Nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency causes age-dependent progression of female osteoporosis.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice. METHODS: Eighteen female wild type (WT) and 16 Nrf2-knockout (KO) mice were sacrificed at different ages (12 weeks = young mature adult and 90 weeks = old) to analyze their femurs. The morphological properties (trabecular and cortical) were evaluated by micro-computed tomography (µCT) and compared to gold standard histochemistry analysis. The quasi-static compression tests were performed to calculate the mechanical properties of bones. Additionally, the population of bone resorbing cells and aromatase expression by osteocytes was immunohistochemically evaluated and empty osteocyte lacunae was counted in cortical bone. RESULTS: Old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness, cortical area, and bone fraction compared to old WT mice, regardless of no significant difference in skeletally mature young adult mice between WT and KO. Specifically, while all old WT mice showed thin metaphyseal trabeculae, trabecular bone was completely absent in 60% of old KO mice. Additionally, old KO mice showed significantly more osteoclast-like cells and fewer aromatase-positive osteocytes than WT mice, whereas the occurrence of empty osteocyte lacunae did not differ between both groups. Nrf2-KO mice further showed an age-dependently reduced fracture resilience compared to age-matched WT mice. CONCLUSION: Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.

Transient Focal Cerebral Ischemia Leads to miRNA Alterations in Different Brain Regions, Blood Serum, Liver, and Spleen.

Ischemic stroke is characterized by an occlusion of a cerebral blood vessel resulting in neuronal cell death due to nutritional and oxygen deficiency. Additionally, post-ischemic cell death is augmented after reperfusion. These events are paralleled by dysregulated miRNA expression profiles in the peri-infarct area. Understanding the underlying molecular mechanism in the peri-infarct region is crucial for developing promising therapeutics. Utilizing a tMCAo (transient Middle Cerebral Artery occlusion) model in rats, we studied the expression levels of the miRNAs (miR) 223-3p, 155-5p, 3473, and 448-5p in the cortex, amygdala, thalamus, and hippocampus of both the ipsi- and contralateral hemispheres. Additionally, the levels in the blood serum, spleen, and liver and the expression of their target genes, namely, Nlrp3, Socs1, Socs3, and Vegfa, were assessed. We observed an increase in all miRNAs on the ipsilateral side of the cerebral cortex in a time-dependent manner and increased miRNAs levels (miR-223-3p, miR-3473, and miR-448-5p) in the contralateral hemisphere after 72 h. Besides the cerebral cortex, the amygdala presented increased expression levels, whereas the thalamus and hippocampus showed no alterations. Different levels of the investigated miRNAs were detected in blood serum, liver, and spleen. The gene targets were altered not only in the peri-infarct area of the cortex but selectively increased in the investigated non-affected brain regions along with the spleen and liver during the reperfusion time up to 72 h. Our results suggest a supra-regional influence of miRNAs following ischemic stroke, which should be studied to further identify whether miRNAs are transported or locally upregulated.

Nrf2 deficiency increases oligodendrocyte loss, demyelination, neuroinflammation and axonal damage in an MS animal model.

Oxidative stress is a pathophysiological hallmark of many CNS diseases, among multiple sclerosis (MS). Accordingly, boosting the astrocytic transcription factor nuclear factor E2-related factor 2 (Nrf2) system in an MS mouse model efficiently ameliorates oligodendrocyte loss, neuroinflammation and axonal damage. Moreover, Dimethylfumarate, an efficient activator of Nrf2, has recently been approved as therapeutic option in MS treatment. Here, we use the cuprizone mouse model of MS to induce oxidative stress, selective oligodendrocyte loss, microglia and astrocyte activation as well as axonal damage in both wild type and Nrf2-deficient mice. We found increased oligodendrocyte apoptosis and loss, pronounced neuroinflammation and higher levels of axonal damage in cuprizone-fed Nrf2-deficient animals when compared to wild type controls. In addition, Nrf2-deficient animals showed a higher susceptibility towards cuprizone within the commissura anterior white matter tract, a structure that is relatively insensitive to cuprizone in wild type animals. Our data highlight the cuprizone model as a suitable tool to study the complex interplay of oxidative stress, neuroinflammation and axonal damage. Further studies will have to show whether distinct expression patterns of Nrf2 are involved in the variable susceptibility towards cuprizone in the mouse.

