Primary Microglia Research Articles

Microglial Activation Results in Inhibition of TGF-β-Regulated Gene Expression.

Chronic inflammation mediated by persistent microglial activation is associated with the pathogenesis of neurodegenerative diseases. The mechanisms underlying chronic microglial activation are poorly understood. We have previously shown that anti-inflammatory TGF-β signaling is inhibited in LPS-treated microglia. In this study, we assessed whether different disease-related microglial activators could downregulate TGF-β induction of gene expression. We examined the effects of amyloid β (Aβ) (1-42)- or heat-killed Listeria monocytogenes (HKLM) on the TGF-β-regulated gene expression in primary rat microglia. We found that Aβ (1-42) oligomers and HKLM, in addition to LPS, suppressed TGF-β-mediated induction of gene expression in part through reducing expression of TβR1 mRNA encoding the TGF-β receptor 1 in primary microglia. Aβ (1-42) and LPS also prevented induction of TGF-β-induced genes in primary microglia. Additionally, Aβ (1-42) rescued primary microglia from TGF-β-mediated cell death without increasing cell proliferation. Blockage of NFκB signaling, but not the ERK or IRF3 pathways, inhibited Aβ (1-42)- and LPS-mediated reduction of TβR1 mRNA. Finally, LPS and Aβ (1-42) induced transient upregulation of mRNAs encoding SnoN and Bambi, inhibitors of TGF-β signaling. Our data indicate that one mechanism through which activators may prolong microglial stimulation is through direct inhibition of anti-inflammatory signaling. A more detailed understanding of the interaction between inflammatory and anti-inflammatory pathways may reveal potential targets for ameliorating chronic inflammation and hence speed the development of therapeutics to address neurodegenerative diseases.

Chronic stress induced disturbances in Laminin: A significant contributor to modulating microglial pro-inflammatory tone?

Over the last decade, evidence supporting a link between microglia enhanced neuro-inflammatory signalling and mood disturbance has continued to build. One issue that has not been well addressed yet are the factors that drive microglia to enter into a higher pro-inflammatory state. The current study addressed the potential role of the extracellular matrix protein Laminin. C57BL6 adult mice were either exposed to chronic stress or handled for 6 consecutive weeks. Changes in Laminin, microglial morphology and pro-inflammatory cytokine expression were examined in tissue obtained from mice exposed to a chronic restraint stress procedure. These in vivo investigations were complemented by an extensive set of in vitro experiments utilising both a primary microglia and BV2 cell line to examine how Laminin influenced microglial pro-inflammatory tone. Chronic stress enhanced the expression of Laminin, microglial de-ramification and pro-inflammatory cytokine signalling. We further identified that microglia when cultured in the presence of Laminin produced and released significantly greater levels of pro-inflammatory cytokines; took longer to return to baseline following stimulation and exhibited enhanced phagocytic activity. These results suggest that chronic restraint stress is capable of modulating Laminin within the CNS, an effect that has implications for understanding environmental mediated disturbances of microglial function.

Androgen alleviates neurotoxicity of β-amyloid peptide (Aβ) by promoting microglial clearance of Aβ and inhibiting microglial inflammatory response to Aβ.

Lower androgen level in elderly men is a risk factor of Alzheimer's disease (AD). It has been reported that androgen reduces amyloid peptides (Aβ) production and increases Aβ degradation by neurons. Activated microglia are involved in AD by either clearing Aβ deposits through uptake of Aβ or releasing cytotoxic substances and pro-inflammatory cytokines. Here, we investigated the effect of androgen on Aβ uptake and clearance and Aβ-induced inflammatory response in microglia, on neuronal death induced by Aβ-activated microglia, and explored underlying mechanisms. Intracellular and extracellular Aβ were examined by immunofluorescence staining and Western blot. Amyloid peptides (Aβ) receptors, Aβ degrading enzymes, and pro-inflammatory cytokines were detected by RT-PCR, real-time PCR, and ELISA. Phosphorylation of MAP kinases and NF-κB was examined by Western blot. We found that physiological concentrations of androgen enhanced Aβ42 uptake and clearance, suppressed Aβ42 -induced IL-1β and TNFα expression by murine microglia cell line N9 and primary microglia, and alleviated neuronal death induced by Aβ42 -activated microglia. Androgen administration also reduced Aβ42 -induced IL-1β expression and neuronal death in murine hippocampus. Mechanistic studies revealed that androgen promoted microglia to phagocytose and degrade Aβ42 through upregulating formyl peptide receptor 2 and endothelin-converting enzyme 1c expression, and inhibited Aβ42 -induced pro-inflammatory cytokines expression via suppressing MAPK p38 and NF-κB activation by Aβ42 , in an androgen receptor independent manner. Our study demonstrates that androgen promotes microglia to phagocytose and clear Aβ42 and inhibits Aβ42 -induced inflammatory response, which may play an important role in reducing the neurotoxicity of Aβ.

