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.

Mesoporous silica particles are phagocytosed by microglia and induce a mild inflammatory response in vitro.

Aim: Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses to MSPs of astrocytes and microglia, the two main cellular players in neuroinflammation. Materials & methods: Primary murine cortical mixed glial cultures were treated with rhodamine B-labeled MSPs. Results: MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. MSPs also do not affect mRNA levels of key proinflammatory genes; however, in combination with lipopolysaccharide, they significantly increase extracellular IL-1ß levels. Conclusion: These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells.

Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses of two types of brain cells, astrocytes and microglia, to MSPs. Mouse astrocytes and microglia were kept alive in cultures and were treated with MSPs that were labeled with a red fluorescent agent to facilitate visualization under the microscope. MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. When given alone, MSPs do not affect mRNA levels of key proinflammatory genes. However, MSPs given in combination with lipopolysaccharide, a strong proinflammatory agent, significantly increase extracellular levels of IL-1ß, one of the proinflammatory mediators studied. These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells.

TRPV2: A Key Player in Myelination Disorders of the Central Nervous System.

Transient potential receptor vanilloid 2 (TRPV2) is widely expressed through the nervous system and specifically found in neuronal subpopulations and some glial cells. TRPV2 is known to be sensitized by methionine oxidation, which results from inflammation. Here we aim to characterize the expression and regulation of TRPV2 in myelination pathologies, such as hypomyelination and demyelination. We validated the interaction between TRPV2 and its putative interactor Opalin, an oligodendrocyte marker, in mixed glial cultures under pro- and anti-inflammatory conditions. Then, we characterized TRPV2 time-course expression in experimental animal models of hypomyelination (jimpy mice) and de-/remyelination (cuprizone intoxication and experimental autoimmune encephalomyelitis (EAE)). TRPV2 showed upregulation associated with remyelination, inflammation in cuprizone and EAE models, and downregulation in hypomyelinated jimpy mice. TRPV2 expression was altered in human samples of multiple sclerosis (MS) patients. Additionally, we analyzed the expression of methionine sulfoxide reductase A (MSRA), an enzyme that reduces oxidated methionines in TRPV2, which we found increased in inflammatory conditions. These results suggest that TRPV2 may be a key player in myelination in accordance with the recapitulation hypothesis, and that it may become an interesting clinical target in the treatment of demyelination disorders.

Altered expression of the immunoregulatory ligand-receptor pair CD200-CD200R1 in the brain of Parkinson's disease patients.

Neuroinflammation, in which activated microglia are involved, appears to contribute to the development of Parkinson's disease (PD). However, the role of microglial activation and the mechanisms governing this process remain uncertain. We focused on one inhibitory mechanism involved in the control of microglial activation, the microglia inhibitory receptor CD200R1, and its ligand CD200, mainly expressed by neurons. The human CD200R1 gene encodes two membrane-associated and two soluble protein isoforms and the human CD200 gene encodes full-length proteins (CD200full) but also truncated (CD200tr) proteins which act as CD200R1 antagonists. Little is known about their expression in the human brain under pathological conditions. We used human peripheral blood monocytes and monocyte-derived microglia-like cells from control subjects to characterize the expression of the CD200R1 mRNA variants, which showed stimulus-specific responses. We provide evidence of increased CD200R1 (mRNA variants and protein isoforms) and CD200 expression (CD200tr mRNA) in brain tissue of PD patients, mainly in the hippocampus, as well as increased CD200 expression (CD200full and CD200tr mRNAs) in iPSCs-derived dopaminergic neurons generated from skin fibroblasts of PD patients. Our results suggest that CD200-CD200R1 signalling is altered in PD, which may affect the microglial function and constitute a potential target in therapeutic strategies for PD.

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.

CCAAT/enhancer binding protein δ is a transcriptional repressor of α-synuclein.

