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.

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.

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.

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.

Bringing Policymakers to Science Through Communication: A Perspective From Latin America.

Scientific knowledge should be shared beyond academic circles in order to promote science in policymaking. Science communication increases the understanding of how the natural world works and the capacity to make informed decisions. However, not every researcher has the ability to master the art of communicating, and even less in a clear, concise, and easy to understand language that society representatives appreciate. Within the huge and extraordinarily diverse Latin American region, science communication has been going on for at least 200 years, when the first science stories appeared in the newspapers, as well as the first science museums and botanical gardens were founded. Nevertheless, resources are limited, and notably time, which researchers spend mostly in mentoring, ensuring funding, publication of their results and laboratory work, while science journalists are an endangered species. This perspective article aims at providing some recommendations to build bridges between science and decision-making parties through communication, by exploring how Latin American diplomats and policymakers engage with scientific knowledge.

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.

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.

Biosafety assessment of conducting nanostructured materials by using co-cultures of neurons and astrocytes.

Neural electrode implants are made mostly of noble materials. We have synthesized a nanostructured material combining the good electrochemical properties of iridium oxide (IrOx) and carbon-nanotubes (CNT) and the properties of poly(3,4-ethylenedioxythiophene) (PEDOT). IrOx-CNT-PEDOT charge storage capacity was lower than that of IrOx and IrOx-CNT, but higher than that of other PEDOT-containing hybrids and Pt. Cyclic voltammetry, SEM, XPS and micro-Raman spectroscopy suggest that PEDOT encapsulates IrOx and CNT. In our search for a cell culture platform that could optimize modelling the in vivo environment, we determined cell viability, neuron and astrocyte functionality and the response of astrocytes to an inflammatory insult by using primary cultures of neurons, of astrocytes and co-cultures of both. The materials tested (based on IrOx, CNT and PEDOT, as well as Pt as a reference) allowed adhesion and proliferation of astrocytes and full compatibility for neurons grown in co-cultures. Functionality assays show that uptake of glutamate in neuron-astrocyte co-culture was significantly higher than the sum of the uptake in astrocytes and neurons. In co-cultures on IrOx, IrOx-CNT and IrOx-CNT-PEDOT, glutamate was released by a depolarizing stimulus and induced a significant increase in intracellular calcium, supporting the expression of functional NMDA/glutamate receptors. LPS-induced inflammatory response in astrocytes showed a decreased response in NOS2 and COX2 mRNA expression for IrOx-CNT-PEDOT. Results indicate that neuron-astrocyte co-cultures are a reliable model for assessing the biocompatibility and safety of nanostructured materials, evidencing also that hybrid IrOx-CNT-PEDOT nanocomposite materials may offer larger resistance to inflammatory insults.

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.