NF-κB Activation In Astrocytes Research Articles

NF-κB and JAK/STAT Signaling Pathways as Crucial Regulators of Neuroinflammation and Astrocyte Modulation in Spinal Cord Injury.

Spinal cord injury (SCI) leads to significant functional impairments below the level of the injury, and astrocytes play a crucial role in the pathophysiology of SCI. Astrocytes undergo changes and form a glial scar after SCI, which has traditionally been viewed as a barrier to axonal regeneration and functional recovery. Astrocytes activate intracellular signaling pathways, including nuclear factor κB (NF-κB) and Janus kinase-signal transducers and activators of transcription (JAK/STAT), in response to external stimuli. NF-κB and STAT3 are transcription factors that play a pivotal role in initiating gene expression related to astrogliosis. The JAK/STAT signaling pathway is essential for managing secondary damage and facilitating recovery processes post-SCI: inflammation, glial scar formation, and astrocyte survival. NF-κB activation in astrocytes leads to the production of pro-inflammatory factors by astrocytes. NF-κB and STAT3 signaling pathways are interconnected: NF-κB activation in astrocytes leads to the release of interleukin-6 (IL-6), which interacts with the IL-6 receptor and initiates STAT3 activation. By modulating astrocyte responses, these pathways offer promising avenues for enhancing recovery outcomes, illustrating the crucial need for further investigation into their mechanisms and therapeutic applications in SCI treatment.

Modest Reduction in CAG Repeat Length Rescues Motor Deficits but Not Purkinje Cell Pathology and Gliosis in Spinocerebellar Ataxia Type 1 Mice

Spinocerebellar ataxia type 1 (SCA1) is a fatal, dominantly inherited neurodegenerative disease caused by the expansion of CAG repeats in the Ataxin-1 (ATXN1) gene. SCA1 is characterized by the early and prominent pathology of the cerebellar Purkinje cells that results in balance and coordination deficits. We previously demonstrated that cerebellar astrocytes contribute to SCA1 pathogenesis in a biphasic, stage of disease-dependent manner. We found that pro-inflammatory transcriptional regulator nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling in astrocytes has a neuroprotective role during early-stage SCA1. Here, we sought to examine whether further inducing NF-κB activation in astrocytes of SCA1 model mice at an early stage of the disease has therapeutic benefits. To perform this task, we created a novel Slc1a3-CreERT/IKKβCA/ATXN1[82Q] triple transgenic mouse model in which TMX injection at 4 weeks of age results in the expression of constitutively active inhibitor of kB kinase beta (IKKβCA), the main activator of NF-κB signaling. As we evaluated SCA1-like phenotypes, we noticed that ATXN1[82Q] mice did not exhibit motor deficits anymore, even at very late stages of the disease. We sequenced the mutant ATXN1 gene and discovered that the CAG repeat number had decreased from 82 to 71. However, despite the loss of motor phenotype, other well-characterized SCA1-changes, including atrophy of Purkinje cell dendrites, hallmarks of cerebellar astrogliosis and microgliosis, and Purkinje cell disease-associated gene expression changes, were still detectable in ATXN1[71Q] mice. We found delayed PC atrophy and calbindin reduction in SCA1 mice expressing IKKβCA in astrocytes implicating beneficial effects of increased NF-κB signaling on Purkinje cell pathology. The change in the motor phenotype of SCA1 mice with CAG reduction prevented us from evaluating the neuroprotective potential of IKKβCA on motor deficits in these mice.

Astrocyte-derived microparticles initiate a neuroinflammatory cycle due to carbon monoxide poisoning