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation.

Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

Esculentoside A exerts anti-inflammatory activity in microglial cells

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. This study was designed to evaluate the pharmacological effects of EsA on lipopolysaccharide (LPS)-stimulated BV2 microglia and primary microglia cells. Our results indicated that EsA pretreatment significantly decreased LPS-induced production of Nitric Oxide (NO) and Prostaglandin E2 (PGE2) and impeded LPS-mediated upregulation of pro-inflammatory mediators' expression such as nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-a (TNF-α) in both BV2 microglia and primary microglia cells. Moreover, EsA markedly suppressed nuclear factor-κB p65 (NF-κB p65) translocation by blocking IκB-α phosphorylation and degradation in LPS-treated BV2 cells. EsA also decreased phosphorylation level of mitogen-activated protein kinases (MAPKs) and inhibited NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome mediated caspase-1 activation in LPS-stimulated BV2 cells. Additionally, EsA decreased β-amyloid1–42 (Aβ1–42)-induced production of TNF-α, IL-1β and IL-6 in primary microglia. Thus, EsA might be a promising therapeutic agent for alleviating neuroinflammatory diseases.

P2X7 Receptor Activation Modulates Autophagy in SOD1-G93A Mouse Microglia.

Autophagy and inflammation play determinant roles in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), an adult-onset neurodegenerative disease characterized by deterioration and final loss of upper and lower motor neurons (MN) priming microglia to sustain neuroinflammation and a vicious cycle of neurodegeneration. Given that extracellular ATP through P2X7 receptor constitutes a neuron-to-microglia alarm signal implicated in ALS, and that P2X7 affects autophagy in immune cells, we have investigated if autophagy can be directly triggered by P2X7 activation in primary microglia from superoxide dismutase 1 (SOD1)-G93A mice. We report that P2X7 enhances the expression of the autophagic marker microtubule-associated protein 1 light chain 3 (LC3)-II, via mTOR pathway and concomitantly with modulation of anti-inflammatory M2 microglia markers. We also demonstrate that the autophagic target SQSTM1/p62 is decreased in SOD1-G93A microglia after a short stimulation of P2X7, but increased after a sustained challenge. These effects are prevented by the P2X7 antagonist A-804598, and the autophagy/phosphoinositide-3-kinase inhibitor wortmannin (WM). Finally, a chronic in vivo treatment with A-804598 in SOD1-G93A mice decreases the expression of SQSTM1/p62 in lumbar spinal cord at end stage of disease. These data identify the modulation of the autophagic flux as a novel mechanism by which P2X7 activates ALS-microglia, to be considered for further investigations in ALS.

Spatiotemporal expression of FOXA1 correlates with reactive gliosis after spinal cord injury

Forkhead box A1 (FOXA1) is a member of the FOX family of transcription factors and involved in various mammalian processes. However, the expression and function of FOXA1 in central nervous system (CNS) are still with limited acquaintance. In present study, we performed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of FOXA1 expression in spinal cord. We found that FOXA1 protein levels were significantly increased after SCI and we observed that the expression of FOXA1 is enhanced in the white matter. Meanwhile, double immunofluorescence staining showed that increased levels of FOXA1 were striking in astrocytes and microglia. We also examined the expression of proliferating cell nuclear antigen (PCNA), whose changes were correlated with the expression profiles of FOXA1. In vitro, FOXA1 depletion by siRNA inhibited astrocyte proliferation and migration. Meanwhile, FOXA1 knockdown also reduce cell cycle related proteins. Which indicated that FOXA1 might modulate cell cycle progression and play a crucial role in cell proliferation. Furthermore, FOXA1 knockdown also inhibited LPS-induced synthesis/secretion of IL-1β and TNF-α in primary microglia. These results indicated that FOXA1 might play an important role in pathophysiology after SCI.

