Emerging role of senescent microglia in brain aging-related neurodegenerative diseases.

Brain aging is a recognized risk factor for neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), but the intricate interplay between brain aging and the pathogenesis of these conditions remains inadequately understood. Cellular senescence is considered to contribute to cellular dysfunction and inflammaging. According to the threshold theory of senescent cell accumulation, the vulnerability to neurodegenerative diseases is associated with the rates of senescent cell generation and clearance within the brain. Given the role of microglia in eliminating senescent cells, the accumulation of senescent microglia may lead to the acceleration of brain aging, contributing to inflammaging and increased vulnerability to neurodegenerative diseases. In this review, we propose the idea that the senescence of microglia, which is notably vulnerable to aging, could potentially serve as a central catalyst in the progression of neurodegenerative diseases. The senescent microglia are emerging as a promising target for mitigating neurodegenerative diseases.

Possible involvement of microglial P2RY12 and peripheral IL-10 in postpartum depression.

Postpartum depression (PPD) is another type of depression, including emotional fluctuation, fatigue, and anxiety. Based on the specific event like giving birth, it can be speculated that PPD might have its specific mechanism. Here, we confirmed that dexamethasone (DEX) administration during pregnancy (gestational days 16-18) induced depressive- and anxiety-like behaviors in dam (DEX-dam) after weaning period (3 weeks). DEX-dam showed anxiety-like behaviors in open-field test (OFT) and light-dark test (LD). In addition, DEX-dam exhibited depressive-like behaviors with the increased immobility time in forced swimming test (TST). Molecular analysis confirmed that microglia, rather than neurons, astrocytes, and oligodendrocytes, are involved in anxiety-/depressive-like behaviors. Notably, P2ry12, homeostatic gene, and purinoceptor, along with hyper-ramified form, were reduced in the hippocampus of DEX-dam. In addition, we found that IL-10 mRNA was reduced in lymph nodes without alteration of pro-inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6. Interestingly, anxiety-/depressive-like behaviors of DEX-dam were restored with the normalization of P2ry12 and IL-10 after 10 weeks postpartum without antidepressants. Our results propose that stress hormone elevation during pregnancy might be associated with PPD via microglial P2RY12 and peripheral IL-10.

A molecular characterization and clinical relevance of microglia-like cells derived from patients with panic disorder.

Few studies report the microglia involvement in the pathogenesis of panic disorder (PD), although the crucial role of microglia in other neuropsychiatric diseases is being emphasized. In addition, there is no report to characterize the phenotypic and functional levels of PD patient-derived microglia to find their clinical relevance. Herein, we used a model to induce patient-derived microglia-like cells (iMGs) to clarify the molecular characteristics and function of PD-iMGs. We established iMGs from 17 PD patients and 16 healthy controls (non-psychiatric controls, HC). PD-iMGs showed increased T-cell death-associated gene-8 expression per the proposal of a previous in vivo study. In addition, we found that patient-derived iMGs showed reduced phagocytosis and increased TREM2 expression. We analyzed the phenotype of the PD-iMGs by RNA sequencing. The PD-iMGs clustered together distinct from HC-iMGs. Gene set enrichment analysis revealed the involvement of cholesterol biosynthesis and steroid metabolism in PD-iMGs. Regarding the cholesterol synthesis pathway, we discovered ACAT2 and DHCR7 as the most impacted genes related to a character of PD-iMGs compared to HC-iMGs. The ACAT2, a major cholesterol esterifier, was increased in PD-iMGs. Nevertheless, PD-iMGs did not show lipid droplet accumulation. Interestingly, ACAT2 expression was inversely correlated with the severity of depression and anxiety sensitivity to publicly observable anxiety reactions. We propose that microglia of PD patients have unique characteristics with dysregulation of cholesterol biosynthesis pathway and impaired phagocytosis, reflecting clinical phenotype.

Fluoxetine Decreases Phagocytic Function via REV-ERBα in Microglia.

