MicroRNA-195 prevents hippocampal microglial/macrophage polarization towards the M1 phenotype induced by chronic brain hypoperfusion through regulating CX3CL1/CX3CR1 signaling.
ABSTRACT: BACKGROUND:Microglial polarization is a dynamic response to acute brain hypoxia induced by stroke and traumatic brain injury (TBI). However, studies on the polarization of microglia in chronic cerebral circulation insufficiency (CCCI) are limited. Our objective was to investigate the effect of CCCI on microglial polarization after chronic brain hypoperfusion (CBH) and explore the underlying molecular mechanisms. METHODS:CBH model was established by bilateral common carotid artery occlusion (2-vessel occlusion, 2VO) in rats. Using the stereotaxic injection technique, lenti-pre-miR-195 and anti-miR-195 oligonucleotide fragments (lenti-pre-AMO-miR-195) were injeted into the CA1 region of the hippocampus to construct animal models with high or low expression of miR-195. Immunofluorescence staining and flow cytometry were conducted to examine the status of microglial polarization. In vitro, Transwell co-culture system was taken to investigate the role of miR-195 on neuronal-microglial communication through CX3CL1-CX3CR1 signaling. Quantitative real-time PCR was used to detect the level of miR-195 and inflammatory factors. The protein levels of CX3CL1 and CX3CR1 were evaluated by both western blot and immunofluorescence staining. RESULTS:CBH induced by 2VO initiated microglial/macrophage activation in the rat hippocampus from 1?week to 8?weeks, as evaluated by increased ratio of (CD68+ and CD206+)/Iba-1 immunofluorescence. And the microglial/macrophage polarization was shifted towards the M1 phenotype at 8 weeks following CBH. The expression of CX3CL1 and CX3CR1 was increased in the hippocampus of 2VO rats at 8 weeks. An in vitro study in a Transwell co-culture system demonstrated that transfection of either primary-cultured neonatal rat neurons (NRNs) or microglial BV2 cells with AMO-195-induced M1 polarization of BV2 cells and increased CX3CL1 and CX3CR1 expression and that these effects were reversed by miR-195 mimics. Furthermore, the upregulation of miR-195 induced by lenti-pre-miR-195 injection prevented microglial/macrophage polarization to M1 phenotype triggered by hippocampal injection of lenti-pre-AMO-miR-195 and 2VO surgery. CONCLUSIONS:Our findings conclude that downregulation of miR-195 in the hippocampus is involved in CBH-induced microglial/macrophage polarization towards M1 phenotype by governing communication between neurons and microglia through the regulation of CX3CL1 and CX3CR1 signaling. This indicates that miR-195 may provide a new strategy for clinical prevention and treatment of CBH.
Project description:Impaired synaptic plasticity and neuron loss are hallmarks of Alzheimer's disease and vascular dementia. Here, we found that chronic brain hypoperfusion (CBH) by bilateral common carotid artery occlusion (2VO) decreased the total length, numbers and crossings of dendrites and caused neuron death in rat hippocampi and cortices. It also led to increase in N-terminal ?-amyloid precursor protein (N-APP) and death receptor-6 (DR6) protein levels and in the activation of caspase-3 and caspase-6. Further study showed that DR6 protein was downregulated by miR-195 overexpression, upregulated by miR-195 inhibition, and unchanged by binding-site mutation and miR-masks. Knockdown of endogenous miR-195 by lentiviral vector-mediated overexpression of its antisense molecule (lenti-pre-AMO-miR-195) decreased the total length, numbers and crossings of dendrites and neuron death, upregulated N-APP and DR6 levels, and elevated cleaved caspase-3 and caspase-6 levels. Overexpression of miR-195 using lenti-pre-miR-195 prevented these changes triggered by 2VO. We conclude that miR-195 is involved in CBH-induced dendritic degeneration and neuron death through activation of the N-APP/DR6/caspase pathway.