Balasubramide derivative 3C modulates microglia activation via CaMKKβ-dependent AMPK/PGC-1α pathway in neuroinflammatory conditions

Neuroinflammation plays a vital role in the pathological process of cerebral ischemic stroke, but currently there is no effective treatment. After ischemia, microglia-produced proinflammatory mediator expression contributes to the aggravation of neuroinflammation, while anti-inflammatory activation of microglia develops an anti-neuroinflammatory effect via secretion of anti-inflammatory factor. Promoting the anti-inflammatory activation of microglia might be an effective treatment of stroke. Previously, we discovered one derivative of the natural product (+)-balasubramide, compound 3C, that exhibits a remarkably anti-neuroinflammatory effect in vitro with unknown mechanisms. Thus in this study, we aimed to clarify its molecular mechanisms and determine whether compound 3C has a neuroprotective effect after ischemia via regulation on microglial inflammation. We found that compound 3C promoted the anti-inflammatory mediator expression and reduced the proinflammatory mediator expression in LPS-stimulated BV2 cells and mouse primary microglia cells, which were reversed by AMP-activated protein kinase (AMPK) inhibition or AMPK upstream calmodulin-dependent protein kinase kinase beta (CaMKKβ) inhibition. Compound 3C also prevented LPS-stimulated JNK activation and enhanced PGC-1α activation in microglia, which was attenuated by AMPK inhibition. Additionally, compound 3C ameliorated depressive behaviors in LPS-induced neuroinflammatory mice by promoting the anti-inflammatory activation of microglia. Furthermore, we found that compound 3C markedly reduced brain infarct volume, improved the neurological deficit in rats with ischemia and reduced the activated microglia/macrophage cells in the ischemic area, which concomitantly enhanced the anti-inflammatory mediator expression. A mechanistic study showed that the compound 3C-mediated activation of CaMKKβ, AMPK and PGC-1α is involved in the anti-neuroinflammatory and neuroprotective effects of 3C in the brain of LPS-treated mice and ischemic rats. Taken together, our results show that compound 3C could suppress neuroinflammation in vitro and in vivo by modulating microglial activation state through the CaMKKβ-dependent AMPK/PGC-1α signaling pathway, and maybe further be developed as a promising new drug candidate for the treatment of brain disorders such as stroke associated with brain inflammation.

Ischemia-responsive protein 94 is a key mediator of ischemic neuronal injury-induced microglial activation.

Neuroinflammation, especially activation of microglia, the key immune cells in the brain, has been proposed to contribute to the pathogenesis of ischemic stroke. However, the dynamics and the potential mediators of microglial activation following ischemic neuronal injury are not well understood. In this study, using oxygen/glucose deprivation and reoxygenation with neuronal and microglial cell cultures as an invitro model of ischemic neuronal injury, we set out to identify neuronal factors released from injured neurons that are capable of inducing microglial activation. Conditioned media (CM) from hippocampal and cortical neurons exposed to oxygen/glucose deprivation and reoxygenation induced significant activation of microglial cells as well as primary microglia, evidenced by up-regulation of inducible nitric oxide synthase, increased production of nitrite and reactive oxygen species, and increased expression of microglial markers. Mechanistically, neuronal ischemia-responsive protein 94 (Irp94) was a key contributor to microglial activation since significant increase in Irp94 was detected in the neuronal CM following ischemic insult and immunodepletion of Irp94 rendered ischemic neuronal CM ineffective in inducing microglial activation. Ischemic insult-augmented oxidative stress was a major facilitator of neuronal Irp94 release, and pharmacological inhibition of NADPH oxidase significantly reduced the ischemic injury-induced neuronal reactive oxygen species production and Irp94 release. Taken together, these results indicate that neuronal Irp94 may play a pivotal role in the propagation of ischemic neuronal damage. Continued studies may help identify Irp94 and/or related proteins as potential therapeutic targets and/or diagnostic/prognostic biomarkers for managing ischemia-associated brain disorders.

