The effects of urolithin A on poly I:C-induced microglial activation.

Neuroinflammation can be triggered by various stimuli, including viral infections. Viruses can directly invade the brain and infect neuronal cells or indirectly trigger a "cytokine storm" in the periphery that eventually leads to microglial activation in the brain. While this initial activation of microglial cells is important for viral clearance, chronic activation leads to excessive inflammation and oxidative stress, which can be neurotoxic. Remarkebly, recent studies have shown that certain viruses such as influenza A virus, coronavirus, herpes virus and Epstein-Barr virus may be involved in the development of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. Therefore, it is important to find therapeutic strategies against chronic neuroinflammation triggered by viral infections. Here, we investigated the effects of urolithin A (UA) on microglial activation in vitro induced by a viral mimetic, poly I:C, in a triple co-culture system of neurons, astrocytes and microglial cells. Immunocytochemistry was used to perform a comprehensive single-cell analysis of the morphological changes of microglia as an indicator of their reactive state. Treatment with UA significantly prevented the poly I:C-induced reactive state of microglia, which was characterized by increased expression of the microglial activation markers CD68 and IBA-1. UA restored the poly I:C-induced morphology by restoring microglial ramification. In addition, UA was able to reduce the release of the pro-inflammatory mediators CCL2, TNF-α, and IL-1ß and showed a trend toward attenuation of cellular ROS production in poly I:C-treated cultures. Overall, this study suggests that UA as a component of a healthy diet may help prevent virus-induced neuroinflammation and may have therapeutic potential for future studies to prevent or treat neurodegenerative diseases by targeting the associated neuroinflammatory processes.

Stretch-injury promotes microglia activation with enhanced phagocytic and synaptic stripping activities.

Microglial cells, as the primary defense line in the central nervous system, play a crucial role in responding to various mechanical signals that can trigger their activation. Despite extensive research on the impact of chemical signaling on brain cells, the understanding of mechanical signaling in microglia remains limited. To bridge this gap, we subjected microglial cells to a singular mechanical stretch and compared their responses with those induced by lipopolysaccharide treatment, a well-established chemical activator. Here we show that stretching microglial cells leads to their activation, highlighting their significant mechanosensitivity. Stretched microglial cells exhibited distinct features, including elevated levels of Iba1 protein, a denser actin cytoskeleton, and increased persistence in migration. Unlike LPS-treated microglial cells, the secretory profile of chemokines and cytokines remained largely unchanged in response to stretching, except for TNF-α. Intriguingly, a single stretch injury resulted in more compacted chromatin and DNA damage, suggesting potential long-term genomic instabilities in stretched microglia. Using compartmentalized microfluidic chambers with neuronal networks, we observed that stretched microglial cells exhibited enhanced phagocytic and synaptic stripping activities. These findings collectively suggest that stretching events can unlock the immune potential of microglial cells, contributing to the maintenance of brain tissue homeostasis following mechanical injury.

Dopamine-mediated striatal activity and function is enhanced in GlyRα2 knockout animals.

The glycine receptor alpha 2 (GlyRα2) is a ligand-gated ion channel which upon activation induces a chloride conductance. Here, we investigated the role of GlyRα2 in dopamine-stimulated striatal cell activity and behavior. We show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1 receptor-expressing striatal projection neurons, but does not alter midbrain dopamine neuron activity. We next show that the locomotor response to d-amphetamine is enhanced in GlyRα2 knockout animals, and that this increase correlates with c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the neuronal ensemble size in the striatum in response to D-amphetamine in GlyRα2 KO mice. Finally, we show enhanced appetitive conditioning in GlyRα2 KO animals that is likely due to increased motivation, but not changes in associative learning or hedonic response. Taken together, we show that GlyRα2 is an important regulator of dopamine-stimulated striatal activity and function.

Extremely Low-Frequency Electromagnetic Stimulation (ELF-EMS) Improves Neurological Outcome and Reduces Microglial Reactivity in a Rodent Model of Global Transient Stroke.

Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.

Novel maternal autoantibodies in autism spectrum disorder: Implications for screening and diagnosis.

