Lyanne C. Schlichter

Mechanisms of Microglia-Mediated Neurotoxicity in a New Model of the Stroke Penumbra

After an ischemic stroke, neurons in the core are rapidly committed to die, whereas neuron death in the slowly developing penumbra is more amenable to therapeutic intervention. Microglia activation contributes to delayed inflammation, but because neurotoxic mechanisms in the penumbra are not well understood, we developed an in vitro model of microglia activation and propagated neuron killing. To recapitulate inflammatory triggers in the core, microglia were exposed to oxygen glucose-deprived neurons and astrocytes. To model the developing penumbra, the microglia were washed and allowed to interact with healthy naive neurons and astrocytes. We found that oxygen–glucose deprivation (OGD)-stressed neurons released glutamate, which activated microglia through their group II metabotropic glutamate receptors (mGluRs). Microglia activation involved nuclear factor κB (NF-κB), a transcription factor that promotes their proinflammatory functions. The activated microglia became neurotoxic, killing naive neurons through an apoptotic mechanism that was mediated by tumor necrosis factor-α (TNF-α), and involved activation of both caspase-8 and caspase-3. In contrast to some earlier models (e.g., microglia activation by lipopolysaccharide), neurotoxicity was not decreased by an inducible nitric oxide synthase (iNOS) inhibitor (S-methylisothiourea) or a peroxynitrite scavenger [5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphinato iron (III) chloride], and did not require p38 mitogen-activated protein kinase (MAPK) activation. The same microglia neurotoxic behavior was evoked without exposure to OGD-stressed neurons, by directly activating microglial group II mGluRs with (2S,2′R,3′R)-2-(2′3′-dicarboxycyclopropyl) glycine or glutamate, which stimulated production of TNF-α (not nitric oxide) and mediated TNF-α-dependent neurotoxicity through activation of NF-κB (not p38 MAPK). Together, these results support potential therapeutic strategies that target microglial group II mGluRs, TNFα overproduction, and NF-κB activation to reduce neuron death in the ischemic penumbra.

The Ca2+-Activated K+ Channel KCNN4/KCa3.1 Contributes to Microglia Activation and Nitric Oxide-Dependent Neurodegeneration

Brain damage and disease involve activation of microglia and production of potentially neurotoxic molecules, but there are no treatments that effectively target their harmful properties. We present evidence that the small-conductance Ca2+/calmodulin-activated K+ channel KCNN4/ KCa3.1/SK4/IK1 is highly expressed in rat microglia and is a potential therapeutic target for acute brain damage. Using a Transwell cell-culture system that allows separate treatment of the microglia or neurons, we show that activated microglia killed neurons, and this was markedly reduced by treating only the microglia with a selective inhibitor of KCa3.1 channels, triarylmethane-34 (TRAM-34). To assess the role of KCa3.1 channels in microglia activation and key signaling pathways involved, we exploited several fluorescence plate-reader-based assays. KCa3.1 channels contributed to microglia activation, inducible nitric oxide synthase upregulation, production of nitric oxide and peroxynitrite, and to consequent neurotoxicity, protein tyrosine nitration, and caspase 3 activation in the target neurons. Microglia activation involved the signaling pathways p38 mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB), which are important for upregulation of numerous proinflammatory molecules, and the KCa3.1 channels were functionally linked to activation of p38 MAPK but not NF-κB. These in vitro findings translated into in vivo neuroprotection, because we found that degeneration of retinal ganglion cells after optic nerve transection was reduced by intraocular injection of TRAM-34. This study provides evidence that KCa3.1 channels constitute a therapeutic target in the CNS and that inhibiting this K+ channel might benefit acute and chronic neurodegenerative disorders that are caused by or exacerbated by inflammation.

Microglia Kv1.3 Channels Contribute to Their Ability to Kill Neurons

Many CNS disorders involve an inflammatory response that is orchestrated by cells of the innate immune system: macrophages, neutrophils, and microglia (the endogenous CNS immune cell). Hence, there is considerable interest in anti-inflammatory strategies that target these cells. Microglia express Kv1.3 (KCNA3) channels, which we showed previously are important for their proliferation and the NADPH-mediated respiratory burst. Here, we demonstrate the potential for targeting Kv1.3 channels to control CNS inflammation. Rat microglia express Kv1.2, Kv1.3, and Kv1.5 transcripts and protein, but only a Kv1.3 current was detected. When microglia were activated with lipopolysaccharide or a phorbol ester, only the Kv1.3 transcript (but not protein) expression changed. Using a Transwell cell-culture system that allows separate drug treatment of microglia or neurons, we found that activated microglia killed postnatal hippocampal neurons through a process that requires Kv1.3 channel activity in microglia but not in neurons. A major neurotoxic molecule in this model was peroxynitrite, which is formed from superoxide and nitric oxide; thus, it is significant that Kv1.3 channel blockers reduced the respiratory burst, but not nitric oxide production, by the activated microglia. In addressing the biochemical pathway affected by Kv1.3 channel activity, we found that Kv1.3 acts via a different cellular mechanism from the broad-spectrum drug minocycline, which is often used in animal models of neuroinflammation. That is, the dose-dependent reduction in neuron killing by minocycline corresponded with a reduction in p38 mitogen-activated protein kinase activation in microglia; however, none of the Kv1.3 blockers affected p38 activation.

