Microglial Phagocytic Activity Research Articles

The microglial innate immune receptor TREM2 participates in fear memory formation through excessive prelimbic cortical synaptic pruning

IntroductionFear memory formation has been implicated in fear- and stress-related psychiatric disorders, including post-traumatic stress disorder (PTSD) and phobias. Synapse deficiency and microglial activation are common among patients with PTSD, and induced in animal models of fear conditioning. Increasing studies now focus on explaining the specific mechanisms between microglia and synapse deficiency. Though newly-identified microglia regulator triggering receptor expressed on myeloid cells 2 (TREM2) plays a role in microglial phagocytic activity, its role in fear-formation remains unknown.MethodsWe successfully constructed a fear- formation model by foot-shock. Four days after foot-shock, microglial capacity of synaptic pruning was investigated via western blotting, immunofluorescence and Golgi-Cox staining. Prelimbic chemical deletion or microglia inhibition was performed to detect the role of microglia in synaptic loss and neuron activity. Finally, Trem2 knockout mice or wild-type mice with Trem2 siRNA injection were exposed to foot-shock to identify the involvement of TREM2 in fear memory formation.ResultsThe results herein indicate that the foot-shock protocol in male mice resulted in a fear formation model. Mechanistically, fear conditioning enhanced the microglial capacity for engulfing synapse materials, and led to glutamatergic neuron activation in the prelimbic cortex. Prelimbic chemical deletion or microglia inhibition improved fear memory formation. Further investigation demonstrated that TREM2 regulates microglial phagocytosis, enhancing synaptic pruning. Trem2 knockout mice showed remarkable reductions in prelimbic synaptic pruning and reduced neuron activation, with decreased fear memory formation.DiscussionOur cumulative results suggest that prelimbic TREM2-mediated excessive microglial synaptic pruning is involved in the fear memory formation process, leading to development of abnormal stress-related behavior.

Microglial TREM2 promotes phagocytic clearance of damaged neurons after status epilepticus

In the central nervous system, triggering receptor expressed on myeloid cells 2 (TREM2) is exclusively expressed by microglia and is critical for microglial proliferation, migration, and phagocytosis. Microglial TREM2 plays an important role in neurodegenerative diseases, such as Alzheimer’s disease and amyotrophic lateral sclerosis. However, little is known about how TREM2 affects microglial function within epileptogenesis. To investigate this, we utilized male TREM2 knockout (KO) mice within the intra-amygdala kainic acid seizure model. Electroencephalographic analysis, immunocytochemistry, and RNA sequencing revealed that TREM2 deficiency significantly promoted seizure-induced pathology. We found that TREM2 KO increased both the severity of acute status epilepticus and the number of spontaneous recurrent seizures characteristic of chronic focal epilepsy. Phagocytic clearance of damaged neurons by microglia was also impaired by TREM2 KO and reduced phagocytic activity correlated with increased spontaneous seizures. Analysis of human tissue from patients who underwent surgical resection for drug resistant temporal lobe epilepsy also showed a negative correlation between expression of the microglial phagocytic marker CD68 and focal to bilateral tonic-clonic generalized seizure history. These results indicate that microglial TREM2 and phagocytic activity are important to epileptogenic pathology.

Ghost cells: Wilder Penfield and the characterization of glia and glial pathology, 1924–1932

ABSTRACT Wilder Penfield is known for his contributions to the structure–function relationship of the brain and for the surgical treatment of focal epilepsy. Less well known are his contributions to the study of glial cells and his investigation of their role in human neuropathology. Penfield learned the gold and silver methods for staining neurons, glial cells, and their projections from Charles Sherrington and Pío del Río-Hortega. He and his colleague William Cone established a laboratory for the study of glial cells and human neuropathology using metallic stains, initially at the Presbyterian Hospital in New York City in 1925, and then at the Montreal Neurological Institute in 1928. Penfield, Cone, and their research fellows, building on the findings of Río-Hortega, confirmed the existence of oligodendrocytes and their relationship with myelin, and investigated the putative mesodermal origin of microglia. They discovered the reaction of oligodendrocytes to pathological stressors, and the phagocytic activity of microglia in human gliomas. In this article, we argue that Penfield’s studies of astrocytes, oligodendrocytes, and microglia, and their responses to craniocerebral trauma, epilepsy, malignant brain tumors, and other pathologies of the central nervous system inaugurated a new era in clinical neurocytology and neuropathology.

