Impaired microglial phagocytosis promotes seizure development.

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. TREM2 plays an important role in neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis. However, little is known about the role TREM2 plays in epileptogenesis. To investigate this, we utilized TREM2 knockout (KO) mice within the murine 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 acute status epilepticus and spontaneous recurrent seizures characteristic of chronic focal epilepsy. Mechanistically, phagocytic clearance of damaged neurons by microglia was impaired in TREM2 KO mice and the reduced phagocytic capacity 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 microglial phagocytic activity and focal to bilateral tonic-clonic generalized seizure history. These results indicate that microglial TREM2 and phagocytic activity may be important to epileptogenesis and the progression of focal temporal lobe epilepsy.

Chemogenetic approaches reveal dual functions of microglia in seizures.

Microglia are key players in maintaining brain homeostasis and exhibit phenotypic alterations in response to epileptic stimuli. However, it is still relatively unknown if these alterations are pro- or anti-epileptic. To unravel this dilemma, we employed chemogenetic manipulation of microglia using the artificial Gi-Dreadd receptor within a kainic acid (KA) induced murine seizure model. Our results indicate that acute Gi-Dreadd activation with Clozapine-N-Oxide can reduce seizure severity. Additionally, we observed increased interaction between microglia and neuronal soma, which correlated with reduced neuronal hyperactivity. Interestingly, prolonged activation of microglial Gi-Dreadds by repeated doses of CNO over 3 days, arrested microglia in a less active, homeostatic-like state, which associated with increased neuronal loss after KA induced seizures. RNAseq analysis revealed that prolonged activation of Gi-Dreadd interferes with interferon ß signaling and microglia proliferation. Thus, our findings highlight the importance of microglial Gi signaling not only during status epilepticus (SE) but also within later seizure induced pathology.

TREM2 mediates MHCII-associated CD4+ T cell response against gliomas.

BACKGROUND: Myeloid cells comprise up to 50% of the total tumor mass in glioblastoma (GBM) and have been implicated in promoting tumor progression and immunosuppression. Modulating the response of myeloid cells to the tumor has emerged as a promising new approach for cancer treatment. In this regard, we focus on the Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which has recently emerged as a novel immune modulator in peripheral tumors. METHODS: We studied the TREM2 expression profile in various patient tumor samples and conducted single-cell transcriptomic analysis in both glioblastoma patients and the GL261 mouse glioma model. We utilized multiple mouse glioma models and employed state-of-the-art techniques such as in vivo two-photon imaging, spectrum flow cytometry, and in vitro co-culture assays to study TREM2 function in myeloid cell-mediated phagocytosis of tumor cells, antigen presentation, and response of CD4+ T cells within the tumor hemispheres. RESULTS: Our research revealed significantly elevated levels of TREM2 expression in brain tumors compared to other types of tumors in patients. TREM2 was predominantly localized in tumor-associated myeloid cells and was highly expressed in nearly all microglia, as well as various subtypes of macrophages. Surprisingly, in pre-clinical glioma models, TREM2 deficiency did not confer a beneficial effect; instead, it accelerated glioma progression. Through detailed investigations, we determined that TREM2 deficiency impaired the ability of tumor-myeloid cells to phagocytose tumor cells and led to reduced expression of MHCII. This deficiency further significantly decreased the presence of CD4+ T cells within the tumor hemispheres. CONCLUSIONS: Our study unveiled a previously unrecognized protective role of tumor-myeloid TREM2. Specifically, we found TREM2 enhance the phagocytosis of tumor cells and promote an immune response by facilitating MHCII-associated CD4+ T cell responses against gliomas.

Chemogenetic manipulation of CX3CR1+ cells transiently induces hypolocomotion independent of microglia.

Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1CreER/+:R26hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1+ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1+ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119CreER/+:R26hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4+ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1CreER/+ mouse line to manipulate microglia.

TREM2 mediates MHCII-associated CD4+ T cell response against gliomas.

