Microglial Processes Research Articles

Mitochondria are absent from microglial processes performing surveillance, chemotaxis, and phagocytic engulfment.

Microglia continually surveil the brain allowing for rapid detection of tissue damage or infection. Microglial metabolism is linked to tissue homeostasis, yet how mitochondria are subcellularly partitioned in microglia and dynamically reorganize during surveillance, injury responses, and phagocytic engulfment in the intact brain are not known. Here, we performed intravital imaging of microglia mitochondria, revealing that microglial processes diverge, with some containing multiple mitochondria while others are completely void. Microglial processes that engage in minute-to-minute surveillance typically do not have mitochondria. Moreover, unlike process surveillance, mitochondrial motility does not change with animal anesthesia. Likewise, the processes that acutely chemoattract to a lesion site or initially engage with a neuron undergoing programmed cell death do not contain mitochondria. Rather, microglia mitochondria have a delayed arrival into the responding cell processes. Thus, there is subcellular heterogeneity of mitochondrial partitioning and asymmetry between mitochondrial localization and cell process motility or acute damage responses.

Discovery and evaluation of novel SHIP-1 inhibitors

Src Homology 2-containing Inositol 5′-Phosphatase-1 (SHIP-1), encoded by INPP5D, has been identified as an Alzheimer’s disease (AD) risk-associated gene through recent genetic and epigenetic studies. SHIP-1 confers AD risk by inhibiting the TREM2 cascade and reducing beneficial microglial cellular processes, including phagocytosis. While several small molecules have been reported to modulate SHIP-1 activity, their limited selectivity and efficacy in advanced models restricted their potential as therapeutic agents or probes for biological studies. Herein, we validated and implemented a high-throughput screening platform to explore new chemotypes that can modulate the phosphatase activity of SHIP-1. We screened 49,260 central nervous system (CNS)-penetrate compounds sourced from commercial vendors using the malachite green-based assay for anti-SHIP-1 activity. Through analysis, prioritization, and validation of the screening hits, we identified three novel types of scaffolds that inhibit the SHIP-1 phosphatase activity with IC50s as low as 46.6 µM. To improve the inhibitory activity of these promising hits, we carried out structure–activity relationship (SAR) studies, resulting in a lead molecule SP3-12 that inhibits SHIP-1 with an IC50 value of 6.1 μM. Kinetic analyses of SP3-12 revealed that its inhibition mechanism is competitive, with a Ki value of 3.2 µM for SHIP-1 and a 7-fold selectivity over SHIP-2. Furthermore, results from testing in a microglial phagocytosis/cell health high content assay indicated that SP3-12 could effectively activate phagocytosis in human microglial clone 3 (HMC3) cells, with an EC50 of 2.0 µM, without cytotoxicity in the dose range. Given its potency, selectivity, and cellular activity, SP3-12 emerges as a promising small molecule inhibitor with potential for investigating the biological functions of SHIP-1.

Microglial Melatonin Receptor 1 Degrades Pathological Alpha-Synuclein Through Activating LC3-Associated Phagocytosis InVitro.

Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs), primarily constituted of α-synuclein (α-Syn). Microglial cells exhibit specific reactivity toward misfolded proteins such as α-Syn. However, the exact clearance mechanism and related molecular targets remain elusive. BV2 cells, primary microglia from wild-type and MT1 knockout mice, and primary cortical neurons were utilized as experimental models. The study investigated relevant mechanisms by modulating microglial MT1 expression through small RNA interference (RNAi) and lentiviral overexpression techniques. Furthermore, pathological aggregation of α-Syn was induced using pre-formed fibrils (PFF) α-Syn. Co-immunoprecipitation, immunofluorescence, Western blot (WB), and quantitative real-time PCR were used to elucidate the mechanisms of molecular regulation. In this study, we elucidated the regulatory role of the melatonin receptor 1 (MT1) in the microglial phagocytic process. Following MT1 knockout, the ability of microglial cells to engulf latex beads and zymosan particles decreased, subsequently affecting the phagocytic degradation of fibrillar α-Syn by microglial cells. Furthermore, the loss of MT1 receptors in microglial cells exacerbates the aggregation of α-Syn in neurons induced by pre-formed fibrils (PFF) α-Syn. Mechanistically, MT1 influences the phagocytic function of microglial cells by regulating the Rubicon-dependent LC3-associated phagocytosis (LAP) pathway. Taken together, the results suggest the neuroprotective function of microglial cells in clearing α-Syn through MT1-mediated LAP, highlighting the potential key role of MT1 in pathogenic mechanisms associated with α-Syn.

Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study

Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury (DAI) and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal swellings, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals with confirmed DAI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.

Weisheng-tang protects against ischemic brain injury by modulating microglia activation through the P2Y12 receptor.

Background: Stroke, a leading cause of death and disability, lacks effective treatments. Post-stroke secondary damage worsens the brain microenvironment, further exacerbating brain injury. Microglia's role in responding to stroke-induced damage in peri-infarct regions is crucial. In this study, we explored Weisheng-tang's potential to enhance ischemic outcomes by targeting microglia. Methods: We induced middle cerebral artery occlusion and reperfusion in mice, followed by behavioral assessments and infarct volume analyses after 48h, and examined the changes in microglial morphology through skeleton analysis. Results: Weisheng-tang (300mg/kg) significantly reduced infarction volume and alleviated neurological and motor deficits. The number of activated microglia was markedly increased within the peri-infarct territory, which was significantly reversed by Weisheng-tang. Microglial morphology analysis revealed that microglial processes were retracted owing to ischemic damage but were restored in Weisheng-tang-treated mice. This restoration was accompanied by the expression of the purinergic P2Y12 receptor (P2Y12R), a key regulator of microglial process extension. Weisheng-tang increased neuronal Kv2.1 clusters while suppressing juxtaneuronal microglial activation. The P2Y12R inhibitor-ticagrelor-eliminated the tissue and functional recovery that had been observed with Weisheng-tang after ischemic damage. Discussion: Weisheng-tang improved experimental stroke outcomes by modulating microglial morphology through P2Y12R, shedding light on its neuroprotective potential in ischemic stroke.

Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study.

Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons following brain injury in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal segments, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals that sustained a TBI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.

A systematic review of the role of TREM2 in Alzheimer's disease.

Given the established genetic linkage between triggering receptors expressed on myeloid cells 2 (TREM2) and Alzheimer's disease (AD), an expanding research body has delved into the intricate role of TREM2 within the AD context. However, a conflicting landscape of outcomes has emerged from both in vivo and in vitro investigations. This study aimed to elucidate the multifaceted nuances and gain a clearer comprehension of the role of TREM2. PubMed database was searched spanning from its inception to January 2022. The search criteria took the form of ("Alzheimer's disease" OR "AD") AND ("transgenic mice model" OR "transgenic mouse model") AND ("Triggering receptor expressed on myeloid cells" OR "TREM2"). Inclusion criteria consisted of the following: (1) publication of original studies in English; (2) utilization of transgenic mouse models for AD research; and (3) reports addressing the subject of TREM2. A total of 43 eligible articles were identified. Our analysis addresses four pivotal queries concerning the interrelation of TREM2 with microglial function, Aβ accumulation, tau pathology, and inflammatory processes. However, the diverse inquiries posed yielded inconsistent responses. Nevertheless, the inconsistent roles of TREM2 within these AD mouse models potentially hinge upon factors such as age, sex, brain region, model type, and detection methodologies. This review substantiates the evolving understanding of TREM2's disease progression-dependent impacts. Furthermore, it reviews the interplay between TREM2 and its effects across diverse tissues and temporal stages.

A microglia-containing cerebral organoid model to study early life immune challenges.

Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

TRIM28 Fosters Microglia Ferroptosis via Autophagy Modulation to Enhance Neuropathic Pain and Neuroinflammation.

This study explores the molecular underpinnings of neuropathic pain (NPP) and neuroinflammation, focusing on the role of TRIM28 in the regulation of autophagy and microglia ferroptosis. Leveraging transcriptomic data associated with NPP, we identified TRIM28 as a critical regulator of ferroptosis. Through comprehensive analysis, including Gene Ontology enrichment and protein-protein interaction network assessments, we unveiled GSK3B as a downstream target of TRIM28. Experimental validation confirmed the capacity of TRIM28 to suppress GSK3B expression and attenuate autophagic processes in microglia. We probed the consequences of autophagy and ferroptosis on microglia physiology, iron homeostasis, oxidative stress, and the release of proinflammatory cytokines. In a murine model, we validated the pivotal role of TRIM28 in NPP and neuroinflammation. Our analysis identified 20 ferroptosis regulatory factors associated with NPP, with TRIM28 emerging as a central orchestrator. Experimental evidence affirmed that TRIM28 governs microglial iron homeostasis and cell fate by downregulating GSK3B expression and modulating autophagy. Notably, autophagy was found to influence oxidative stress and proinflammatory cytokine release through the iron metabolism pathway, ultimately fueling neuroinflammation. In vivo experiments provided conclusive evidence of TRIM28-mediated pathways contributing to heightened pain sensitivity in neuroinflammatory states. The effect of TRIM28 on autophagy and microglia ferroptosis drives NPP and neuroinflammation. These findings offer promising avenues for identifying novel therapeutic targets to manage NPP and neuroinflammation.

