KCa 3.1-a microglial target ready for drug repurposing?

Over the past decade, glial cells have attracted attention for harboring unexploited targets for drug discovery. Several glial targets have attracted de novo drug discovery programs, as highlighted in this GLIA Special Issue. Drug repurposing, which has the objective of utilizing existing drugs as well as abandoned, failed, or not yet pursued clinical development candidates for new indications, might provide a faster opportunity to bring drugs for glial targets to patients with unmet needs. Here, we review the potential of the intermediate-conductance calcium-activated potassium channels KCa 3.1 as the target for such a repurposing effort. We discuss the data on KCa 3.1 expression on microglia in vitro and in vivo and review the relevant literature on the two KCa 3.1 inhibitors TRAM-34 and Senicapoc. Finally, we provide an outlook of what it might take to harness the potential of KCa 3.1 as a bona fide microglial drug target. GLIA 2016;64:1733-1741.

Critical data-based re-evaluation of minocycline as a putative specific microglia inhibitor.

Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of microglia. It is, however, unclear how this "inhibition" is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788-1794.

TRPM7 regulates proliferation and polarisation of macrophages.

Ion channels play pivotal roles in regulating important functions of macrophages, such as cytokine and chemokine production, migration, proliferation, phagocytosis and others. In this study, we have identified the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) for the first time in macrophages. TRPM7 activity is differentially regulated in macrophages, i.e. current density in TRPM7 is significantly larger in anti-inflammatory M2-type macrophages than in untreated and in pro-inflammatory M1-type macrophages, whereas mRNA levels of TRPM7 remain unchanged upon cell polarisation. The specific TRPM7 inhibitors NS8593 and FTY720 abolish proliferation of macrophages induced by interleukin-4 (IL-4) and macrophage colony-stimulating factor (M-CSF), respectively, whereas proliferation arrest was not accompanied by induction of apoptosis or necrosis in macrophages. Furthermore, NS8593 and FTY720 prevented polarisation of macrophages towards the anti-inflammatory M2 phenotype. Inhibition of TRPM7 reduced IL-4-induced upregulation of arginase-1 (Arg1) mRNA levels and Arg1 activity, and abolished the inhibitory effects of IL-4 or M-CSF on LPS-induced TNF-α production by macrophages. In summary, our data suggest a main role of TRPM7 in the regulation of macrophage proliferation and polarisation.

Does intrawound application of vancomycin influence bone healing in spinal surgery?

PURPOSE: Surgical site infections represent a major complication of spinal surgery. The application of lyophilised vancomycin into the wound is reported to significantly decrease infection rates. As concentrations applied locally can exceed the minimal bacterial inhibitory concentration for more than a 1000-fold, toxic side effects on local tissue may be possible. METHODS: Primary osteoblast cell cultures were generated from bone tissue samples of 10 patients. Samples were incubated in absence or presence of either 3, 6 or 12 mg/cm(2) vancomycin according to a planned phase I clinical trial protocol. Changes in pH, osteoblast migration, proliferation and viability were analysed. Alkaline phosphatase as well as mineralisation patterns was studied. RESULTS: The application of more than 3 mg/cm(2) vancomycin induced a decline of pH. The migration potential of osteoblasts was decreased from 100% (control samples) to zero (12 mg/cm(2) vancomycin) in a dose-dependant manner. Cell proliferation was significantly inhibited at dosages above 3 mg/cm(2). Significant cell death was observed if the dosage applied exceeded 6 mg/cm(2). The synthesis of alkaline phosphatase was markedly reduced in all dosages applied and calcium deposition was significantly decreased in dosages above 3 mg/cm(2). CONCLUSION: As bone remodelling requires the immigration, proliferation and differentiation of osteoblasts at the fusion site, high dosages of intrawound vancomycin might interfere with regenerative processes and increase the risk of non-union. To allow an appropriate balance of infection risk and the risk of non-union, the minimal local concentration required should be determined by controlled in vivo studies.

Microglial K(+) channel expression in young adult and aged mice.

