Microglia perform surveillance and phagocytosis to maintain the homeostasis of the central nervous system (CNS). These processes are energetically demanding, and given the critical roles of mitochondria in providing ATP, the characteristics of the mitochondrial network can modulate microglial behavior. Although the Ca2+-permeable Transient Receptor Potential Vanilloid 4 (TRPV4) is known for regulating microglial morphology and migration, and it is implicated in mitochondrial calcium uptake, it is unknown whether TRPV4 affects the mitochondrial network in microglia. Our study provides evidence that TRPV4 plays a role in the integrity and complexity of the mitochondrial network in microglia. Quantification of the Mitochondrial Fragmentation and Complexity Index (MFCI) and increased pDrp1 (Ser616) showed a shift towards mitochondrial network fragmentation, and lowered complexity in Trpv4 knockout versus wild-type primary murine microglia in vitro. The distribution of mitochondria within microglia showed significant differences in density at 10–32 µm away from the nucleus. Furthermore, acute pharmacological TRPV4 inhibition with GSK2193874 did not induce significant mitochondria network fragmentation. Our findings establish TRPV4 as a regulator of mitochondrial dynamics and adaptive responses, highlighting its importance for maintaining homeostasis in microglia and the entire CNS.

In multiple sclerosis (MS), the chronic, unresolved nature of neuroinflammation within the central nervous system (CNS) remains a major obstacle for effective therapeutic intervention. This challenge arises primarily due to an incomplete understanding of the dysregulated inflammatory and pro-resolving pathways underlying MS lesion progression. Bioactive lipid mediators (LMs), biosynthesized through the coordinated actions of specific enzymes like lipoxygenases (LOX) and cyclooxygenases (COX), are key regulators of both the initiation and resolution of an inflammatory response; however, their spatial organization and functional role during MS pathology have not been fully elucidated. Here, by using pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry imaging and immunohistochemistry, we reveal an increase in the LM leukotriene B4 (LTB4) in human MS white matter compared to controls, with further enrichment in MS lesions relative to perilesional areas, alongside elevated microglial 5-LOX activating protein (FLAP) expression. Pharmacological antagonism of FLAP suppresses LTB4 biosynthesis in human-induced pluripotent stem cell (iPSC)-derived microglia with only marginal effects on the microglia transcriptional phenotype as determined by RNA sequencing. Moreover, in vivo FLAP antagonism ameliorates disease severity and spinal cord inflammatory gene expression in the experimental autoimmune encephalomyelitis (EAE) model, an animal model of MS, in both a prophylactic and therapeutic settings. This coincided with reduced local LTB4 biosynthesis and reduced levels of inflammatory monocytes within the spinal cord during EAE. Together these findings establish the FLAP/LTB4 axis as a driver of neuroinflammation and a druggable therapeutic target for chronic inflammatory CNS disorders like MS.

In AD, amyloid pathology (A) precedes progressive development of tau pathology (T) and neurodegeneration (N), with the latter (T/N) processes associated with symptom progression. Recent anti-amyloid beta (Aβ) clinical trials raise hope but indicate the need for multi-targeted therapies, to effectively halt clinical AD and ATN pathology progression. APOE-related putative protective mutations (including APOE3Christchurch, RELN-COLBOS) were recently identified in case reports with exceptionally high resilience to autosomal dominant AD. In these cases, Nature provided proof of concept for halting autosomal dominant AD and ATN progression in humans, despite a high amyloid load, and pointing to the APOE pathway as a potential target. This is further supported by the recent identification of APOE4 homozygosity as genetic AD. Here we studied the role of APOE in a preclinical model that robustly mimics amyloid-facilitated (A) tau pathology (T) and subsequent neurodegeneration (N), denoted as ATN model, generated by crossing 5xFAD (F +) and TauP301S (T +) mice. We show that APOE deficiency, markedly inhibited progression to tau pathology and tau-induced neurodegeneration in this ATN model, despite a high Aβ load, reminiscent of the high resilience ADAD case reports. Further study identified, despite increased Aβ load (W02 stained), a significant decrease in compacted, dense core plaques stained by ThioS in APOE deficient ATN mice. Furthermore, single-cell RNA sequencing (scRNA-seq) showed a crucial role of APOE in microglial conversion beyond homeostatic microglia to reactive and disease associated microglia (DAM) in this ATN preclinical model. Microglial elimination significantly decreased amyloid-driven tau pathology, in the presence of APOE, but not in APOE deficient mice. Together the data demonstrate that APOE deficiency inhibits amyloid-driven tau pathology and subsequent neurodegeneration, by pleiotropic effects including prevention of dense core plaque formation and halting conversion of homeostatic microglia. We here present a model recapitulating inhibition of amyloid-facilitated tau pathology by APOE deficiency despite high Aβ load, important for understanding the role of APOE, and APOE-dependent processes in ATN progression and its therapeutic exploitation in AD.

