Human Microglia-Like Cells Differentiated from Monocytes with GM-CSF and IL-34 Show Phagocytosis of α-Synuclein Aggregates and C/EBPß-Dependent Proinflammatory Activation.

Microglia, the main resident immune cells in the central nervous system, are implicated in the pathogenesis of various neurological disorders. Much of our knowledge on microglial biology was obtained using rodent microglial cultures. To understand the role of microglia in human disease, reliable in vitro models of human microglia are necessary. Monocyte-derived microglia-like cells (MDMi) are a promising approach. This study aimed to characterize MDMi cells generated from adult human monocytes using granulocyte-macrophage colony-stimulating factor and interleukin-34. To this end, 49 independent cultures of MDMI were prepared, and various methodological and functional studies were performed. We show that with this protocol, adult human monocytes develop into microglia-like cells, a coating is unnecessary, and high cell density seeding is preferable. When compared to monocytes, MDMi upregulate the expression of many, but not all, microglial markers, indicating that, although these cells display a microglia-like phenotype, they cannot be considered bona fide human microglia. At the functional level, MDMi phagocytose α-synuclein aggregates and responds to lipopolysaccharide (LPS) by nuclear translocation of the transcription factor nuclear factor-kappaB (NFkappaB) and the upregulation of proinflammatory genes. Finally, a long-lasting silencing of the transcription factor CCAAT/enhancer protein ß (C/EBPß) was achieved by small interfering RNA, resulting in the subsequent downregulation of proinflammatory genes. This supports the hypothesis that C/EBPß plays a key role in proinflammatory gene program activation in human microglia. Altogether, this study sheds new light on the properties of MDMi cells and supports these cells as a promising in vitro model for studying adult human microglia-like cells.

The CD200R1 microglial inhibitory receptor as a therapeutic target in the MPTP model of Parkinson's disease.

BACKGROUND: It is suggested that neuroinflammation, in which activated microglial cells play a relevant role, contributes to the development of Parkinson's disease (PD). Consequently, the modulation of microglial activation is a potential therapeutic target to be taken into account to act against the dopaminergic neurodegeneration occurring in this neurological disorder. Several soluble and membrane-associated inhibitory mechanisms contribute to maintaining microglial cells in a quiescent/surveillant phenotype in physiological conditions. However, the presence of activated microglial cells in the brain in PD patients suggests that these mechanisms have been somehow overloaded. We focused our interest on one of the membrane-associated mechanisms, the CD200-CD200R1 ligand-receptor pair. METHODS: The acute MPTP experimental mouse model of PD was used to study the temporal pattern of mRNA expression of CD200 and CD200R1 in the context of MPTP-induced dopaminergic neurodegeneration and neuroinflammation. Dopaminergic damage was assessed by tyrosine hydroxylase (TH) immunoreactivity, and neuroinflammation was evaluated by the mRNA expression of inflammatory markers and IBA1 and GFAP immunohistochemistry. The effect of the modulation of the CD200-CD200R1 system on MPTP-induced damage was determined by using a CD200R1 agonist or CD200 KO mice. RESULTS: MPTP administration resulted in a progressive decrease in TH-positive fibres in the striatum and TH-positive neurons in the substantia nigra pars compacta, which were accompanied by transient astrogliosis, microgliosis and expression of pro- and anti-inflammatory markers. CD200 mRNA levels rapidly decreased in the ventral midbrain after MPTP treatment, while a transient decrease of CD200R1 mRNA expression was repeatedly observed in this brain area at earlier and later phases. By contrast, a transient increase in CD200R1 expression was observed in striatum. The administration of a CD200R1 agonist resulted in the inhibition of MPTP-induced dopaminergic neurodegeneration, while microglial cells showed signs of earlier activation in CD200-deficient mice. CONCLUSIONS: Collectively, these findings provide evidence for a correlation between CD200-CD200R1 alterations, glial activation and neuronal loss. CD200R1 stimulation reduces MPTP-induced loss of dopaminergic neurons, and CD200 deficiency results in earlier microglial activation, suggesting that the potentiation of CD200R1 signalling is a possible approach to controlling neuroinflammation and neuronal death in PD.

Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair.