The K(+) channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K(+) channels and their regulation in vivo is limited. Here, we have investigated K(+) currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K(+) currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K(+) currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K(+) current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K(+) currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK-type, but not IK- or SK-type, Ca(2+) -activated K(+) channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K(+) channel pattern nor K(+) channel expression levels differed markedly between the three brain regions investigated. It is concluded that age-related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage-activated, but not Ca(2+) -activated, K(+) channels.

Resistance profiles of Neisseria gonorrhoeae isolates in Vienna, Austria: a phenotypic and genetic characterization from 2013 to 2020.

OBJECTIVES: International surveillance data show a constant rise in the number of Neisseria gonorrhoeae infections and an increase in drug resistance of N. gonorrhoeae. As recent N. gonorrhoeae surveillance data in Austria are scarce, this study investigated phenotypic and genotypic antimicrobial resistance in N. gonorrhoeae isolates. METHODS: In total, 440 N. gonorrhoeae samples were collected at the Medical University of Vienna, and the minimal inhibitory concentrations (MICs) for a range of different antibiotics were determined. Sampling sites and treatments were recorded, and whole-genome sequencing of N. gonorrhoeae isolates was performed using allele libraries to determine genotypic resistance. RESULTS: The median MICs for ceftriaxone, cefixime, azithromycin, ciprofloxacin, tetracycline and penicillin were <0.002 µg/mL, <0.016 µg/mL, 0.25 µg/mL, 2.0 µg/mL, 1.5 µg/mL and 0.25 µg/mL, respectively. Annual comparison showed that MICs were generally stable for all antimicrobial agents except azithromycin, for which an increase in median MIC was observed from 2017 (0.25 µg/mL). There was no genetic resistance to ceftriaxone; 8% of samples displayed resistance mutations against cefixime, primarily located in the penA gene. Resistance to azithromycin increased from 2% in 2013 to 12% in 2020. MtrD mosaic had the highest impact on azithromycin susceptibility; 47% of the resistant isolates showed this mutation. The majority of cases of gonorrhoea were treated successfully with either ceftriaxone or a ceftriaxone/azithromycin regime. Two treatment failures occurred under monotherapy with doxycycline. Overall, genotypic resistance corresponded significantly to all respective MICs. CONCLUSIONS: The resistance rate of N. gonorrhoeae to antibiotics has remained stable in Vienna over the last decade, except for azithromycin. The strong correlation found between genetic and phenotypic patterns in this study holds promise for future diagnostics of N. gonorrhoeae resistance based on genotypes.

Microdosing as a Potential Tool to Enhance Clinical Development of Novel Antibiotics: A Tissue and Plasma PK Feasibility Study with Ciprofloxacin.

BACKGROUND AND OBJECTIVE: In microdose studies, drug pharmacokinetics is measured in humans after administration of subtherapeutic doses. While previous microdose studies focused primarily on plasma pharmacokinetics, we set out to evaluate the feasibility of microdosing for a pharmacokinetic assessment in subcutaneous tissue and epithelial lining fluid. METHODS: Healthy subjects received a single intravenous bolus injection of a microdose of [14C]ciprofloxacin (1.1 µg, 7 kBq) with (cohort A, n = 9) or without (cohort B, n = 9) a prior intravenous infusion of a therapeutic dose of unlabeled ciprofloxacin (400 mg). Microdialysis and bronchoalveolar lavage were applied for determination of subcutaneous and intrapulmonary drug concentrations. Microdose [14C]ciprofloxacin was quantified by accelerator mass spectrometry and therapeutic-dose ciprofloxacin by liquid chromatography-tandem mass spectrometry. RESULTS: The pharmacokinetics of therapeutic-dose ciprofloxacin (cohort A) in plasma, subcutaneous tissue, and epithelial lining fluid was in accordance with previous data. In plasma and subcutaneous tissue, the dose-adjusted area under the concentration-time curve of microdose ciprofloxacin was similar in cohorts A and B and within an 0.8-fold to 1.1-fold range of the area under the concentration-time curve of therapeutic-dose ciprofloxacin. Penetration of microdose ciprofloxacin into subcutaneous tissue was similar in cohorts A and B and comparable to that of therapeutic-dose ciprofloxacin with subcutaneous tissue-to-plasma area under the concentration-time curve ratios of 0.44, 0.44, and 0.38, respectively. Penetration of microdose ciprofloxacin into epithelial lining fluid was highly variable and failed to predict the epithelial lining fluid penetration of therapeutic-dose ciprofloxacin. CONCLUSIONS: Our study confirms the feasibility of microdosing for pharmacokinetic measurements in plasma and subcutaneous tissue. Microdosing combined with microdialysis is a potentially useful tool in clinical antimicrobial drug development, but its applicability for the assessment of pulmonary pharmacokinetics with bronchoalveolar lavage requires further studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03177720 (registered 6 June, 2017).