Although fluoxetine (FLX) is a commonly used drug in psychiatric disorders, such as major depressive disorder, anxiety disorder, panic disorder, and obsessive-compulsive disorder, the mechanism by which FLX exerts its therapeutic effect is not completely understood. In this study, we aimed to determine the possible mechanism by which FLX focuses on microglial phagocytosis. FLX reduced phagocytic function in BV2 cells and increased REV-ERBα without affecting other microglia-related genes, such as inflammation and phagocytosis. Although FLX did not change BMAL1 protein levels, it restricted the nucleocytoplasmic transport (NCT) of BMAL1, leading to its cytosolic accumulation. REV-ERBα antagonist SR8278 rescued the decreased phagocytic activity and restricted NCT of BMAL1. We also found that REV-ERBα mediates the effect of FLX via the inhibition of phospho-ERK (pERK). The ERK inhibitor FR180204 was sufficient to reduce phagocytic function in BV2 cells and restrict the NCT of BMAL1. These results were recapitulated in the primary microglia. In conclusion, we propose that FLX decreases phagocytic function and restricts BMAL1 NCT via REV-ERBα. In addition, ERK inhibition mimics the effects of FLX on microglia.

A Step-by-step Protocol for Obtaining Mature Microglia from Mice.

In mice, microglial precursors in the yolk sac migrate to the brain parenchyma through the head neuroepithelial layer between embryonic days 8.5 (E8.5)-E16.5 and acquire their unique identity with a ramified form. Based on the microglial developmental process, we dissected the neuroepithelial layer (NEL) of E13.5 mice, which is composed of microglial progenitor and neuroepithelial cells. The NEL was bankable and expandable. In addition, microglial precursors were matured according to NEL culture duration. The matured microglia (MG; CD11b-positive cells) were easily isolated from the cultured NEL using a magnetic-activated cell sorting system and named NEL-MG. In conclusion, we obtained higher yields of adult-like microglia (mature microglia: NEL-MG) compared to previous in vitro surrogates such as neonatal microglia and microglial cell lines. Graphical abstract.

Microglia involvement in sex-dependent behaviors and schizophrenia occurrence in offspring with maternal dexamethasone exposure.

Fetal microglia that are particularly sensitive cells to the changes in utero environment might be involved in the sex-biased onset and vulnerability to psychiatric disorders. To address this issue, we administered a 50 µg/kg dexamethasone (DEX) to dams subcutaneously from gestational days 16 to 18 and a series of behavioral assessments were performed in the offspring. Prenatal exposure to dexamethasone (PN-DEX) induced schizophrenia (SCZ)-relevant behaviors in male mice and depressive-like behavior in female mice. SCZ-relevant behavioral patterns occurred in 10-week-old (10 W) male mice but not in 4-week-old (4 W) male mice. Microglia in the medial prefrontal cortex (mPFC) and the striatum (STR) of 10 W males prenatally treated with dexamethasone (10 W PN-DEX-M) showed hyper-ramified morphology and dramatically reduced spine density in mPFC. Immunofluorescence studies indicated that microglia in the mPFC of the 10 W PN-DEX-M group interacted with pre-synaptic Bassoon and post-synaptic density 95 (PSD95) puncta. PN-DEX-M also showed significantly changed dopamine system proteins. However, a testosterone surge during adolescence was not a trigger on SCZ-relevant behavior occurrence in 10 W PN-DEX-M. Furthermore, females prenatally treated with dexamethasone (PN-DEX-F) displayed depressive-like behavior, in addition to HPA-axis activation and inflammatory microglial phenotypes in their hippocampus (HPC). We propose that altered microglial function, such as increased synaptic pruning, may be involved in the occurrence of SCZ-relevant behavior in PN-DEX-M and sex-biased abnormal behavior in the PN-DEX model.

Persistent Acidic Environment Induces Impaired Phagocytosis via ERK in Microglia.

Acidic environment evoked by stroke, traumatic brain injury, and Alzheimer's disease may change the functional properties of microglia. Nevertheless, the underlying mechanisms of functional changes in microglia remain unclear. In this study, we found that acidic stimuli (pH 6.8) increased rapidly interleukin (IL)-1ß and IL-6 mRNA levels and subsequently reduced IL-10, transforming growth factor (TGF)-ß1, Cx3cr1, and P2ry12 as the exposure time to acidic environment increase in BV2 cells. In addition, persistent acidic environment (pH 6.8 for 6 h) induced impaired phagocytic function in BV2 cells. Short-term acidic exposure (pH 6.8 for 30 min) increased cyclic AMP (cAMP) and phospho-protein kinase A (PKA) but inhibited phospho-extracellular signal-regulated kinase (p-ERK). However, under persistent acidic environment (pH 6.8 for 6 h), cyclic AMP and PKA were normalized and p-ERK was increased with TDAG8 (T cell death associated gene 8; GPR65) reduction. FR 180,204, an ERK inhibitor, rescued the persistent acidic environment-induced functional changes in BV2 cells and its effect was recapitulated in primary neonatal microglia. Thus, we propose that ERK targeting may be an alternative strategy to restore microglial dysfunction in the central nervous system (CNS) acidic environment in various neurological disorders.