α-Synuclein is the main component of Lewy bodies, the intracellular protein aggregates representing the histological hallmark of Parkinson's disease. Elevated α-synuclein levels and mutations in SNCA gene are associated with increased risk for Parkinson's disease. Despite this, little is known about the molecular mechanisms regulating SNCA transcription. CCAAT/enhancer binding protein (C/EBP) ß and δ are b-zip transcription factors that play distinct roles in neurons and glial cells. C/EBPß overexpression increases SNCA expression in neuroblastoma cells and putative C/EBPß and δ binding sites are present in the SNCA genomic region suggesting that these proteins could regulate SNCA transcription. Based on these premises, the goal of this study was to determine if C/EBPß and δ regulate the expression of SNCA. We first observed that α-synuclein CNS expression was not affected by C/EBPß deficiency but it was markedly increased in C/EBPδ-deficient mice. This prompted us to characterize further the role of C/EBPδ in SNCA transcription. C/EBPδ absence led to the in vivo increase of α-synuclein in all brain regions analyzed, both at mRNA and protein level, and in primary neuronal cultures. In agreement with this, CEBPD overexpression in neuroblastoma cells and in primary neuronal cultures markedly reduced SNCA expression. ChIP experiments demonstrated C/EBPδ binding to the SNCA genomic region of mice and humans and luciferase experiments showed decreased expression of a reporter gene attributable to C/EBPδ binding to the SNCA promoter. Finally, decreased CEBPD expression was observed in the substantia nigra and in iPSC-derived dopaminergic neurons from Parkinson patients resulting in a significant negative correlation between SNCA and CEBPD levels. This study points to C/EBPδ as an important repressor of SNCA transcription and suggests that reduced C/EBPδ neuronal levels could be a pathogenic factor in Parkinson's disease and other synucleinopathies and C/EBPδ activity a potential pharmacological target for these neurological disorders.

Cerebrospinal fluid cytokines in multiple system atrophy: A cross-sectional Catalan MSA registry study.

INTRODUCTION: Neuroinflammation is a potential player in neurodegenerative conditions, particularly the aggressive ones, such as multiple system atrophy (MSA). Previous reports on cytokine levels in MSA using serum or cerebrospinal fluid (CSF) have been inconsistent, including small samples and a limited number of cytokines, often without comparison to Parkinson's disease (PD), a main MSA differential diagnosis. METHODS: Cross-sectional study of CSF levels of 38 cytokines using a multiplex assay in 73 participants: 39 MSA patients (19 with parkinsonian type [MSAp], 20 with cerebellar type [MSAc]; 31 probable, 8 possible), 19 PD patients and 15 neurologically unimpaired controls. None of the participants was under non-steroidal anti-inflammatory drugs at the time of the lumbar puncture. RESULTS: There were not significant differences in sex and age among participants. In global non-parametric comparisons FDR-corrected for multiple comparisons, CSF levels of 5 cytokines (FGF-2, IL-10, MCP-3, IL-12p40, MDC) differed among the three groups. In pair-wise FDR-corrected non-parametric comparisons 12 cytokines (FGF-2, eotaxin, fractalkine, IFN-α2, IL-10, MCP-3, IL-12p40, MDC, IL-17, IL-7, MIP-1ß, TNF-α) were significantly higher in MSA vs. non-MSA cases (PD + controls pooled together). Of these, MCP-3 and MDC were the most significant ones, also differed in MSA vs. PD, and were significant MSA-predictors in binary logistic regression models and ROC curves adjusted for age. CSF levels of fractalkine and MIP-1α showed a strong and significant positive correlation with UMSARS-2 scores. CONCLUSION: Increased CSF levels of cytokines such as MCP-3, MDC, fractalkine and MIP-1α deserve consideration as potential diagnostic or severity biomarkers of MSA.

Evaluación del aprendizaje de los estudiantes en los tres primeros cursos del Grado de Medicina de la Universitat de Barcelona / Student's learning assessment in the first three years of the Degree of Medicine in the University of Barcelona

Introducción: El proceso de adaptación al Espacio Europeo de Educación Superior ha comportado cambios en los sistemas de evaluación de los aprendizajes en las facultades de medicina y ha introducido los conceptos de evaluación de competencias y evaluación continuada. Objetivos: Describir y analizar, después de seis años de implementación del nuevo plan de estudios de medicina, cómo se está realizando el proceso de evaluación continuada en las diferentes asignaturas de los tres primeros cursos del Grado de Medicina de la Universitat de Barcelona, comparándolo con lo recomendado por los expertos en evaluación en educación médica, y establecer posibles mejoras. Sujetos y métodos: Se describen las actividades evaluativas e instrumentos utilizados por las diferentes asignaturas que demuestran una gran variabilidad y diversificación y una evaluación compartimentada. Se recogen las opiniones y el grado de satisfacción de los coordinadores sobre las actividades evaluativas utilizadas. Asimismo, se evidencia el esfuerzo realizado por los profesores de las distintas asignaturas para promover mejoras en el sistema. Resultados: Se comparan los resultados obtenidos con las recomendaciones establecidas por los expertos en educación médica, con especial referencia a los programas de formación institucional y al paradigma de la evaluación para el aprendizaje. Se discuten las dificultades existentes para desarrollar un mejor sistema de evaluación continuada. Conclusiones: A pesar del esfuerzo realizado y de las mejoras introducidas en las actividades de evaluación, estas no se ajustan totalmente a lo que debería ser una evaluación continuada real; se aboga por promover programas de formación del profesorado sobre evaluación en educación médica