Project description:BACKGROUND:Chronic brain hypoperfusion (CBH) is closely related to Alzheimer's disease (AD) and vascular dementia (VaD). Meanwhile, synaptic pathology plays a prominent role in the initial stage of AD and VaD. However, whether and how CBH impairs presynaptic plasticity is currently unclear. METHODS:In the present study, we performed a battery of techniques, including primary neuronal culture, patch clamp, stereotaxic injection of the lentiviral vectors, morris water maze (MWM), dual luciferase reporter assay, FM1-43 fluorescence dye evaluation, qRT-PCR and western blot, to investigate the regulatory effect of miR-153 on hippocampal synaptic vesicle release both in vivo and in vitro. The CBH rat model was generated by bilateral common carotid artery ligation (2VO). RESULTS:Compared to sham rats, 2VO rats presented decreased field excitatory postsynaptic potential (fEPSP) amplitude and increased paired-pulse ratios (PPRs) in the CA3-CA1 pathway, as well as significantly decreased expression of multiple vesicle fusion-related proteins, including SNAP-25, VAMP-2, syntaxin-1A and synaptotagmin-1, in the hippocampi. The levels of microRNA-153 (miR-153) were upregulated in the hippocampi of rats following 2VO surgery, and in the plasma of dementia patients. The expression of the vesicle fusion-related proteins affected by 2VO was inhibited by miR-153, elevated by miR-153 inhibition, and unchanged by binding-site mutation or miR masks. FM1-43 fluorescence images showed that miR-153 blunted vesicle exocytosis, but this effect was prevented by either 2'-O-methyl antisense oligoribonucleotides to miR-153 (AMO-153) and miR-masking of the miR-153 binding site in the 3' untranslated region (3'UTR) of the Snap25, Vamp2, Stx1a and Syt1 genes. Overexpression of miR-153 by lentiviral vector-mediated miR-153 mimics (lenti-pre-miR-153) decreased the fEPSP amplitude and elevated the PPR in the rat hippocampus, whereas overexpression of the antisense molecule (lenti-AMO-153) reversed these changes triggered by 2VO. Furthermore, lenti-AMO-153 attenuated the cognitive decline of 2VO rats. CONCLUSIONS:Overexpression of miR-153 controls CBH-induced presynaptic vesicle release impairment by posttranscriptionally regulating the expression of four vesicle release-related proteins by targeting the 3'UTRs of the Stx1a, Snap25, Vamp2 and Syt1 genes. These findings identify a novel mechanism of presynaptic plasticity impairment during CBH, which may be a new drug target for prevention or treatment of AD and VaD. Video Abstract.
Project description:Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Nav?2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Nav?2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Nav?2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Nav?2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Nav?2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Nav?2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.
Project description:Myelin is closely associated with cognitive function and is extremely vulnerable to damage in ischemic cerebrovascular diseases. The failure of remyelination is mainly due to limitations in oligodendrocyte progenitor cells (OPCs) differentiation in the damaged area. Previous studies have shown that physical exercise can improve vascular cognitive impairment, but whether it can reverse the defect in remyelination during ischemic injury and the underlying mechanism remains unclear. In this study, we observed the effects of physical exercise on chronic cerebral hypoperfusion (CCH) established by bilateral carotid artery occlusion. The cognitive function, myelin integrity, OPCs proliferation and differentiation, as well as microglia polarization were analyzed at 28 days after CCH. Besides, the expression of CX3CL1/CX3CR1 axis and activation of mitogen-activated protein kinase (MAPK) signal cascades were also evaluated. We found that physical exercise improved the cognitive function of rats with CCH, alleviated myelin injury, triggered OPCs proliferation and differentiation, facilitated microglia polarization toward M2, augmented the expression of CX3CL1/CX3CR1 axis, and reduced ERK and JNK phosphorylation. The results indicated that physical exercise improved the cognitive function of rats with CCH, possibly through microglial phenotype modulation and enhancement of oligodendrocytegenesis and remyelination. Moreover, the CX3CL1/CX3CR1 axis played an important role in this process by mediating ERK- and JNK-dependent pathways.
Project description:BACKGROUND:Fractalkine (CX3CL1) and its receptor (CX3CR1) play an important role in regulating microglial function. We have previously shown that Cx3cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX3CL1 is essential in regulating neuronal tau pathology. METHODS:We used transgenic mice lacking endogenous Cx3cl1 (Cx3cl1-/-) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX3CL1 (referred to as Cx3cl1105? mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy. RESULTS:First, increased basal tau levels accompanied microglial activation in Cx3cl1105? mice compared to control groups. Second, increased CD45+ and F4/80+ neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx3cl1-/-, and hTau/Cx3cl1105? mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX3CR1 was reduced in Cx3cl1105? mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx3cr1 deletion), which likely contributes to the elevated tau pathology. CONCLUSIONS:Collectively, our data suggest that overexpression of only chemokine domain of CX3CL1 does not protect against tau pathology.
