Sex- and Development-Dependent Responses of Rat Microglia to Pro- and Anti-inflammatory Stimulation.

Addressing potential sex differences in pre-clinical studies is crucial for developing therapeutic interventions. Although sex differences have been reported in epidemiological studies and from clinical experience, most pre-clinical studies of neuroinflammation use male rodents; however, sexual dimorphisms in microglia might affect the CNS inflammatory response. Developmental changes are also important and, in rodents, there is a critical period of sexual brain differentiation in the first 3 weeks after birth. We compared rat microglia from sex-segregated neonates (P1) and at about the time of weaning (P21). To study transitions from a basal homeostatic state (untreated), microglia were subjected to a pro-inflammatory (IFNγ + TNFα) or anti-inflammatory (IL-4) stimulus. Responses were compared by quantifying changes in nitric oxide production, migration, and expression of nearly 70 genes, including inflammatory mediators and receptors, inflammasome molecules, immune modulators, and genes that regulate microglial physiological functions. No sex differences were seen in transcriptional responses in either age group but the IL-4-evoked migration increase was larger in male cells at both ages. Protein changes for the hallmark molecules, NOS2, COX-2, PYK2 and CD206 correlated with mRNA changes. P1 and P21 microglia showed substantial differences, including expression of genes related to developmental roles. That is, P21 microglia had a more mature phenotype, with higher basal and stimulated levels of many inflammatory genes, while P1 cells had higher expression of phagocytosis-related molecules. Nevertheless, cells of both ages responded to IL-4 and IFNγ + TNFα. We examined the Kv1.3 potassium channel (a potential target for modulating neuroinflammation) and the Kir2.1 channel, which regulate several microglia functions. Kv1.3 mRNA (Kcna3) was higher at P21 under all conditions and male P21 cells had higher mRNA and Kv currents in response to IFNγ + TNFα. Overall, numerous transcriptional and functional responses of microglia changed during the first 3 weeks after birth but few sex-dependent changes were seen.

Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10).

Microglia respond to CNS injuries and diseases with complex reactions, often called "activation." A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.

Comparing Effects of Transforming Growth Factor ß1 on Microglia From Rat and Mouse: Transcriptional Profiles and Potassium Channels.

The cytokine, transforming growth factor ß1 (TGFß1), is up-regulated after central nervous system (CNS) injuries or diseases involving microglial activation, and it has been proposed as a therapeutic agent for treating neuroinflammation. Microglia can produce and respond to TGFß1. While rats and mice are commonly used for studying neuroinflammation, very few reports directly compare them. Such studies are important for improving pre-clinical studies and furthering translational progress in developing therapeutic interventions. After intracerebral hemorrhage (ICH) in the rat striatum, the TGFß1 receptor was highly expressed on microglia/macrophages within the hematoma. We recently found species similarities and differences in response to either a pro-inflammatory (interferon-γ, IFN-γ, +tumor necrosis factor, TNF-α) or anti-inflammatory interleukin-4 (IL-4) stimulus. Here, we assessed whether rat and mouse microglia differ in their responses to TGFß1. Microglia were isolated from Sprague-Dawley rats and C57BL/6 mice and treated with TGFß1. We quantified changes in expression of >50 genes, in their morphology, proliferation, apoptosis and in three potassium channels that are considered therapeutic targets. Many inflammatory mediators, immune receptors and modulators showed species similarities, but notable differences included that, for some genes, only one species responded (e.g., Il4r, Il10, Tgfbr2, colony-stimulating factor receptor (Csf1r), Itgam, suppressor of cytokine signaling 1 (Socs1), toll-like receptors 4 (Tlr4), P2rx7, P2ry12), and opposite responses were seen for others (Tgfb1, Myc, Ifngr1). In rat only, TGFß1 affected microglial morphology and proliferation, but there was no apoptosis in either species. In both species, TGFß1 dramatically increased Kv1.3 channel expression and current (no effects on Kir2.1). KCa3.1 showed opposite species responses: the current was low in unstimulated rat microglia and greatly increased by TGFß1 but higher in control mouse cells and decreased by TGFß1. Finally, we compared TGFß1 and IL10 (often considered similar anti-inflammatory stimuli) and found many different responses in both species. Overall, the numerous species differences should be considered when characterizing neuroinflammation and microglial activation in vitro and in vivo, and when targeting potassium channels.

Responses of rat and mouse primary microglia to pro- and anti-inflammatory stimuli: molecular profiles, K+ channels and migration.