Amyloid-ß-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia.

Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimer's disease. Although it is recognized that amyloid-ß (Aß) plays a major role in inducing and regulating microglial ROS production in Alzheimer's disease, to date little is known about cellular mechanisms underlying Aß-stimulated ROS production. Here, we identified ion channels involved in Aß-induced microglial ROS production and in Aß-induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aß(1-42) (fAß(1-42) ) or soluble Aß(1-42) (sAß(1-42) ) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5-iodo-resiniferatoxin (I-RTX), but were unaffected by inhibition of K(+) channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAß(1-42) or sAß(1-42) induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAß(1-42) or sAß(1-42) remained unaffected by TRPV1 channel inhibition with I-RTX. However, sAß(1-42) -induced priming was inhibited by CTX and margatoxin, but not by TRAM-34 or paxilline, indicating a role of Kv1.3 voltage-gated K(+) channels, but not of Ca(2+) -activated K(+) channels, in the priming process. In summary, our data suggest that in microglia Aß-induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K(+) channels may provide potential therapeutic targets to reduce microglia-induced oxidative stress in Alzheimer's disease.

Autografts for spinal fusion: osteogenic potential of laminectomy bone chips and bone shavings collected via high speed drill.

In case of revision or minimal invasive spinal surgery, the amount of autograft possibly harvested from the lamina and the spinous processes is limited. Ekanayake and Shad (Acta Neurochir 152:651-653, 2010) suggest the application of bone shavings harvested via high speed burr additionally or instead, but so far no data regarding their osteogenic potential exist. Aim of the study was to compare the osteogenic potential of bone chips and high speed burr shavings, and to evaluate the applicability of bone shavings as an autograft for spinal fusion. Bone chips and shavings from 14 patients undergoing spinal decompression surgery were analyzed using in vitro tissue culture methods. Osteoblast emigration and proliferation, viability and mineralization were investigated and histological evaluation was performed. Bone chips from all patients showed successful osteoblast emigration after average 5.5 days. In contrast, only 57% of the corresponding bone shavings successfully demonstrated osteoblast emigration within an average time span of 14.8 days. Average osteoblast mobilisation was 1.25 × 10(6) cells per gram from bone chips and 1.73 × 10(5) cells per gram from the corresponding bone shavings. No difference was observed regarding cell viability, but population doubling times of bone chip cultures were significantly lower (50.5 vs. 121 h) and mineralization was observed in osteoblasts derived from bone chips only. Although some authors suggest the general applicability of laminectomy bone shavings as autografts for spinal fusion, autologous bone grafts obtained from laminectomy bone chips are superior in terms of cell delivery, cell proliferation and mineralization.

Importance of lipid rafts for lysophosphatidylcholine-induced caspase-1 activation and reactive oxygen species generation.

Lipid rafts play an important role in regulating cellular processes and functions. Here, we demonstrate that in microglia stimulated with the pro-inflammatory lipid lysophosphatidylcholine (LPC), caspase-1 activation and NADPH oxidase activity depend on intact lipid rafts. Disruption of lipid rafts with methyl-ß-cyclodextrin, fumonisin B1 or nystatin prevented LPC-stimulated caspase-1 activation and reactive oxygen species (ROS) production, whereas LPC-induced Na(+) influx remained unaffected. Since ROS regulate caspase-1 activity in LPC-stimulated microglia, the effects of lipid raft-disrupting agents on caspase-1 activation can be related to their inhibition of NADPH oxidase-mediated ROS production.