We hypothesized that carbon monoxide (CO) establishes an inflammatory cycle mediated by microparticles (MPs). Mice exposed to a CO protocol (1000 ​ppm for 40 ​min and then 3000 ​ppm for 20 ​min) that causes neuroinflammation exhibit NF-κB activation in astrocytes leading to generation of MPs expressing thrombospondin-1(TSP-1) that collect in deep cervical lymph nodes draining the brain glymphatic system. TSP-1 bearing MPs gain access to the blood stream where they activate neutrophils to generate a new family of MPs, and also stimulate endothelial cells as documented by leakage of intravenous 2000 ​kDa dextran. At the brain microvasculature, neutrophil and MPs sequestration, and myeloperoxidase activity result in elevations of the p65 subunit of NF-κB, serine 536 phosphorylated p65, CD36, and loss of astrocyte aquaporin-4 that persist for at least 7 days. Knock-out mice lacking the CD36 membrane receptor are resistant to all CO inflammatory changes. Events triggered by CO are recapitulated in naïve wild type mice injected with cervical node MPs from CO-exposed mice, but not control mice. All MPs-mediated events are inhibited with a NF-κB inhibitor, a myeloperoxidase inhibitor, or anti-TSP-1 antibodies. We conclude that astrocyte-derived MPs expressing TSP-1 establish a feed-forward neuroinflammatory cycle involving endothelial CD36-to-astrocyte NF-κB crosstalk. As there is currently no treatment for CO-induced neurological sequelae, these findings pose several possible sites for therapeutic interventions.

IKK2/NF-κB Activation in Astrocytes Reduces amyloid β Deposition: A Process Associated with Specific Microglia Polarization.

Alzheimer’s disease (AD) is a common neurodegenerative disease that is accompanied by pronounced neuroinflammatory responses mainly characterized by marked microgliosis and astrogliosis. However, it remains open as to how different aspects of astrocytic and microglial activation affect disease progression. Previously, we found that microglia expansion in the spinal cord, initiated by IKK2/NF-κB activation in astrocytes, exhibits stage-dependent beneficial effects on the progression of amyotrophic lateral sclerosis. Here, we investigated the impact of NF-κB-initiated neuroinflammation on AD pathogenesis using the APP23 mouse model of AD in combination with conditional activation of IKK2/NF-κB signaling in astrocytes. We show that NF-κB activation in astrocytes triggers a distinct neuroinflammatory response characterized by striking astrogliosis as well as prominent microglial reactivity. Immunohistochemistry and Congo red staining revealed an overall reduction in the size and number of amyloid plaques in the cerebral cortex and hippocampus. Interestingly, isolated primary astrocytes and microglia cells exhibit specific marker gene profiles which, in the case of microglia, point to an enhanced plaque clearance capacity. In contrast, direct IKK2/NF-κB activation in microglia results in a pro-inflammatory polarization program. Our findings suggest that IKK2/NF-κB signaling in astrocytes may activate paracrine mechanisms acting on microglia function but also on APP processing in neurons.

Serum miR-221-3p as a new potential biomarker for depressed mood in perioperative patients

MicroRNAs (miRNAs) modulate various genes associated with brain disorders and circulating miRNAs may therefore serve as biomarkers for these neurological diseases. We previously found that the miRNA miR-221-3p was highly expressed in cerebrospinal fluid and the serum of major depressive disorder (MDD) patients. Here, we examined whether miR-221-3p could be used as a biomarker for depressed mood in perioperative patients. We first examined the relative expression of serum miR-221-3p by real-time quantitative PCR in perioperative patients with different degrees of depressive mood assessed by the Patient Health Questionnaire-9 (PHQ-9) diagnostic form. We found that miR-221-3p expression in the mild depressive mood group (PHQ-9 scores 5–9) was 2.21 fold that of the normal group (PHQ-9 scores 0–4) and the moderate＆severe depressive mood group (PHQ-9 scores ≥ 10) showed miR-221-3p expression levels 3.66 fold that of the normal group. Then the absolute quantification of serum miR-221-3p was obtained using an miRNA standard curve. We found that the amount of serum miR-221-3p was positively correlated with depressed mood; when serum miR-221-3p > 1.7 × 107 copies/μg RNA, all indicated PHQ-9 scores were higher than 6. Subsequently, we found that miR-221-3p could indirectly increase the expression of IFN-α (Interferon alpha) in astrocytes by targeting IRF2 (Interferon Regulatory Factor 2) and that miR-221-3p participated in the anti-neuroinflammatory signaling cascades induced by ketamine and paroxetine via the IRF2/IFN-α pathway. Our results indicate that elevated serum miR-221-3p can be used as a biomarker for depressed mood in perioperative patients and that IFN-α-induced NF-κB activation in astrocytes mediated by miR-221-3p targeting of IRF2 may be one of the potential mechanisms.