BACKGROUND: Acute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans. Microglia can undergo complex molecular and functional changes, often studied by in vitro responses to discrete activating stimuli. There is considerable evidence that pro-inflammatory (M1) activation can exacerbate tissue damage, while anti-inflammatory (M2) states help resolve inflammation and promote tissue repair. However, in assessing potential therapeutic targets for controlling inflammation, it is crucial to determine whether rat and mouse microglia respond the same. METHODS: Primary microglia from Sprague-Dawley rats and C57BL/6 mice were cultured, then stimulated with interferon-γ + tumor necrosis factor-α (I + T; M1 activation), interleukin (IL)-4 (M2a, alternative activation), or IL-10 (M2c, acquired deactivation). To profile their activation responses, NanoString was used to monitor messenger RNA (mRNA) expression of numerous pro- and anti-inflammatory mediators, microglial markers, immunomodulators, and other molecules. Western analysis was used to measure selected proteins. Two potential targets for controlling inflammation-inward- and outward-rectifier K+ channels (Kir2.1, Kv1.3)-were examined (mRNA, currents) and specific channel blockers were applied to determine their contributions to microglial migration in the different activation states. RESULTS: Pro-inflammatory molecules increased after I + T treatment but there were several qualitative and quantitative differences between the species (e.g., iNOS and nitric oxide, COX-2). Several molecules commonly associated with an M2a state differed between species or they were induced in additional activation states (e.g., CD206, ARG1). Resting levels and/or responses of several microglial markers (Iba1, CD11b, CD68) differed with the activation state, species, or both. Transcripts for several Kir2 and Kv1 family members were detected in both species. However, the current amplitudes (mainly Kir2.1 and Kv1.3) depended on activation state and species. Treatment-induced changes in morphology and migratory capacity were similar between the species (migration reduced by I + T, increased by IL-4 or IL-10). In both species, Kir2.1 block reduced migration and Kv1.3 block increased it, regardless of activation state; thus, these channels might affect microglial migration to damage sites. CONCLUSIONS: Caution is recommended in generalizing molecular and functional responses of microglia to activating stimuli between species.

Molecular and Cellular Responses to Interleukin-4 Treatment in a Rat Model of Transient Ischemia.

Within hours after stroke, potentially cytotoxic pro-inflammatory mediators are elevated within the brain; thus, one potential therapeutic strategy is to reduce them and skew the brain toward an anti-inflammatory state. Because interleukin-4 (IL-4) treatment induces an anti-inflammatory, "alternative-activation" state in microglia and macrophages in vitro, we tested the hypothesis that early supplementation of the brain with IL-4 can shift it toward an anti-inflammatory state and reduce damage after transient focal ischemia. Adult male rat striata were injected with endothelin-1, with or without co-injection of IL-4. Inflammation, glial responses and damage to neurons and white matter were quantified from 1 to 7 days later. At 1 day, IL-4 treatment increased striatal expression of several anti-inflammatory markers (ARG1, CCL22, CD163, PPARγ), increased phagocytic (Iba1-positive, CD68-positive) microglia/macrophages, and increased VEGF-A-positive infiltrating neutrophils in the infarcts. At 7 days, there was evidence of sustained, propagating responses. IL-4 increased CD206, CD200R1, IL-4Rα, STAT6, PPARγ, CD11b, and TLR2 expression and increased microglia/macrophages in the infarct and astrogliosis outside the infarct. Neurodegeneration and myelin damage were not reduced, however. The sustained immune and glial responses when resolution and repair processes have begun warrant further studies of IL-4 treatment regimens and long-term outcomes.

Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

BACKGROUND: Microglia are the "professional" phagocytes of the CNS. Phagocytosis is crucial for normal CNS development and maintenance, but it can be either beneficial or detrimental after injury or disease. For instance, white matter damage releases myelin debris that must be cleared by microglia in order for re-myelination to occur. However, phagocytosis can also produce damaging reactive oxygen species (ROS). Furthermore, microglia can acquire pro-inflammatory (M1) or anti-inflammatory (M2) activation states that affect cell functions. Although microglia are exposed to a changing cytokine environment after injury or disease, little is known about the molecular and functional consequences. Therefore, we applied several microglial activation paradigms, with or without myelin debris. We assessed (i) gene expression changes reflecting microglial activation and inflammatory states, and receptors and enzymes related to phagocytosis and ROS production, (ii) myelin phagocytosis and production of ROS, and (iii) expression and contributions of several ion channels that are considered potential targets for regulating microglial behavior. METHODS: Primary rat microglia were exposed to cytokines, individually or sequentially. First, responses to individual M1 or M2 stimuli were compared: IFN-γ plus TNF-α ("I + T"; M1 activation), interleukin-4 (M2a/alternative activation), and interleukin-10 (M2c/acquired deactivation). Second, sequential cytokine addition was used to assess microglia repolarization and cell functions. The paradigms were M2a→M1, M2c→M1, M1→M2a, and M1→M2c. RESULTS: M1 stimulation increased pro-inflammatory genes, phagocytosis, and ROS, as well as expression of Kv1.3, KCa3.1, and Kir2.1 channels. M2a stimulation increased anti-inflammatory genes, ROS production, and Kv1.3 and KCa3.1 expression. Myelin phagocytosis enhanced the M1 profile and dampened the M2a profile, and both phagocytosis and ROS production were dependent on NOX enzymes and Kir2.1 and CRAC channels. Importantly, microglia showed some capacity for re-polarization between M1 and M2a states, based on gene expression changes, myelin phagocytosis, and ROS production. CONCLUSIONS: In response to polarizing and re-polarizing cytokine treatments, microglia display complex changes in gene transcription profiles, phagocytic capacity, NOX-mediated ROS production, and in ion channels involved in microglial activation. Because these changes might affect microglia-mediated CNS inflammation, they should be considered in future experimental, pre-clinical studies.

After Intracerebral Hemorrhage, Oligodendrocyte Precursors Proliferate and Differentiate Inside White-Matter Tracts in the Rat Striatum.

Damage to myelinated axons contributes to neurological deficits after acute CNS injury, including ischemic and hemorrhagic stroke. Potential treatments to promote re-myelination will require fully differentiated oligodendrocytes, but almost nothing is known about their fate following intracerebral hemorrhage (ICH). Using a rat model of ICH in the striatum, we quantified survival, proliferation, and differentiation of oligodendrocyte precursor cells (OPCs) (at 1, 3, 7, 14, and 28 days) in the peri-hematoma region, surrounding striatum, and contralateral striatum. In the peri-hematoma, the density of Olig2(+) cells increased dramatically over the first 7 days, and this coincided with disorganization and fragmentation of myelinated axon bundles. Very little proliferation (Ki67(+)) of Olig2(+) cells was seen in the anterior subventricular zone from 1 to 28 days. However, by 3 days, many were proliferating in the peri-hematoma region, suggesting that local proliferation expands their population. By 14 days, the density of Olig2(+) cells declined in the peri-hematoma region, and, by 28 days, it reached the low level seen in the contralateral striatum. At these later times, many surviving axons were aligned into white-matter bundles, which appeared less swollen or fragmented. Oligodendrocyte cell maturation was prevalent over the 28-day period. Densities of immature OPCs (NG2(+)Olig2(+)) and mature (CC-1(+)Olig2(+)) oligodendrocytes in the peri-hematoma increased dramatically over the first week. Regardless of the maturation state, they increased preferentially inside the white-matter bundles. These results provide evidence that endogenous oligodendrocyte precursors proliferate and differentiate in the peri-hematoma region and have the potential to re-myelinate axon tracts after hemorrhagic stroke.

Expression and contributions of the Kir2.1 inward-rectifier K(+) channel to proliferation, migration and chemotaxis of microglia in unstimulated and anti-inflammatory states.

When microglia respond to CNS damage, they can range from pro-inflammatory (classical, M1) to anti-inflammatory, alternative (M2) and acquired deactivation states. It is important to determine how microglial functions are affected by these activation states, and to identify molecules that regulate their behavior. Microglial proliferation and migration are crucial during development and following damage in the adult, and both functions are Ca(2+)-dependent. In many cell types, the membrane potential and driving force for Ca(2+) influx are regulated by inward-rectifier K(+) channels, including Kir2.1, which is prevalent in microglia. However, it is not known whether Kir2.1 expression and contributions are altered in anti-inflammatory states. We tested the hypothesis that Kir2.1 contributes to Ca(2+) entry, proliferation and migration of rat microglia. Kir2.1 (KCNJ2) transcript expression, current amplitude, and proliferation were comparable in unstimulated microglia and following alternative activation (IL-4 stimulated) and acquired deactivation (IL-10 stimulated). To examine functional roles of Kir2.1 in microglia, we first determined that ML133 was more effective than the commonly used blocker, Ba(2+); i.e., ML133 was potent (IC50 = 3.5 µM) and voltage independent. Both blockers slightly increased proliferation in unstimulated or IL-4 (but not IL-10)-stimulated microglia. Stimulation with IL-4 or IL-10 increased migration and ATP-induced chemotaxis, and blocking Kir2.1 greatly reduced both but ML133 was more effective. In all three activation states, blocking Kir2.1 with ML133 dramatically reduced Ca(2+) influx through Ca(2+)-release-activated Ca(2+) (CRAC) channels. Thus, Kir2.1 channel activity is necessary for microglial Ca(2+) signaling and migration under resting and anti-inflammatory states but the channel weakly inhibits proliferation.