Triggering receptor expressed on myeloid cells 2 (TREM2) was recently highlighted as a novel immune suppressive marker in peripheral tumors. The aim of this study was to characterize TREM2 expression in gliomas and investigate its contribution in glioma progression by using Trem2-/- mouse line. Our results showed that higher TREM2 expression was correlated with poor prognosis in glioma patients. Unexpectedly, TREM2 deficiency did not have a beneficial effect in a pre-clinical model of glioma. The increased TREM2 expression in glioma was likely due to increased myeloid cell infiltration, as evidenced by our single-cell analysis showing that almost all microglia and macrophages in gliomas were TREM2+. Furthermore, we found that deficiency of TREM2 impaired tumor-myeloid phagocytosis and MHCII presentation, and significantly reduced CD4+ T cells in tumor hemispheres. Our results revealed a previously unrecognized protective role of tumor-myeloid TREM2 in promoting MHCII-associated CD4+ T cell response against gliomas.

Ginkgo biloba L. Prevents Hypobaric Hypoxia-Induced Spatial Memory Deficit Through Small Conductance Calcium-Activated Potassium Channel Inhibition: The Role of ERK/CaMKII/CREB Signaling.

Hypobaric hypoxia (HH) is a stressful condition, which is more common at high altitudes and can impair cognitive functions. Ginkgo biloba L. leaf extract (GBE) is widely used as herbal medicine against different disorders. Its ability to improve cognitive functions, reduce oxidative stress, and promote cell survival makes it a putative therapeutic candidate against HH. The present study has been designed to explore the effect of GBE on HH-induced neurodegeneration and memory impairment as well as possible signaling mechanisms involved. 220-250 gm (approximately 6- to 8-week-old) Sprague Dawley rats were randomly divided into different groups. GBE was orally administered to respective groups at a dose of 100 mg/kg/day throughout the HH exposure, i.e., 14 days. Memory testing was performed followed by hippocampus isolation for further processing of different molecular and morphological parameters related to cognition. The results indicated that GBE ameliorates HH-induced memory impairment and oxidative damage and reduces apoptosis. Moreover, GBE modulates the activity of the small conductance calcium-activated potassium channels, which further reduces glutamate excitotoxicity and apoptosis. The exploration of the downstream signaling pathway demonstrated that GBE administration prevents HH-induced small conductance calcium-activated potassium channel activation, and that initiates pro-survival machinery by activating extracellular signal-regulated kinase (ERK)/calmodulin-dependent protein kinase II (CaMKII) and the cAMP response element-binding protein (CREB) signaling pathway. In summary, the current study demonstrates the beneficial effect of GBE on conditions like HH and provides various therapeutic targets involved in the mechanism of action of GBE-mediated neuroprotection.

HDAC inhibition prevents hypobaric hypoxia-induced spatial memory impairment through PΙ3K/GSK3ß/CREB pathway.

Hypobaric hypoxia at higher altitudes usually impairs cognitive function. Previous studies suggested that epigenetic modifications are the culprits for this condition. Here, we set out to determine how hypobaric hypoxia mediates epigenetic modifications and how this condition worsens neurodegeneration and memory loss in rats. In the current study, different duration of hypobaric hypoxia exposure showed a discrete pattern of histone acetyltransferases and histone deacetylases (HDACs) gene expression in the hippocampus when compared with control rat brains. The level of acetylation sites in histone H2A, H3 and H4 was significantly decreased under hypobaric hypoxia exposure compared to the control rat's hippocampus. Additionally, inhibiting the HDAC family with sodium butyrate administration (1.2 g/kg body weight) attenuated neurodegeneration and memory loss in hypobaric hypoxia-exposed rats. Moreover, histone acetylation increased at the promoter regions of brain-derived neurotrophic factor (BDNF); thereby its protein expression was enhanced significantly in hypobaric hypoxia exposed rats treated with HDAC inhibitor compared with hypoxic rats. Thus, BDNF expression upregulated cAMP-response element binding protein (CREB) phosphorylation by stimulation of PI3K/GSK3ß/CREB axis, which counteracts hypobaric hypoxia-induced spatial memory impairment. In conclusion, these results suggested that sodium butyrate is a novel therapeutic agent for the treatment of spatial memory loss associated with hypobaric hypoxia, and also further studies are warranted to explore specific HDAC inhibitors in this condition.

Optogenetic activation of spinal microglia triggers chronic pain in mice.

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1ß production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

The complement C3-C3aR pathway mediates microglia-astrocyte interaction following status epilepticus.