Introduction: The process of adaptation to the European Higher Education Area has brought changes in the assessment of learning outcomes in medical schools and has introduced the concepts of competence assessment and continuous assessment. Aims: To describe and analyze, six years after the implementation of the new curriculum, how the continuous assessment process is being carried out in the different subjects of the first three years of the Degree of Medicine at the University of Barcelona, comparing it with what is recommended by the experts in assessment and to propose ways to improve it. Subjects and methods: The different assessment activities and tools used in the different subjects are described, showing a great variability and diversification and a compartmentalized assessment. The opinions and level of satisfaction of the coordinators of different subjects regarding the assessment activities used are also shown. Likewise, it is evidenced the effort made by the teachers in promoting improvements in the system. Results: The results obtained are compared with the recommendations established in the literature by the experts in medical education, with special reference to the concept of programmatic and institutional assessment and the paradigm of the assessment for learning. The existing difficulties to develop a better continuous assessment are discussed. Conclusions: Despite the efforts made and the improvements introduced in the assessment activities, these do not totally conform to what a real continuous assessment must be and we advocate to promote teacher's training programs on assessment in medical education

RNA-Seq transcriptomic profiling of primary murine microglia treated with LPS or LPS + IFNγ.

Microglia, the main resident immune cells in the CNS, are thought to participate in the pathogenesis of various neurological disorders. LPS and LPS + IFNγ are stimuli that are widely used to activate microglia. However, the transcriptomic profiles of microglia treated with LPS and LPS + IFNγ have not been properly compared. Here, we treated murine primary microglial cultures with LPS or LPS + IFNγ for 6 hours and then performed RNA-Sequencing. Gene expression patterns induced by the treatments were obtained by WGCNA and 11 different expression profiles were found, showing differential responses to LPS and LPS + IFNγ in many genes. Interestingly, a subset of genes involved in Parkinson's, Alzheimer's and Huntington's disease were downregulated by both treatments. By DESeq analysis we found differentially upregulated and downregulated genes that confirmed LPS and LPS + IFNγ as inducers of microglial pro-inflammatory responses, but also highlighted their involvement in specific cell functions. In response to LPS, microglia tended to be more proliferative, pro-inflammatory and phagocytic; whereas LPS + IFNγ inhibited genes were involved in pain, cell division and, unexpectedly, production of some inflammatory mediators. In summary, this study provides a detailed description of the transcriptome of LPS- and LPS + IFNγ treated primary microglial cultures. It may be useful to determine whether these in vitro phenotypes resemble microglia in in vivo pathological conditions.

Receptor-heteromer mediated regulation of endocannabinoid signaling in activated microglia. Role of CB1 and CB2 receptors and relevance for Alzheimer's disease and levodopa-induced dyskinesia.

Endocannabinoids are important regulators of neurotransmission and, acting on activated microglia, they are postulated as neuroprotective agents. Endocannabinoid action is mediated by CB1 and CB2 receptors, which may form heteromeric complexes (CB1-CB2Hets) with unknown function in microglia. We aimed at establishing the expression and signaling properties of cannabinoid receptors in resting and LPS/IFN-γ-activated microglia. In activated microglia mRNA transcripts increased (2 fold for CB1 and circa 20 fold for CB2), whereas receptor levels were similar for CB1 and markedly upregulated for CB2; CB1-CB2Hets were also upregulated. Unlike in resting cells, CB2 receptors became robustly coupled to Gi in activated cells, in which CB1-CB2Hets mediated a potentiation effect. Hence, resting cells were refractory while activated cells were highly responsive to cannabinoids. Interestingly, similar results were obtained in cultures treated with ß-amyloid (Aß1-42). Microglial activation markers were detected in the striatum of a Parkinson's disease (PD) model and, remarkably, in primary microglia cultures from the hippocampus of mutant ß-amyloid precursor protein (APPSw,Ind) mice, a transgenic Alzheimer's disease (AD) model. Also of note was the similar cannabinoid receptor signaling found in primary cultures of microglia from APPSw,Ind and in cells from control animals activated using LPS plus IFN-γ. Expression of CB1-CB2Hets was increased in the striatum from rats rendered dyskinetic by chronic levodopa treatment. In summary, our results showed sensitivity of activated microglial cells to cannabinoids, increased CB1-CB2Het expression in activated microglia and in microglia from the hippocampus of an AD model, and a correlation between levodopa-induced dyskinesia and striatal microglial activation in a PD model. Cannabinoid receptors and the CB1-CB2 heteroreceptor complex in activated microglia have potential as targets in the treatment of neurodegenerative diseases.