KCa3.1/IK1 Channel Regulation by cGMP-Dependent Protein Kinase (PKG) via Reactive Oxygen Species and CaMKII in Microglia: An Immune Modulating Feedback System?

The intermediate conductance Ca(2+)-activated K(+) channel, KCa3.1 (IK1/SK4/KCNN4) is widely expressed in the innate and adaptive immune system. KCa3.1 contributes to proliferation of activated T lymphocytes, and in CNS-resident microglia, it contributes to Ca(2+) signaling, migration, and production of pro-inflammatory mediators (e.g., reactive oxygen species, ROS). KCa3.1 is under investigation as a therapeutic target for CNS disorders that involve microglial activation and T cells. However, KCa3.1 is post-translationally regulated, and this will determine when and how much it can contribute to cell functions. We previously found that KCa3.1 trafficking and gating require calmodulin (CaM) binding, and this is inhibited by cAMP kinase (PKA) acting at a single phosphorylation site. The same site is potentially phosphorylated by cGMP kinase (PKG), and in some cells, PKG can increase Ca(2+), CaM activation, and ROS. Here, we addressed KCa3.1 regulation through PKG-dependent pathways in primary rat microglia and the MLS-9 microglia cell line, using perforated-patch recordings to preserve intracellular signaling. Elevating cGMP increased both the KCa3.1 current and intracellular ROS production, and both were prevented by the selective PKG inhibitor, KT5823. The cGMP/PKG-evoked increase in KCa3.1 current in intact MLS-9 microglia was mediated by ROS, mimicked by applying hydrogen peroxide (H2O2), inhibited by a ROS scavenger (MGP), and prevented by a selective CaMKII inhibitor (mAIP). Similar results were seen in alternative-activated primary rat microglia; their KCa3.1 current required PKG, ROS, and CaMKII, and they had increased ROS production that required KCa3.1 activity. The increase in current apparently did not result from direct effects on the channel open probability (P o) or Ca(2+) dependence because, in inside-out patches from transfected HEK293 cells, single-channel activity was not affected by cGMP, PKG, H2O2 at normal or elevated intracellular Ca(2+). The regulation pathway we have identified in intact microglia and MLS-9 cells is expected to have broad implications because KCa3.1 plays important roles in numerous cells and tissues.

PKA reduces the rat and human KCa3.1 current, CaM binding, and Ca2+ signaling, which requires Ser332/334 in the CaM-binding C terminus.

The Ca(2+)-dependent K(+) channel, KCa3.1 (KCNN4/IK/SK4), is widely expressed and contributes to cell functions that include volume regulation, migration, membrane potential, and excitability. KCa3.1 is now considered a therapeutic target for several diseases, including CNS disorders involving microglial activation; thus, we need to understand how KCa3.1 function is regulated. KCa3.1 gating and trafficking require calmodulin binding to the two ends of the CaM-binding domain (CaMBD), which also contains three conserved sites for Ser/Thr kinases. Although cAMP protein kinase (PKA) signaling is important in many cells that use KCa3.1, reports of channel regulation by PKA are inconsistent. We first compared regulation by PKA of native rat KCa3.1 channels in microglia (and the microglia cell line, MLS-9) with human KCa3.1 expressed in HEK293 cells. In all three cells, PKA activation with Sp-8-Br-cAMPS decreased the current, and this was prevented by the PKA inhibitor, PKI14-22. Inhibiting PKA with Rp-8-Br-cAMPS increased the current in microglia. Mutating the single PKA site (S334A) in human KCa3.1 abolished the PKA-dependent regulation. CaM-affinity chromatography showed that CaM binding to KCa3.1 was decreased by PKA-dependent phosphorylation of S334, and this regulation was absent in the S334A mutant. Single-channel analysis showed that PKA decreased the open probability in wild-type but not S334A mutant channels. The same decrease in current for native and wild-type expressed KCa3.1 channels (but not S334A) occurred when PKA was activated through the adenosine A2a receptor. Finally, by decreasing the KCa3.1 current, PKA activation reduced Ca(2+)-release-activated Ca(2+) entry following activation of metabotropic purinergic receptors in microglia.

Expression and contributions of TRPM7 and KCa2.3/SK3 channels to the increased migration and invasion of microglia in anti-inflammatory activation states.

