The pro- and anti-inflammatory activity of fatty acids

Inflammation is crucial to maintain homeostasis in the body. The contribution of fatty acids to the inflammatory process is exerted through a variety of mechanisms leading to cell surface modifications, activation of intracellular receptors that control inflammatory signaling processes, and changes in gene expression patterns. While long-chain saturated fatty acids induce NFkB pathway activation through TLR-4 binding, unsaturated fatty acids, such as monounsaturated, polyunsaturated, and conjugated fatty acids’ antiinflammatory ability is mediated through PPARs or GPR120. Moreover, these unsaturated fatty acids, especially omega-3 fatty acids, have immunomodulatory and cytoprotective potential, which is highly relevant for diseases with a neuroinflammatory component, such as obesity, Alzheimer’s disease, multiple sclerosis, and schizophrenia.info:eu-repo/semantics/acceptedVersio

CLA and CLNA ameliorate neuroinflammation and cellular oxidation related with western diets

High-fat diet has been associated with a chronic-low grade inflammation in both adipose tissue and central nervous system. Fatty acids are known to cross the blood-brain barrier and reach the central nervous system where they can accumulate. Microglia express a wide range of lipid-sensitive receptors, potentially triggering inflammatory responses. Since fatty acids can exert pro and anti-inflammatory effects in the hypothalamus, in this work, through live cell imaging and FRET technology, we assessed the potential role of omega-3 fatty acids and CFAs in modulating microglia inflammation triggered by obesogenic nutrients. First, the combined action of fructose and saturated fatty acid palmitic acid (to mimic western pattern diet), induced NFkB pathway activation and oxidative stress, by reactive oxygen species production, in HMC3 human microglia. Such results suggest that western pattern diet may induce microglia inflammatory processes in the central nervous system, ultimately resulting in neuroinflammation. On the other hand, exposure of HMC3 cells to polyunsaturated fatty acids (omega-3 – EPA and DHA- and CLA and CLNA isomers) showed a preventive potential, since they were able to abolish the palmitic acid+fructose induced-NFkB pathway activation. Moreover, omega-3 and CLA also showed antioxidant potential by inhibition of reactive oxygen species production. Although the mechanisms of action have not been fully described yet, GPR120/FFA4 is known to bind some omega-3 fatty acids. By using chemical agonists and antagonists of GPR120/FFA4 it was demonstrated that while omega-3, CLA and CLNA effect on NFkB pathway inhibition is mediated by this receptor, the antioxidant ability of omega-3 and CLA occurs through different signaling mechanisms. It was suggested, for the first time, that CLA and CLNA have a similar action to omega-3 on microglia, probably via GPR120 activation and modulation of NFkB-associated inflammatory pathways.info:eu-repo/semantics/publishedVersio

CLA and CLNA ameliorate neuroinflammation and cellular oxidation related with western diets

info:eu-repo/semantics/publishedVersio

Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein — MBP — and proteolipid protein — PLP — (respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination. Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination. Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium. OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA. Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes

Myelin is dependent on the Charcot-Marie-Tooth Type 4H disease culprit protein FRABIN/FGD4 in Schwann cells

Studying the function and malfunction of genes and proteins associated with inherited forms of peripheral neuropathies has provided multiple clues to our understanding of myelinated nerves in health and disease. Here, we have generated a mouse model for the peripheral neuropathy Charcot-Marie-Tooth disease type 4H by constitutively disrupting the mouse orthologue of the suspected culprit gene FGD4 that encodes the small RhoGTPase Cdc42-guanine nucleotide exchange factor Frabin. Lack of Frabin/Fgd4 causes dysmyelination in mice in early peripheral nerve development, followed by profound myelin abnormalities and demyelination at later stages. At the age of 60 weeks, this was accompanied by electrophysiological deficits. By crossing mice carrying alleles of Frabin/Fgd4 flanked by loxP sequences with animals expressing Cre recombinase in a cell type-specific manner, we show that Schwann cell-autonomous Frabin/Fgd4 function is essential for proper myelination without detectable primary contributions from neurons. Deletion of Frabin/Fgd4 in Schwann cells of fully myelinated nerve fibres revealed that this protein is not only required for correct nerve development but also for accurate myelin maintenance. Moreover, we established that correct activation of Cdc42 is dependent on Frabin/Fgd4 function in healthy peripheral nerves. Genetic disruption of Cdc42 in Schwann cells of adult myelinated nerves resulted in myelin alterations similar to those observed in Frabin/Fgd4-deficient mice, indicating that Cdc42 and the Frabin/Fgd4-Cdc42 axis are critical for myelin homeostasis. In line with known regulatory roles of Cdc42, we found that Frabin/Fgd4 regulates Schwann cell endocytosis, a process that is increasingly recognized as a relevant mechanism in peripheral nerve pathophysiology. Taken together, our results indicate that regulation of Cdc42 by Frabin/Fgd4 in Schwann cells is critical for the structure and function of the peripheral nervous system. In particular, this regulatory link is continuously required in adult fully myelinated nerve fibres. Thus, mechanisms regulated by Frabin/Fgd4-Cdc42 are promising targets that can help to identify additional regulators of myelin development and homeostasis, which may crucially contribute also to malfunctions in different types of peripheral neuropathie

Essential and distinct roles for cdc42 and rac1 in the regulation of Schwann cell biology during peripheral nervous system development

During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue-specific conditional gene targeting to show that members of the Rho GTPases, cdc42 and rac1, have different and essential roles in axon sorting by Schwann cells. Our results indicate that although cdc42 is required for normal Schwann cell proliferation, rac1 regulates Schwann cell process extension and stabilization, allowing efficient radial sorting of axon bundles

Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado–Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of a5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.National Institutes of Health (NIH) ‘(R01NS038712)Fundação para a Ciência e a Tecnologia (FCT) and COMPETE through the project ‘(PTDC/SAU-GMG/ 101572/2008)Fundação para a Ciência e a Tecnologia (FCT) - fellowships SFRH/BD/51059/2010, SFRH/BD/ 78388/2011 and SFRH/BPD/91562/201

Profiling microglia in a mouse model of Machado-Joseph disease

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/ 127828/2016). S.P.N. was also supported by FCT (PD/BD/114120/2015). Work in the JBR laboratory was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the Project POCI-01-0145- FEDER030647 (PTDC/MED-NEU/31318/2017). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Microglia dysfunction caused by the loss of Rhoa disrupts neuronal physiology and leads to neurodegeneration

© 2020 The Author(s). Creative Commons Attribution (CC BY 4.0)Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.The authors acknowledge the support of the following i3S Scientific Platforms: Animal Facility, Translational Cytometry Unit (TraCy), BioSciences Screening (BS) and Advanced Light Microscopy (ALM), and members of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01–0145-FEDER-022122). FCT Portugal ( PTDC/MED-NEU/31318/2017-031318 ) supported work in the J.B.R. lab. FCT Portugal , PEst ( UID/NEU/04539/2013 ), COMPETE-FEDER ( POCI-01-0145-FEDER-007440 ), Centro 2020 Regional Operational Programme ( CENTRO-01-0145-FEDER-000008 : BrainHealth 2020), and Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB) supported work in the A.F.A. lab. C.C.P. and R.S. hold employment contracts financed by national funds through FCT (Fundação para a Ciência e a Tecnologia, I.P.) in the context of the program contract described in paragraphs 4, 5, and 6 of article 23 of law no. 57/2016, of August 29th, as amended by law no. 57/2017 of July 19th.info:eu-repo/semantics/publishedVersio