Glial fibrillary acidic protein expression alters astrocytic chemokine release and protects mice from cuprizone-induced demyelination.

Enhanced glial fibrillary acidic protein (GFAP) expression occurs in most diseases of the central nervous system. Thus far, little is known about the effect that GFAP exerts on astrocyte cell signaling. In the present study, we observed that silencing GFAP expression in isolated astrocytes leads to enhanced CCL2 and CXCL10 release, whereas overexpression of GFAP in astrocytes results in a significantly reduced CXCL10 release in vitro. Additionally, we analyzed transgenic mice carrying a full-length copy of the wild-type human GFAP gene. We demonstrate that a persistent GFAP increase alters the astrocytic cell signaling profile, thereby protecting oligodendrocytes, myelin and, subsequently, axons from cuprizone-induced demyelination. Our study revealed that reduced CXCL10 mRNA was accompanied by reduced NF-κB expression in astrocytes. Furthermore, analysis of human tissue from a patient with Alexander disease showed NF-κB activation in astrocytes to be almost completely absent. Our findings indicate that regulation of GFAP expression in astrocytes is crucial for astrocyte signaling and function. Understanding the role of the cytoskeletal protein, GFAP is thus of importance as it is highly regulated in diseases of the central nervous system.

Astrocytic NF-κB brings the best and worst out of microglia.