Gonadal Hormones E2 and P Mitigate Cerebral Ischemia-Induced Upregulation of the AIM2 and NLRC4 Inflammasomes in Rats.

Acute ischemic stroke (AIS) is a devastating neurological condition with a lack of neuroprotective therapeutic options, despite the reperfusion modalities thrombolysis and thrombectomy. Post-ischemic brain damage is aggravated by an excessive inflammatory cascade involving the activation and regulation of the pro-inflammatory cytokines IL-1ß and IL-18 by inflammasomes. However, the role of AIM2 and NLRC4 inflammasomes and the influence of the neuroprotective steroids 17ß-estradiol (E2) and progesterone (P) on their regulation after ischemic stroke have not yet been conclusively elucidated. To address the latter, we subjected a total of 65 rats to 1 h of transient Middle Cerebral Artery occlusion (tMCAO) followed by a reperfusion period of 72 h. Moreover, we evaluated the expression and regulation of AIM2 and NLRC4 in glial single-cell cultures (astroglia and microglia) after oxygen-glucose deprivation (OGD). The administration of E2 and P decreased both infarct sizes and neurological impairments after cerebral ischemia in rats. We detected a time-dependent elevation of gene and protein levels (Western Blot/immunohistochemistry) of the AIM2 and NLRC4 inflammasomes in the post-ischemic brains. E2 or P selectively mitigated the stroke-induced increase of AIM2 and NLRC4. While both inflammasomes seemed to be exclusively abundant in neurons under physiological and ischemic conditions in vivo, single-cell cultures of cortical astrocytes and microglia equally expressed both inflammasomes. In line with the in vivo data, E and P selectively reduced AIM2 and NLRC4 in primary cortical astrocytes and microglial cells after OGD. In conclusion, the post-ischemic elevation of AIM2 and NLRC4 and their down-regulation by E2 and P may shed more light on the anti-inflammatory effects of both gonadal hormones after stroke.

Impact of Uniaxial Stretching on Both Gliding and Traction Areas of Tendon Explants in a Novel Bioreactor.

The effects of mechanical stress on cells and their extracellular matrix, especially in gliding sections of tendon, are still poorly understood. This study sought to compare the effects of uniaxial stretching on both gliding and traction areas in the same tendon. Flexor digitorum longus muscle tendons explanted from rats were subjected to stretching in a bioreactor for 6, 24, or 48 h, respectively, at 1 Hz and an amplitude of 2.5%. After stimulation, marker expression was quantified by histological and immunohistochemical staining in both gliding and traction areas. We observed a heightened intensity of scleraxis after 6 and 24 h of stimulation in both tendon types, though it had declined again 48 h after stimulation. We observed induced matrix metalloproteinase-1 and -13 protein expression in both tendon types. The bioreactor produced an increase in the mechanical structural strength of the tendon during the first half of the loading time and a decrease during the latter half. Uniaxial stretching of flexor tendon in our set-up can serve as an overloading model. A combination of mechanical and histological data allows us to improve the conditions for cultivating tendon tissues.

Effects of Strontium-Doped ß-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

It was hypothesized that strontium (Sr)-doped ß-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