Introduction: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which early recognition is a major challenge. Autoantibodies against fetal brain antigens have been found in the blood of mothers of children with ASD (m-ASD) and can be transferred to the fetus where they can impact neurodevelopment by binding to fetal brain proteins. This study aims to identify novel maternal autoantibodies reactive against human fetal brain antigens, and explore their use as biomarkers for ASD screening and diagnosis. Methods: A custom-made human fetal brain cDNA phage display library was constructed, and screened for antibody reactivity in m-ASD samples from the Simons Simplex Collection (SSC) of the Simons Foundation Autism Research Initiative (SFARI). Antibody reactivity against 6 identified antigens was determined in plasma samples of 238 m-ASD and 90 mothers with typically developing children (m-TD). Results: We identified antibodies to 6 novel University Hasselt (UH)-ASD antigens, including three novel m-ASD autoantigens, i.e., ribosomal protein L23 (RPL23), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calmodulin-regulated spectrin-associated protein 3 (CAMSAP3). Antibody reactivity against a panel of four of these targets was found in 16% of m-ASD samples, compared to 4% in m-TD samples (p = 0.0049). Discussion: Maternal antibodies against 4 UH-ASD antigens could therefore provide a novel tool to support the diagnosis of ASD in a subset of individuals.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

Hetero-pentamerization determines mobility and conductance of Glycine receptor α3 splice variants.

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases.

Transcutaneous electrical nerve inhibition using medium frequency alternating current.

Transcutaneous medium-frequency alternating electrical current is defined as an alternating current between 1 and 10 kHz and is capable of producing an instant, reversible block. This study aims to evaluate the efficacy of sensory perception and force production of the index and middle finger after transcutaneous medium-frequency alternating electrical current stimulation of the distal median nerve. A single-center prospective interventional cohort study was conducted in adult healthy volunteers at the Jessa Hospital, Hasselt, Belgium. Two different electrodes (PALS & 3M) were placed on the distal median nerve, which was located using a Sonosite X-Porte Ultrasound transducer, with the first electrode being placed on the skin at the level of the transverse carpal ligament and the second electrode 7 cm proximally to the first electrode. The tactile sensation was evaluated with Semmes-Weinstein monofilament test and sensation of pressure/pain was evaluated with an algometer. Peak force production was assessed with an electronic dynamometer. All measurements were performed at baseline and tMFAEC stimulation frequencies of 2 and 10 kHz in a randomized manner. Statistical analysis was performed with a one-way ANOVA with repeated measures test or a Friedman rank sum test, followed by the Wilcoxon signed rank test adjusted with Bonferroni correction. A p-value < 0.05 was considered statistically significant. From 9 to 13th of April 2021, 25 healthy volunteers were included in the Jessa Hospital, Hasselt, Belgium. A statistically significant reduction in tactile sensation during 2 kHz and 10 kHz stimulation compared to baseline was observed (2.89 ± 0.22 (PALS2); 3.35 ± 0.25 (3M2) and 2.14 ± 0.12 (PALS10); 2.38 ± 0.12 (3M10) versus - 1.75 ± 0.09 (baseline), p < 0.0001). 3M electrodes showed a tendency towards the elevation of pressure pain threshold compared to baseline. No significant difference in mean peak forces of the index and middle fingers after transcutaneous medium-frequency alternating electrical current stimulation with 2 and 10 kHz was found. This study demonstrates that transcutaneous medium-frequency alternating electrical current stimulation on the distal median nerve inhibits tactile sensory nerve activity in the index and middle finger when stimulation of 2 kHz and, to a lesser extent, 10 kHz was applied. A reduction of motor nerve activity was not observed but force production measurements may be prone to error.Trial registration: clinicaltrials.gov on 01/04/2021. NCT-Number: NCT04827173.

p27kip1 Modulates the Morphology and Phagocytic Activity of Microglia.

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.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

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.

Experimental early-life febrile seizures cause a sustained increase in excitatory neurotransmission in newborn dentate granule cells.