Exposure to pesticides such as rotenone is a risk factor for Parkinson's disease. Dopaminergic neurons are especially sensitive to the toxicity of compounds that inhibit the mitochondrial respiratory chain such as rotenone and 1-methyl-4-phenylpyridinium (MPP+). However, there is scarce information on their effects on glia. To evaluate whether these neurotoxicants affect the immune response of glia, primary mouse mixed glial and microglial cultures were treated with interleukin (IL) 4 in the absence and presence of MPP+ or rotenone. Using qRTPCR or western blot, we determined the expression of anti-inflammatory markers, the CD200R1 microglial receptor and its ligand CD200, and genes regulating glycolysis and oxidative metabolism. ATP and lactate levels were additionally determined as an index of cell metabolism. Microglial phagocytosis was also evaluated. MPP+ and rotenone clearly abrogated the IL4-induced expression of anti-inflammatory markers in mixed glial cultures. CD200 and CD200R1 expression and microglia phagocytosis were also affected by the neurotoxicants. Changes in the mRNA expression of the molecules regulating glycolysis and oxidative metabolism, as well as in ATP levels and lactate release suggested that metabolic reprogramming in response to MPP+ and rotenone differs between microglial and mixed glial cultures. These findings support the hypothesis that parkinsonian neurotoxicants may impair brain immune response altering glial cell metabolism.

Role of p27Kip1 as a transcriptional regulator.

The protein p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies. The carboxyl moiety of p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression. We summarize in this review our current knowledge on the role of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.

p27Kip1 regulates alpha-synuclein expression.

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies.

Parkinsonian Neurotoxins Impair the Pro-inflammatory Response of Glial Cells.

In the case of Parkinson's disease (PD), epidemiological studies have reported that pesticide exposure is a risk factor for its pathology. It has been suggested that some chemical agents, such as rotenone and paraquat, that inhibit the mitochondrial respiratory chain (in the same way as the PD mimetic toxin 1-methyl-4-phenylpyridinium, MPP+) are involved in the development of PD. However, although the neurotoxic effect of such compounds has been widely reported using in vivo and in vitro experimental approaches, their direct effect on the glial cells remains poorly characterized. In addition, the extent to which these toxins interfere with the immune response of the glial cells, is also underexplored. We used mouse primary mixed glial and microglial cultures to study the effect of MPP+ and rotenone on glial activation, in the absence and the presence of a pro-inflammatory stimulus (lipopolysaccharide plus interferon-γ, LPS+IFN-γ). We determined the mRNA expression of the effector molecules that participate in the inflammatory response (pro-inflammatory cytokines and enzymes), as well as the nitric oxide (NO) and cytokine production. We also studied the phagocytic activity of the microglial cells. In addition, we evaluated the metabolic changes associated with the observed effects, through the measurement of adenosine triphosphate (ATP) production and the expression of genes involved in the control of metabolic pathways. We observed that exposure of the glial cultures to the neurotoxins, especially rotenone, impaired the pro-inflammatory response induced by LPS/IFN-γ. MPP+ and rotenone also impaired the phagocytic activity of the microglial cells, and this effect was potentiated in the presence of LPS/IFN-γ. The deficit in ATP production that was detected, mainly in MPP+ and rotenone-treated mixed glial cultures, may be responsible for the effects observed. These results show that the response of glial cells to a pro-inflammatory challenge is altered in the presence of toxins inhibiting mitochondrial respiratory chain activity, suggesting that the glial immune response is impaired by such agents. This may have relevant consequences for brain function and the central nervous system's (CNS's) response to insults.

Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages.