Amyloid β-Induced Redistribution of Transcriptional Factor EB and Lysosomal Dysfunction in Primary Microglial Cells.

Impaired clearance of Amyloid β (Aβ) by microglia in the brain may be associated with the senile plaque formation, a pathological hallmark relevant to Alzheimer's disease. Microglial cells in the brain are not able to efficiently degrade Aβ, suggesting that microglial lysosome impairment may occur. However, the mechanism of Aβ-induced impairment of microglia remains poorly understood. We observed the effects of Aβ on the trafficking of nuclear transcriptional factor EB (TFEB), a master regulator of lysosome biogenesis, and the expression of a downstream osteoporosis-associated transmembrane protein 1 (OSTM1), a vital molecule involved in lysosome acidification in primary microglial cells. Aβ1−42 but not Aβ42−1 resulted in a significant release of tumor necrosis factor-α in primary microglia, but the total cellular TFEB was not changed. Further, Aβ induced a dose-dependent reduction of the TFEB in the nucleus of primary microglial cells, coincident with the increase in the plasma, as revealed by Western blot and confocal microscopy. In addition, a dramatic decrease of OSTM1 expression was observed in the Aβ-challenged microglial cells, along with the intracellular pH steady state, indicating the inadequate lysosomal acidification. These data suggest that Aβ might result in a lysosomal dysfunction via inhibiting nuclear TFEB translocation in microglial cells.

Synaptotagmin‐11 inhibits cytokine secretion and phagocytosis in microglia

Cytokine secretion and phagocytosis are key functions of activated microglia. However, the molecular mechanisms underlying their regulation in microglia remain largely unknown. Here, we report that synaptotagmin-11 (Syt11), a non-Ca2+ -binding Syt implicated in Parkinson disease and schizophrenia, inhibits cytokine secretion and phagocytosis in microglia. We found Syt11 expression in microglia in brain slices and primary microglia. Interestingly, Syt11-knockdown (KD) increased cytokine secretion and NO release in primary microglia both in the absence and presence of lipopolysaccharide. NF-κB was activated in untreated KD microglia together with enhanced synthesis of IL-6, TNF-α, IL-1β, and iNOS. When the release capacity was assessed by the ratio of extracellular to intracellular levels, only the IL-6 and TNF-α secretion capacity was increased in Syt11-KD cells, suggesting that Syt11 specifically regulates conventional secretion. Consistently, Syt11 localized to the trans-Golgi network and recycling endosomes. In addition, Syt11 was recruited to phagosomes and its deficiency enhanced microglial phagocytosis. All the KD phenotypes were rescued by expression of an shRNA-resistant Syt11, while overexpression of Syt11 suppressed cytokine secretion and phagocytosis. Importantly, Syt11 also inhibited microglial phagocytosis of α-synuclein fibrils, supporting its association with Parkinson disease. Taken together, we propose that Syt11 suppresses microglial activation under both physiological and pathological conditions through the inhibition of cytokine secretion and phagocytosis.

Sevoflurane suppresses microglial M2 polarization.