Microglia rapidly respond to CNS injury and disease and can assume a spectrum of activation states. While changes in gene expression and production of inflammatory mediators have been extensively described after classical (LPS-induced) and alternative (IL4-induced) microglial activation, less is known about acquired de-activation in response to IL10. It is important to understand how microglial activation states affect their migration and invasion; crucial functions after injury and in the developing CNS. We reported that LPS-treated rat microglia migrate very poorly, while IL4-treated cells migrate and invade much better. Having discovered that the lamellum of migrating microglia contains a large ring of podosomes--microscopic structures that are thought to mediate adhesion, migration and invasion--we hypothesized that IL4 and IL10 would differentially affect podosome expression, gene induction, migration and invasion. Further, based on the enrichment of the KCa2.3/SK3 Ca2+-activated potassium channel in microglial podosomes, we predicted that it regulates migration and invasion. We found both similarities and differences in gene induction by IL4 and IL10 and, while both cytokines increased migration and invasion, only IL10 affected podosome expression. KCa2.3 currents were recorded in microglia under all three activation conditions and KCNN3 (KCa2.3) expression was similar. Surprisingly then, of three KCa2.3 inhibitors (apamin, tamapin, NS8593), only NS8593 abrogated the increased migration and invasion of IL4 and IL10-treated microglia (and invasion of unstimulated microglia). This discrepancy was explained by the observed block of TRPM7 currents in microglia by NS8593, which occurred under all three activation conditions. A similar inhibition of both migration and invasion was seen with a TRPM7 inhibitor (AA-861) that does not block KCa2.3 channels. Thus, we conclude that TRPM7 (not KCa2.3) contributes to the enhanced ability of microglia to migrate and invade when in anti-inflammatory states. This will be an important consideration in developing TRPM7 inhibitors for treating CNS injury.

IL-4 type 1 receptor signaling up-regulates KCNN4 expression, and increases the KCa3.1 current and its contribution to migration of alternative-activated microglia.

The Ca(2+)-activated K(+) channel, KCa3.1 (KCNN4/IK1/SK4), contributes to "classical," pro-inflammatory activation of microglia, and KCa3.1 blockers have improved the outcome in several rodent models of CNS damage. For instance, blocking KCa3.1 with TRAM-34 rescued retinal ganglion neurons after optic nerve damage in vivo and, reduced p38 MAP kinase activation, production of reactive oxygen and nitrogen species, and neurotoxicity by microglia in vitro. In pursuing the therapeutic potential of KCa3.1 blockers, it is crucial to assess KCa3.1 contributions to other microglial functions and activation states, especially the IL-4-induced "alternative" activation state that can counteract pro-inflammatory states. We recently found that IL-4 increases microglia migration - a crucial function in the healthy and damaged CNS - and that KCa3.1 contributes to P2Y2 receptor-stimulated migration. Here, we discovered that KCa3.1 is greatly increased in alternative-activated rat microglia and then contributes to an enhanced migratory capacity. IL-4 up-regulated KCNN4 mRNA (by 6 h) and greatly increased the KCa3.1 current by 1 day, and this required de novo protein synthesis. The increase in current was sustained for at least 6 days. IL-4 increased microglial migration and this was reversed by blocking KCa3.1 with TRAM-34. A panel of inhibitors of signal-transduction mediators was used to analyze contributions of IL-4-related signaling pathways. Induction of KCNN4 mRNA and KCa3.1 current was mediated specifically through IL-4 binding to the type I receptor and, surprisingly, it required JAK3, Ras/MEK/ERK signaling and the transcription factor, activator protein-1, rather than JAK2, STAT6, or phosphatidylinositol 3-kinase.The same receptor subtype and pathway were required for the enhanced KCa3.1-dependent migration. In providing the first direct signaling link between an IL-4 receptor, expression and roles of an ion channel, this study also highlights the potential importance of KCa3.1 in alternative-activated microglia.

Regulation of hERG and hEAG channels by Src and by SHP-1 tyrosine phosphatase via an ITIM region in the cyclic nucleotide binding domain.