In the brain of patients with multiple sclerosis, activated microglia/macrophages appear in active lesions and in normal appearing white matter. However, whether they play a beneficial or a detrimental role in the development of the pathology remains a controversial issue. The production of pro-inflammatory molecules by chronically activated microglial cells is suggested to contribute to the progression of neurodegenerative processes in neurological disease. In the healthy brain, neurons control glial activation through several inhibitory mechanisms, such as the CD200-CD200R1 interaction. Therefore, we studied whether alterations in the CD200-CD200R1 system might underlie the neuroinflammation in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. We determined the time course of CD200 and CD200R1 expression in the brain and spinal cord of an EAE mouse model from presymptomatic to late symptomatic stages. We also assessed the correlation with associated glial activation, inflammatory response and EAE severity. Alterations in CD200 and CD200R1 expression were mainly observed in spinal cord regions in the EAE model, mostly a decrease in CD200 and an increase in CD200R1 expression. A decrease in the expression of the mRNA encoding a full CD200 protein was detected before the onset of clinical signs, and remained thereafter. A decrease in CD200 protein expression was observed from the onset of clinical signs. By contrast, CD200R1 expression increased at EAE onset, when a glial reaction associated with the production of pro- and anti-inflammatory markers occurred, and continued to be elevated during the pathology. Moreover, the magnitude of the alterations correlated with severity of the EAE mainly in spinal cord. These results suggest that neuronal-microglial communication through CD200-CD200R1 interaction is compromised in EAE. The early decreases in CD200 expression in EAE suggest that this downregulation might also occur in the initial phases of multiple sclerosis, and that this early neuronal dysfunction might facilitate the development of neuroinflammation. The increased CD200R1 expression in the EAE model highlights the potential use of targeted agonist molecules as therapeutic tools to control neuroinflammation. In summary, the CD200-CD200R1 system is a potential therapeutic target in multiple sclerosis, and CD200R1 agonists are molecules that may be worth developing in this context.

Myeloid C/EBPß deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.

BACKGROUND: CCAAT/enhancer binding protein ß (C/EBPß) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPß show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPß-expressing cell types is not solved. Since C/EBPß-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPß deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPß deficiency. METHODS: Mice with myeloid C/EBPß deficiency were generated by crossing LysMCre and C/EBPßfl/fl mice. Primary microglial cultures from C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPß deletion were analyzed in vivo in microglia isolated from the brains of C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPßfl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used. RESULTS: LysMCre-C/EBPßfl/fl mice showed an efficiency of C/EBPß deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPß deficiency. Transcriptomic analysis of C/EBPß-deficient primary microglia revealed C/EBPß-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPß deletion. CNS expression of C/EBPß was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPßfl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis. CONCLUSION: This study provides new data that support a central role for C/EBPß in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPß inhibition in multiple sclerosis.

The proteasome inhibitor bortezomib reduced cholesterol accumulation in fibroblasts from Niemann-Pick type C patients carrying missense mutations.

Niemann-Pick disease type C (NPC) is a lipid storage disorder mainly caused by mutations in the NPC1 gene. Approximately 60% of these mutations are missense changes that may induce reduced NPC1 protein levels by increased degradation via ubiquitin-proteasome. This is the case for the most prevalent worldwide mutation, p.Ile1061Thr, as well as for other three missense changes. In the present study, we analyzed the NPC1 levels in fibroblasts from eighteen NPC patients presenting missense mutations. We found that fourteen of these cells lines showed decreased levels of NPC1. Six of these cell lines were homozygous, whereas the other eight were associated with a frame shifting mutation. We focused our attention in the NPC homozygous samples and demonstrated that, in most of the cases, NPC1 reduction was a consequence of a decrease of its half-life. NPC cells were treated not only with the proteasome inhibitors carbobenzoxy-l-leucyl-l-leucyl-l-leucinal or N-acetyl-leucyl-leucyl-norleucinal, both widely used as a research tools, but also with bortezomib, the first proteasome inhibitor to reach clinical applications, although it has never been used in NPC disease. We observed that, after treatment, the mutant NPC1 protein levels were partially recovered in most of the cell lines. Importantly, these mutant proteins partially recovered their activity and substantially reduced free cholesterol levels. These results suggest that by enhancing the NPC1 protein stability with the use of proteasome inhibitors, their functionality might be recovered and this might represent a therapeutical approach for future treatments of NPC disease resulting from specific missense mutations.

CD200R1 and CD200 expression are regulated by PPAR-γ in activated glial cells.

