NF-κB Signaling In Astrocytes Research Articles

Gender-based differences in neuroprotective effects of hydrogen gas against intracerebral hemorrhage-induced depression

BackgroundPost-stroke depression (PSD) severely affects recovery in patients with intracerebral hemorrhage (ICH). Although hydrogen gas (H2) exerts excellent neuroprotective effects in patients with ICH, there are sex-based differences in H2 efficacy in several diseases. Herein, we determined whether estrogen increases susceptibility to the neuroprotective effects of H2 in males with ICH-induced depression. MethodsA rodent model of ICH in the basal ganglia was established using autologous blood injection (30 μL). Mice were treated with 2.9% H2 for 2 h daily for 3 days post-ICH. Estrogen (1 mg/kg) was administered by subcutaneous injection daily for 3 days to male mice post-ICH. Thirty days post-ICH, PSD was evaluated by sucrose preference, forced swimming, and 3-chamber social tests. Following the completion of behavioral tests, levels of superoxide dismutase (SOD) and reactive oxygen species (ROS), astrocytic activation, phosphorylated (p)–NF–κB-positive astrocytes, p–NF–κB, p-IKKβ, IL-1β, and IL-6 expression were determined. ResultsCompared with female mice, H2 administration post-ICH exhibited fewer neuroprotective effects, including decreased sucrose consumption and time spent sniffing a novel mouse, increased immobility time, downregulated total SOD content, upregulated ROS content and p–NF–κB levels, and elevated astrocyte branches, whereas estrogen enhanced the neuroprotective effects of H2 in male mice. A reduced number of p–NF–κB-positive astrocytes, downregulated expression of p–NF–κB, p-IKKβ, IL-1β, and IL-6 in the amygdala were demonstrated in ICH-males treated with estrogen plus H2. ConclusionsEstrogen was responsible for increased H2 sensitivity in male mice with ICH. The underlying mechanism may be associated with the suppression of NF-κB signaling in astrocytes.

NF-κB Signaling in Astrocytes Modulates Brain Inflammation and Neuronal Injury Following Sequential Exposure to Manganese and MPTP During Development and Aging.

Chronic exposure to manganese (Mn) is associated with neuroinflammation and extrapyramidal motor deficits resembling features of Parkinson's disease. Activation of astrocytes and microglia is implicated in neuronal injury from Mn but it is not known whether early life exposure to Mn may predispose glia to more severe inflammatory responses during aging. We therefore examined astrocyte nuclear factor kappa B (NF-κB) signaling in mediating innate immune inflammatory responses during multiple neurotoxic exposures spanning juvenile development into adulthood. MnCl2 was given in drinking water for 30-day postweaning to both wildtype mice and astrocyte-specific knockout (KO) mice lacking I kappa B kinase 2, the central upstream activator of NF-κB. Following juvenile exposure to Mn, mice were subsequently administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 4 months of age. Animals were evaluated for behavioral alterations and brain tissue was analyzed for catecholamine neurotransmitters. Stereological analysis of neuronal and glial cell counts from multiple brain regions indicated that juvenile exposure to Mn amplified glial activation and neuronal loss from MPTP exposure in the caudate-putamen and globus pallidus, as well as increased the severity of neurobehavioral deficits in open field activity assays. These alterations were prevented in astrocyte-specific I kappa B kinase 2 KO mice. Juvenile exposure to Mn increased the number of neurotoxic A1 astrocytes expressing C3 as well as the number of activated microglia in adult mice following MPTP challenge, both of which were inhibited in KO mice. These results demonstrate that exposure to Mn during juvenile development heightens the innate immune inflammatory response in glia during a subsequent neurotoxic challenge through NF-κB signaling in astrocytes.

Oxycodone suppresses the lipopolysaccharide-induced neuroinflammation by downregulating nuclear factor-κB in hippocampal astrocytes of Sprague-Dawley rats.

Neuroinflammation is a common pathogenic mechanism in several neurodegenerative diseases, and glial cells are the primary inflammatory mediators of the central nervous system (CNS). Acute neuronal injury, infection, and chronic neurodegeneration may induce astrocyte activation, which is a response characterized by hyperproliferation and release of multiple inflammatory signaling factors. The opioid analgesic oxycodone has demonstrated anti-inflammatory efficacy in peripheral tissue, but its effects on the CNS have not been studied. We evaluated the inhibitory effects of oxycodone on astrocyte activation and proinflammatory mediator production in response to lipopolysaccharide (LPS). Our results showed that oxycodone (5-20 μg/ml) dose-dependently inhibited the LPS-induced astrocytosis, as measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide and bromodeoxyuridine assays, as well as the overexpression of glial fibrillary acidic protein, which are two hallmarks of reactive astrogliosis in neurodegenerative diseases. Oxycodone also decreased both the mRNA and protein expression levels of proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β. Besides, oxycodone increased the expression of the nuclear factor kappa-B (NF-κB) endogenous inhibitor IκB-α, and blocked NF-κB translocation to the nucleus. The anti-inflammatory efficacy of oxycodone on rat astrocytes increased with pretreatment duration. These results suggest that oxycodone can suppress neuroinflammation by inhibiting NF-κB signaling in astrocytes. Targeting the astrocytic NF-κB-mediated inflammatory response may be an effective therapeutic strategy against diseases involving neuroinflammatory damage.