A new method for obtaining bankable and expandable adult-like microglia in mice.

BACKGROUND: The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice. METHODS: The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG. RESULTS: The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid ß, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term passage culture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells. CONCLUSIONS: This new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience.

Chronically infused angiotensin II induces depressive-like behavior via microglia activation.

Brain inflammation is one of hypotheses explaining complex pathomechanisms of depression. Angiotensin II (ANGII), which is associated with hypertension, also induces brain inflammation. However, there is no animal study showing the direct relationship between ANGII and depression. To address this issue, ANGII-containing osmotic pumps were implanted into adult male C57BL/6 mice subcutaneously for subacute (7 days) and chronic (at least 21 days) periods and behavioral and molecular analyses were conducted. Chronic infusion of ANGII into mice induced depressive-like behaviors, including the tail suspension test and forced swimming test, which were reversed by imipramine. Chronic infusion of ANGII also induced microglial activation in the hippocampus with increase of Il-1ß mRNA and decrease of Arg1 mRNA. In addition, chronic ANGII infusion activated the hypothalamic-pituitary-adrenal axis (HPA axis) and resulted in decreased hippocampal glucocorticoid receptor level. However, subacute ANGII infusion did not induce significant molecular and behavioral changes in mice compared to that of control. The molecular and behavioral changes by chronic ANGII infusion were reversed by co-treatment of minocycline or telmisartan. In addition, ANGII treatment also induced the pro-inflammatory changes in BV-2 microglial cells. Our results indicate that ANGII can induce depressive-like behaviors via microglial activation in the hippocampus and HPA axis hyperactivation in mice. These might suggest possible mechanism on depressive symptom in chronic hypertensive state.

Human umbilical cord-derived mesenchymal stem cells alleviate schizophrenia-relevant behaviors in amphetamine-sensitized mice by inhibiting neuroinflammation.

At present, therapeutic options available for treating schizophrenia are limited to monoamine-based antipsychotic drugs. Recent genome wide association study (GWAS) indicated a close relationship between immune system and schizophrenia. To leverage the GWAS finding for therapeutic strategy, we conducted a mechanism and effect study on application of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with potent immune-modulatory effect in an animal model useful for the study of schizophrenia. Schizophrenia-relevant behaviors were induced by amphetamine administration (amphetamine-sensitized mice) and the effect of a single intravenous administration of hUC-MSC was examined in the amphetamine-sensitized mice. Schizophrenia-relevant behaviors were assessed by open field test, light/dark box, social interaction test, latent inhibition, prepulse inhibition, tail suspension test, and forced swimming test. Our results indicated that neuroinflammation along with peripheral TNF-α elevation is associated with schizophrenia-relevant behaviors in amphetamine-sensitized mice. In addition, hUC-MSC inhibited schizophrenia-relevant and the neuroinflammatory changes. The main mechanism of hUC-MSC was associated with the induction of Treg and production of the anti-inflammatory cytokine, IL-10 in periphery. In vitro study revealed that amphetamine did not directly induce a neuroinflammatory reaction, while recombinant TNF-α (rTNF-α) increased mRNA expression of TNF-α, KMO, and IL-1ß in several microglial cell lines. Moreover, recombinant IL-10 (rIL-10) and MSC conditioned media inhibited the inflammatory response in rTNF-α-treated microglial cells. Assuming that hUC-MSCs rarely reach the CNS and do not remain in the body for an extended time, these findings suggest that a single hUC-MSC infusion have long-term beneficial effect via regulatory T cell induction and secretion of IL-10 in amphetamine-sensitized mice.