The mechanisms that control microglial activation are of interest, since neuroinflammation, which involves reactive microglia, may be an additional target in the search for therapeutic strategies to treat neurodegenerative diseases. Neuron-microglia interaction through contact-dependent or independent mechanisms is involved in the regulation of the microglial phenotype in both physiological and pathological conditions. The interaction between CD200, which is mainly present in neurons but also in astrocytes, and CD200R1, which is mainly present in microglia, is one of the mechanisms involved in keeping the microglial proinflammatory phenotype under control in physiological conditions. Alterations in the expression of CD200 and CD200R1 have been described in neurodegenerative diseases, but little is known about the mechanism of regulation of these proteins under physiological or pathological conditions. The aim of this work was to study the modulation of CD200 and CD200R1 expression by peroxisome proliferator-activated receptor gamma (PPAR-γ), a transcription factor involved in the control of the inflammatory response. Mouse primary neuronal and glial cultures and neuron-microglia cocultures were treated with the PPAR-γ endogenous ligand 15-deoxy-Δ(12, 14) -prostaglandin J2 (15d-PGJ2 ) in the presence and absence of lipopolysaccharide plus interferon-γ (LPS/IFN-γ)-induced glial activation. We show that 15d-PGJ2 inhibits the pro-inflammatory response and prevents both CD200R1 downregulation and CD200 upregulation in reactive glial cells. In addition, 15d-PGJ2 abrogates reactive-microglia induced neurotoxicity in neuron-microglia cultures through a CD200-CD200R1 dependent mechanism. These results suggest that PPAR-γ modulates CD200 and CD200R1 gene expression and that CD200-CD200R1 interaction is involved in the anti-inflammatory and neuroprotective action of PPAR-γ agonists.

CCAAT/enhancer binding protein δ regulates glial proinflammatory gene expression.

The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ) is expressed in activated astrocytes and microglia and can regulate the expression of potentially detrimental proinflammatory genes. The objective of this study was to determine the role of C/EBPδ in glial activation. To this end, glial activation was analyzed in primary glial cultures and in the central nervous system from wild type and C/EBPδ(-/-) mice. In vitro studies showed that the expression of proinflammatory genes nitric oxide (NO)synthase-2, cyclooxygenase-2, and interleukin (IL)-6 in glial cultures, and the neurotoxicity elicited by microglia in neuron-microglia cocultures, were decreased in the absence of C/EBPδ when cultures were treated with lipopolysaccharide (LPS) and interferon γ, but not with LPS alone. In C/EBPδ(-/-) mice, systemic LPS-induced brain expression of NO synthase-2, tumor necrosis factor-α, IL-1ß, and IL-6 was attenuated. Finally, increased C/EBPδ nuclear expression was observed in microglial cells from amyotrophic lateral sclerosis patients and G93A-SOD1 mice spinal cord. These results demonstrate that C/EBPδ plays a key role in the regulation of proinflammatory gene expression in glial activation and suggest that C/EBPδ inhibition has potential for the treatment of neurodegenerative disorders, in particular, amyotrophic lateral sclerosis.

Modelling neuroinflammation in vitro: a tool to test the potential neuroprotective effect of anti-inflammatory agents.

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.

Inhibition of CD200R1 expression by C/EBP ß in reactive microglial cells.