Lack of PINK1 alters glia innate immune responses and enhances inflammation-induced, nitric oxide-mediated neuron death

Neuroinflammation is involved in the pathogenesis of Parkinson’s disease (PD) and other neurodegenerative disorders. We show that lack of PINK1- a mitochondrial kinase linked to recessive familial PD – leads to glia type-specific abnormalities of innate immunity. PINK1 loss enhances LPS/IFN-γ stimulated pro-inflammatory phenotypes of mixed astrocytes/microglia (increased iNOS, nitric oxide and COX-2, reduced IL-10) and pure astrocytes (increased iNOS, nitric oxide, TNF-α and IL-1β), while attenuating expression of both pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory (IL-10) cytokines in microglia. These abnormalities are associated with increased inflammation-induced NF-κB signaling in astrocytes, and cause enhanced death of neurons co-cultured with inflamed PINK1−/− mixed glia and neuroblastoma cells exposed to conditioned medium from LPS/IFN-γ treated PINK1−/− mixed glia. Neuroblastoma cell death is prevented with an iNOS inhibitor, implicating increased nitric oxide production as the cause for enhanced death. Finally, we show for the first time that lack of a recessive PD gene (PINK1) increases α-Synuclein-induced nitric oxide production in all glia types (mixed glia, astrocytes and microglia). Our results describe a novel pathogenic mechanism in recessive PD, where PINK1 deficiency may increase neuron death via exacerbation of inflammatory stimuli-induced nitric oxide production and abnormal innate immune responses in glia cells.

Transglutaminase 2 modulation of NF-κB signaling in astrocytes is independent of its ability to mediate astrocytic viability in ischemic injury

Transglutaminase 2 (TG2) is a multifunctional protein that can contribute to cell death and cell survival processes in a variety of disease contexts. Within the brain, TG2 has been shown to promote cell death in ischemic injury when expressed in astrocytes (Colak and Johnson, 2012). However, the specific functions and characteristics of astrocytic TG2 that mediate this effect are largely unknown. Therefore, the goal of this study was to investigate the role of astrocytic TG2 in mediating cellular viability processes in the context of ischemic injury, with a specific focus on its contributions to intracellular signaling cascades. We show that, in response to oxygen/glucose deprivation (OGD), acute lentiviral-mediated knockdown of TG2, as well as inhibition with an irreversible TG2 inhibitor, enhances cell survival. We also show that TG2 depletion increases nuclear factor-κB (NF-κB) signaling, whereas inhibition reduces NF-κB activity. Despite its clear contribution to NF-κB signaling, however, TG2 modulation of NF-κB signaling is not likely to be a major contributor to its ability to mediate astrocytic viability in this context. Overall, the results of this study provide insight into the role of TG2 in astrocytes and suggest possible avenues for future study of the relationship between astrocytic TG2 and ischemic injury.

Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice.

ObjectiveIntroduction of a high-fat diet to mice results in a period of voracious feeding, known as hyperphagia, before homeostatic mechanisms prevail to restore energy intake to an isocaloric level. Acute high-fat diet hyperphagia induces astrocyte activation in the rodent hypothalamus, suggesting a potential role of these cells in the homeostatic response to the diet. The objective of this study was to determine physiologic role of astrocytes in the acute homeostatic response to high-fat feeding.MethodsWe bred a transgenic mouse model with doxycycline-inducible inhibition of NFkappaB (NFκB) signaling in astrocytes to determine the effect of loss of NFκB-mediated astrocyte activation on acute high-fat hyperphagia. ELISA was used to measure the levels of markers of astrocyte activation, glial-fibrillary acidic protein (GFAP) and S100B, in the medial basal hypothalamus.ResultsInhibition of NFκB signaling in astrocytes prevented acute high-fat diet-induced astrocyte activation and resulted in a 15% increase in caloric intake (P < 0.01) in the first 24 h after introduction of the diet.ConclusionsThese data reveal a novel homeostatic role for astrocytes in the acute physiologic regulation of food intake in response to high-fat feeding.