Members of the EAG K(+) channel superfamily (EAG/Kv10.x, ERG/Kv11.x, ELK/Kv12.x subfamilies) are expressed in many cells and tissues. In particular, two prototypes, EAG1/Kv10.1/KCNH1 and ERG1/Kv11.1/KCNH2 contribute to both normal and pathological functions. Proliferation of numerous cancer cells depends on hEAG1, and in some cases, hERG. hERG is best known for contributing to the cardiac action potential, and for numerous channel mutations that underlie 'long-QT syndrome'. Many cells, particularly cancer cells, express Src-family tyrosine kinases and SHP tyrosine phosphatases; and an imbalance in tyrosine phosphorylation can lead to malignancies, autoimmune diseases, and inflammatory disorders. Ion channel contributions to cell functions are governed, to a large degree, by post-translational modulation, especially phosphorylation. However, almost nothing is known about roles of specific tyrosine kinases and phosphatases in regulating K(+) channels in the EAG superfamily. First, we show that tyrosine kinase inhibitor, PP1, and the selective Src inhibitory peptide, Src40-58, reduce the hERG current amplitude, without altering its voltage dependence or kinetics. PP1 similarly reduces the hEAG1 current. Surprisingly, an 'immuno-receptor tyrosine inhibitory motif' (ITIM) is present within the cyclic nucleotide binding domain of all EAG-superfamily members, and is conserved in the human, rat and mouse sequences. When tyrosine phosphorylated, this ITIM directly bound to and activated SHP-1 tyrosine phosphatase (PTP-1C/PTPN6/HCP); the first report that a portion of an ion channel is a binding site and activator of a tyrosine phosphatase. Both hERG and hEAG1 currents were decreased by applying active recombinant SHP-1, and increased by the inhibitory substrate-trapping SHP-1 mutant. Thus, hERG and hEAG1 currents are regulated by activated SHP-1, in a manner opposite to their regulation by Src. Given the widespread distribution of these channels, Src and SHP-1, this work has broad implications in cell signaling that controls survival, proliferation, differentiation, and other ERG1 and EAG1 functions in many cell types.

The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion.

BACKGROUND: Microglial cells are highly mobile under many circumstances and, after central nervous system (CNS) damage, they must contend with the dense extracellular matrix (ECM) in order to reach their target sites. In response to damage or disease, microglia undergo complex activation processes that can be modulated by environmental cues and culminate in either detrimental or beneficial outcomes. Thus, there is considerable interest in comparing their pro-inflammatory ('classical' activation) and resolving 'alternative' activation states. Almost nothing is known about how these activation states affect the ability of microglia to migrate and degrade ECM, or the enzymes used for substrate degradation. This is the subject of the present study. METHODS: Primary cultured rat microglial cells were exposed to lipopolysaccharide (LPS) to evoke classical activation or IL4 to evoke alternative activation. High-resolution microscopy was used to monitor changes in cell morphology and aspects of the cytoskeleton. We quantified migration in a scratch-wound assay and through open filter holes, and invasion through Matrigel™. A panel of inhibitors was used to analyze contributions of different matrix-degrading enzymes to migration and invasion, and quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to assess changes in their expression. RESULTS: Vinculin- and F-actin-rich lamellae were prominent in untreated and IL4-treated microglia (but not after LPS). IL4 increased the migratory capacity of microglia but eliminated the preferential anterior nuclear-centrosomal axis polarity and location of the microtubule organizing center (MTOC). Microglia degraded fibronectin, regardless of treatment, but LPS-treated cells were relatively immobile and IL4-treated cells invaded much more effectively through Matrigel™. For invasion, untreated microglia primarily used cysteine proteases, but IL4-treated cells used a wider range of enzymes (cysteine proteases, cathepsin S and K, heparanase, and matrix metalloproteases). Untreated microglia expressed MMP2, MMP12, heparanase, and four cathepsins (B, K, L1, and S). Each activation stimulus upregulated a different subset of enzymes. IL4 increased MMP2 and cathepsins S and K; whereas LPS increased MMP9, MMP12, MMP14 (MT1-MMP), heparanase, and cathepsin L1. CONCLUSIONS: Microglial cells migrate during CNS development and after CNS damage or disease. Thus, there are broad implications of the finding that classically and alternatively activated microglia differ in morphology, cytoskeleton, migratory and invasive capacity, and in the usage of ECM-degrading enzymes.

Selective activation of KCa3.1 and CRAC channels by P2Y2 receptors promotes Ca(2+) signaling, store refilling and migration of rat microglial cells.