BACKGROUND: In physiological conditions, it is postulated that neurons control microglial reactivity through a series of inhibitory mechanisms, involving either cell contact-dependent, soluble-factor-dependent or neurotransmitter-associated pathways. In the current study, we focus on CD200R1, a microglial receptor involved in one of these cell contact-dependent mechanisms. CD200R1 activation by its ligand, CD200 (mainly expressed by neurons in the central nervous system),is postulated to inhibit the pro-inflammatory phenotype of microglial cells, while alterations in CD200-CD200R1 signalling potentiate this phenotype. Little is known about the regulation of CD200R1 expression in microglia or possible alterations in the presence of pro-inflammatory stimuli. METHODS: Murine primary microglial cultures, mixed glial cultures from wild-type and CCAAT/enhancer binding protein ß (C/EBPß)-deficient mice, and the BV2 murine cell line overexpressing C/EBPß were used to study the involvement of C/EBPß transcription factor in the regulation of CD200R1 expression in response to a proinflammatory stimulus (lipopolysaccharide (LPS)). Binding of C/EBPß to the CD200R1 promoter was determined by quantitative chromatin immunoprecipitation (qChIP). The involvement of histone deacetylase 1 in the control of CD200R1 expression by C/EBPß was also determined by co-immunoprecipitation and qChIP. RESULTS: LPS treatment induced a decrease in CD200R1 mRNA and protein expression in microglial cells, an effect that was not observed in the absence of C/EBPß. C/EBPß overexpression in BV2 cells resulted in a decrease in basal CD200R1 mRNA and protein expression. In addition, C/EBPß binding to the CD200R1 promoter was observed in LPS-treated but not in control glial cells, and also in control BV2 cells overexpressing C/EBPß. Finally, we observed that histone deacetylase 1 co-immunoprecipitated with C/EBPß and showed binding to a C/EBPß consensus sequence of the CD200R1 promoter in LPS-treated glial cells. Moreover, histone deacetylase 1 inhibitors reversed the decrease in CD200R1 expression induced by LPS treatment. CONCLUSIONS: CD200R1 expression decreases in microglial cells in the presence of a pro-inflammatory stimulus, an effect that is regulated, at least in part, by C/EBPß. Histone deacetylase 1 may mediate C/EBPß inhibition of CD200R1 expression, through a direct effect on C/EBPß transcriptional activity and/or on chromatin structure.

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27 Kip1.

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

C/EBPß expression in activated microglia in amyotrophic lateral sclerosis.

Neuroinflammation is thought to play a pathogenic role in many neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). In this study we demonstrate that the expression of nitric oxide (NO) synthase-2 (NOS2), and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) with interferon-γ is higher in microglial-enriched cultures from G93A-SOD1 mice, an ALS animal model, than from wild type mice. The levels of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor that regulates proinflammatory gene expression, are also upregulated in activated G93A-SOD1 microglial cells. In vivo, systemic lipopolysaccharide also induces an exacerbated neuroinflammatory response in G93A-SOD1 mice versus wild type mice, with increased expression of glial fibrillary acidic protein (GFAP), CD11b, nitric oxide synthase-2, cyclooxygenase-2, proinflammatory cytokines, and C/EBPß. Finally, we report that C/EBPß is expressed by microglia in the spinal cord of ALS patients. This is the first demonstration to our knowledge of microglial C/EBPß expression in human disease. Altogether these findings indicate that G93A-SOD1 expression results in an exacerbated pattern of neuroinflammation and suggest that C/EBPß is a candidate to regulate the expression of potentially neurotoxic genes in microglial cells in ALS.

Tissue plasminogen activator induces microglial inflammation via a noncatalytic molecular mechanism involving activation of mitogen-activated protein kinases and Akt signaling pathways and AnnexinA2 and Galectin-1 receptors.

Inflammatory responses mediated by glial cells play a critical role in many pathological situations related to neurodegeneration such as Alzheimer's disease. Tissue plasminogen activator (tPA) is a serine protease which best-known function is fibrinolysis, but it is also involved in many other physiological and pathological events as microglial activation. Here, we found that tPA is required for Aß-mediated microglial inflammatory response and tumor necrosis factor-α release. We further investigated the molecular mechanism responsible for tPA-mediated microglial activation. We found that tPA induces a catalytic-independent rapid and sustained activation of extracellular signal-regulated kinase (ERK)1/2, Jun N-terminal kinase (JNK), Akt, and p38 signaling pathways. Inhibition of ERK1/2 and JNK resulted in a strong inhibition of microglial activation, whereas Akt inhibition led to increased inflammatory response, suggesting specific functions for each signaling pathway in the regulation of microglial activation. Furthermore, we demonstrated that AnnexinA2 and Galectin-1 receptors are involved in tPA signaling and inflammatory response in glial cells. This study provides new evidences supporting that tPA plays a cytokine-like role in glial activation by triggering receptor-mediated intracellular signaling circuits and opens new therapeutic strategies for the treatment of neurological disorders in which neuroinflammation plays a pathogenic role.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein ß.

BACKGROUND: Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. METHODS: Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. RESULTS: C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon γ (IFNγ). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFNγ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFNγ-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia. CONCLUSIONS: These findings show involvement of C/EBPß in the regulation of pro-inflammatory gene expression in glial activation, and demonstrate for the first time a key role for C/EBPß in the induction of neurotoxic effects by activated microglia.

