and its reuptake inhibitor reboxetine were analyzed in microglial cultures and mice respectively. Our results indicate that in AD CX3CR1 production is increased in the brain cortex and that reboxetine administration further increases it and enhances microglial reactivity toward amyloid beta plaques. However, direct administration of NA to primary rat microglia or human HMC3 cells inhibits CX3CR1 production, suggesting that microglia responses to NA may be altered in the absence of CX3CL1-producing neurons or other nonmicroglial external factors.

Reboxetine Treatment Reduces Neuroinflammation and Neurodegeneration in the 5xFAD Mouse Model of Alzheimer's Disease: Role of CCL2.

The reduction of brain noradrenaline levels is associated to the initiation of Alzheimer's disease and contributes to its progression. This seems to be due mainly to the anti-neuroinflammatory actions of noradrenaline. The analysis of noradrenaline effects on brain cells demonstrates that it also regulates the production of the chemokine CCL2. In the present study, we analyzed the effect of the selective noradrenaline reuptake inhibitor, reboxetine, on the inflammatory and neurodegenerative alterations present in 5xFAD mice, and how the genetic removal of CCL2 affects reboxetine actions. We observed that the removal of CCL2 reduced the memory impairments in 5xFAD mice as well as the neuroinflammatory response, the accumulation of amyloid beta plaques, and the degeneration of neurons in the brain cortex. The administration of reboxetine with osmotic pumps for 28 days also resulted in anti-inflammatory and neuroprotective changes in 5xFAD mice, even in the absence of CCL2. Yet, 6-month-old CCL2KO mice presented a significant degree of neuroinflammation and neuronal damage. These findings indicate that reboxetine treatment prevents the brain alterations caused by prolonged overproduction of amyloid beta, being these effects independent of CCL2, which is a mediator of the damage caused by amyloid beta in the brain cortex, but necessary for the prevention of the development of neurodegeneration in normal healthy conditions.

Alternative Method to Detect Neuronal Degeneration and Amyloid ß Accumulation in Free-Floating Brain Sections With Fluoro-Jade.

Fluoro-Jade is a fluorescein-derived fluorochrome which specifically binds to damaged neurons. Due to this characteristic, it is commonly used for the histochemical detection and quantification of neurodegeneration in mounted brain sections. Here, we describe an alternative and simpler histochemistry protocol based on the use of free-floating brain sections. For this purpose, we have used brain slices from wild-type and 5xFAD mice as well as from mice that received an intracerebral injection of oligomeric amyloid beta peptides. We observed that our histochemistry staining procedure allows for a well-defined labeling of degenerating neurons providing a better signal-to-noise ratio staining than the commonly used one. In addition, our modified protocol demonstrates the ability of Fluoro-Jade C to also fluorescently label amyloid beta plaques.

CCL2 Induces the Production of ß2 Adrenergic Receptors and Modifies Astrocytic Responses to Noradrenaline.

The decline in brain noradrenaline levels is associated with the progression of certain neurodegenerative diseases. This seems to be due, at least in part, to the ability of noradrenaline to limit glial activation and to reduce the damage associated with it. Our previous studies of the mechanisms involved in this process indicate that noradrenaline induces the production of the chemokine CCL2 in astrocytes. While CCL2 can protect neurons against certain injuries, its overproduction has also proven to be harmful and to prevent noradrenaline neuroprotective effects. Therefore, in this study, we analyze if the modifications caused to astrocytes by an excessive production of CCL2 may alter their response to noradrenaline. Using primary cultures of rat cortical astrocytes, we observed that CCL2 enhances the production of beta 2 adrenergic receptors in these cells. While this potentiates noradrenaline signaling through cAMP, the activation of the transcription factor CREB is inhibited by CCL2. Furthermore, although CCL2 potentiates noradrenaline induction of glycogenolysis, this does not translate into an augmented release of lactate, one of the processes through which astrocytes help support neurons. Additionally, other neuroprotective actions of noradrenaline, such as the production of brain derived neurotrophic factor and the inhibition of the inducible nitric oxide synthase in astrocytes were modified by CCL2. These data suggest that some of the central nervous system alterations related to CCL2 could be due to its effects on adrenergic receptors and its interference with noradrenaline signaling.

Noradrenaline induces CX3CL1 production and release by neurons.

CX3CL1 is a chemokine for which neurons constitute its primary source within the brain. Besides acting as a chemokine, CX3CL1 regulates multiple processes and is known to inhibit microglial activation. Because of this, CX3CL1 is considered as a messenger used by neurons to communicate with microglia. Similarly, the neurotransmitter noradrenaline reduces microglial activation and production of neurotoxic agents. Based on this, the regulation of neuronal CX3CXL1 by noradrenaline was analyzed. In primary cortical neurons, noradrenaline induced the accumulation of CX3CL1 protein and mRNA. Noradrenaline also increased CX3CL1 in its soluble form despite the inhibition of the activity and synthesis of ADAM10 and ADAM17, the main proteases known to cleave CX3CL1 from the neuronal membrane. Noradrenaline-treated neurons displayed a higher degree of dendritic arborization and a characteristic accumulation of CX3CL1 in the dendritic bifurcation zones. The soluble CX3CL1 produced by neurons after noradrenaline treatment, reduced the accumulation of nitrites in microglia. These findings indicate that NA anti-inflammatory actions are mediated by neuronal CX3CL1. In addition, CX3CL1 seems to be involved in the development of neuronal processes stimulated by noradrenaline.