Prolonged febrile seizures (FS) are a risk factor for the development of hippocampal-associated temporal lobe epilepsy. The dentate gyrus is the major gateway to the hippocampal network and one of the sites in the brain where neurogenesis continues postnatally. Previously, we found that experimental FS increase the survival rate and structural integration of newborn dentate granule cells (DGCs). In addition, mature post-FS born DGCs express an altered receptor panel. Here, we aimed to study if these molecular and structural changes are accompanied by an altered cellular functioning. Experimental FS were induced by hyperthermia in 10-days-old Sprague-Dawley rats. Proliferating progenitor cells were labeled the next day by injecting green fluorescent protein expressing retroviral particles bilaterally in the dentate gyri. Eight weeks later, spontaneous excitatory and inhibitory postsynaptic events (sEPSCs and sIPSCs, respectively) were recorded from labeled DGCs using the whole-cell patch-clamp technique. Experimental FS resulted in a robust decrease of the inter event interval (p < .0001) and a small decrease of the amplitude of sEPSCs (p < .001). Collectively the spontaneous excitatory charge transfer increased (p < .01). Experimental FS also slightly increased the frequency of sIPSCs (p < .05), while the amplitude of these events decreased strongly (p < .0001). The net inhibitory charge transfer remained unchanged. Experimental, early-life FS have a long-term effect on post-FS born DGCs, as they display an increased spontaneous excitatory input when matured. It remains to be established if this presents a mechanism for FS-induced epileptogenesis.

Non-invasive brain stimulation as therapeutic approach for ischemic stroke: Insights into the (sub)cellular mechanisms.

Although spontaneous recovery can occur following ischemic stroke due to endogenous neuronal reorganization and neuroplastic events, the degree of functional improvement is highly variable, causing many patients to remain permanently impaired. In the last decades, non-invasive brain stimulation (NIBS) techniques have emerged as potential add-on interventions to the standard neurorehabilitation programs to improve post-stroke recovery. Due to their ability to modulate cortical excitability and to induce neuroreparative processes in the brain, multiple studies have assessed the safety, efficacy and (sub)cellular mechanisms of NIBS following ischemic stroke. In this review, an overview will be provided of the different NIBS techniques that are currently being investigated in (pre)clinical stroke studies. The NIBS therapies that will be discussed include transcranial magnetic stimulation, transcranial direct current stimulation and extremely low frequency electromagnetic stimulation. First, an overview will be given of the cellular mechanisms induced by NIBS that are associated with enhanced stroke outcome in preclinical models. Furthermore, the current knowledge on safety and efficacy of these NIBS techniques in stroke patients will be reviewed.

Extremely low frequency electromagnetic stimulation reduces ischemic stroke volume by improving cerebral collateral blood flow.

Extremely low frequency electromagnetic stimulation (ELF-EMS) has been considered as a neuroprotective therapy for ischemic stroke based on its capacity to induce nitric oxide (NO) signaling. Here, we examined whether ELF-EMS reduces ischemic stroke volume by stimulating cerebral collateral perfusion. Moreover, the pathway responsible for ELF-EMS-induced NO production was investigated. ELF-EMS diminished infarct growth following experimental stroke in collateral-rich C57BL/6 mice, but not in collateral-scarce BALB/c mice, suggesting that decreased lesion sizes after ELF-EMS results from improved collateral blood flow. In vitro analysis demonstrated that ELF-EMS increased endothelial NO levels by stimulating the Akt-/eNOS pathway. Furthermore, ELF-EMS augmented perfusion in the hind limb of healthy mice, which was mediated by enhanced Akt-/eNOS signaling. In healthy C57BL/6 mouse brains, ELF-EMS treatment increased cerebral blood flow in a NOS-dependent manner, whereas no improvement in cerebrovascular perfusion was observed in collateral-sparse BALB/c mice. In addition, ELF-EMS enhanced cerebral blood flow in both the contra- and ipsilateral hemispheres of C57BL/6 mice subjected to experimental ischemic stroke. In conclusion, we showed that ELF-EMS enhances (cerebro)vascular perfusion by stimulating NO production, indicating that ELF-EMS could be an attractive therapeutic strategy for acute ischemic stroke by improving cerebral collateral blood flow.

ATP-citrate lyase promotes axonal transport across species.

Microtubule (MT)-based transport is an evolutionary conserved process finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, primarily catalyzed by a vesicular pool of α-tubulin N-acetyltransferase 1 (Atat1), promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanism that controls Atat1 activity remains poorly understood. Here, we show that ATP-citrate lyase (Acly) is enriched in vesicles and provide Acetyl-Coenzyme-A (Acetyl-CoA) to Atat1. In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in a pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine light on the pathophysiological mechanisms underlying FD.