EPO and TMBIM3/GRINA Promote the Activation of the Adaptive Arm and Counteract the Terminal Arm of the Unfolded Protein Response after Murine Transient Cerebral Ischemia.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to impairment of endoplasmic reticulum (ER) function. The unfolded protein response (UPR) is an ER-located and cytoprotective pathway that aims to resolve ER stress. Transmembrane BAX inhibitor-1 motif-containing (TMBIM) protein family member TMBIM3/GRINA is highly expressed in the brain and mostly located at the ER membrane suppressing ER calcium release by inositol-1,4,5-trisphosphate receptors. GRINA confers neuroprotection and is regulated by erythropoietin (EPO) after murine cerebral ischemia. However, the role of GRINA and the impact of EPO treatment on the post-ischemic UPR have not been elucidated yet. We subjected GRINA-deficient (Grina-/-) and wildtype mice to transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 6 h or 72 h of reperfusion. We administered EPO or saline 0, 24 and 48 h after tMCAo/sham surgery. Oxygen-glucose deprivation (OGD) and pharmacological stimulation of the UPR using Tunicamycin and Thapsigargin were carried out in primary murine cortical mixed cell cultures. Treatment with the PERK-inhibitor GSK-2606414, IRE1a-RNase-inhibitor STF-083010 and EPO was performed 1 h prior to either 1 h, 2 h or 3 h of OGD. We found earlier and larger infarct demarcations in Grina-/- mice compared to wildtype mice, which was accompanied by a worse neurological outcome and an abolishment of EPO-mediated neuroprotection after ischemic stroke. In addition, GRINA-deficiency increased apoptosis and the activation of the corresponding PERK arm of the UPR after stroke. EPO enhanced the post-ischemic activation of pro-survival IRE1a and counteracted the pro-apoptotic PERK branch of the UPR. Both EPO and the PERK-inhibitor GSK-2606414 reduced cell death and regulated Grina mRNA levels after OGD. In conclusion, GRINA plays a crucial role in post-ischemic UPR and the use of both GSK-2606414 and EPO might lead to neuroprotection.

α1-antitrypsin mitigates NLRP3-inflammasome activation in amyloid ß1-42-stimulated murine astrocytes.

BACKGROUND: Neuroinflammation has an essential impact on the pathogenesis and progression of Alzheimer's disease (AD). Mostly mediated by microglia and astrocytes, inflammatory processes lead to degeneration of neuronal cells. The NLRP3-inflammasome (NOD-like receptor family, pyrin domain containing 3) is a key component of the innate immune system and its activation results in secretion of the proinflammatory effectors interleukin-1ß (IL-1ß) and interleukin-18 (IL-18). Under physiological conditions, cytosolic NLRP3-inflammsome is maintained in an inactive form, not able to oligomerize. Amyloid ß1-42 (Aß1-42) triggers activation of NLRP3-inflammasome in microglia and astrocytes, inducing oligomerization and thus recruitment of proinflammatory proteases. NLRP3-inflammasome was found highly expressed in human brains diagnosed with AD. Moreover, NLRP3-deficient mice carrying mutations associated with familial AD were partially protected from deficits associated with AD. The endogenous protease inhibitor α1-antitrypsin (A1AT) is known for its anti-inflammatory and anti-apoptotic properties and thus could serve as therapeutic agent for NLRP3-inhibition. A1AT protects neurons from glutamate-induced toxicity and reduces Aß1-42-induced inflammation in microglial cells. In this study, we investigated the effect of Aß1-42-induced NLRP3-inflammasome upregulation in primary murine astrocytes and its regulation by A1AT. METHODS: Primary cortical astrocytes from BALB/c mice were stimulated with Aß1-42 and treated with A1AT. Regulation of NLRP3-inflammasome was examined by immunocytochemistry, PCR, western blot and ELISA. Our studies included an inhibitor of NLRP3 to elucidate direct interactions between A1AT and NLRP3-inflammasome components. RESULTS: Our study revealed that A1AT reduces Aß1-42-dependent upregulation of NLRP3 at the mRNA and protein levels. Furthermore, A1AT time-dependently mitigated the expression of caspase 1 and its cleavage product IL-1ß in Aß1-42-stimulated astrocytes. CONCLUSION: We conclude that Aß1-42-stimulation results in an upregulation of NLRP3, caspase 1, and its cleavage products in astrocytes. A1AT time-dependently hampers neuroinflammation by downregulation of Aß1-42-mediated NLRP3-inflammasome expression and thus may serve as a pharmaceutical opportunity for the treatment of Alzheimer's disease.

Poststroke Inflammasome Expression and Regulation in the Peri-Infarct Area by Gonadal Steroids after Transient Focal Ischemia in the Rat Brain.