Non-selective cation channel activity is required for lysophosphatidylcholine-induced monocyte migration.

Lysophosphatidylcholine (LPC) is a major atherogenic lipid which stimulates the recruitment of monocytes to atherosclerotic lesions. The physiological mechanisms underlying LPC-induced monocyte migration are poorly understood. Here we demonstrate that LPC activates non-selective cation channels, which are significantly involved in LPC-induced chemotaxis of monocytes. External LPC elicited the activation of non-selective cation currents in THP-1 monocytes, which occurred in a G protein and phospholipase C-independent manner. LPC-activated currents were almost completely inhibited by Gd(3+), La(3+), and TRAM-34. Furthermore, currents were partially reduced by either 2-aminoethoxydiphenyl borate (2-APB) or ruthenium red, while combined application of 2-APB and ruthenium red abolished LPC-activated currents. The 2-APB-sensitive current component was potentiated by flufenamic acid and Ca(2+)-free extracellular solution, while the ruthenium red-sensitive current component was abolished by capsazepine. This pharmacological profile suggests that LPC simultaneously activates TRPC6 and TRPV1 channels in monocytes. Furthermore, in the presence of Gd(3+), La(3+), TRAM-34, 2-APB, ruthenium red or capsazepine, LPC-induced chemotaxis of monocytes was substantially inhibited, indicating that activation of both channel types is required for optimal migration of LPC-stimulated monocytes. Thus, ion channel inhibition may represent a powerful strategy to attenuate the progression of atherosclerosis by reducing monocyte infiltration.

Lysophosphatidylcholine- and MCP-1-induced chemotaxis of monocytes requires potassium channel activity.

One of the earliest cellular responses in atherogenesis is the focal recruitment of circulating monocytes, while the most important atherogenic chemoattractants are monocyte chemoattractant protein-1 (MCP-1) and lysophosphatidylcholine (LPC). Invading monocytes transform into activated macrophages and foam cells, which stimulate inflammatory processes and promote atherosclerosis. In this study, we have searched for common mechanisms involved in MCP-1- and LPC-stimulated monocyte migration. We have found that migration of THP-1 monocytes stimulated with MCP-1 was reduced upon inhibition of G(i/o) proteins with pertussis toxin and upon inhibition of platelet activating factor receptors with BN52021, whereas LPC-stimulated monocyte chemotaxis remained unaffected by both inhibitors. Furthermore, Cl(-) channels were only required for MCP-1-induced chemotaxis. However, activity of voltage-gated K+ channels and of Ca2+-activated K+ channels was found to be involved in migration of monocytes stimulated with either MCP-1 or LPC. Inhibition of voltage-gated K+ channels with 4-aminopyridine or margatoxin partially inhibited MCP-1- and LPC-stimulated migration of monocytes. Blockade of Ca2+-activated K+ channels with TRAM-34 also partially reduced migration of MCP-1- and LPC-stimulated monocytes. Simultaneous inhibition of voltage-gated and Ca2+-activated K+ channels abolished MCP-1- and LPC-induced chemotaxis of monocytes. Thus, K+ channel inhibition may represent a novel powerful strategy to reduce monocyte infiltration and subsequent inflammation in atherosclerosis.

Technology forecast: advanced therapies in late clinical research, EMA approval or clinical application via hospital exemption.