News & Views23 July 2018free access Astrocytic NF-κB brings the best and worst out of microglia Nikolaos Dokalis Institute of Neuropathology, University Freiburg, Freiburg, Germany Search for more papers by this author Marco Prinz [email protected] orcid.org/0000-0002-0349-1955 Institute of Neuropathology, University Freiburg, Freiburg, Germany BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Nikolaos Dokalis Institute of Neuropathology, University Freiburg, Freiburg, Germany Search for more papers by this author Marco Prinz [email protected] orcid.org/0000-0002-0349-1955 Institute of Neuropathology, University Freiburg, Freiburg, Germany BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Author Information Nikolaos Dokalis1 and Marco Prinz1,2 1Institute of Neuropathology, University Freiburg, Freiburg, Germany 2BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany EMBO J (2018)37:e100130https://doi.org/10.15252/embj.2018100130 See also NO Alami et al (August 2018) PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The role of astrocytes and microglia in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) remains incompletely understood. In this issue of The EMBO Journal, Alami et al (2018) employ a sophisticated genetic system that allows precise temporal control of NF-κB activation in astrocytes to demonstrate that the timing of astrocyte activation is a key determinant of disease progression. Their results suggest that astrocyte activation drives microglia proliferation and that this can amplify not only the protective microglial effects in the presymptomatic phase of ALS, but also the detrimental microglial effects in the symptomatic phase. While neurons are the protagonists of the central nervous system (CNS), the supporting glial cells such as astrocytes and microglia have an essential role in maintaining CNS homeostasis. Glial cells are the first responders to central nervous system (CNS) insults, and their coordinated and highly regulated action is thought to be essential for successful recovery from local neural lesions. However, excessive or inappropriate activation of these cells can promote the progression of neurodegenerative diseases. We are just beginning to understand how the coordinated activity of different glial cell populations maintains CNS homeostasis (Rothhammer et al, 2018), and even less is known about how glial crosstalk influences the progression of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Amyotrophic lateral sclerosis is a terminal neurodegenerative disease characterized by progressive, non-cell autonomous death of CNS motor neurons and thereby loss of voluntary muscle control. In most patients, the cause of ALS is unknown, but 5–10% of cases are familial. Approximately one fifth of the familial ALS cases are linked to mutations in superoxide dismutase 1 (SOD1), a copper/zinc enzyme that normally detoxifies highly reactive superoxide radicals. The pathophysiology of ALS is complex and has been investigated in several animal models including the widespread SOD1(G39A) transgenic mouse. Evidence from these disease models and from ALS patients suggests that astrocytes and microglia are key players in the initiation and progression of disease. NF-κB is a highly conserved and broadly expressed transcription factor with a major role in regulating immunity, inflammation and cellular stress responses. NF-κB signaling has been strongly implicated to various CNS disorders, including neurodegeneration (Kaltschmidt et al, 1997), autoimmunity (Goldmann et al, 2013) and spinal cord injury (Bethea et al, 1998). Modulation of microglial NF-κB has been shown to affect the clinical manifestation of the disease (Frakes et al, 2014). Although activation of NF-κB in astrocytes has been observed during ALS (Haidet-Phillips et al, 2011), repression of the inhibitor of NF-κB kinase subunit beta IKK2 in astrocytes did not change the course of ALS (Frakes et al, 2014). However, astrocytic NF-κB signaling does play a role in other neurodegenerative disorders. For instance, it has been shown that astrocytic NF-κB activation plays a crucial pathogenic role during toxic (Raasch et al, 2011) and also neuroinflammatory (van Loo et al, 2006) demyelination in the CNS, regulating both the severity of myelin loss and concomitant neuronal damage. The current study by Alami et al (2018) demonstrates that astrocyte activation determines the course and severity of ALS by controlling microglia proliferation. In order to reveal the role of astrocyte activation in ALS, the authors employed a genetic model of tetracycline-regulated, astrocyte-specific IKK2/NF-κB activation in combination with the SOD1(G39A) mouse model. First, they demonstrated that NF-κB activation specifically in astrocytes leads to progressive gliosis and enhanced cell proliferation in the spinal cord. Astrocytic NF-κB activation also compromised the integrity of the blood–spinal cord barrier, which was accompanied by a progressive infiltration of lymphocytes. The proliferative cell fraction was mainly comprised of microglia cells. Interestingly, they observed phenotypic heterogeneity in the expanded microglia population that reflected resting and disease-associated cells, which can be differentiated on the basis of surface markers such as TMEM119, CD11c, and CD169. Furthermore, they observed that the switch from presymptomatic to the symptomatic disease phase was accompanied by a decrease in the proportion of resting microglia and an increase in the proportion of pro-inflammatory microglia. The authors then sought to determine how the timing of astrocyte NF-κB activation affects the course and severity of ALS. Although prolonged activation of the pathway (i.e., starting at postnatal day P25) had no net effect on overall survival time, it did change the course of disease progression. As compared to SOD1 mice, SOD1/IKK mice displayed delayed onset of the disease, but faster and more severe progression. Indeed, markers of disease progression such as misfolded SOD1, autophagy activation, and oxidative damage in the motor neurons were significantly reduced in mice with astrocytic NF-κB activation during the presymptomatic stage, but dramatically increased in the later stages. Given the differential impact of astrocyte NF-κB on microglial responses in early and late disease phases, Alami et al (2018) sought to determine how temporal dissociation of astrocyte NF-κB influences disease course. When astrocyte NF-κB was activated only in the early, presymptomatic phase of disease progression (P25-P80), mice displayed a delay in disease onset and longer survival. However, the mice did eventually succumb to disease after a delayed but markedly accelerated progression phase. In contrast, activation of astrocyte NF-κB after disease onset (i.e., from P80 onwards) reduced survival time. Despite the clinical manifestation, astrogliosis and microglia expansion and polarization were comparable at the late