Microglia, the primary phagocytes activated after spinal cord injury (SCI), play a key role in containing the lesion and protecting the glial scar from infiltrating immune cells. Although these responses are initially protective, excessive microglial proliferation and sustained pro-inflammatory activation can worsen secondary damage and limit recovery. Modulating microglial activity has been proposed as a potential therapeutic approach to enhance SCI repair. Previous studies suggest that constitutive loss of the mechanosensory channel TRPV4 (transient receptor potential vanilloid 4) reduces microgliosis and inflammation at the lesion site, improving functional outcomes. TRPV4 is a Ca2+-permeable channel implicated in several microglial characteristics, like morphology, motility, proliferation, and phagocytosis. Whereas endogenous TRPV4-activating stimuli are abundant at the lesion site, the microglia-specific contribution of TRPV4 in SCI recovery remains unknown. To investigate this, we used phagocyte-specific Trpv4 conditional knockout mice and phagocyte-specific TRPV4-deficient bone marrow chimeras subjected to contusion SCI. We achieved robust and efficient spinal cord monocyte-derived cell repopulation after bone marrow transplantation and PLX treatment. Surprisingly, TRPV4 deficiency in phagocytes did not enhance functional recovery, reduce microgliosis, or diminish scar formation after SCI. Furthermore, contrary to prior studies, constitutive TRPV4 deficiency did not improve SCI outcomes, indicating that the role of TRPV4 in this context is complex and potentially redundant with other pathways.

Synaptic dysfunction is a hallmark of neurodevelopmental disorders (NDDs), often linked to genes involved in cytoskeletal regulation. While the role of these genes has been extensively studied in neurons, microglial functions such as phagocytosis are also dependent on cytoskeletal dynamics. We demonstrate that disturbance of actin cytoskeletal regulation in microglia, modeled by genetically impairing the scaffold protein Disrupted-in-Schizophrenia 1 (DISC1), which integrates actin-binding proteins, causes a shift in actin regulatory balance favoring filopodial versus lamellipodial actin organization. The resulting microglia-specific dysregulation of actin dynamics leads to excessive uptake of synaptic proteins. Genetically engineered DISC1-deficient mice show diminished hippocampal excitatory transmission and associated spatial memory deficits. Reintroducing wild-type microglia-like cells via bone marrow transplantation in adult DISC1-deficient mice restores the synaptic function of neurons and rescues cognitive performance. These findings reveal a pivotal role for microglial actin cytoskeletal remodeling in preserving synaptic integrity and cognitive health. Targeting microglial cytoskeletal dynamics may effectively address cognitive impairments associated with NDDs, even in adulthood.

Microglia activity can drive excessive synaptic loss during the prodromal phase of Alzheimer’s disease (AD) and is associated with lowered cyclic adenosine monophosphate (cAMP) due to cAMP phosphodiesterase 4B (PDE4B). This study aimed to investigate whether long-term inhibition of PDE4B by A33 (3 mg/kg/day) can prevent synapse loss and its associated cognitive decline in APPswe/PS1dE9 mice. This model is characterized by a chimeric mouse/human APP with the Swedish mutation and human PSEN1 lacking exon 9 (dE9), both under the control of the mouse prion protein promoter. The effects on cognitive function of prolonged A33 treatment from 20 days to 4 months of age, was assessed at 7–8 months. PDE4B inhibition significantly improved both the working and spatial memory of APPswe/PSdE9 mice after treatment ended. At the cellular level, in vitro inhibition of PDE4B induced microglial filopodia formation, suggesting that regulation of PDE4B activity can counteract microglia activation. Further research is needed to investigate if this could prevent microglia from adopting their ‘disease-associated microglia (DAM)’ phenotype in vivo. These findings support the possibility that PDE4B is a potential target in combating AD pathology and that early intervention using A33 may be a promising treatment strategy for AD.