Background: The umbrella term ATMPs (Advanced Therapy Medicinal Products) comprises cell therapies, gene therapeutics and tissue engineered products. After implementation of the Regulation 1394/2007, only a couple of products have obtained a centralized European marketing authorisation. Objectives: The aim of the presented study is to give an overview on ATMPs available within the European Union either via centralized marketing authorisation or via national Hospital exemption. Additionally, a forecast on innovative ATMPs in the process of EMA approval as well as in phase III and IV clinical trial is provided. Methods: Systematic literature search including 'grey literature' and database reviews as well as manual search following pre-defined search terms. Results: 8 ATMPs are currently available via centralized marketing authorisation. 6 new product launches are expected before 2020. At least 32 additional ATMPs are available in individual European Union member states via Hospital exemption. Another 31 potential ATMP candidates could be identified in industry-driven phase III research projects. Conclusion: Advanced therapeutic medicinal therapies are still in their early days, but constantly evolving. By 2020, innovative therapies targeting retinal dystrophy, ß-thalassemia, scleroderma, sickle-cell anaemia, adrenoleukodystrophy and leukaemia shall be available on the market.

Ion channel expression in resting and activated microglia of hippocampal slices from juvenile mice.

Ion channels have frequently been studied in isolated cultured microglia, whereas only little is known about the physiological properties of microglia in situ. In this study, expression of voltage-gated and Ca(2+)-activated ion channels was investigated in microglial cells of acutely prepared and briefly cultured hippocampal slices from juvenile mice. In contrast to microglia of adult brain slices, microglia of slices from juvenile mice exhibited a complex ion channel pattern. Resting microglial cells of acute slices expressed inward rectifier and outward rectifier K(+) channels as well as Zn(2+)-sensitive voltage-activated H(+) channels. In activated microglia of briefly cultured slices, expression levels of both K(+) channel types were upregulated, while H(+) channel expression remained unchanged. In addition, microglia of cultured slices exhibited Ca(2+)-activated K(+) currents, which were not seen in microglia of acute slices, and differed from those expressed in isolated cultured murine microglia. In situ, microglial Ca(2+)-activated K(+) channels were voltage- and Ca(2+)-dependent, had a large single-channel conductance and were inhibited by tetraethylammonium. In summary, our data indicate that the physiological properties of microglia in acute and cultured hippocampal slices from juvenile mice differ markedly from those of microglia cultured in vitro and from those of microglia in adult brain slices.

Lysophosphatidylcholine activates caspase-1 in microglia via a novel pathway involving two inflammasomes.

Inflammasomes regulate microglial caspase-1 activation and subsequent neuroinflammatory processes in brain pathology. In the present study, we have identified inflammasomes causing caspase-1 activation following stimulation of microglia with lysophosphatidylcholine (LPC), a proinflammatory lipid generated under pathological conditions in the brain. LPC-induced caspase-1 activation in microglia was found to depend on LPS prestimulation, inflammasome NLRP3 and adaptor molecule ASC. Furthermore, knockdown of inflammasome NLRC4 inhibited LPC-stimulated caspase-1 activity in microglia, suggesting the requirement of two inflammasomes for optimal caspase-1 activity.

Safety and tolerability of topically administered autologous, apoptotic PBMC secretome (APOSEC) in dermal wounds: a randomized Phase 1 trial (MARSYAS I).

Developing effective therapies against chronic wound healing deficiencies is a global priority. Thus we evaluated the safety of two different doses of topically administered autologous APOSEC, the secretome of apoptotic peripheral blood mononuclear cells (PBMCs), in healthy male volunteers with artificial dermal wounds. Ten healthy men were enrolled in a single-center, randomized, double-blinded, placebo-controlled phase 1 trial. Two artificial wounds at the upper arm were generated using a 4-mm punch biopsy. Each participant was treated with both topically applied APOSEC and placebo in NuGel for 7 consecutive days. The volunteers were randomized into two groups: a low-dose group (A) receiving the supernatant of 12.5 × 106 PBMCs and a high-dose group (B) receiving an equivalent of 25 × 106 PBMCs resuspended in NuGel Hydrogel. Irradiated medium served as placebo. The primary outcome was the tolerability of the topical application of APOSEC. All adverse events were recorded until 17 days after the biopsy. Local tolerability assessment was measured on a 4-point scale. Secondary outcomes were wound closure and epithelization at day 7. No therapy-related serious adverse events occurred in any of the participants, and both low- and high-dose treatments were well tolerated. Wound closure was not affected by APOSEC therapy.