Microglia mediate the early-life programming of adult glucose control.

Mammalian glucose homeostasis is, in part, nutritionally programmed during early neonatal life, a critical window for the formation of synapses between hypothalamic glucoregulatory centers. Although microglia are known to prune synapses throughout the brain, their specific role in refining hypothalamic glucoregulatory circuits remains unknown. Here, we show that microglia in the mediobasal hypothalamus (MBH) of mice actively engage in synaptic pruning during early life. Microglial phagocytic activity is induced following birth, regresses upon weaning from maternal milk, and is exacerbated by feeding dams a high-fat diet while lactating. In particular, we show that microglia refine perineuronal nets (PNNs) within the neonatal MBH. Indeed, transiently depleting microglia before weaning (P6-16), but not afterward (P21-31), remarkably increased PNN abundance in the MBH. Furthermore, mice lacking microglia only from P6-16 had glucose intolerance due to impaired glucose-responsive pancreatic insulin secretion in adulthood, a phenotype not seen if microglial depletion occurred after weaning. Viral retrograde tracing revealed that this impairment is linked to a reduction in the number of neurons in specific hypothalamic glucoregulatory centers that synaptically connect to the pancreatic β-cell compartment. These findings show that microglia facilitate synaptic plasticity in the MBH during early life through a process that includes PNN refinement, to establish hypothalamic circuits that regulate adult glucose homeostasis.

MicroRNAs in microglia: deciphering their role in neurodegenerative diseases.

This review presents a comprehensive analysis of the role of microRNAs in microglia and their implications in the pathogenesis of neurodegenerative diseases. Microglia, as the resident immune cells of the central nervous system (CNS), are pivotal in maintaining neural homeostasis and responding to pathological changes. Recent studies have highlighted the significance of miRNAs, small non-coding RNA molecules, in regulating microglial functions. In neurodegenerative diseases, such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), and Multiple Sclerosis (MS), dysregulated miRNA expression in microglia contributes to disease progression through various mechanisms such regulation of gene expression, as modulation of cytokine response and phagocytosis. This review synthesizes current knowledge on how miRNAs influence microglial activation, cytokine production, and phagocytic activity. Specific miRNAs, such as miR-155, are explored for their roles in modulating microglial responses in the context of neuroinflammation and neurodegeneration. The study also discusses the impact of miRNA dysregulation on the transition of microglia from a neuroprotective to a neurotoxic phenotype, a critical aspect in the progression of neurodegenerative diseases.

Mitochondrial Dysfunction as the Major Basis of Brain Aging.

The changes in the properties of three biological events that occur with cerebral aging are discussed. These adverse changes already begin to develop early in mid-life and gradually become more pronounced with senescence. Essentially, they are reflections of the progressive decline in effectiveness of key processes, resulting in the deviation of essential biochemical trajectories to ineffective and ultimately harmful variants of these programs. The emphasis of this review is the major role played by the mitochondria in the transition of these three important processes toward more deleterious variants as brain aging proceeds. The immune system: the shift away from an efficient immune response to a more unfocused, continuing inflammatory condition. Such a state is both ineffective and harmful. Reactive oxygen species are important intracellular signaling systems. Additionally, microglial phagocytic activity utilizing short lived reactive oxygen species contribute to the removal of aberrant or dead cells and bacteria. These processes are transformed into an excessive, untargeted, and persistent generation of pro-oxidant free radicals (oxidative stress). The normal efficient neural transmission is modified to a state of undirected, chronic low-level excitatory activity. Each of these changes is characterized by the occurrence of continuous activity that is inefficient and diffused. The signal/noise ratio of several critical biological events is thus reduced as beneficial responses are gradually replaced by their impaired and deleterious variants.