CNS ischemia results in locally confined and rapid tissue damage accompanied by a loss of neurons and their circuits. Early and time-delayed inflammatory responses are critical variables determining the extent of neural disintegration and regeneration. Inflammasomes are vital effectors in innate immunity. Their activation in brain-intrinsic immune cells contributes to ischemia-related brain damage. The steroids 17ß-estradiol (E2) and progesterone (P) are neuroprotective and anti-inflammatory. Using a transient focal rat ischemic model, we evaluated the time response of different inflammasomes in the peri-infarct zone from the early to late phases after poststroke ischemia. We show that the different inflammasome complexes reveal a specific time-oriented sequential expression pattern with a maximum at approximately 24 h after the infarct. Within the limits of antibody availability, immunofluorescence labeling demonstrated that microglia and neurons are major sources of the locally activated inflammasomes NOD-like receptor protein-3 (NLRP3) and associated speck-like protein (ASC), respectively. E2 and P given for 24 h immediately after ischemia onset reduced hypoxia-induced mRNA expression of the inflammasomes NLRC4, AIM2 and ASC, and decreased the protein levels of ASC and NLRP3. In addition, mRNA protein levels of the cytokines interleukin-1ß (IL1ß), IL18 and TNFα were reduced by the steroids. The findings provide for the first time a detailed flow chart of hypoxia-driven inflammasome regulation in the peri-infarct cerebral cortex. Further, we demonstrate that E2 and P alleviate the expression of certain inflammasome components, sometimes in a hormone-specific way. Besides directly regulating other cellular neuroprotective pathways, the control of inflammasomes by these steroids might contribute to its neuroprotective potency.

Hypoxia-induced gene expression of aquaporin-4, cyclooxygenase-2 and hypoxia-inducible factor 1α in rat cortical astroglia is inhibited by 17ß-estradiol and progesterone.

17ß-Estradiol (E2) and progesterone (P) are neuroprotective in acute brain injury by attenuating neuropathophysiological processes and regulating local glial function. Besides controlling brain-intrinsic immune responses, astrocytes are cellular targets for sex steroids in health and disease and typically resist to hypoxic damage. In this in vitro study, we aimed at uncovering astroglia-specific reactions to sublethal hypoxic conditions and astroglia-specific effects of both sex steroid hormones on these parameters. Short-term hypoxia for 3 h increased reactive oxygen species production, but had no influence on cell viability of cerebral cortical rat astroglia. Astrocytes expressed classical estrogen receptors (ER), progesterone receptor (PR), and a set of nonclassical steroid hormone receptors. Hypoxia specifically induced ERα and PR isoform A gene expression. Oxygen deprivation increased gene expression of aquaporin-4 (AQP4), hypoxia-inducible factor 1α (Hif1α), and cyclooxygenase-2 (COX2). The application of E2 and P selectively prevented this induction. Effects on protein levels of these genes appeared to be delayed. These data show that astrocytes change their receptivity for sex steroid hormones by switching steroid hormone receptor expression and that E2 and P modify or antagonize proinflammatory COX2 synthesis, edema-promoting AQP4 expression, and the Hif1α increase. In vivo studies have to address whether these cell responses contribute to steroid-mediated neuroprotection in stroke.

Platelets display potent antimicrobial activity and release human beta-defensin 2.

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/10(9) platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.

Erythropoietin Enhances Post-ischemic Migration and Phagocytosis and Alleviates the Activation of Inflammasomes in Human Microglial Cells.