Differential effect of LPS and paclitaxel on microglial functional phenotypes and circulating cytokines: the possible role of CX3CR1 and IL-4/10 in blocking persistent inflammation.

Neuroinflammation plays a role in cancer chemotherapy-induced chronic pain. Thus far, most studies have focused on neuroinflammation suppression. However, there are limited reports of which factor is involved in the transition from acute inflammation to chronic inflammation, resulting in neuroinflammation and chronic pain. Here, we compared the inflammatory reaction and pain response induced by LPS and paclitaxel. LPS (0.5 mg/kg) or paclitaxel (2 mg/kg/day for 5 days) was administered intraperitoneally to mice, and mechanical allodynia was examined by von Frey test. LPS induced transient mechanical allodynia, whereas paclitaxel induced persistent mechanical allodynia. The CD86/CX3CR1 ratio remained unchanged due to CX3CR1 elevation following LPS injection, whereas the ratio was increased on day 1 after paclitaxel injection. LPS also increased CD45, CCL2, and CCL5 mRNA in the spinal cord and circulating pro- and anti-inflammatory cytokines 1 day after injection; however, the pattern was not consistent. Paclitaxel gradually increased inflammatory cytokines in the spinal cord. CX3CR1 might be involved in blocking the transition from acute pain to persistent pain in the LPS group. In addition, serum IL-4 and IL-10 elevation in the LPS group may be associated with chronic pain prevention. Therefore, targeting CX3CR1, IL-4, and IL-10 might be an alternative therapeutic strategy.

Dexamethasone Induces a Specific Form of Ramified Dysfunctional Microglia.

The functional status of dynamic microglial cells plays an important role in maintaining homeostasis of microenvironment in CNS. In a previous study, we reported that microglia phenotype might be involved in stress vulnerability and depression recurrence. Here, we aimed to clarify a character of microglia exposed persistently to glucocorticoid (GC), which is representative a stress hormone, in primary cultured microglial cells. Five nanomolars of dexamethasone (DEX, GC agonist) for 72 h decreased CX3CR1 and CD200R expression and induced ramified form of microglial cells in similar morphology to in vivo resident microglia. However, the ramified form of microglia did not increase microglia signature genes such as P2RY12, OLFML3, TMEM119, and TGFBR1. In addition, DEX-treated microglia showed a reduction of phagocytosis function, pro-and anti-inflammatory cytokine production, and cell proliferation. DEX washout did not restore these changes. Based on transcriptomic analysis and functional characters of DEX-treated microglia, we performed senescence-associated beta-galactosidase (SA-ß gal) assay in DEX-treated microglia and DEX-treated microglia showed more SA-ß gal activity with alteration of cell cycle-related genes. Thus, our results suggest that DEX can induce a specific phenotype of microglia (like-senescence).

Chronic high dose of captopril induces depressive-like behaviors in mice: possible mechanism of regulatory T cell in depression.

Major depression has various types of symptoms and disease courses with inconsistent response to monoamine-related antidepressants. Thus, monoamine theory may not be the only pathophysiologic pathway relevant to depression. Recently, it has been suggested that regulatory T cell (Treg) is associated with depression. Based on our previous study that showed decreased regulatory T cell (Treg) population following chronic high-dose captopril (CHC, 40 mg/kg/day * 21 days) administration, we examined whether CHC alone can induce depressive-like behaviors in mice even without stressful stimuli. In this study, we found that CHC induced depressive-like behaviors in tail suspension test (TST) and forced swimming test (FST) without systemic illness, while it did not induce anhedonic behavior, anxiety-like behaviors, or sociality-related behavior. The depressive-like behaviors were rescued by either CHC washout or antidepressant. CHC caused reduction in foxp3 and gata3 mRNA expression in the lymph nodes with elevation in plasma IL-1ß and IL-6. Interestingly, CHC increased serum angiotensin II level. In the hippocampus, CHC increased TNF-α and IL-6 mRNA expression with microglia activation while reduced glucocorticoid receptor expression. However, CHC did not affect to hippocampal kynurenine pathway, serotonin level, hypothalamic corticotropin-releasing hormone mRNA level, or serum corticosterone level. Consequently, we propose that CHC may induce a specific form of depressive-like behaviors via Treg reduction and microglial activation.