Microglia can be polarized into the classical (M1) or alternative (M2) activation states in response to various stimuli. The M2 phenotype has its own set of receptor profiles, cytokine production, and chemokine secretion, of which arginase 1 (Arg1), chitinase 3-like 3 (Chi3l3, Ym1), and IL-10 have neuroprotective properties. Sevoflurane is one of the most commonly used volatile anesthetics in clinics. Previous studies have shown that sevoflurane promotes microglial M1 activation. However, it remains unclear whether sevoflurane regulates microglial M2 activation. In this study, we found that sevoflurane treatment abolished interleukin 4 (IL-4)-induced M2 microglial activation. Our results indicate that IL-4-mediated induction of the characteristic M2 marker genes and proteins Arg1, Ym1, and IL-10 was significantly attenuated by sevoflurane pretreatment in primary microglia. Microglial M2 polarization induced by incubation with culture supernatant from human umbilical cord mesenchymal stromal cells (HUC-MSCs) was abolished by treatment with 2% or 4% sevoflurane. Upregulation of SOCS1 and suppression of SOCS3 were shown to play a crucial role in the process of M2 microglial polarization. Our results also indicate that SOCS1 expression was induced by IL-4, but it was inhibited by pretreatment with sevoflurane. In contrast, IL-4 suppressed SOCS3 expression, which was restored by pretreatment with sevoflurane. Mechanistically, it was shown that sevoflurane suppresses STAT6 phosphorylation in primary microglia.

TAU SECRETION AND PROPAGATION

One of the neuropathological hallmarks of AD is an increase in the intracellular amount of tau. In order to revert that increase, one mechanism used by cells could be the secretion of tau to the extracellular space. It has been postulated that under physiological conditions, an increase in the intracellular amount of tau facilitates its secretion to the extracellular space. Extracellular tau can be toxic and can propagate through its interaction with neuronal or glia cells. In this presentation we will comment on: a) the regions of tau molecule that could be involved in the protein secretion when it is in monomeric form. Those tau regions could be different to those needed for the secretion of truncated or aggregated tau; b) some mechanisms for the interaction of extracellular tau with neuronal and microglia cells. a) COS-7 cell line was used as an in vitro model. Cells were transfected with different tau constructs. Purified tau was added to primary neuron or microglia cultures to study the interaction between tau and these cell types a) N-terminal region of tau molecule could be required for the secretion of soluble intracellular tau. b) Extracellular monomeric tau could react with specific cell receptors present in neurons or in microglia cells A possible mechanism for monomeric tau secretion and further interactions of tau with neuron or microglia cells is proposed.

RAGE-Specific Inhibitor FPS-ZM1 Attenuates AGEs-Induced Neuroinflammation and Oxidative Stress in Rat Primary Microglia.

Advanced glycation end products (AGEs) enhance microglial activation and intensify the inflammatory response and oxidative stress in the brain. This process may occur due to direct cytotoxicity or interacting with AGEs receptors (RAGE), which are expressed on the surface of microglia. FPS-ZM1 is a high-affinity but nontoxic RAGE-specific inhibitor that has been recently shown to attenuate the Aβ-induced inflammatory response by blocking the ligation of Aβ to RAGE. In this study, we further investigated the effect of FPS-ZM1 on the AGEs/RAGE interaction and downstream elevation of neuroinflammation and oxidative stress in primary microglia cells. The results suggested that FPS-ZM1 significantly suppressed AGEs-induced RAGE overexpression, RAGE-dependent microglial activation, nuclear translocation of nuclear factor kappaB p65 (NF-κB p65), and the expression of downstream inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS)/nitric oxide (NO). Furthermore, FPS-ZM1 attenuated AGEs-stimulated NADPH oxidase (NOX) activation and reactive oxygen species (ROS) expression. Finally, FPS-ZM1 elevated the levels of transcription factors nuclear-factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1), as well as decreased antioxidant capacity and increased production of oxidative species. Our results suggest that FPS-ZM1 may be neuroprotective through attenuating microglial activation, oxidative stress and inflammation by blocking RAGE.

Activated Microglia Desialylate and Phagocytose Cells via Neuraminidase, Galectin-3, and Mer Tyrosine Kinase.