Monocytic involvement in the neuroinflammatory response after retinal injury

Aims/Purpose: Inflammation and disease lead to the recruitment of blood monocyte‐derived macrophages (MDM) to the central nervous system. The role of MDMs following a neuroinflammatory process after a lesion remains elusive because MDMs that enter the neural parenchyma are difficult to distinguish from microglia. The aim of this study is to elucidate the response of recruited MDMs after selective retinal ganglion cell (RGC) injury.Methods: Unilateral complete intra‐orbital optic nerve (ON) crush was performed in adult C57BL/6J LysM‐GFP mice carrying the selective marker green fluorescent protein (GFP) in myeloid MDM but not microglia, 1 week after retrograde labelling of RGCs with the neuronal tracer hydroxystilbamidine (OHSt) from the superior colliculi. This injury model allows to investigate how the retinal mononuclear phagocytic system responds to a neurodegenerative process via the phagocytic activity of its main cellular components, resident microglia and recruited MDM, both in the injured retina and its contralateral uninjured fellow retina, at 5‐, 7‐ or 10‐days post‐lesion.Results: At all‐time points after injury, microglial phagocytic activity was statistically significant in the injured retina compared to the uninjured fellow retina. In comparison to naïve retinas, the number of MDMs increases significantly after injury in both injured and uninjured retinas. In injured retinas the increase is statistically higher. However, only a subset of the recruited MDMs shows phagocytic activity in the injured retinas 10 days after the lesion but not before. In contrast, the population of MDMs in the contralateral uninjured retina shows no signs of phagocytic activity after the lesion.Conclusions: In the injured retina, recruited MDMs show an initial quiescent stage, with no phagocytic activity compatible with a neuroprotective state, followed by a delayed phase with detrimental behaviour for RGC survival. In the uninjured fellow retinas however, MDMs retain a quiescent stage. It is tempting to suggest that following ON injury, recruited MDMs adopt an initial neuroprotective stage followed by a detrimental phagocytic activity in the injured retina, whereas in the uninjured retina MDMs remain in the initial stage.

Sleep restoration by optogenetic targeting of GABAergic neurons reprograms microglia and ameliorates pathological phenotypes in an Alzheimer’s disease model

BackgroundAlzheimer’s disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation.MethodsWe generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression.ResultsAPP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid β (Aβ) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice.ConclusionsIn summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aβ by microglia in an AD mouse model.

Loss of oligodendrocyte‐derived IL‐33 results in increased amyloid plaque deposition in APP/PS1 mice

AbstractBackgroundGlial cells in the central nervous system (CNS) express IL‐33, a member of the IL‐1 family. IL‐33 is known to regulate microglial phagocytic activity toward amyloid β. In the aged brain, unlike the young counterpart, IL‐33 is predominantly expressed by oligodendrocytes (OLs), but the role of OL‐derived IL‐33 in Alzheimer’s disease (AD) is unknown.MethodOL‐specific IL‐33 conditional knockout (cKO) mice were further crossed with APPswe/Psen1 dE9 (APP/PS1) and Mobp‐EGFP transgenic mice. Cortical lysates from one‐year‐old male APP/PS1 (n = 15) and APP/PS1 + IL‐33 cKO (n = 16) mice were used for the quantitative analysis (MSD and ELISA) of amyloid β 1‐42 (Aβ1‐42) and Aβ1‐40. Immunofluorescence and morphometric analysis were also performed to measure Aβ‐covered or LAMP1‐covered areas in different regions of the mouse brain. EGFP+ OLs were also quantified.ResultsBoth Aβ1‐42 and Aβ1‐40 levels were significantly increased in the cortex (p<0.0001), and so were Aβ plaque‐covered areas in the cortex, corpus callosum, and hippocampus of APP/PS1 + IL‐33 cKO mice compared with control APP/PS1 (p<0.01). OL‐derived IL‐33 loss also resulted in increased accumulation of LAMP1+ dystrophic neurites (p<0.05). However, cortical EGFP+ OL density was unaltered by IL‐33 cKO.ConclusionOL‐derived IL‐33 may play a regulatory role in Aβ clearance during amyloidogenesis, probably via promoting microglial activity, but not in OL survival in the AD brain. These findings support a novel mechanism of instructive interaction between OLs and microglia in disease‐associated neuroinflammation.