Inhibition of CCAAT/enhancer binding protein δ expression by chrysin in microglial cells results in anti-inflammatory and neuroprotective effects.

The control of neuroinflammation is a potential target to be considered in the treatment of neurodegenerative diseases. It is therefore important to find anti-inflammatory drugs and study new targets that inhibit neuroinflammation. We designed an experimental model of neuroinflammation in vitro to study the anti-inflammatory and neuroprotective effects of the flavonoid chrysin and the involvement of nuclear factor-κB p65 and CCAAT/enhancer binding proteins (C/EBPs) ß and δ transcription factors in its mechanism of action. We used primary cultures of mouse embryonic cortical neurons and cultures of BV2 (murine microglial cell line) or mouse primary microglia. We induced neuronal death in neuronal-BV2/microglial co-cultures using lipopolysaccharide of Escherichia coli and interferon-γ. Chrysin pre-treatment inhibited nitric oxide and tumor necrosis factor-α production, as well as inducible nitric oxide synthase expression in lipopolysaccharide E. coli and interferon-γ-treated microglial cells, but did not affect cyclooxygenase-2 expression. Chrysin pre-treatment also protected neurons against the neurotoxicity induced by reactive microglial cells. These effects were associated to a decrease in C/EBPδ protein level, mRNA expression, and DNA-binding activity, with no effect on C/EBPß and p65 nuclear protein levels or DNA-binding activity, pointing out C/EBPδ as a possible mediator of chrysin effects. Consequently, C/EBPδ is a possible target to act against neuroinflammation in neurodegenerative processes.

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase is regulated by acetylation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping glycolitic enzyme that recently has been implicated in cell signaling. Under apoptotic stresses, cells activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The GAPDH-Siah interaction depends on the integrity of lysine 227 in human GAPDH, being the mutant K227A unable to associate with Siah. As lysine residues are susceptible to be modified by acetylation, we aimed to analyze whether acetylation could mediate transport of GAPDH from cytoplasm to the nucleus. We observed that the acetyltransferase P300/CBP-associated factor (PCAF) interacts with and acetylates GAPDH. We also found that over-expression of PCAF induces the nuclear translocation of GAPDH and that for this translocation its intact acetylase activity is needed. Finally, the knocking down of PCAF reduces nuclear translocation of GAPDH induced by apoptotic stimuli. By spot mapping analysis we first identified Lys 117 and 251 as the putative GAPDH residues that could be acetylated by PCAF. We further demonstrated that both Lys were necessary but not sufficient for nuclear translocation of GAPDH after apoptotic stimulation. Finally, we identified Lys 227 as a third GAPDH residue whose acetylation is needed for its transport from cytoplasm to the nucleus. Thus, results reported here indicate that nuclear translocation of GAPDH is mediated by acetylation of three specific Lys residues (117, 227 and 251 in human cells). Our results also revealed that PCAF participates in the GAPDH acetylation that leads to its translocation to the nucleus.

CCAAT/enhancer binding protein delta in microglial activation.

The transcription factor CCAAT/enhancer binding protein delta (C/EBP delta) regulates transcription of genes that play important roles in glial activation. Previous studies have shown the astroglial expression of C/EBP delta but the microglial expression of C/EBP delta remains virtually unexplored, with the exception of two microarray studies. In this report, using murine primary cultures and BV2 cells we clearly demonstrate that C/EBP delta is expressed by microglia and it is upregulated in microglial activation. Lipopolysaccharide upregulates C/EBP delta both in microglia and in astrocytes. This effect is time-dependent, with a maximum effect at 3 hr at mRNA level and at 4-8 hr at protein level, and concentration-dependent, with a maximum effect at 100 ng/mL. The lipopolysaccharide-induced C/EBP delta upregulation in BV2 microglia is mimicked by agonists of the toll-like receptors 2, 3 and 9 and can be prevented by an inhibitor of extracellular signal-regulated kinase activation. C/EBP delta from activated BV2 microglia binds to the cyclooxygenase-2 promoter and forms complexes with C/EBP beta isoforms. These results point to C/EBP delta as a putative key regulator of proinflammatory gene expression in microglial activation.