Project description:Female mice are less susceptible to the negative metabolic consequences of high-fat diet feeding than male mice, for reasons that are incompletely understood. Here we identify sex-specific differences in hypothalamic microglial activation via the CX3CL1-CX3CR1 pathway that mediate the resistance of female mice to diet-induced obesity. Female mice fed a high-fat diet maintain CX3CL1-CX3CR1 levels while male mice show reductions in both ligand and receptor expression. Female Cx3cr1 knockout mice develop 'male-like' hypothalamic microglial accumulation and activation, accompanied by a marked increase in their susceptibility to diet-induced obesity. Conversely, increasing brain CX3CL1 levels in male mice through central pharmacological administration or virally mediated hypothalamic overexpression converts them to a 'female-like' metabolic phenotype with reduced microglial activation and body-weight gain. These data implicate sex differences in microglial activation in the modulation of energy homeostasis and identify CX3CR1 signalling as a potential therapeutic target for the treatment of obesity.
Project description:<h4>Background</h4>Neuron-microglia communication plays a crucial role in the motor neurons (MNs) death in amyotrophic lateral sclerosis (ALS). Neurons can express chemokine (C-X3-C motif) ligand 1 (CX3CL1), which mediates microglial activation via interacting with its sole receptor CX3CR1 in microglia. In the present study, we aimed to investigate the dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and MNs loss in SOD1<sup>G93A</sup> mouse model of ALS.<h4>Methods</h4>qPCR, western blot and immunofluorescent staining were used to examine the mRNA and protein levels and localization of CX3CL1/CX3CR1 in the anterior horn region of spinal cord in both SOD1<sup>G93A</sup> mice and their age-matched wild type (WT) littermates at 40, 60, 90 and 120?days of age. The M1/M2 microglial activation in the spinal cord tissues of SOD1<sup>G93A</sup> mice and WT mice were evaluated by immunofluorescent staining of M1/M2 markers and further confirmed by qPCR analysis of M1/M2-related cytokines.<h4>Results</h4>The immunofluorescent staining revealed that CX3CL1 was predominately expressed in MNs, while CX3CR1 was highly expressed in microglia in the anterior horn region of spinal cord. Compared with age-matched WT mice, CX3CL1 mRNA level was elevated at 40?days but decreased at 90 and 120?days in the anterior horn region of spinal cords in ALS mice. Consistently, CX3CR1 mRNA level was increased at 90 and 120?days. Western blot assay further confirmed the dynamic changes of CX3CL1/CX3CR1 axis in ALS mice. Additionally, the levels of M1/M2 markers of microglia and their related cytokines in the anterior horn region of spinal cord in ALS mice were increased at 90 and 120?days. Moreover, while M1-related cytokines in ALS mice were persistently increased at 120?days, the upregulated M2-related cytokines started to decline at 120?days, suggesting an altered microglial activation.<h4>Conclusions</h4>Our data revealed the dynamic changes of CX3CL1/CX3CR1 axis and an imbalanced M1/M2 microglial activation during ALS pathological progression. These findings may help identify potential molecular targets for ALS therapy.