Amyloid‐beta immunotherapy induces novel methylomic changes in Alzheimer’s disease brain

AbstractBackgroundPost‐mortem neuropathological examinations following the first active immunotherapy strategy (AN‐1792, Elan Pharmaceuticals, 2000) for Alzheimer’s disease (AD) have evidenced amyloid‐β (Aβ) plaque clearance and increased microglial phagocytic activity in immunised individuals. This study characterises the epigenetic profiles of individuals who underwent Aβ immunotherapy with the aim of discovering novel therapeutic targets and biomarkers.MethodDNA was isolated from post‐mortem prefrontal cortex tissue of 16 immunised AD cases who received varying doses (ug) and number of doses during the trial period, including placebos. DNA methylation was quantified using methylation arrays and the raw intensity values processed and normalised for subsequent statistical analysis to identify differentially methylated positions (DMPs) across the genome associated with Aβ immunotherapy. Additional analyses included regions of enrichment analysis for identification of differentially methylated regions (DMRs) and weighted gene co‐expression network analysis to identify gene clusters with highly correlated methylation levels.ResultAfter correcting for known (age, sex, cell type composition) and unknown variables, a DMP located within Chromosome 19 Open Reading Frame 12 (C19Orf12) at the genome‐wide significance level (P < 9.00E−08) was associated with Aβ immunisation. 10 DMRs were further associated with immunisation, with three regions consisting of ≥ 3 CpG sites and having a Sidak‐corrected P < 0.05. These regions were located within the CRISP2 (chr6; 49681178:49681391 [8 probes], Sidak‐corrected P = 8.29E‐05), CTTNA1 (chr5; 138210906:138211184 [8 probes], Sidak‐corrected P = 5.96E‐06), and RNF39 (chr6; 30038859:30039025 [12 probes], Sidak‐Corrected P = 2.60E‐05) genes.ConclusionIndicating better inflammatory markers can help inform of effective preventative care strategies for elders. This study provides evidence for altered epigenetic processes in the pathophysiology of AD and identifies novel processes specific to Aβ immunotherapy. The next step for this project includes analysing RNA sequencing data, to determine if observed methylomic changes correlate with gene expression, and genotyping array data.

Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone

Microglia are the primary phagocytes in the central nervous system and clear dead cells generated during development or disease. The phagocytic process shapes the microglia phenotype, which affects the local environment. A unique population of microglia resides in the ventricular-subventricular zone (V-SVZ) of neonatal mice, but how they influence the neurogenic niche is not well understood. Here, we demonstrate that phagocytosis contributes to a pro-neurogenic microglial phenotype in the V-SVZ and that these microglia phagocytose apoptotic cells via the engulfment receptor Jedi-1. Deletion of Jedi-1 decreases apoptotic cell clearance, triggering a neuroinflammatory microglia phenotype that resembles dysfunctional microglia in neurodegeneration and aging and that reduces neural precursor proliferation via elevated interleukin-1β signaling; interleukin-1 receptor inhibition rescues precursor proliferation invivo. Together, these results reveal a critical role for Jedi-1 in connecting microglial phagocytic activity to the maintenance of a pro-neurogenic phenotype in the developing V-SVZ.

Microglia contribute to cognitive decline in hypercholesterolemic LDLr-/- mice.