stages regardless of the time-point of the pathway activation. How does astrocyte NF-κB activation cause microglia proliferation? The authors rationalized that WNT proteins might mediate this effect, since they are secreted by astrocytes, induced by NF-κB, and are known to trigger proliferation of microglia. They observed that Wnt5a was significantly upregulated in astrocytes after activation of NF-κB pathway. Moreover, chemical inhibition of WNT release reduced microglia expansion. The authors then sought to influence disease outcome by controlling microglial proliferation in a therapeutic setting using full agonists and inverse agonists of the microglia-specific cannabinoid receptor 2 (CB2). Whereas full agonists of CB2 prevented microglial expansion and worsened disease progression, inverse agonists altered microglial morphological complexity, which was accompanied by a reduction in motor neuron degeneration. Collectively, these findings suggest that astrocyte Wnt5a promotes microglial proliferation and that pharmacological interventions directly promoting microglial proliferation during the early phases of ALS can reduce disease progression. Taken together, the study by Alami et al (2018) demonstrates that the timing of astrocyte activation crucially regulates neuroimmunological responses in ALS, affecting both disease onset and progression (Fig 1). Furthermore, this elegant study provides valuable insight into the cellular interplay of CNS-resident cells during ALS. Their results suggest that astrocyte NF-κB activation promotes microglial proliferation and that this can amplify the protective microglial effects in the presymptomatic phase of ALS, but also the detrimental microglial effects in the symptomatic phase. Although targeting microglial proliferation may represent a valid therapeutic strategy, the Alami et al (2018) study suggests that the timing of such interventions is critical. Specifically, promoting microglial expansion at late disease stages where microglia have already acquired a reactive, pro-inflammatory phenotype is likely to accelerate disease progression. Further studies deciphering the complexity of the neuroinflammatory environment and the interaction between both peripheral and resident cells (Prinz & Priller, 2017) will be required. Figure 1. The timing of astrocytic NF-κB activation determines microglia-dependent ALS pathologyDuring ALS progression, astrocytes (green) are suggested to regulate microglia (red) proliferation and shift from an anti-inflammatory to a pro-inflammatory phenotype contributing to the disease progression. Prolonged (P25 onwards) or early (P25–P80) astrocytic NF-κB activation extends the presymptomatic phase through Wnt5a-dependent microglia activation and expansion, but dramatically increases the severity of the symptomatic stage. In contrast, late activation of astrocytic NF-κB (P80 onwards) has no effect on disease onset, but rather exacerbates the disease progression. Download figure Download PowerPoint References Alami NO, Schurr C, Olde Heuvel F, Tang L, Li Q, Tasdogan A, Kimbara A, Nettekoven M, Ottaviani G, Raposo C, Röver S, Rogers-Evans M, Rothenhäusler B, Ullmer C, Fingerle J, Grether U, Knuesel I, Boeckers TM, Ludolph A, Wirth T et al (2018) NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS. EMBO J 37: e98697Google Scholar Bethea JR, Castro M, Keane RW, Lee TT, Dietrich WD, Yezierski RP (1998) Traumatic spinal cord injury induces nuclear factor-kappaB activation. J Neurosci 18: 3251–3260CrossrefCASPubMedWeb of Science®Google Scholar Frakes AE, Ferraiuolo L, Haidet-Phillips AM, Schmelzer L, Braun L, Miranda CJ, Ladner KJ, Bevan AK, Foust KD, Godbout JP, Popovich PG, Guttridge DC, Kaspar BK (2014) Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis. Neuron 81: 1009–1023CrossrefCASPubMedWeb of Science®Google Scholar Goldmann T, Wieghofer P, Müller PF, Wolf Y, Varol D, Yona S, Brendecke SM, Kierdorf K, Staszewski O, Datta M, Luedde T, Heikenwalder M, Jung S, Prinz M (2013) A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat Neurosci 16: 1618–1626CrossrefCASPubMedWeb of Science®Google Scholar Haidet-Phillips AM, Hester ME, Miranda CJ, Meyer K, Braun L, Frakes A, Song S, Likhite S, Murtha MJ, Foust KD, Rao M, Eagle A, Kammesheidt A, Christensen A, Mendell JR, Burghes AH, Kaspar BK (2011) Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nat Biotechnol 29: 824–828CrossrefCASPubMedWeb of Science®Google Scholar Kaltschmidt B, Uherek M, Volk B, Baeuerle PA, Kaltschmidt C (1997) Transcription factor NF-κB is activated in primary neurons by amyloid β peptides and in neurons surrounding early plaques from patients with Alzheimer's disease. Proc Natl Acad Sci USA 94: 2642–2647CrossrefCASPubMedWeb of Science®Google Scholar van Loo G, De Lorenzi R, Schmidt H, Huth M, Mildner A, Schmidt-Supprian M, Lassmann H, Prinz MR, Pasparakis M (2006) Inhibition of transcription factor NF-kappaB in the central nervous system ameliorates autoimmune encephalomyelitis in mice. Nat Immunol 7: 954–961CrossrefCASPubMedWeb of Science®Google Scholar Prinz M, Priller J (2017) The role of peripheral immune cells in the CNS in steady state and disease. Nat Neurosci 20: 136–144CrossrefCASPubMedWeb of Science®Google Scholar Raasch J, Zeller N, van Loo G, Merkler D, Mildner A, Erny D, Knobeloch KP, Bethea JR, Waisman A, Knust M, Del Turco D, Deller T, Blank T, Priller J, Brück W, Pasparakis M, Prinz M (2011) IkappaB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-kappaB in the central nervous system. Brain 134: 1184–1198CrossrefPubMedWeb of Science®Google Scholar Rothhammer V, Borucki DM, Tjon EC, Takenaka MC, Chao CC, Ardura-Fabregat A, de Lima KA, Gutiérrez-Vázquez C, Hewson P, Staszewski O, Blain M, Healy L, Neziraj T, Borio M, Wheeler M, Dragin LL, Laplaud DA, Antel J, Alvarez JI, Prinz M et al (2018) Microglial control of astrocytes in response to microbial metabolites. Nature 557: 724–728CrossrefCASPubMedWeb of Science®Google Scholar Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 37,Issue 16,15 August 2018Caption: Cell size control is a fundamental process for multicellular systems development. In the Arabidopsis root meristem, characteristic changes in the size of the distal meristematic cells identify cells that initiated the differentiation program. Cell wall remodelling due to acidification and expansin‐dependent loosening brings about a change in cell shape that has an important role in controlling the cell differentiation program. The cover shows the AHA2 proton pump expression domain in the Arabidopsis root tip (AHA2‐GFP in cyan). Cell walls are stained with propidium iodide (grey). From Elena Pacifici, Riccardo Di Mambro, Raffaele Dello Ioio, Paolo Costantino and Sabrina Sabatini: Acidic cell elongation drives cell differentiation in the Arabidopsis root. For detail see Article e99134. Scientific Image by Elena Pacifici. Volume 37Issue 1615 August 2018In this issue FiguresReferencesRelatedDetailsLoading ...

NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS.

Astrocytes are involved in non‐cell‐autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF‐κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF‐κB activation. While NF‐κB activation in astrocytes induced a Wnt‐dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF‐κB‐dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB2R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage‐dependent microglia modulation may be an effective therapeutic strategy in ALS.

Deficiency of IκB Kinase β in Myeloid Cells Reduces Severity of Experimental Autoimmune Encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase β (IKKβ) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKβ in MS, we developed strains in which IKKβ was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKβ in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKβ also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKβ-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKβ wild-type EAE mice. Thus, deficiency of myeloid IKKβ attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKβ may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Inhibition of astroglial NF-kappaB enhances oligodendrogenesis following spinal cord injury

BackgroundAstrocytes are taking the center stage in neurotrauma and neurological diseases as they appear to play a dominant role in the inflammatory processes associated with these conditions. Previously, we reported that inhibiting NF-κB activation in astrocytes, using a transgenic mouse model (GFAP-IκBα-dn mice), results in improved functional recovery, increased white matter preservation and axonal sparing following spinal cord injury (SCI). In the present study, we sought to determine whether this improvement, due to inhibiting NF-κB activation in astrocytes, could be the result of enhanced oligodendrogenesis in our transgenic mice.MethodsTo assess oligodendrogenesis in GFAP-IκBα-dn compared to wild-type (WT) littermate mice following SCI, we used bromodeoxyuridine labeling along with cell-specific immuno-histochemistry, confocal microscopy and quantitative cell counts. To further gain insight into the underlying molecular mechanisms leading to increased white matter, we performed a microarray analysis in naïve and 3 days, 3 and 6 weeks following SCI in GFAP-IκBα-dn and WT littermate mice.ResultsInhibition of astroglial NF-κB in GFAP-IκBα-dn mice resulted in enhanced oligodendrogenesis 6 weeks following SCI and was associated with increased levels of myelin proteolipid protein compared to spinal cord injured WT mice. The microarray data showed a large number of differentially expressed genes involved in inflammatory and immune response between WT and transgenic mice. We did not find any difference in the number of microglia/leukocytes infiltrating the spinal cord but did find differences in their level of expression of toll-like receptor 4. We also found increased expression of the chemokine receptor CXCR4 on oligodendrocyte progenitor cells and mature oligodendrocytes in the transgenic mice. Finally TNF receptor 2 levels were significantly higher in the transgenic mice compared to WT following injury.ConclusionsThese studies suggest that one of the beneficial roles of blocking NF-κB in astrocytes is to promote oligodendrogenesis through alteration of the inflammatory environment.