Gliosis is a histopathological characteristic of epilepsy that comprises activated microglia and astrocytes. It is unclear whether or how crosstalk occurs between microglia and astrocytes in the evolution of epilepsy. Here, we report in a mouse model of status epilepticus, induced by intracerebroventricular injection of kainic acid (KA), sequential activation of microglia and astrocytes and their close spatial interaction in the hippocampal CA3 region. Microglial ablation reduced astrocyte activation and their upregulation of complement C3. When compared to wild-type mice, both C3-/- and C3aR-/- mice had significantly less microglia-astrocyte interaction in response to KA-induced status epilepticus. Additionally, KA-injected C3-/- mice had significantly less histochemical evidence of neurodegeneration. The results suggest that the C3-C3aR pathway contributes to KA-induced neurodegeneration by mediating microglia-astrocyte communication. The C3-C3aR pathway may prove to be a potential therapeutic target for epilepsy treatment.

Hypobaric Hypoxia-Induced Learning and Memory Impairment: Elucidating the Role of Small Conductance Ca2+-Activated K+ Channels.

Hypobaric Hypoxia (HH) is well-known to cause cognitive impairment and synaptic dysfunction which results in neurodegeneration. Although the role of small conductance calcium-activated potassium channels (SK channels) has been reported in synaptic plasticity, cognition and different neurological disorders; however, the precise role of SK channels in HH-induced memory impairment remains yet to be explored. We, therefore, hypothesized the pivotal role of SK channels in HH-induced cognitive decline and investigated the SK channel expression during different duration of HH exposure (Control, 1, 3, 7 and 14 days) at mRNA and protein level in male Sprague-Dawley rats. Further the role of SK channels in spatial memory and neurodegeneration were explored by inhibiting SK channel through Apamin (a known SK channel blocker). Results from the present study revealed that acute exposure of HH for 3 days leads to significant increase in expression of SK1 and SK3 channels at mRNA and protein levels, which upon chronic exposure restored to normal. Remarkably, SK2 channel expression showed gradual increase from 3 days till 14 days. Immunohistochemical analysis revealed similar pattern in different regions of the hippocampus. Additionally, SK channel inhibition with Apamin prevented HH-induced neurodegeneration and memory impairment as evident from decreased number of Fluoro Jade-positive cells, pyknotic cells, and caspase-3 expression and improved performance in the Morris water maze task. Thus, the present study demonstrates that SK channels play a crucial role in HH-induced cognitive decline and neurodegeneration.

Role of DNA Methylation in Hypobaric Hypoxia-Induced Neurodegeneration and Spatial Memory Impairment.

Hypobaric hypoxia (HH) is a major stress factor that is associated with physiological, biochemical, molecular and genomic alterations. Brain is the organ that reacts sensitively to oxygen deprivation, which leads to oxidative stress and cognitive function impairment. Our previous studies have reported that downregulation of brain derived neurotrophic factor (BDNF) leads to neurodegeneration and memory impairment. The aim of the present study was to investigate the effect of HH exposure on DNA methylation and its regulation in BDNF expression, neurodegeneration and spatial memory impairment. For this purpose, Sprague Dawley rats were exposed to HH at a simulated altitude of 25,000 feet for 14 days. Real-time polymerase chain reaction was used for transcriptional expression of DNA Methyltransferases (DNMTs) including DNMT1, DNMT3a and -DNMT3b, and immunoblotting was used for the translational expression of DNMT1, DNMT3a, DNMT3b, Methyl CpG binding protein 2 (MeCP2), pMeCP2 and BDNF in rat hippocampus. Additionally, neuronal morphology alteration and neurodegeneration in CA1 region of hippocampus were investigated though Cresyl violet (CV) staining and Fluoro-Jade C staining respectively. Results obtained suggested that HH exposure increased the expression of DNMT1 DNMT3b at the mRNA as well as protein level, whereas no significant change was observed in the level of DNMT3a. Furthermore, the level of pMeCP2 and BDNF were significantly decreased; however, the expression level of MeCP2 was significantly increased. The CV and Fluoro-Jade C-positive cells were significantly enhanced in the CA1 region of hippocampus in the HH exposed group as compared to unexposed rats. Thus, the present study concluded that HH decreases neuronal activation by the upregulation of DNA methylation and MeCP2 and decreased the expression of pMeCP2, which result in the downregulation of BDNF. The decreased BDNF expression is associated with neuronal loss and spatial memory impairment. This study highlights that DNMT inhibition could be an important therapeutic target for neurodegenerative diseases.