APOE4 genotype and aging impair injury-induced microglial behavior in brain slices, including toward Aβ, through P2RY12

Microglia are highly dynamic cells that play a critical role in tissue homeostasis through the surveillance of brain parenchyma and response to cues associated with damage. Aging and APOE4 genotype are the strongest risk factors for Alzheimer’s disease (AD), but how they affect microglial dynamics remains unclear. Using ex vivo confocal microscopy, we analyzed microglial dynamic behaviors in the entorhinal cortex (EC) and hippocampus CA1 of 6-, 12-, and 21-month-old mice APOE3 or APOE4 knock-in mice expressing GFP under the CX3CR1 promoter. To study microglia surveillance, we imaged microglia baseline motility for 20 min and measured the extension and retraction of processes. We found that APOE4 microglia exhibited significantly less brain surveillance (27%) compared to APOE3 microglia in 6-month-old mice; aging exacerbated this deficit. To measure microglia response to damage, we imaged process motility in response to ATP, an injury-associated signal, for 30 min. We found APOE4 microglia extended their processes significantly slower (0.9 µm/min, p < 0.005) than APOE3 microglia (1.1 μm/min) in 6-month-old animals. APOE-associated alterations in microglia motility were observed in 12- and 21-month-old animals, and this effect was exacerbated with aging in APOE4 microglia. We measured protein and mRNA levels of P2RY12, a core microglial receptor required for process movement in response to damage. We found that APOE4 microglia express significantly less P2RY12 receptors compared to APOE3 microglia despite no changes in P2RY12 transcripts. To examine if the effect of APOE4 on the microglial response to ATP also applied to amyloid β (Aβ), we infused locally Hi-Lyte Fluor 555-labeled Aβ in acute brain slices of 6-month-old mice and imaged microglia movement for 2 h. APOE4 microglia showed a significantly slower (p < 0.0001) process movement toward the Aβ, and less Aβ coverage at early time points after Aβ injection. To test whether P2RY12 is involved in process movement in response to Aβ, we treated acute brain slices with a P2RY12 antagonist before Aβ injection; microglial processes no longer migrated towards Aβ. These results provide mechanistic insights into the impact of APOE4 genotype and aging in dynamic microglial behaviors prior to gross Aβ pathology and could help explain how APOE4 brains are more susceptible to AD pathogenesis.

High-Dimensional Methods of Single-Cell Microglial Profiling to Enhance Understanding of Neuropathological Disease.

Microglia are the innate myeloid cells of the central nervous system (CNS) parenchyma, functionally implicated in almost every defined neuroinflammatory and neurodegenerative disorder. Current understanding of disease pathogenesis for many neuropathologies is limited and/or lacks reliable diagnostic markers, vaccines, and treatments. With the increasing aging of society and rise in neurogenerative diseases, improving our understanding of their pathogenesis is essential. Analysis of microglia from murine disease models provides an investigative tool to unravel disease processes. In many neuropathologies, bone-marrow-derived monocytes are recruited to the CNS, adopting a phenotype similar to that of microglia. This significantly confounds the accurate identification of cell-type-specific functions and downstream therapeutic targeting. The increased capacity to analyze more phenotypic markers using spectral-cytometry-based technologies allows improved separation of microglia from monocyte-derived cells. Full-spectrum profiling enables enhanced marker resolution, time-efficient analysis of >40 fluorescence parameters, and extraction of cellular autofluorescence parameters. Coupling this system with additional cytometric technologies, including cell sorting and high-parameter imaging, can improve the understanding of microglial phenotypes in disease. To this end, we provide detailed, step-by-step protocols for the analysis of murine brain tissue by high-parameter ex vivo cytometric analysis using the Aurora spectral cytometer (Cytek), including best practices for unmixing and autofluorescence extraction, cell sorting for single-cell RNA analysis, and imaging mass cytometry. Together, this provides a toolkit for researchers to comprehensively investigate microglial disease processes at protein, RNA, and spatial levels for the identification of therapeutic targets in neuropathology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Processing the mouse brain into a single-cell suspension for microglia isolation Basic Protocol 2: Staining single-cell mouse brain suspensions for microglial phenotyping by spectral cytometry Basic Protocol 3: Flow cytometric sorting of mouse microglia for ex vivo analysis Basic Protocol 4: Processing the mouse brain for imaging mass cytometry for spatial microglia analysis.