Microglial activation involves Ca(2+) signaling, and numerous receptors can evoke elevation of intracellular Ca(2+). ATP released from damaged brain cells can activate ionotropic and metabotropic purinergic receptors, and act as a chemoattractant for microglia. Metabotropic P2Y receptors evoke a Ca(2+) rise through release from intracellular Ca(2+) stores and store-operated Ca(2+) entry, and some have been implicated in microglial migration. This Ca(2+) rise is expected to activate small-conductance Ca(2+)-dependent K(+) (SK) channels, if present. We previously found that SK3 (KCa2.3) and KCa3.1 (SK4/IK1) are expressed in rat microglia and contribute to LPS-mediated activation and neurotoxicity. However, neither current has been studied by elevating Ca(2+) during whole-cell recordings. We hypothesized that, rather than responding only to Ca(2+), each channel type might be coupled to different receptor-mediated pathways. Here, our objective was to determine whether the channels are differentially activated by P2Y receptors, and, if so, whether they play differing roles. We used primary rat microglia and a rat microglial cell line (MLS-9) in which riluzole robustly activates both SK3 and KCa3.1 currents. Using electrophysiological, Ca(2+) imaging and pharmacological approaches, we show selective functional coupling of KCa3.1 to UTP-mediated P2Y2 receptor activation. KCa3.1 current is activated by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC/Orai1) channels, and both CRAC/Orai1 and KCa3.1 channels facilitate refilling of Ca(2+) stores. The Ca(2+) dependence of KCa3.1 channel activation was skewed to abnormally high concentrations, and we present evidence for a close physical association of the two channel types. Finally, migration of primary rat microglia was stimulated by UTP and inhibited by blocking either KCa3.1 or CRAC/Orai1 channels. This is the first report of selective coupling of one type of SK channel to purinergic stimulation of microglia, transactivation of KCa3.1 channels by CRAC/Orai1, and coordinated roles for both channels in store refilling, Ca(2+) signaling and microglial migration.

Regulation of podosome formation, microglial migration and invasion by Ca(2+)-signaling molecules expressed in podosomes.

BACKGROUND: Microglia migrate during brain development and after CNS injury, but it is not known how they degrade the extracellular matrix (ECM) to accomplish this. Podosomes are tiny structures with the unique ability to adhere to and dissolve ECM. Podosomes have a two-part architecture: a core that is rich in F-actin and actin-regulatory molecules (for example, Arp2/3), surrounded by a ring with adhesion and structural proteins (for example, talin, vinculin). We recently discovered that the lamellum at the leading edge of migrating microglia contains a large F-actin-rich superstructure ('podonut') composed of many podosomes. Microglia that expressed podosomes could degrade ECM molecules. Finely tuned Ca(2+) signaling is important for cell migration, cell-substrate adhesion and contraction of the actomyosin network. Here, we hypothesized that podosomes contain Ca(2+)-signaling machinery, and that podosome expression and function depend on Ca(2+) influx and specific ion channels. METHODS: High-resolution immunocytochemistry was used on rat microglia to identify podosomes and novel molecular components. A pharmacological toolbox was applied to functional assays. We analyzed roles of Ca(2+)-entry pathways and ion channels in podosome expression, microglial migration into a scratch-wound, transmigration through pores in a filter, and invasion through Matrigel™-coated filters. RESULTS: Microglial podosomes were identified using well-known components of the core (F-actin, Arp2) and ring (talin, vinculin). We discovered four novel podosome components related to Ca(2+) signaling. The core contained calcium release activated calcium (CRAC; Orai1) channels, calmodulin, small-conductance Ca(2+)-activated SK3 channels, and ionized Ca(2+) binding adapter molecule 1 (Iba1), which is used to identify microglia in the CNS. The Orai1 accessory molecule, STIM1, was also present in and around podosomes. Podosome formation was inhibited by removing external Ca(2+) or blocking CRAC channels. Blockers of CRAC channels inhibited migration and invasion, and SK3 inhibition reduced invasion. CONCLUSIONS: Microglia podosome formation, migration and/or invasion require Ca(2+) influx, CRAC, and SK3 channels. Both channels were present in microglial podosomes along with the Ca(2+)-regulated molecules, calmodulin, Iba1 and STIM1. These results suggest that the podosome is a hub for sub-cellular Ca(2+)-signaling to regulate ECM degradation and cell migration. The findings have broad implications for understanding migration mechanisms of cells that adhere to, and dissolve ECM.

Podosomes in migrating microglia: components and matrix degradation.