Activated microglia can phagocytose dying, stressed, or excess neurons and synapses via the phagocytic receptor Mer tyrosine kinase (MerTK). Galectin-3 (Gal-3) can cross-link surface glycoproteins by binding galactose residues that are normally hidden below terminal sialic acid residues. Gal-3 was recently reported to opsonize cells via activating MerTK. We found that LPS-activated BV-2 microglia rapidly released Gal-3, which was blocked by calcineurin inhibitors. Gal-3 bound to MerTK on microglia and to stressed PC12 (neuron-like) cells, and it increased microglial phagocytosis of PC12 cells or primary neurons, which was blocked by inhibition of MerTK. LPS-activated microglia exhibited a sialidase activity that desialylated PC12 cells and could be inhibited by Tamiflu, a neuraminidase (sialidase) inhibitor. Sialidase treatment of PC12 cells enabled Gal-3 to bind and opsonize the live cells for phagocytosis by microglia. LPS-induced microglial phagocytosis of PC12 was prevented by small interfering RNA knockdown of Gal-3 in microglia, lactose inhibition of Gal-3 binding, inhibition of neuraminidase with Tamiflu, or inhibition of MerTK by UNC569. LPS-induced phagocytosis of primary neurons by primary microglia was also blocked by inhibition of MerTK. We conclude that activated microglia release Gal-3 and a neuraminidase that desialylates microglial and PC12 surfaces, enabling Gal-3 binding to PC12 cells and their phagocytosis via MerTK. Thus, Gal-3 acts as an opsonin of desialylated surfaces, and inflammatory loss of neurons or synapses may potentially be blocked by inhibiting neuraminidases, Gal-3, or MerTK.

Cathepsin B plays a critical role in inducing Alzheimer’s disease-like phenotypes following chronic systemic exposure to lipopolysaccharide from Porphyromonas gingivalis in mice

A number of clinical and experimental studies have revealed a strong association between periodontitis and accelerated cognitive decline in Alzheimer’s disease (AD); however, the mechanism of the association is unknown. In the present study, we tested the hypothesis that cathepsin (Cat) B plays a critical role in the initiation of neuroinflammation and neural dysfunction following chronic systemic exposure to lipopolysaccharide from Porphyromonas gingivalis (PgLPS) in mice (1mg/kg, daily, intraperitoneally). Young (2months old) and middle-aged (12months old) wild-type (WT; C57BL/6N) or CatB-deficient (CatB−/−) mice were exposed to PgLPS daily for 5 consecutive weeks. The learning and memory function were assessed using the passive avoidance test, and the expression of amyloid precursor protein (APP), CatB, TLR2 and IL-1β was analyzed in brain tissues by immunohistochemistry and Western blotting. We found that chronic systemic exposure to PgLPS for five consecutive weeks induced learning and memory deficits with the intracellular accumulation of Aβ in neurons in the middle-aged WT mice, but not in young WT or middle-aged CatB−/− mice. PgLPS significantly increased the expression of CatB in both microglia and neurons in middle-aged WT mice, while increased expression of mature IL-1β and TLR2 was restricted to microglia in the hippocampus of middle-aged WT mice, but not in that of the middle-aged CatB−/− ones. In in vitro studies, PgLPS (1µg/ml) stimulation upregulated the mean mRNA expression of IL-1β, TLR2 and downregulated the protein levels of IκBα in the cultured MG6 microglia as well as in the primary microglia from WT mice, which were significantly inhibited by the CatB-specific inhibitor CA-074Me as well as by the primary microglia from CatB−/− mice. Furthermore, the mean mRNA expression of APP and CatB were significantly increased in the primary cultured hippocampal neurons after treatment with conditioned medium from PgLPS-treated WT primary microglia, but not after treatment with conditioned medium neutralized with anti-IL-1beta, and not after treatment with conditioned medium from PgLPS-treated CatB−/− primary microglia or with PgLPS directly. Taken together, these findings indicate that chronic systemic exposure to PgLPS induces AD-like phenotypes, including microglia-mediated neuroinflammation, intracellular Aβ accumulation in neurons and impairment of the learning and memory functions in the middle-aged mice in a CatB-dependent manner. We propose that CatB may be a therapeutic target for preventing periodontitis-associated cognitive decline in AD.

Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity

BackgroundAs the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood.MethodsExperiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes.ResultsImmunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1β, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-κB in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes.ConclusionsThese results provide evidence that NF-κB signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.

Teriflunomide Impacts Primary Microglia and Astrocyte Functions In Vitro (P2.348)

Teriflunomide Impacts Primary Microglia and Astrocyte Functions In Vitro (P2.348)

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson's, Alzheimer's, and Huntington's Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.