Introduction Chronic cassava-derived cyanide poisoning is associated with the appearance of konzo, a tropical spastic paraparesis due to selective upper motor neuron damage. Whether the disease is caused by a direct action of cyanide or its metabolites is still an open question. This preliminary study assessed the neurotoxic effects of thiocyanate (SCN) and cyanate (OCN), two cyanide metabolites hypothesized to be plausible toxic agents in konzo. Methods Cultured mouse neuroblastoma (Neuro-2A) and human neuroblastoma (SH-SY5Y) cell lines were incubated (24, 48, and 72hours) in sodium OCN or sodium SCN in a disease-relevant concentration range. Cell viability, caspase (3, 8, and 9) activities, and reactive oxygen species (ROS) generation were evaluated using appropriate assay kits. Additionally, electrophysiological responses induced by OCN and SCN in primary spinal cord neurons (from Sprague Dawley rats) were assessed by whole-cell patch-clamp techniques. Results Both OCN and SCN were toxic in a dose-dependent way, even if SCN toxicity appeared at very high concentrations (30mM, corresponding to more than 100-fold higher than normal plasmatic levels), contrary to OCN (0.3-3mM). OCN was markedly more toxic in a poor culture medium (MEM; IC50 = 3.2mM) compared to a glucose- and amino acid-rich medium (DMEM; IC50=7.6mM). OCN treatment increased the ROS generation by 8.9 folds, as well as the Caspase-3, Caspase-8, and Caspase-9 activities by 3.2, 2.5, and 2.6 folds, respectively. Finally, OCN (and SCN to a lesser extent) induced depolarizing currents in primary spinal cord neurons, through an activation of ionotropic glutamate receptors. Conclusion Our results suggest OCN as the most plausible neurotoxic agent involved in konzo, while SCN toxicity could be questioned at such high concentrations. Also, they support apoptosis, oxidative stress, and excitotoxicity as probable mechanisms of OCN neurotoxicity.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

p27kip1 is a multifunctional protein that promotes cell cycle exit by blocking the activity of cyclin/cyclin-dependent kinase complexes as well as migration and motility via signaling pathways that converge on the actin and microtubule cytoskeleton. Despite the broad characterization of p27kip1 function in neural cells, little is known about its relevance in microglia. Here, we studied the role of p27kip1 in microglia using a combination of in vitro and in situ approaches. While the loss of p27kip1 did not affect microglial density in the cerebral cortex, it altered their morphological complexity in situ. However, despite the presence of p27kip1 in microglial processes, as shown by immunofluorescence in cultured cells, loss of p27kip1 did not change microglial process motility and extension after applying laser-induced brain damage in cortical brain slices. Primary microglia lacking p27kip1 showed increased phagocytic uptake of synaptosomes, while a cell cycle dead variant negatively affected phagocytosis. These findings indicate that p27kip1 plays specific roles in microglia.

Synapses are the functional units of the brain.They form specific contact points that drive neuronal communication and are highly plastic in their strength, density, and shape.A carefully orchestrated balance between synaptogenesis and synaptic pruning, i.e., the elimination of weak or redundant synapses, ensures adequate synaptic density.An imbalance between these two processes lies at the basis of multiple neuropathologies.Recent evidence has highlighted the importance of glia-neuron interactions in the synaptic unit, emphasized by glial phagocytosis of synapses and local excretion of inflammatory mediators.These findings warrant a closer look into the molecular basis of cell-signaling pathways in the different brain cells that are related to synaptic plasticity.In neurons, intracellular second messengers, such as cyclic guanosine or adenosine monophosphate (cGMP and cAMP, respectively), are known mediators of synaptic homeostasis and plasticity.Increased levels of these second messengers in glial cells slow down inflammation and neurodegenerative processes.These multi-faceted effects provide the opportunity to counteract excessive synapse loss by targeting cGMP and cAMP pathways in multiple cell types.Phosphodiesterases (PDEs) are specialized degraders of these second messengers, rendering them attractive targets to combat the detrimental effects of neurological disorders.Cellular and subcellular compartmentalization of the specific isoforms of PDEs leads to divergent downstream effects for these enzymes in the various central nervous system resident cell types.This review provides a detailed overview on the role of PDEs and their inhibition in the context of glia-neuron interactions in different neuropathologies characterized by synapse loss.In doing so, it provides a framework to support future research towards finding combinational therapy for specific neuropathologies.

Abstract Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial‐derived extracellular vesicles (M‐EVs) to the maintenance of microglia homeostasis and how M‐EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M‐EVs is limited. Here, we analysed the effects of M‐EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non‐activated microglia BV2 cells. We showed that M‐EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M‐EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M‐EVs increased the protein expression of the autophagy marker, microtubule‐associated proteins 1A/1B light chain 3B isoform II (LC3B‐II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M‐EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M‐EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia‐microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis.