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.

C/EBPß and C/EBPδ transcription factors: Basic biology and roles in the CNS.

CCAAT/enhancer binding protein (C/EBP) ß and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPß mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPß and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPß and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPß and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPß and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPß and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.

Correlates of cerebrospinal fluid levels of oligomeric- and total-α-synuclein in premotor, motor and dementia stages of Parkinson's disease.

High-oligomeric and low-total-α-synuclein cerebrospinal fluid (CSF) levels have been found in Parkinson's disease (PD), but with inconsistent or limited data, particularly on their clinical and structural correlates in earliest (premotor) or latest (dementia) PD stages. We determined CSF oligomeric- and total-α-synuclein in 77 subjects: 23 with idiopathic REM-sleep behaviour disorder (iRBD, a condition likely to include a remarkable proportion of subjects in the premotor stage of PD) and 41 with PD [21 non-demented (PDND) + 20 demented (PDD)], intended to reflect the premotor-motor-dementia PD continuum, along with 13 healthy controls. The study protocol also included the Unified PD Rating Scale motor-section (UPDRS-III), mini mental state examination (MMSE), neuropsychological cognitive testing, 3T brain MRI for cortical-thickness analyses, CSF τ and CSF Aß. CSF oligomeric-α-synuclein was higher in PDND than iRBD and in PDD than iRBD and controls, and correlated with UPDRS-III, MMSE, semantic fluency and visuo-perceptive scores across the proposed premotor-motor-dementia PD continuum (iRBD + PDND + PDD). CSF total-α-synuclein positively correlated with age, CSF Aß, and, particularly, CSF τ, tending towards lower levels in PD (but not iRBD) vs. controls only when controlling for CSF τ. Low CSF total-α-synuclein was associated with dysfunction in phonetic-fluency (a frontal-lobe function) in PD and with frontal cortical thinning in iRBD and PDND independently of CSF τ. Conversely, the associations of high (instead of low) CSF total-α-synuclein with posterior-cortical neuropsychological deficits in PD and with posterior cortical thinning in PDD were driven by high CSF τ. These findings suggest that CSF oligomeric- and total-α-synuclein have different clinical, neuropsychological and MRI correlates across the proposed premotor-motor-dementia PD continuum. CSF total-α-synuclein correlations with CSF τ and Aß support the hypothesis of an interaction among these proteins in PD, with CSF τ probably influencing the presence of high (instead of low) CSF total-α-synuclein and its correlates mostly in the setting of PD-related dementia.

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 ß regulates prostaglandin E synthase expression and prostaglandin E2 production in activated microglial cells.

The eicosanoid prostaglandin E2 (PGE2 ) plays important roles in neuroinflammation and it is produced by the sequential action of the enzymes cyclooxygenase-2 (COX-2) and prostaglandin E synthase (PTGES). The expression of both enzymes and the production of PGE2 are increased in neuroinflammation. The objective of this study was to elucidate whether the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) regulates the expression of prostaglandin synthesis enzymes in neuroinflammation. To this aim, the expression of these enzymes in wild-type and C/EBPß-null mice was analyzed in vitro and in vivo. In mixed glial cultures, lipopolysaccharide (LPS) ± interferon γ (IFN-γ) induced C/EBPß binding to COX-2 and PTGES promoters. LPS ± IFN-γ-induced increases in PTGES expression and in PGE2 production in mixed glial and microglial cultures were abrogated in the absence of C/EBPß. Also, increased brain PTGES expression induced by systemic LPS administration was markedly reduced in C/EBPß-null mice. In contrast to PTGES, the induction of COX-2 expression in vitro or in vivo was not markedly affected by the absence of C/EBPß. These results demonstrate that C/EBPß regulates PTGES expression and PGE2 production by activated microglial cells in vitro and point to C/EBPß as a regulator of PTGES expression in vivo in the inflamed central nervous system. Altogether, these findings strengthen the proposed role of C/EBPß as a key player in the orchestration of neuroinflammatory gene response.

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.