Nuclear Factor- B Contributes to Neuron-Dependent Induction of Glutamate Transporter-1 Expression in Astrocytes

The glutamate transporter-1 [GLT-1 (excitatory amino acid transporter 2)] subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild-type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at -583 or -251 relative to the transcription start site were required for neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.

Angiotensin II Type 1 receptor (AT1) signaling in astrocytes regulates synaptic degeneration-induced leukocyte entry to the central nervous system

Astrocytes are the major cellular component of the blood–brain barrier glia limitans and act as regulators of leukocyte infiltration via chemokine expression. We have studied angiotensin-II receptor Type 1 (AT1) and related NF-κB signaling in astrocytes. Angiotensin II derives from cleavage of angiotensin I by angiotensin converting enzyme (ACE), angiotensin I deriving from angiotensinogen via cleavage by renin. Level of expression of ACE was slightly increased in transgenic mice that express dominant-negative IκBα in astrocytes (GFAP-IκBα-dn mice), whereas angiotensinogen and renin, also constitutively expressed in the CNS, were unaffected by NF-κB inhibition. Leukocytes infiltrate the hippocampus of mice after unilateral stereotactic lesion of afferent perforant path axons in the entorhinal cortex. Upregulation of the chemokine CXCL10 that normally occurs in response to synaptic degeneration in the dentate gyrus following axonal transection was totally abrogated in GFAP-IκBα-dn mice. Whereas angiotensin II was upregulated in microglia and astrocytes in the dentate gyrus post-lesion, AT1 was exclusively expressed on astrocytes. Blocking AT1 with Candesartan led to significant increase in numbers of infiltrating macrophages in the hippocampus 2 days post-lesion. Lesion-induced increases in T-cell infiltration and morphologic glial response were unaffected, and the blood-brain barrier remained intact to horseradish peroxidase. These findings show that angiotensin II signaling to astrocytes via AT1 plays an important role in regulation of leukocyte infiltration to the CNS in response to a neurodegenerative stimulus, and identify potential targets for therapies directed at adaptive immune responses in the CNS.

NF-κB-Driven STAT2 and CCL2 Expression in Astrocytes in Response to Brain Injury

Tissue response to injury includes expression of genes encoding cytokines and chemokines. These regulate entry of immune cells to the injured tissue. The synthesis of many cytokines and chemokines involves NF-kappaB and signal transducers and activators of transcription (STAT). Injury to the CNS induces glial response. Astrocytes are the major glial population in the CNS. We examined expression of STATs and the chemokine CCL2 and their relationship to astroglial NF-kappaB signaling in the CNS following axonal transection. Double labeling with Mac-1/CD11b and glial fibrillary acidic protein revealed that STAT2 up-regulation and phosphorylation colocalized exclusively to astrocytes, suggesting the involvement of STAT2 activating signals selectively in astroglial response to injury. STAT1 was also up-regulated and phosphorylated but not exclusively in astrocytes. Both STAT2 up-regulation and phosphorylation were NF-kappaB -dependent since they did not occur in the lesion-reactive hippocampus of transgenic mice with specific inhibition of NF-kappaB activation in astrocytes. We further showed that lack of NF-kappaB signaling significantly reduced injury-induced CCL2 expression as well as leukocyte infiltration. Our results suggest that NF-kappaB signaling in astrocytes controls expression of both STAT2 and CCL2, and thus regulates infiltration of leukocytes into lesion-reactive hippocampus after axonal injury. Taken together, these findings indicate a central role for astrocytes in directing immune-glial interaction in the CNS injury response.

Inhibition of astroglial nuclear factor κB reduces inflammation and improves functional recovery after spinal cord injury

In the central nervous system (CNS), the transcription factor nuclear factor (NF)-κB is a key regulator of inflammation and secondary injury processes. After trauma or disease, the expression of NF-κB–dependent genes is highly activated, leading to both protective and detrimental effects on CNS recovery. We demonstrate that selective inactivation of astroglial NF-κB in transgenic mice expressing a dominant negative (dn) form of the inhibitor of κBα under the control of an astrocyte-specific promoter (glial fibrillary acidic protein [GFAP]–dn mice) leads to a dramatic improvement in functional recovery 8 wk after contusive spinal cord injury (SCI). Histologically, GFAP mice exhibit reduced lesion volume and substantially increased white matter preservation. In parallel, they show reduced expression of proinflammatory chemokines and cytokines, such as CXCL10, CCL2, and transforming growth factor–β2, and of chondroitin sulfate proteoglycans participating in the formation of the glial scar. We conclude that selective inhibition of NF-κB signaling in astrocytes results in protective effects after SCI and propose the NF-κB pathway as a possible new target for the development of therapeutic strategies for the treatment of SCI.