Familial hypercholesterolemia (FH) is caused by mutations in the gene that encodes the low-density lipoprotein (LDL) receptor, which leads to an excessive increase in plasma LDL cholesterol levels. Previous studies have shown that FH is associated with gliosis, blood-brain barrier dysfunction, and memory impairment, but the mechanisms associated with these events are still not fully understood. Therefore, we aimed to investigate the role of microgliosis in the neurochemical and behavioral changes associated with FH using LDL receptor knockout (LDLr-/-) mice. We noticed that microgliosis was more severe in the hippocampus of middle-aged LDLr-/- mice, which was accompanied by microglial morphological changes and alterations in the immunocontent of synaptic protein markers. At three months of age, the LDLr-/- mice already showed increased microgliosis and decreased immunocontent of claudin-5 in the prefrontal cortex (PFC). Subsequently, 6-month-old male C57BL/6 wild-type and LDLr-/- mice were treated once daily for 30 days with minocycline (a pharmacological inhibitor of microglial cell reactivity) or vehicle (saline). Adult LDLr-/- mice displayed significant hippocampal memory impairment, which was ameliorated by minocycline treatment. Non-treated LDLr-/- mice showed increased microglial density in all hippocampal regions analyzed, a process that was not altered by minocycline treatment. Region-specific microglial morphological analysis revealed different effects of genotype or minocycline treatment on microglial morphology, depending on the hippocampal subregion analyzed. Moreover, 6-month-old LDLr-/- mice exhibited a slight but not significant increase in IBA-1 immunoreactivity in the PFC, which was reduced by minocycline treatment without altering microglial morphology. Minocycline treatment also reduced the presence of microglia within the perivascular area in both the PFC and hippocampus of LDLr-/- mice. However, no significant effects of either genotype or minocycline treatment were observed regarding the phagocytic activity of microglia in the PFC and hippocampus. Our results demonstrate that hippocampal microgliosis, microglial morphological changes, and the presence of these glial cells in the perivascular area, but not increased microglial phagocytic activity, are associated with cognitive deficits in a mouse model of FH.

Microglia depletion ameliorates neuroinflammation, anxiety-like behavior, and cognitive deficits in a sex-specific manner in Rev-erbα knockout mice

The circadian system is an evolutionarily adaptive system that synchronizes biological and physiological activities within the body to the 24 h oscillations on Earth. At the molecular level, circadian clock proteins are transcriptional factors that regulate the rhythmic expression of genes involved in numerous physiological processes such as sleep, cognition, mood, and immune function. Environmental and genetic disruption of the circadian clock can lead to pathology. For example, global deletion of the circadian clock gene Rev-erbα (RKO) leads to hyperlocomotion, increased anxiety-like behaviors, and cognitive impairments in male mice; however, the mechanisms underlying behavioral changes remain unclear. Here we hypothesized that RKO alters microglia function leading to neuroinflammation and altered mood and cognition, and that microglia depletion can resolve neuroinflammation and restore behavior. We show that microglia depletion (CSF1R inhibitor, PLX5622) in 8-month-old RKO mice ameliorated hyperactivity, memory impairments, and anxiety/risky-like behaviors. RKO mice exhibited striking increases in expression of pro-inflammatory cytokines (e.g., IL-1β and IL-6). Surprisingly, these increases were only fully reversed by microglia depletion in the male but not female RKO hippocampus. In contrast, male RKO mice showed greater alterations in microglial morphology and phagocytic activity than females. In both sexes, microglia depletion reduced microglial branching and decreased CD68 production without altering astrogliosis. Taken together, we show that male and female RKO mice exhibit unique perturbations to the neuroimmune system, but microglia depletion is effective at rescuing aspects of behavioral changes in both sexes. These results demonstrate that microglia are involved in Rev-erbα-mediated changes in behavior and neuroinflammation.