Electromagnetic Field as a Treatment for Cerebral Ischemic Stroke.

Cerebral stroke is a leading cause of death and adult-acquired disability worldwide. To this date, treatment options are limited; hence, the search for new therapeutic approaches continues. Electromagnetic fields (EMFs) affect a wide variety of biological processes and accumulating evidence shows their potential as a treatment for ischemic stroke. Based on their characteristics, they can be divided into stationary, pulsed, and sinusoidal EMF. The aim of this review is to provide an extensive literature overview ranging from in vitro to even clinical studies within the field of ischemic stroke of all EMF types. A thorough comparison between EMF types and their effects is provided, as well as an overview of the signal pathways activated in cell types relevant for ischemic stroke such as neurons, microglia, astrocytes, and endothelial cells. We also discuss which steps have to be taken to improve their therapeutic efficacy in the frame of the clinical translation of this promising therapy.

CSF1R inhibition rescues tau pathology and neurodegeneration in an A/T/N model with combined AD pathologies, while preserving plaque associated microglia.

Alzheimer's disease (AD) is characterized by a sequential progression of amyloid plaques (A), neurofibrillary tangles (T) and neurodegeneration (N), constituting ATN pathology. While microglia are considered key contributors to AD pathogenesis, their contribution in the combined presence of ATN pathologies remains incompletely understood. As sensors of the brain microenvironment, microglial phenotypes and contributions are importantly defined by the pathologies in the brain, indicating the need for their analysis in preclinical models that recapitulate combined ATN pathologies, besides their role in A and T models only. Here, we report a new tau-seed model in which amyloid pathology facilitates bilateral tau propagation associated with brain atrophy, thereby recapitulating robust ATN pathology. Single-cell RNA sequencing revealed that ATN pathology exacerbated microglial activation towards disease-associated microglia states, with a significant upregulation of Apoe as compared to amyloid-only models (A). Importantly, Colony-Stimulating Factor 1 Receptor inhibition preferentially eliminated non-plaque-associated versus plaque associated microglia. The preferential depletion of non-plaque-associated microglia significantly attenuated tau pathology and neuronal atrophy, indicating their detrimental role during ATN progression. Together, our data reveal the intricacies of microglial activation and their contributions to pathology in a model that recapitulates the combined ATN pathologies of AD. Our data may provide a basis for microglia-targeting therapies selectively targeting detrimental microglial populations, while conserving protective populations.

PDE inhibition in distinct cell types to reclaim the balance of synaptic plasticity.

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.

Ethanol consumption and sedation are altered in mice lacking the glycine receptor α2 subunit.

BACKGROUND AND PURPOSE: The precise mechanism/s of action of ethanol, although studied for many years, are not well understood. Like other drugs of abuse, ethanol affects dopamine levels in the nucleus accumbens (nAc), an important region of the mesolimbic system, causing a reinforcing effect. It has been shown that glycine receptors (GlyRs) present in the nAc are potentiated by clinically relevant concentrations of ethanol, where α1 and α2 are the predominant subunits expressed. EXPERIMENTAL APPROACH: Using a combination of electrophysiology and behavioural assays, we studied the involvement of GlyR α2 subunits on the effects of low and high doses of ethanol, as well as on consumption using mice lacking the GlyR α2 subunit (male Glra2-/Y and female Glra2-/- ). KEY RESULTS: GlyR α2 subunits exist in accumbal neurons, since the glycine-evoked currents and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in Glra2-/Y mice were drastically decreased. In behavioural studies, differences in ethanol consumption and sedation were observed between wild-type (WT) and Glra2 knockout (KO) mice. Using the drinking in the dark (DID) paradigm, we found that Glra2-/Y mice presented a binge-like drinking behaviour immediately when exposed to ethanol rather than the gradual consumption seen in WT animals. Interestingly, the effect of knocking out Glra2 in female (Glra2-/- ) mice was less evident, since WT female mice already showed higher DID. CONCLUSION AND IMPLICATIONS: The differences in ethanol consumption between WT and KO mice provide additional evidence supporting the conclusion that GlyRs are biologically relevant targets for the sedative and rewarding properties of ethanol.

Microglial derived extracellular vesicles activate autophagy and mediate multi-target signaling to maintain cellular homeostasis.

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.