Nuclear Factor κB Activation Impairs Ependymal Ciliogenesis and Links Neuroinflammation to Hydrocephalus Formation

Hydrocephalus formation is a frequent complication of neuropathological insults associated with neuroinflammation. However, the mechanistic role of neuroinflammation in hydrocephalus development is unclear. We have investigated the function of the proinflammatory acting inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling system in neuroinflammatory processes and generated a novel mouse model that allows conditional activation of the IKK/NF-κB system in astrocytes. Remarkably, NF-κB activation in astrocytes during early postnatal life results in hydrocephalus formation and additional defects in brain development. NF-κB activation causes global neuroinflammation characterized by a strong, astrocyte-specific expression of proinflammatory NF-κB target genes as well as a massive infiltration and activation of macrophages. In this animal model, hydrocephalus formation is specifically induced during a critical time period of early postnatal development, in which IKK/NF-κB-induced neuroinflammation interferes with ependymal ciliogenesis. Our findings demonstrate for the first time that IKK/NF-κB activation is sufficient to induce hydrocephalus formation and provides a potential mechanistic explanation for the frequent association of neuroinflammation and hydrocephalus formation during brain development, namely impairment of ependymal cilia formation. Therefore, our study might open up new perspectives for the treatment of certain types of neonatal and childhood hydrocephalus associated with hemorrhages and infections.

Reduced astrocytic NF-κB activation by laquinimod protects from cuprizone-induced demyelination

Laquinimod (LAQ) is a new oral immunomodulatory compound that reduces relapse rate, brain atrophy and disability progression in multiple sclerosis (MS). LAQ has well-documented effects on inflammation in the periphery, but little is known about its direct activity within the central nervous system (CNS). To elucidate the impact of LAQ on CNS-intrinsic inflammation, we investigated the effects of LAQ on cuprizone-induced demyelination in mice in vivo and on primary CNS cells in vitro. Demyelination, inflammation, axonal damage and glial pathology were evaluated in LAQ-treated wild type and Rag-1-deficient mice after cuprizone challenge. Using primary cells we tested for effects of LAQ on oligodendroglial survival as well as on cytokine secretion and NF-κB activation in astrocytes and microglia. LAQ prevented cuprizone-induced demyelination, microglial activation, axonal transections, reactive gliosis and oligodendroglial apoptoses in wild type and Rag-1-deficient mice. LAQ significantly decreased pro-inflammatory factors in stimulated astrocytes, but not in microglia. Oligodendroglial survival was not affected by LAQ in vitro. Astrocytic, but not microglial, NF-κB activation was markedly reduced by LAQ as evidenced by NF-κB reporter assay. LAQ also significantly decreased astrocytic NF-κB activation in cuprizone-treated mice. Our data indicate that LAQ prevents cuprizone-induced demyelination by attenuating astrocytic NF-κB activation. These effects are CNS-intrinsic and not mediated by peripheral immune cells. Therefore, LAQ downregulation of the astrocytic pro-inflammatory response may be an important mechanism underlying its protective effects on myelin, oligodendrocytes and axons. Modulation of astrocyte activation may be an attractive therapeutic target to prevent tissue damage in MS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1009-1) contains supplementary material, which is available to authorized users.