GSDMD knockdown attenuates phagocytic activity of microglia and exacerbates seizure susceptibility in TLE mice

BackgroundTemporal lobe epilepsy (TLE) is often characterized pathologically by severe neuronal loss in the hippocampus. Phagocytic activity of microglia is essential for clearing apoptotic neuronal debris, allowing for repair and regeneration. Our previous research has shown that gasdermin D (GSDMD)-mediated pyroptosis is involved in the pathogenesis of TLE. However, whether GSDMD-mediated pyroptosis influences the accumulation of apoptotic neurons remains unclear. Therefore, the present study was designed to investigate whether phagocytic activity of microglia is involved in GSDMD-mediated pyroptosis and the pathogenesis of TLE.MethodsTo establish a TLE model, an intra-amygdala injection of kainic acid (KA) was performed. The Racine score and local field potential (LFP) recordings were used to assess seizure severity. Neuronal death in the bilateral hippocampus was assessed by Nissl staining and TUNEL staining. Microglial morphology and phagocytic activity were detected by immunofluorescence and verified by lipopolysaccharide (LPS) and the P2Y12R agonist 2MeSADP.ResultsGSDMD knockdown augmented the accumulation of apoptotic neurons and seizure susceptibility in TLE mice. Microglia activated and transition to the M1 type with increased pro-inflammatory cytokines. Furthermore, GSDMD knockdown attenuated the migration and phagocytic activity of microglia. Of note, LPS-activated microglia attenuated seizure susceptibility and the accumulation of apoptotic neurons in TLE after GSDMD knockdown. A P2Y12R selective agonist, 2MeSADP, enhanced the migration and phagocytic activity of microglia.ConclusionsOur results demonstrate that GSDMD knockdown exacerbates seizure susceptibility and the accumulation of apoptotic neurons by attenuating phagocytic activity of microglia. These findings suggest that GSDMD plays a protective role against KA-induced seizure susceptibility.

Activation of TNF Receptor 2 Improves Synaptic Plasticity and Enhances Amyloid-β Clearance in an Alzheimer's Disease Mouse Model with Humanized TNF Receptor 2.

Tumor necrosis factor-alpha (TNF-α) is a master cytokine involved in a variety of inflammatory and neurological diseases, including Alzheimer's disease (AD). Therapies that block TNF-α proved ineffective as therapeutic for neurodegenerative diseases, which might be explained by the opposing functions of the two receptors of TNF (TNFRs): while TNFR1 stimulation mediates inflammatory and apoptotic pathways, activation of TNFR2 is related to neuroprotection. Despite the success of targeting TNFR2 in a transgenic AD mouse model, research that better mimics the human context is lacking. The aim of this study is to investigate whether stimulation of TNFR2 with a TNFR2 agonist is effective in activating human TNFR2 and attenuating AD neuropathology in the J20xhuTNFR2-k/i mouse model. Transgenic amyloid-β (Aβ)-overexpressing mice containing a human extracellular TNFR2 domain (J20xhuTNFR2-k/i) were treated with a TNFR2 agonist (NewStar2). After treatment, different behavioral tests and immunohistochemical analysis were performed to assess different parameters, such as cognitive functions, plaque deposition, synaptic plasticity, or microglial phagocytosis. Treatment with NewStar2 in J20xhuTNFR2-k/i mice resulted in a drastic decrease in plaque load and beta-secretase 1 (BACE-1) compared to controls. Moreover, TNFR2 stimulation increased microglial phagocytic activity, leading to enhanced Aβ clearance. Finally, activation of TNFR2 rescued cognitive impairments and improved synaptic plasticity. Our findings demonstrate that activation of human TNFR2 ameliorates neuropathology and improves cognitive functions in an AD mouse model. Moreover, our study confirms that the J20xhuTNFR2-k/i mouse model is suitable for testing human TNFR2-specific compounds.