Project description:Retinitis pigmentosa (RP), a disease characterized by the progressive degeneration of mutation-bearing photoreceptors, is a significant cause of incurable blindness in the young worldwide. Recent studies have found that activated retinal microglia contribute to photoreceptor demise via phagocytosis and proinflammatory factor production, however mechanisms regulating these contributions are not well-defined. In this study, we investigate the role of CX3CR1, a microglia-specific receptor, in regulating microglia-mediated degeneration using the well-established rd10 mouse model of RP. We found that in CX3CR1-deficient (CX3CR1(GFP/GFP) ) rd10 mice microglial infiltration into the photoreceptor layer was significantly augmented and associated with accelerated photoreceptor apoptosis and atrophy compared with CX3CR1-sufficient (CX3CR1(GFP/+) ) rd10 littermates. CX3CR1-deficient microglia demonstrated increased phagocytosis as evidenced by (1) having increased numbers of phagosomes in vivo, (2) an increased rate of phagocytosis of fluorescent beads and photoreceptor cellular debris in vitro, and (3) increased photoreceptor phagocytosis dynamics on live cell imaging in retinal explants, indicating that CX3CR1 signaling in microglia regulates the phagocytic clearance of at-risk photoreceptors. We also found that CX3CR1 deficiency in retinal microglia was associated with increased expression of inflammatory cytokines and microglial activation markers. Significantly, increasing CX3CL1-CX3CR1 signaling in the rd10 retina via exogenous intravitreal delivery of recombinant CX3CL1 was effective in (1) decreasing microglial infiltration, phagocytosis and activation, and (2) improving structural and functional features of photoreceptor degeneration. These results indicate that CX3CL1-CX3CR1 signaling is a molecular mechanism capable of modulating microglial-mediated degeneration and represents a potential molecular target in therapeutic approaches to RP. GLIA 2016;64:1479-1491.
Project description:TAU protein aggregation is the main characteristic of neurodegenerative diseases known as tauopathies. Low-grade chronic inflammation is also another hallmark that indicates crosstalk between damaged neurons and glial cells. Previously, we have demonstrated that neurons overexpressing TAUP301L release CX3CL1, which activates the transcription factor NRF2 signalling to limit over-activation in microglial cells in vitro and in vivo. However, the connection between CX3CL1/CX3CR1 and NRF2 system and its functional implications in microglia are poorly described. We evaluated CX3CR1/NRF2 axis in the context of tauopathies and its implication in neuroinflammation. Regarding the molecular mechanisms that connect CX3CL1/CX3CR1 and NRF2 systems, we observed that in primary microglia from Cx3cr1-/- mice the mRNA levels of Nrf2 and its related genes were significantly decreased, establishing a direct linking between both systems. To determine functional relevance of CX3CR1, migration and phagocytosis assays were evaluated. CX3CR1-deficient microglia showed impaired cell migration and deficiency of phagocytosis, as previously described for NRF2-deficient microglia, reinforcing the idea of the relevance of the CX3CL1/CX3CR1 axis in these events. The importance of these findings was evident in a tauopathy mouse model where the effects of sulforaphane (SFN), an NRF2 inducer, were examined on neuroinflammation in Cx3cr1+/+ and Cx3cr1-/- mice. Interestingly, the treatment with SFN was able to modulate astrogliosis but failed to reduce microgliosis in Cx3cr1-/- mice. These findings suggest an essential role of the CX3CR1/NRF2 axis in microglial function and in tauopathies. Therefore, polymorphisms with loss of function in CX3CR1 or NRF2 have to be taken into account for the development of therapeutic strategies.
Project description:Extracellular Tau is toxic for neighboring cells, and it contributes to the progression of AD. The CX3CL1/CX3CR1 axis is an important neuron/microglia communication mechanism.We studied Tau clearance by microglia both in vitro (microglia primary cultures treated with Cy5-Tau, affinity chromatography to study the binding of Tau to CX3CR1, and Tau-CX3CL1 competition assays) and in vivo (stereotaxic injection of Cy5-Tau into WT and CX3CR1-/- mice). The expression of CX3CR1, CX3CL1 and the microglial phagocytic phenotype were studied in brain tissue samples from AD patients.Tau binding to CX3CR1 triggers the internalization of the former by microglia, whereas S396 Tau phosphorylation decreases the binding affinity of this protein to CX3CR1. Of note, the progressive increase in the levels of phosho-Tau occurred in parallel with an increase in CX3CR1. In addition, our studies suggest that the phagocytic capacity of microglia in brain tissue samples from AD patients is decreased. Furthermore, the CX3CR1/CX3CL1 axis may be impaired in late stages of the disease.Our data suggest that the CX3CR1/CX3CL1 axis plays a key role in the phagocytosis of Tau by microglia in vitro and in vivo and that it is affected as AD progresses. Taken together, our results reveal CX3CR1 as a novel target for the clearance of extracellular Tau.