•Microglia are immigrant CNS immune cells that show motility and mobility, especially in the developing brain.•Microglial motility and mobility is likely steered by the constantly changing microenvironment of the CNS.•Microglia populate the developing CNS by proliferation and intensive migration, of which the molecular mechanisms have just started to be unravelled.•Microglia actively participate in CNS development and homeostasis, even at early developmental stages.•Defective microglial functions during development can be related to neurodevelopmental disorders.

Brains of Alzheimer’s disease patients are characterized by the presence of amyloid plaques and neurofibrillary tangles, both invariably associated with neuroinflammation. A crucial role for NLRP3–ASC inflammasome [NACHT, LRR and PYD domains-containing protein 3 (NLRP3)–Apoptosis-associated speck-like protein containing a CARD (ASC)] in amyloid-beta (Aβ)-induced microgliosis and Aβ pathology has been unequivocally identified. Aβ aggregates activate NLRP3–ASC inflammasome (Halle et al. in Nat Immunol 9:857–865, 2008) and conversely NLRP3–ASC inflammasome activation exacerbates amyloid pathology in vivo (Heneka et al. in Nature 493:674–678, 2013), including by prion-like ASC-speck cross-seeding (Venegas et al. in Nature 552:355–361, 2017). However, the link between inflammasome activation, as crucial sensor of innate immunity, and Tau remains unexplored. Here, we analyzed whether Tau aggregates acting as prion-like Tau seeds can activate NLRP3–ASC inflammasome. We demonstrate that Tau seeds activate NLRP3–ASC-dependent inflammasome in primary microglia, following microglial uptake and lysosomal sorting of Tau seeds. Next, we analyzed the role of inflammasome activation in prion-like or templated seeding of Tau pathology and found significant inhibition of exogenously seeded Tau pathology by ASC deficiency in Tau transgenic mice. We furthermore demonstrate that chronic intracerebral administration of the NLRP3 inhibitor, MCC950, inhibits exogenously seeded Tau pathology. Finally, ASC deficiency also decreased non-exogenously seeded Tau pathology in Tau transgenic mice. Overall our findings demonstrate that Tau-seeding competent, aggregated Tau activates the ASC inflammasome through the NLRP3–ASC axis, and we demonstrate an exacerbating role of the NLRP3–ASC axis on exogenously and non-exogenously seeded Tau pathology in Tau mice in vivo. The NLRP3–ASC inflammasome, which is an important sensor of innate immunity and intensively explored for its role in health and disease, hence presents as an interesting therapeutic approach to target three crucial pathogenetic processes in AD, including prion-like seeding of Tau pathology, Aβ pathology and neuroinflammation.

Background: Microglia, the resident phagocytic cells of the brain, have recently been the subject of intense investigation given their role in pathology and normal brain physiology. In general, phagocytic cells are hard to transfect with plasmid DNA. The BV2 cell line is a murine cell line of microglial origin which is often used to study this cell type in vitro. Unfortunately, this microglial cell line is, like other phagocytic cells, resistant to transfection. New method: Magnetofection is a well-established transfection method that combines DNA with magnetic particles which, under the influence of a magnetic field, ensures a high concentration of particles in proximity of cultured cells. Only recently, Glial-Mag was specifically developed for efficient transfection of microglia and microglial cell lines. Results: Magnetofection with Glial-Mag yielded a transfection efficiency of 34.95% in BV2 cells, 24 h after transfection with an eGFP-expressing plasmid. Efficient gene delivery caused a modest and short-lived cell activation (as measured by IL6 secretion) that ceased by 24 h after transfection. Comparison with existing methods: Here we show that Glial-Mag magnetofection of BV2 cells yielded a significantly higher transfection efficiency (34.95%) compared to other chemical transfection methods including calcium-phoshate precipitation (0.34%), X-tremeGENE (3.30%) and Lipofectamine 2000 (12.51%). Conclusion: Transfection of BV2 cells using Glial-Mag magnetofection is superior compared to other chemical transfection methods and could be considered as the method of choice to chemically transfect microglial cell lines. (C) 2017 Elsevier B.V. All rights reserved.

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix–cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix–integrin interactions during microglial migration in the embryonic brain ex vivo . We focused on microglia–fibronectin interactions mediated through the fibronectin receptor α5β1 integrin because in vitro work indirectly suggested a role for this ligand–receptor pair. Using 2‐photon time‐lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age‐specific function of the α5β1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5β1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5β1 integrin in microglial migration during colonization of the embryonic brain.