Recombinant human erythropoietin (rhEPO) has been shown to exert anti-apoptotic and anti-inflammatory effects after cerebral ischemia. Inflammatory cytokines interleukin-1ß and -18 (IL-1ß and IL-18) are crucial mediators of apoptosis and are maturated by multiprotein complexes termed inflammasomes. Microglia are the first responders to post-ischemic brain damage and are a main source of inflammasomes. However, the impact of rhEPO on microglial activation and the subsequent induction of inflammasomes after ischemia remains elusive. To address this, we subjected human microglial clone 3 (HMC-3) cells to various durations of oxygen-glucose-deprivation/reperfusion (OGD/R) to assess the impact of rhEPO on cell viability, metabolic activity, oxidative stress, phagocytosis, migration, as well as on the regulation and activation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes. Administration of rhEPO mitigated OGD/R-induced oxidative stress and cell death. Additionally, it enhanced metabolic activity, migration and phagocytosis of HMC-3. Moreover, rhEPO attenuated post-ischemic activation and regulation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes as well as their downstream effectors CASPASE1 and IL-1ß. Pharmacological inhibition of NLRP3 via MCC950 had no effect on the activation of CASPASE1 and maturation of IL-1ß after OGD/R, but increased protein levels of NLRP1, NLRC4, and AIM2, suggesting compensatory activities among inflammasomes. We provide evidence that EPO-conveyed anti-inflammatory actions might be mediated via the regulation of the inflammasomes.

Erythropoietin Abrogates Post-Ischemic Activation of the NLRP3, NLRC4, and AIM2 Inflammasomes in Microglia/Macrophages in a TAK1-Dependent Manner.

Inflammasomes are known to contribute to brain damage after acute ischemic stroke (AIS). TAK1 is predominantly expressed in microglial cells and can regulate the NLRP3 inflammasome, but its impact on other inflammasomes including NLRC4 and AIM2 after AIS remains elusive. EPO has been shown to reduce NLRP3 protein levels in different disease models. Whether EPO-mediated neuroprotection after AIS is conveyed via an EPO/TAK1/inflammasome axis in microglia remains to be clarified. Subjecting mice deficient for TAK1 in microglia/macrophages (Mi/MΦ) to AIS revealed a significant reduction in infarct sizes and neurological impairments compared to the corresponding controls. Post-ischemic increased activation of TAK1, NLRP3, NLRC4, and AIM2 inflammasomes including their associated downstream cascades were markedly reduced upon deletion of Mi/MΦ TAK1. EPO administration improved clinical outcomes and dampened stroke-induced activation of TAK1 and inflammasome cascades, which was not evident after the deletion of Mi/MΦ TAK1. Pharmacological inhibition of NLRP3 in microglial BV-2 cells did not influence post-OGD IL-1ß levels, but increased NLRC4 and AIM2 protein levels, suggesting compensatory activities among inflammasomes. Overall, we provide evidence that Mi/MΦ TAK1 regulates the expression and activation of the NLRP3, NLRC4, AIM2 inflammasomes. Furthermore, EPO mitigated stroke-induced activation of TAK1 and inflammasomes, indicating that EPO conveyed neuroprotection might be mediated via an EPO/TAK1/inflammasome axis.

The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis.

BACKGROUND: Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. METHODS: Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP) and Neisseria meningitides (NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined. RESULTS: We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1ß expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction. CONCLUSIONS: We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.

Alteration of miRNA Biogenesis Regulating Proteins in the Human Microglial Cell Line HMC-3 After Ischemic Stress.

MicroRNAs (miRNA) are small noncoding sequences that control apoptosis, proliferation, and neuroinflammatory pathways in microglia cells. The expression of distinct miRNAs is altered after ischemia in the brain. Only minor information is available about the biogenesis and maturation of miRNAs after ischemia. We aimed at examining the impact of oxygen-glucose deprivation (OGD) and hydrogen peroxide (H2O2)-induced stress on the expression of miRNA regulating proteins such as DROSHA, DGCR8, XPO5, DICER, TARBP2, and AGO2 in the cultured human microglial cell line HMC-3 (human microglial cell line clone 3). OGD duration of 2.5 h or H2O2 stimulation at a concentration of 100 µM for 24 h resulted in a marked increase of the hypoxia sensor hypoxia-inducible factor1-α in HMC-3 cells. These treatments also led to an upregulation of DROSHA, DICER1, and AGO2 detected by semiquantitative real-time PCR (qrtPCR). XPO5 and TARBP2 were only upregulated after stimulation with H2O2, while DGCR8 responded only to OGD. We found elevated DICER1, DROSHA, and AGO2 protein levels by western blot and immunohistochemistry staining. Interestingly, the latter also exposed a colocalization of AGO2 with stress granules (G3BP1) after OGD. Our data indicate that DICER, DROSHA, and AGO2 are induced in microglial cells under hypoxia-like conditions. It might be speculated that their inductions might increase the miRNA synthesis rate. Future studies should investigate this correlation to determine which miRNAs are preferably expressed by microglia cells after ischemia and which functions they could exert.