Microglia enhance post-anesthesia neuronal activity by shielding inhibitory synapses.

Microglia are resident immune cells of the central nervous system and play key roles in brain homeostasis. During anesthesia, microglia increase their dynamic process surveillance and interact more closely with neurons. However, the functional significance of microglial process dynamics and neuronal interaction under anesthesia is largely unknown. Using in vivo two-photon imaging in mice, we show that microglia enhance neuronal activity after the cessation of isoflurane anesthesia. Hyperactive neuron somata are contacted directly by microglial processes, which specifically colocalize with GABAergic boutons. Electron-microscopy-based synaptic reconstruction after two-photon imaging reveals that, during anesthesia, microglial processes enter into the synaptic cleft to shield GABAergic inputs. Microglial ablation or loss of microglial β2-adrenergic receptors prevents post-anesthesia neuronal hyperactivity. Our study demonstrates a previously unappreciated function of microglial process dynamics, which enable microglia to transiently boost post-anesthesia neuronal activity by physically shielding inhibitory inputs.

Does retinal microglia respond to peripheral nervous system injury?

The nervous system is the principal integration and coordination centre of the organism receiving, processing and transmitting information to produce a response that regulates and maintains homeostasis. In the central nervous system (CNS), homeostasis is regulated by haemato‐neuronal barriers that prevent free entry to immune system cells, although there is increasing evidence that neuro‐immunological interactions during injury, inflammation and chronic peripheral disease may contribute to the establishment and development of chronic neurodegenerative pathologies at the CNS level.Inflammation plays a significant role in the resolution and sequelae of CNS damage. In recent years, special attention has been dedicated to the role and phenotype of phagocytic cells acting in the CNS and, in particular, to the study of which macrophage subpopulations contribute to excessive or detrimental inflammation or, on the contrary, to beneficial repair or resolution of the lesion.All this raises the question of how local peripheral neuroimmunological interaction induces an inflammatory response in the CNS and how this can be better understood. The study of this phenomenon in the retina is of particular interest, as the retina does not receive neural afferents from other areas of the CNS.The use of experimental models of peripheral lesions of the peripheral nervous system, such as sciatic nerve trauma, or inflammatory lesions, such as the induction of a peripheral arthritic joint process, aims to investigate the time course of the response in the CNS to local peripheral inflammation in the retina of adult mice. The results reveal qualitative and quantitative cellular changes compatible with microglial activation processes in the retina, but no significant changes occur in its neuronal population. The presence and activation of haematic mononuclear‐phagocytic cells of myelopoietic origin are also observed in the retina, where they differentiate into microglia.In the absence of local retinal injury, we postulate that the detected neuroinflammatory reaction may be due to a preventive CNS defence reaction favouring neuroprotection since microglial cells derived from the haematic monocyte/macrophage cell population may consider within the M2 macrophage subtype, characterized by their ability to suppress proinflammatory immune responses and promote the expression of neuroprotective and repair genes.On the other hand, we must consider that activation of the mononuclear phagocytic system in the retina caused by a peripheral inflammatory process could potentially be detrimental to patients with neurodegenerative retinal diseases or contribute to their development.

Is it possible to reduce glial activation in an old taupathia mouse model by deletion of microglial Hemoxygenase‐1?