Extracellular tau stimulates phagocytosis of living neurons by activated microglia via Toll-like 4 receptor–NLRP3 inflammasome–caspase-1 signalling axis

In tauopathies, abnormal deposition of intracellular tau protein followed by gradual elevation of tau in cerebrospinal fluids and neuronal loss has been documented, however, the mechanism how actually neurons die under tau pathology is largely unknown. We have previously shown that extracellular tau protein (2N4R isoform) can stimulate microglia to phagocytose live neurons, i.e. cause neuronal death by primary phagocytosis, also known as phagoptosis. Here we show that tau protein induced caspase-1 activation in microglial cells via ‘Toll-like’ 4 (TLR4) receptors and neutral sphingomyelinase. Tau-induced neuronal loss was blocked by caspase-1 inhibitors (Ac-YVAD-CHO and VX-765) as well as by TLR4 antibodies. Inhibition of caspase-1 by Ac-YVAD-CHO prevented tau-induced exposure of phosphatidylserine on the outer leaflet of neuronal membranes and reduced microglial phagocytic activity. We also show that suppression of NLRP3 inflammasome, which is down-stream of TLR4 receptors and mediates caspase-1 activation, by a specific inhibitor (MCC550) also prevented tau-induced neuronal loss. Moreover, NADPH oxidase is also involved in tau-induced neurotoxicity since neuronal loss was abolished by its pharmacological inhibitor. Overall, our data indicate that extracellular tau protein stimulates microglia to phagocytose live neurons via Toll-like 4 receptor–NLRP3 inflammasome–caspase-1 axis and NADPH oxidase, each of which may serve as a potential molecular target for pharmacological treatment of tauopathies.

Severity- and Time-Dependent Activation of Microglia in Spinal Cord Injury.

A spinal cord injury (SCI) initiates a number of cascades of biochemical reactions and intercellular interactions, the outcome of which determines the regenerative potential of the nervous tissue and opens up capacities for preserving its functions. The key elements of the above-mentioned processes are microglia. Many assumptions have been put forward, and the first evidence has been obtained, suggesting that, depending on the severity of SCI and the post-traumatic period, microglia behave differently. In this regard, we conducted a study to assess the microglia behavior in the model of mild, moderate and severe SCI in vitro for various post-traumatic periods. We reported for the first time that microglia make a significant contribution to both anti- and pro-inflammatory patterns for a prolonged period after severe SCI (60 dpi), while reduced severities of SCI do not lead to prolonged activation of microglia. The study also revealed the following trend: the greater the severity of the SCI, the lower the proliferative and phagocytic activity of microglia, which is true for all post-traumatic periods of SCI.

Abstract 2472: Interdisciplinary method to elucidate the interaction between brain microenvironment and cancer cells

Abstract Cancer cells may escape from host immunity to form distant metastases. We have made an original imaging model of brain metastasis (IBrM) to show a heterogeneous interaction between microglia, the tissue macrophages in the brain, and cancer cells in the brain niche. Here we report that enhanced microglial elimination of cancer cells is a new potential treatment for metastatic brain tumors by inhibiting their engraftment. First, to explore factors that favor survival of cancer cells in the brain microenvironment, mouse lung cancer cells (CMT167) were injected through the internal carotid artery, and after 4, 7, 10, and 13 days, surviving cancer cells in the mouse brain were collected and RNAseq was performed. In particular, we focused on CD24/CD7/PD-L1, a known factor that escapes phagocytosis by macrophages. The expression of CD24/CD47 in cancer cells gradually increased, while that of PD-L1 decreased. Next, we injected adenocarcinoma lung cancer cells (LUCs, CMT167) or breast cancer cells (BCCs, E0711) expressing mCherry via the internal carotid artery of the CX3CR1-EGFP mice, whose microglia had been specifically labeled with EGFP. The microglia and LUCs/BCCs were visualized in vivo simultaneously by two-photon microscopy for 14 days. The tumor fate and microglial response against cancer cells were evaluated at single cell resolution in the IBrM. Microglial phagocytosis was significantly increased by single deletion of CD47 or CD24 compared with WT cells. Combined deletion of CD47 and CD24 also synergically increased microglial phagocytosis, resulting in reduced IBrM and prolonged mouse survival. By contrast, deletion of PD-L1 did not enhance microglial phagocytosis or prolong survival. The pharmacological (PLX3397) or genetic conditional (Siglechdtr het/CX3CR1GFP het) conditional depletion of microglia canceled both the increased phagocytosis and the extended survival time, suggesting that the effect of “don't eat me” signals was microglia dependent. The extended survival time was also observed in IBrM using BALB-c nu/nu, that suggest the effect is not dependent on matured T-lymphocytes.These results indicate that novel function of microglia against cancer cells, and suppression of both CD24 and CD47 in cancer cells and enhances microglial phagocytic activity and suppresses the metastatic formation. We are further developing a method for isolating LUCs fluorescently labeled in vivo using holographic 2-photon microscopy for evaluating single cell gene expression profiles. Using the novel techniques, the spatio-temporal information can be integrated across the molecular, cellular and tissue levels that allow us to further investigate the dynamic behavior of not only tumors, but also microglia and neurons. Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Rahadian Y. Hartantio, Misuzu Horikoshi, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Interdisciplinary method to elucidate the interaction between brain microenvironment and cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2472.