Aggregated Tau-PHF6 (VQIVYK) Potentiates NLRP3 Inflammasome Expression and Autophagy in Human Microglial Cells.

Intra-neuronal misfolding of monomeric tau protein to toxic ß-sheet rich neurofibrillary tangles is a hallmark of Alzheimer's disease (AD). Tau pathology correlates not only with progressive dementia but also with microglia-mediated inflammation in AD. Amyloid-beta (Aß), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1ß (IL1ß) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1ß and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer's PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.

Inflammatory Responses of Astrocytes Are Independent from Lipocalin 2.

The central nervous system (CNS) responds to diverse neurologic injuries with a vigorous activation of astrocytes. In addition to their role in the maintenance of CNS homeostasis and neuronal function, astrocytes are thought to participate in the regulation of innate and adaptive immune responses in the CNS. Following antigen recognition, reactive astrocytes may participate in the initiation of innate immune responses, and modulate adaptive immune response leading to the recruitment of peripheral immune cells. Among activation, astrocytes undergo morphological changes and express several molecules, e.g., chemokines. Lipocalin 2 (LCN2) is involved in the control of innate immune responses, regulation of excess iron, and reactive oxygen production. Here, we investigated the influence of LCN2 on basic astrocytic functions linked to inflammatory responses. In vitro studies revealed a similar chemokine expression pattern in wild-type and Lcn2-deficient astrocyte cultures after treatment with lipopolysaccharides (LPS). Increased wound closure and morphological changes upon LPS treatment are independent of Lcn2 expression. We conclude that LCN2 is not necessary for basic astrocytic functions in the context of inflammation. However, CNS-derived LCN2 might have a regulatory effect on other cells, e.g., endothelial cells of the blood-brain barrier.

NLRP3 Depletion Fails to Mitigate Inflammation but Restores Diminished Phagocytosis in BV-2 Cells After In Vitro Hypoxia.

Post-hypoxic/ischemic neuroinflammation is selectively driven by sterile inflammation, which implies the interplay of brain-intrinsic immune cells with other neural cells and immigrated peripheral immune cells. The resultant inflammatory cascade evolves extra- and intracellular pathogen and danger-associated receptors. The latter interacts with multiprotein complexes termed inflammasomes. The NLRP3 inflammasome is one of the best-described inflammasomes. However, its impact on post-ischemic neuroinflammation and its role in neuroprotection after ischemic stroke are still under debate. Microglial cells are known to be the main source of neuroinflammation; hence, we depleted NLRP3 in BV-2 microglial cells using shRNA to investigate its role in IL-1ß maturation and phagocytosis after hypoxia (oxygen-glucose-deprivation (OGD)). We also examined the expression profiles of other inflammasomes (NLRC4, AIM2, ASC) and caspase-1 activity after OGD. OGD triggered caspase-1 activity and increased IL-1ß secretion in BV-2 cells with no alteration after NLRP3 depletion. The expression of the AIM2 inflammasome was significantly higher after OGD in NLRP3-depleted cells, whereas NLRC4 was unaltered in all groups. Interestingly, OGD induced a complete inactivation of phagocytic activity in wild-type cells, while in NLRP3-depleted BV-2, this inactivity was restored after hypoxia. Our findings indicate a minor role of NLRP3 in the inflammatory response after hypoxic/ischemic stimulus. However, NLRP3 seems to play a pivotal role in the regulation of post-ischemic phagocytosis. This might be a prerequisite for the putative neuroprotective effect.

Microglial-specific depletion of TAK1 is neuroprotective in the acute phase after ischemic stroke.