Boron-Based Inhibitors of the NLRP3 Inflammasome.

NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.

Mechanisms Underlying Interferon-γ-Induced Priming of Microglial Reactive Oxygen Species Production.

Microglial priming and enhanced reactivity to secondary insults cause substantial neuronal damage and are hallmarks of brain aging, traumatic brain injury and neurodegenerative diseases. It is, thus, of particular interest to identify mechanisms involved in microglial priming. Here, we demonstrate that priming of microglia with interferon-γ (IFN γ) substantially enhanced production of reactive oxygen species (ROS) following stimulation of microglia with ATP. Priming of microglial ROS production was substantially reduced by inhibition of p38 MAPK activity with SB203580, by increases in intracellular glutathione levels with N-Acetyl-L-cysteine, by blockade of NADPH oxidase subunit NOX2 activity with gp91ds-tat or by inhibition of nitric oxide production with L-NAME. Together, our data indicate that priming of microglial ROS production involves reduction of intracellular glutathione levels, upregulation of NADPH oxidase subunit NOX2 and increases in nitric oxide production, and suggest that these simultaneously occurring processes result in enhanced production of neurotoxic peroxynitrite. Furthermore, IFNγ-induced priming of microglial ROS production was reduced upon blockade of Kir2.1 inward rectifier K+ channels with ML133. Inhibitory effects of ML133 on microglial priming were mediated via regulation of intracellular glutathione levels and nitric oxide production. These data suggest that microglial Kir2.1 channels may represent novel therapeutic targets to inhibit excessive ROS production by primed microglia in brain pathology.

Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer's disease in rodent models.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase-1 (COX-1) and COX-2 enzymes. The NLRP3 inflammasome is a multi-protein complex responsible for the processing of the proinflammatory cytokine interleukin-1ß and is implicated in many inflammatory diseases. Here we show that several clinically approved and widely used NSAIDs of the fenamate class are effective and selective inhibitors of the NLRP3 inflammasome via inhibition of the volume-regulated anion channel in macrophages, independently of COX enzymes. Flufenamic acid and mefenamic acid are efficacious in NLRP3-dependent rodent models of inflammation in air pouch and peritoneum. We also show therapeutic effects of fenamates using a model of amyloid beta induced memory loss and a transgenic mouse model of Alzheimer's disease. These data suggest that fenamate NSAIDs could be repurposed as NLRP3 inflammasome inhibitors and Alzheimer's disease therapeutics.

Microglial Kv1.3 Channels and P2Y12 Receptors Differentially Regulate Cytokine and Chemokine Release from Brain Slices of Young Adult and Aged Mice.

Brain tissue damage following stroke or traumatic brain injury is accompanied by neuroinflammatory processes, while microglia play a central role in causing and regulating neuroinflammation via production of proinflammatory substances, including cytokines and chemokines. Here, we used brain slices, an established in situ brain injury model, from young adult and aged mice to investigate cytokine and chemokine production with particular focus on the role of microglia. Twenty four hours after slice preparation, higher concentrations of proinflammatory cytokines, i.e. TNF-α and IL-6, and chemokines, i.e. CCL2 and CXCL1, were released from brain slices of aged mice than from slices of young adult mice. However, maximal microglial stimulation with LPS for 24 h did not reveal age-dependent differences in the amounts of released cytokines and chemokines. Mechanisms underlying microglial cytokine and chemokine production appear to be similar in young adult and aged mice. Inhibition of microglial Kv1.3 channels with margatoxin reduced release of IL-6, but not release of CCL2 and CXCL1. In contrast, blockade of microglial P2Y12 receptors with PSB0739 inhibited release of CCL2 and CXCL1, whereas release of IL-6 remained unaffected. Cytokine and chemokine production was not reduced by inhibitors of Kir2.1 K+ channels or adenosine receptors. In summary, our data suggest that brain tissue damage-induced production of cytokines and chemokines is age-dependent, and differentially regulated by microglial Kv1.3 channels and P2Y12 receptors.