Early life stress induces visual dysfunction and retinal structural alterations in adult mice.

Early life stress (ELS) is defined as a period of severe and/or chronic trauma, as well as environmental/social deprivation or neglect in the prenatal/early postnatal stage. Presently, the impact of ELS on the retina in the adult stage is unknown. The long-term consequences of ELS at retinal level were analyzed in an animal model of maternal separation with early weaning (MSEW), which mimics early life maternal neglect. For this purpose, mice were separated from the dams for 2h at postnatal days (PNDs) 4-6, for 3h at PNDs 7-9, for 4h at PNDs 10-12, for 6h at PNDs 13-16, and weaned at PND17. At the end of each separation period, mothers were subjected to movement restriction for 10min. Control pups were left undisturbed from PND0, and weaned at PND21. Electroretinograms, visual evoked potentials, vision-guided behavioral tests, retinal anterograde transport, and retinal histopathology were examined at PNDs 60-80. MSEW induced long-lasting functional and histological effects at retinal level, including decreased retinal ganglion cell function and alterations in vision-guided behaviors, likely associated to decreased synaptophysin content, retina-superior colliculus communication deficit, increased microglial phagocytic activity, and retinal ganglion cell loss through a corticoid-dependent mechanism. A treatment with mifepristone, injected every 3 days between PNDs 4 and16, prevented functional and structural alterations induced by MSEW. These results suggest that retinal alterations might be included among the childhood adversity-induced threats to life quality, and that an early intervention with mifepristone avoided ELS-induced retinal disturbances.

Phloretin decreases microglia-mediated synaptic engulfment to prevent chronic mild stress-induced depression-like behaviors in the mPFC.

Background: Stress is an important risk factor to induce psychiatric disorders such as depression. Phloretin (PHL), a natural dihydrochalcone compound, has been shown to exhibit anti-inflammatory and anti-oxidative effects. However, the impact of PHL on the depression and the underlying mechanism remain unclear. Methods: The animal behavior tests were used to determine the protective of PHL on the chronic mild stress (CMS)-induced depression-like behaviors. The Magnetic Resonance Imaging (MRI), electron microscopy analysis, fiber photometry, electrophysiology, and Structure Illumination Microscopy (SIM) were used to investigate the protective of PHL on the structural and functional impairments induced by CMS exposure in the mPFC. The RNA sequencing, western blot, reporter gene assay, and chromatin immunoprecipitation were adopted to investigate the mechanisms. Results: We showed that PHL efficiently prevented the CMS-induced depressive-like behaviors. Moreover, PHL not only attenuated the decrease of synapse losses but also improved the dendritic spine density and neuronal activity in the mPFC after CMS exposure. Furthermore, PHL remarkably inhibited the CMS-induced microglial activation and phagocytic activity in the mPFC. In addition, we demonstrated that PHL decreased the CMS-induced synapse losses by inhibiting the deposition of complement C3 deposition onto synapses and subsequent microglia-mediated synaptic engulfment. Finally, we revealed that PHL inhibited the NF-κB-C3 axis to display neuroprotective effects. Conclusions: Our results indicate that PHL represses the NF-κB-C3 axis and subsequent microglia-mediated synaptic engulfment to protect against CMS-induced depression in the mPFC.