Transforming growth factor-ß-activated kinase 1 (TAK1) is upregulated after cerebral ischemia and contributes to an aggravation of brain injury. TAK1 acts as a key regulator of NF-ΚB and the MAP kinases JNK and p38 and modulates post-ischemic neuroinflammation and apoptosis. Microglia are the main TAK1-expressing immunocompetent cells of the brain. However, little is known about the function and regulation of microglial TAK1 after cerebral ischemia. Tamoxifen-dependent conditional depletion of TAK1 in microglial cells was induced in Cx3cr1creER-Tak1fl/fl mice. The creER-negative Tak1fl/fl mice and vehicle-treated (corn oil) mice served as control groups. A transient intraluminal middle cerebral artery occlusion of 30 min followed by 6 h and 72 h of reperfusion was performed in male mice. Oxygen-glucose-deprivation (OGD) was performed with primary cortical glial cell cultures to examine the effect of microglial-specific and general (5Z-7-Oxozeaenol) TAK1 inhibition after different reperfusion times (1 h, 6 h, and 72 h). Cx3cr1creER-Tak1fl/fl mice showed reduced infarct sizes and improved neurological outcomes compared to the control group. The mRNA and protein levels of pro-inflammatory Il1b/IL-1ß and Tnf/TNF-α in the peri-infarct zones of microglial-specific TAK1-depleted mice were significantly reduced. Furthermore, TAK1 depletion in vitro led to reduced cell death rates after OGD. Moreover, hypoxia-mediated activation of TAK1 and its downstream signalling proteins, JNK and p38, were dampened by microglial TAK1 depletion. In contrast, 5Z-7-Oxozeaenol-induced pharmacological inhibition of TAK1 completely diminished MAPK-signalling including the kinases JNK and p38 in all cells. Microglial TAK1 depletion abrogates post-ischemic neuroinflammation and apoptosis in the acute phase, hence might be considered as a potential target in the treatment of cerebral hypoxia. KEY MESSAGES: TAK1 is activated after cerebral ischemia and induces MAP kinases p38 and JNK. Activated TAK1 increases apoptosis rate and the level pro-inflammatory cytokines IL-1ß and TNF-α. Microglial cells seem to be the main source of TAK1-mediated post-ischemic neuroinflammation. Microglial-specific TAK1-depletion mediates sustainable neuroprotective effects, which might be superior to global TAK1 inhibition.

EPO regulates neuroprotective Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury.

BACKGROUND AND PURPOSE: Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members exert inhibitory activities in apoptosis and necroptosis. FAIM2 (TMBIM-2) is neuroprotective against murine focal ischemia and is regulated by erythropoietin (EPO). Similar to FAIM2, GRINA (TMBIM-3) is predominantly expressed in the brain. The role of GRINA in transient brain ischemia, its potential synergistic effects with FAIM2 and its regulation by EPO treatment were assessed. METHODS: We performed transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 72 h of reperfusion in GRINA-deficient (GRINA-/-), FAIM2-deficient (FAIM2-/-), double-deficient (GRINA-/-FAIM2-/-) and wildtype littermates (WT) mice. We administered EPO or saline 0, 24 and 48 h after tMCAo. We subjected primary murine cortical neurons (pMCN) of all mouse strains to oxygen-glucose deprivation (OGD) after GRINA and/or FAIM2 gene transfection. RESULTS: Compared to wildtype controls GRINA deficiency led to a similar increase in infarct volumes as FAIM2 deficiency (p < .01). We observed the highest neurological deficits and largest infarct sizes in double-deficient mice. EPO administration upregulated GRINA and FAIM2 mRNA levels in wildtype littermates. EPO decreased infarct sizes and abrogated neurological impairments in wildtype controls. GRINA and/or FAIM2 deficient mice showed increased expression levels of cleaved-caspase 3 and of pro-apoptotic BAX mRNA. Further, caspase 8 was upregulated in FAIM2-/- and caspase 9 in GRINA-/- mice. Overexpression of GRINA and FAIM2 in wildtype and in double deficient pMCN decreased cell death rate after OGD. CONCLUSIONS: GRINA and FAIM2 are highly expressed in the brain and convey EPO-mediated neuroprotection after ischemic stroke involving different caspases.