BACKGROUND: To perform their functions during development and after central nervous system injury, the brain's immune cells (microglia) must migrate through dense neuropil and extracellular matrix (ECM), but it is not known how they degrade the ECM. In several cancer cell lines and peripheral cells, small multi-molecular complexes (invadopodia in cancer cells, podosomes in nontumor cells) can both adhere to and dissolve the ECM. Podosomes are tiny multi-molecular structures (0.4 to 1 µm) with a core, rich in F-actin and its regulatory molecules, surrounded by a ring containing adhesion and structural proteins. METHODS: Using rat microglia, we performed several functional assays: live cell imaging for chemokinesis, degradation of the ECM component, fibronectin, and chemotactic invasion through Matrigel™, a basement membrane type of ECM. Fluorescent markers were used with high-resolution microscopy to identify podosomes and their components. RESULTS: The fan-shaped lamella at the leading edge of migrating microglia contained a large F-actin-rich superstructure composed of many tiny (<1 µm) punctae that were adjacent to the substrate, as expected for cell-matrix contact points. This superstructure (which we call a podonut) was restricted to cells with lamellae, and conversely almost every lamella contained a podonut. Each podonut comprised hundreds of podosomes, which could also be seen individually adjacent to the podonut. Microglial podosomes contained hallmark components of these structures previously seen in several cell types: the plaque protein talin in the ring, and F-actin and actin-related protein (Arp) 2 in the core. In microglia, podosomes were also enriched in phosphotyrosine residues and three tyrosine-kinase-regulated proteins: tyrosine kinase substrate with five Src homology 3 domains (Tks5), phosphorylated caveolin-1, and Nox1 (nicotinamide adenine dinucleotide phosphate oxidase 1). When microglia expressed podonuts, they were able to degrade the ECM components, fibronectin, and Matrigel™. CONCLUSION: The discovery of functional podosomes in microglia has broad implications, because migration of these innate immune cells is crucial in the developing brain, after damage, and in disease states involving inflammation and matrix remodeling. Based on the roles of invadosomes in peripheral tissues, we propose that microglia use these complex structures to adhere to and degrade the ECM for efficient migration.

Age-related comparisons of evolution of the inflammatory response after intracerebral hemorrhage in rats.

UNLABELLED: In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response within the brain characterized by the infiltration of peripheral neutrophils and macrophages and the activation of brain-resident microglia and astrocytes. Despite the strong correlation of aging and ICH incidence, and increasing information about cellular responses, little is known about the temporal- and age-related molecular responses of the brain after ICH. Here, we monitored a panel of 27 genes at 6 h and 1, 3, and 7 days after ICH was induced by injecting collagenase into the striatum of young adult and aged rats. Several molecules (CR3, TLR2, TLR4, IL-1ß, TNFα, iNOS, IL-6) were selected to reflect the classical activation of innate immune cells (macrophages, microglia) and the potential to exacerbate inflammation and damage brain cells. Most of the others are associated with the resolution of innate inflammation, alternative pathways of macrophage/microglial activation, and the repair phase after acute injury (TGFß, IL-1ra, IL-1r2, IL-4, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22). In young animals, the up-regulation of 26 in 27 genes (not IL-4) was detected within the first week. Differences in timing or levels between young and aged animals were detected for 18 of 27 genes examined (TLR2, GFAP, IL-1ß, IL-1ra, IL-1r2, iNOS, IL-6, TGFß, MMP9, MMP12, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22), with a generally less pronounced or delayed inflammatory response in the aged animals. Importantly, within this complex response to experimental ICH, the induction of pro-inflammatory, potentially harmful mediators often coincided with resolving and beneficial molecules. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12975-012-0151-3) contains supplementary material, which is available to authorized users.

SC1/hevin identifies early white matter injury after ischemia and intracerebral hemorrhage in young and aged rats.

The progression of white matter damage after ischemic and hemorrhagic strokes can exacerbate the initial injury, but little is known about the processes involved. We show that the antiadhesive matricellular glycoprotein SC1 is a novel early marker of white matter damage in 3 models of acute injury in the rat striatum: transient focal ischemia, intracerebral hemorrhage, and a needle penetration wound. SC1 was restricted to the damaged portions of axon bundles that bordered stroke lesions in young-adult and aged rats. SC1 peaked at 1 and 3 days after intracerebral hemorrhage and at 7 days after ischemia. The SC1-positive bundles usually expressed degraded myelin basic protein and amyloid precursor protein, a marker of axonal injury. At the hematoma edge, SC1 was seen in a few axon bundles that retained myelin basic protein staining. In these bundles, punctate SC1 staining filled individual axons, extended beyond a core of pan-axonal neurofilament and NF200 and was inside or overlapped with myelin basic protein staining when it was present. Aged rats had less SC1 (and amyloid precursor protein) after both types of stroke, suggesting a reduced axonal response. SC1 also labeled amyloid precursor protein-positive axon bundles along the needle penetration tract of saline-injected rats; thus, SC1 appears to characterize damaged striatal white matter damage after multiple types of injury.

Inhibition of the Ca²âº-dependent K⁺ channel, KCNN4/KCa3.1, improves tissue protection and locomotor recovery after spinal cord injury.

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K⁺ channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1ß in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca²âº signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.