Tauopathies such as Alzheimer's disease are characterized by the accumulation of neurotoxic aggregates of tau protein. With aging and, especially, in Alzheimer's patients, the inducible enzyme heme oxygenase 1 (HO‐1) progressively increases in microglia, causing iron accumulation, neuroinflammation, and neurodegeneration. The retina is an organ that can be readily accessed and can reflect changes that occur in the brain. In this context, we evaluated how the lack of microglial HO‐1, using mice that do not express HO‐1 in microglia (HMO‐KO), impacts retinal macro and microgliosis of aged subjects (18 months old mice) subjected to tauopathy by intrahippocampal delivery of AAV‐hTauP301L (TAU). Our results show that although tauopathy, measured as anti‐TAUY9 and anti‐AT8 positive immunostaining, was not observed in the retina of WT‐TAU or HMO‐KO + TAU mice, a morphometric study of retinal microglia and macroglia showed significant retinal changes in the TAU group compared to the WT group, such as: (i) increased number of activated microglia, (ii) retraction of microglial processes, (iii) increased number of CD68+ microglia, and (iv) increased retinal area occupied by GFAP (AROA) and C3 (AROC3). This retinal inflammatory profile was reduced in HMO‐KO + TAU mice. Therefore, a reduction of microglial HO‐1 could be beneficial to prevent tauopathy‐induced neuroinflammation.

Differential effects of sleep deprivation on behavior and microglia in a brain-region-specific manner in young and aged male mice

Microglia, resident immune cells in the central nervous system, constantly monitor the state of the surrounding brain activity. The animal model induced by sleep deprivation (SD) is widely used to study the pathophysiological mechanisms of insomnia and bipolar disorder. However, it remains unclear whether SD affects behaviors in young and aged male mice and microglia in various brain regions. In this study, we confirmed brain region-specific changes in microglial density and morphology in the accumbens nucleus (Acb), amygdala (AMY), cerebellum (Cb), corpus callosum (cc), caudate putamen, hippocampus (HIP), hypothalamus (HYP), medial prefrontal cortex (mPFC), and thalamus (TH) of young mice. In addition, the density of microglia in old mice was higher than that in young mice. Compared with young mice, old mice showed a markedly increased microglial size, decreased total length of microglial processes, and decreased maximum length. Importantly, we found that 48-h SD decreased microglial density and morphology in old mice, whereas SD increased microglial density and morphology in most observed brain regions in young mice. SD-induced hyperactivity was observed only in young mice but not in old mice. Moreover, microglial density (HIP, AMY, mPFC, CPu) was significantly positively correlated with behaviors in SD- and vehicle-treated young mice. Contrarily, negative correlations were shown between the microglial density (cc, Cb, TH, HYP, Acb, AMY) and behaviors in vehicle-treated young and old mice. These results suggest that SD dysregulates the homeostatic state of microglia in a region- and age-dependent manner. Microglia may be involved in regulating age-related behavioral responses to SD.

Morphological features of microglial cells in a 5xFAD mouse model of Alzheimer's disease

BACKGROUND: Aging is an inevitable and irreversible process associated with increased risk of developing various neurodegenerative diseases, one of which is Alzheimer's disease. Currently, the role of glial cells, in particular microglia, in the pathogenesis of Alzheimer's disease is being actively studied. However, only a few studies have correlated the morphological features of microglia and their spatial arrangement in relation to β-amyloid plaques. AIM: Describe the main morphological parameters of microglia in the 5xFAD mouse model of Alzheimer's disease at a late stage of pathology development. METHODS: As the studied object, mice were chosen by the age of 15–16 months of the 5xFAD line, as a model of acceleid amyloidosis. The immunohistochemical staining of the study of the morphological diversity of microglia was carried out on the cuts of the cortex of the mouse brain. The obtained confocal images performed an immunogystological analysis of the cuts of the cerebral cortex when analyzed using the Imagej application using the plugins of Skeleton, AnalyzeSkeleton (2D/3D) and FracLac. RESULTS: During the study, 5xFAD mice were divided into two groups (n=3 each). Carriers of the app and psen1 transgenes were assigned to the “FAD” group, and wild-type mice were assigned to the “Wt” group (control). We analyzed 3–4 sagittal sections (50 µm) of the brain from each mouse. The results showed that microglial cells from mice with signs of Alzheimer's disease have smaller fractal dimension, lacunarity and branching. CONCLUSION: The presence of β-amyloid plaques contributes to the migration of microglia to the focus of inflammation, its proliferation and transition to the phagocytic and dystrophic subtype. According to fractal analysis, there is a significant (p ≤0.05) decrease in the average branching of microglial processes, a decrease in fractal dimension and lacunarity.

Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an “inflammatory signaling cascade” in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

The influence of rs75932628 and rs2234253 polymorphisms of the TREM2 gene and the mTOR signaling pathway in the development of Alzheimer's disease

Annotation. According to the data of the World Health Organization, as of March 15, 2023, more than 55 million cases of dementia disorders were recorded in the world. Among them, Alzheimer’s disease remains a global medical challenge, which is particularly relevant for low- and middle-income countries. This pathology already ranks 7th among the causes of death among the elderly population and has a significant social and economic impact. The purpose of the study – to determine the role of the TREM2 gene and its polymorphisms, in particular rs75932628 and rs2234253, in susceptibility to Alzheimer’s disease. To analyze the role of the mTOR signaling pathway in the neurodegeneration of microglia. Literature sources were searched using the key words “Alzheimer’s disease”, “microglia”, “dementia”, etc. The study was conducted based on an interactive group approach using a comprehensive literature review in the Web of Science, Scopus, Pubmed, Elsevier, and Springer databases. As a result of the analysis, it was found that one of the key participants in the process of neuroinflammation is microglia, which plays an important role in phagocytosis, transmission of neuronal signals and local immune response. The TREM2 gene, which normally promotes the phagocytosis of β-amyloid protein, was found to be a significant factor associated with dementia. Due to its effect on galectin-3, it causes the activation of microglia, which is of key importance in the development of neurodegenerative diseases. Another important factor turned out to be the mTOR signaling pathway, which is considered a key regulator of the main cellular and metabolic processes in microglia, however, the data of practical works on this pathway remain controversial. Thus, the study confirms the importance of the TREM2 gene and the mTOR signaling pathway in the development of Alzheimer's disease, which is related to their effects on microglia. The obtained results can be used for further research and development of strategies for the treatment of Alzheimer's disease.

The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function – Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLCγ2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLCγ2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLCγ2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLCγ2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLCγ2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLCγ2 GOF on human microglia. PLCγ2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLCγ2 wild type. Despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLCγ2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLCγ2 in the CNS. Drug candidates targeting PLCγ2 will need to precisely mimic the effects of the PLCγ2 P522R variant on microglial function, but not those of the PLCγ2 S707Y variant.

The NADPH Oxidase Inhibitor, Mitoapocynin, Mitigates DFP-Induced Reactive Astrogliosis in a Rat Model of Organophosphate Neurotoxicity.

NADPH oxidase (NOX) is a primary mediator of superoxides, which promote oxidative stress, neurodegeneration, and neuroinflammation after diisopropylfluorophosphate (DFP) intoxication. Although orally administered mitoapocynin (MPO, 10 mg/kg), a mitochondrial-targeted NOX inhibitor, reduced oxidative stress and proinflammatory cytokines in the periphery, its efficacy in the brain regions of DFP-exposed rats was limited. In this study, we encapsulated MPO in polyanhydride nanoparticles (NPs) based on 1,6-bis(p-carboxyphenoxy) hexane (CPH) and sebacic anhydride (SA) for enhanced drug delivery to the brain and compared with a high oral dose of MPO (30 mg/kg). NOX2 (GP91phox) regulation and microglial (IBA1) morphology were analyzed to determine the efficacy of MPO-NP vs. MPO-oral in an 8-day study in the rat DFP model. Compared to the control, DFP-exposed animals exhibited significant upregulation of NOX2 and a reduced length and number of microglial processes, indicative of reactive microglia. Neither MPO treatment attenuated the DFP effect. Neurodegeneration (FJB+NeuN) was significantly greater in DFP-exposed groups regardless of treatment. Interestingly, neuronal loss in DFP+MPO-treated animals was not significantly different from the control. MPO-oral rescued inhibitory neuronal loss in the CA1 region of the hippocampus. Notably, MPO-NP and MPO-oral significantly reduced astrogliosis (absolute GFAP counts) and reactive gliosis (C3+GFAP). An analysis of inwardly rectifying potassium channels (Kir4.1) in astroglia revealed a significant reduction in the brain regions of the DFP+VEH group, but MPO had no effect. Overall, both NP-encapsulated and orally administered MPO had similar effects. Our findings demonstrate that MPO effectively mitigates DFP-induced reactive astrogliosis in several key brain regions and protects neurons in CA1, which may have long-term beneficial effects on spontaneous seizures and behavioral comorbidities. Long-term telemetry and behavioral studies and a different dosing regimen of MPO are required to understand its therapeutic potential.