Astroglial NF‐κB mediates oxidative stress by regulation of NADPH oxidase in a model of retinal ischemia reperfusion injury

Astrocytes undergo rapid activation after injury, which is mediated in part by the transcription factor nuclear factor-kappaB (NF-κB). Consequently, activated astrocytes have been shown to induce the NF-κB regulated phagocyte NADPH oxidase (PHOX), resulting in elevated production of reactive oxygen species. We investigated the regulatory mechanisms of PHOX-induced oxidative stress in astrocytes and its non-cell-autonomous effects on retinal ganglion cell loss following retinal ischemia-reperfusion (IR) injury. To study PHOX activity and neurotoxicity mediated by glial NF-κB, we employed GFAP-IκBα-dn transgenic mice, where the NF-κB canonical pathway is suppressed specifically in astrocytes. Our analysis showed that NF-κB activation in astrocytes correlated with an increased expression of PHOX and reactive oxygen species production in primary cells and whole retinas subjected to oxygen-glucose deprivation or IR injury. Selective blockade of NF-κB in astrocytes or application of NADPH oxidase inhibitors suppressed retinal ganglion cell loss in co-cultures with astroglia challenged by oxygen-glucose deprivation. Furthermore, genetic suppression of astroglial NF-κB reduced oxidative stress in ganglion layer neurons in vivo in retinal IR. Collectively, our results suggest that astroglial NF-κB-regulated PHOX activity is a crucial toxicity pathway in the pathogenesis of retinal IR injury.

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.

Extracellular HIV-1 Tat enhances monocyte adhesion by up-regulation of ICAM-1 and VCAM-1 gene expression via ROS-dependent NF-κB activation in astrocytes

One of characteristic features of AIDS-related encephalitis and dementia is the infiltration of monocytes into the CNS. HIV-1 Tat was demonstrated to facilitate monocyte entry into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of adhesion molecules, generation of reactive oxygen species (ROS) and NF-kappaB activation in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein and mRNA levels of ICAM-1 and VCAM-1, as measured by Western blot analysis and RT-PCR, indicating that Tat increases these protein levels at an mRNA level. In addition, Tat induced the activation of NF-kappaB in astrocytes. Treatment of CRT-MG with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of ICAM-1 and VCAM-1. Furthermore, HIV-1 Tat protein increased ROS generation. Inhibition of Tat-induced ROS generation by N-acetyl cysteine, vitamin C and diphenyl iodonium suppressed Tat-induced NF-kappaB activation, ICAM-1 and VCAM-1 expression, and monocyte adhesion in CRT-MG. These data indicate that HIV-1 Tat can modulate monocyte adhesiveness by increasing expression of adhesion molecules such as ICAM-1 and VCAM-1 via ROS- and NF-kappaB-dependent mechanisms in astrocytes.

Neuronal (type I) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type II) nitric oxide synthase expression.

Nitric oxide subserves diverse physiologic roles in the nervous system. NO is produced from at least three different NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS, and immunologic NOS (iNOS). We show that nNOS is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits the transcription factor nuclear factor kappaB (NF kappaB) as NOS inhibitors enhance basal NF kappaB activation. Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of NF kappaB in most cells; however, we show that PDTC is also a potent scavenger of NO through formation of mononitrosyl iron complexes with PDTC. In Jurkat cells, a human T-cell lymphoma cell line, tumor necrosis factor-alpha (TNF-alpha) induces NF kappaB activation that is inhibited by PDTC. Contrary to the results in Jurkat cells, PDTC did not inhibit tumor necrosis factor-alpha-induced NF kappaB activation in astrocytes; instead PDTC itself induces NF kappaB activation in astrocytes, and this may be related to scavenging of endogenously produced NO by the PDTC iron complex. In astrocytes PDTC also dramatically induces the NF kappaB-dependent enzyme, iNOS, supporting the physiologic relevance of endogenous NO regulation of NF kappaB. NF kappaB activation in glia from mice lacking nNOS responds more rapidly to PDTC compared with astrocytes from wild-type mice. Our data suggest that nNOS in astrocytes regulates NF kappaB activity and iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NF kappaB-regulated genes.