Regulation of a TRPM7-like Current in Rat Brain Microglia

Non-excitable cells use Ca2+ influx for essential functions but usually lack voltage-gated Ca2+ channels. The main routes of Ca2+ entry appear to be store-operated channels or Ca2+-permeable non-selective cation channels, of which the magnesium-inhibited cation (or magnesium-nucleotide-regulated metal cation) current has received considerable recent attention. This current appears to be produced by one of the recently cloned transient receptor potential (TRP) channels, TRPM7. In this study of rat microglia, we identified TRPM7 transcripts and a prevalent current with the hallmark biophysical and pharmacological features of TRPM7. This is the first identification of a TRPM7-like current in the brain. There is little known about how members of the TRPM sub-family normally become activated. Using whole-cell patch clamp recordings from rat microglia, we found that the TRPM7-like current activates spontaneously after break-in and that the current and its activation are inhibited by elevated intracellular Mg2+ but not affected by cell swelling or a wide range of intracellular Ca2+ concentrations. The TRPM7-like current in microglia appears to depend on tyrosine phosphorylation. It was inhibited by several tyrosine kinase inhibitors, including a peptide (Src 40–58) that was shown previously to inhibit Src actions, but not by inactive drugs or peptide analogues. The current did not depend on the cell activation state; i.e. it was the same in microglia recently removed from the brain or when cultured under a wide range of conditions that favor the resting or activated state. Because TRPM7 channels are permeable to Ca2+, this current may be important for microglia functions that depend on elevations in intracellular Ca2+.

Minocycline protects the blood-brain barrier and reduces edema following intracerebral hemorrhage in the rat

Intracerebral hemorrhage (ICH) results from rupture of a blood vessel in the brain. After ICH, the blood-brain barrier (BBB) surrounding the hematoma is disrupted, leading to cerebral edema. In both animals and humans, edema coincides with inflammation, which is characterized by production of pro-inflammatory cytokines, activation of resident brain microglia and migration of peripheral immune cells into the brain. Accordingly, inflammation is an attractive target for reducing edema following ICH. In the present study, BBB damage was assessed by quantifying intact microvessels surrounding the hematoma, monitoring extravasation of IgG and measuring brain water content 3 days after ICH induced by collagenase injection into the rat striatum. In the injured brain, the water content increased in both ipsilateral and contralateral hemispheres compared with the normal brain. Quantitative real-time RT-PCR revealed an up-regulation of inflammatory genes associated with BBB damage; IL1beta, TNFalpha and most notably, MMP-12. Immunostaining showed MMP-12 in damaged microvessels and their subsequent loss from tissue surrounding the hematoma. MMP-12 was also observed for the first time in neurons. Dual-antibody labeling demonstrated that neutrophils were the predominant source of TNFalpha protein. Intraperitoneal injection of the tetracycline derivative, minocycline, beginning 6 h after ICH ameliorated the damage by reducing microvessel loss, extravasation of plasma proteins and edema; decreasing TNFalpha and MMP-12 expression; and reducing the numbers of TNFalpha-positive cells and neutrophils in the brain. Thus, minocycline, administered at a clinically relevant time, appears to target the inflammatory processes involved in edema development after ICH.

Internalization of the Kv1.4 Potassium Channel Is Suppressed by Clustering Interactions with PSD-95

The contribution of voltage-dependent ion channels to nerve function depends upon their cell-surface distributions. Nevertheless, the mechanisms underlying channel localization are poorly understood. Two phenomena appear particularly important: the clustering of channels by membrane-associated guanylate kinases (MAGUKs), such as PSD-95, and the regional stabilization of cell-surface proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined the effect of PSD-95 on the internalization rate of Kv1.4 K+ channels in transfected HEK293 cells using cell-surface biotinylation assays. When expressed alone Kv1.4 was internalized with a half-life of 87 min, but, in the presence of PSD-95, Kv1.4 internalization was completely suppressed. Immunochemistry and electrophysiology showed PSD-95 had little effect on total or cell-surface levels of Kv1.4 or on current amplitude, activation, or inactivation kinetics. Clustering was necessary and sufficient to suppress Kv1.4 internalization since C35S-PSD-95, a mutant reported to bind but not cluster Kv1.4, (confirmed by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4) restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t 1 2 = 16 min). These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role for PSD-95, and perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.

Differential Recruitment of Kv1.4 and Kv4.2 to Lipid Rafts by PSD-95*

The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation. Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell. Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts. Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains. Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts. We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raft-associated. Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons. Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts. We then assessed the contribution of PSD-95 to the raft association of these channels. Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts. Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant. This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.

Evolution of the inflammatory response in the brain following intracerebral hemorrhage and effects of delayed minocycline treatment

There are no effective treatments for intracerebral hemorrhage (ICH). Although inflammation is a potential therapeutic target, there is a dearth of information about time-dependent and cell-specific changes in the expression of inflammation-related genes. Using the collagenase-induced ICH model in rats and real-time quantitative RT-PCR we monitored mRNA levels of markers of glial activation, pro- and anti-inflammatory cytokines, enzymes responsible for cytokine activation and several matrix metalloproteases at 6 h and 1, 3 and 7 days after ICH onset. For the most highly up-regulated genes, immunohistochemistry was then used to identify cell-specific protein expression. Finally, minocycline, a drug widely reported to reduce damage in several models of brain injury, was used to test the hypothesis that it can reduce up-regulation of inflammation-related genes when administered using a clinically relevant dosing regime: intraperitoneal injection beginning 6 h after ICH. Our results show a complex inflammatory response, with different brain cell types producing several pro- and anti-inflammatory molecules for at least 7 days after ICH onset. Included is the first demonstration that astrocytes are an important source of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6) and MMP-12. Importantly, our results demonstrate that while delayed minocycline treatment effectively reduces early up-regulation of TNFalpha and MMP-12, its efficacy is lost when treatment is extended for up to a week, and it does not reduce several other genes associated with microglia activation. These results suggest caution in extrapolating to ICH the promising results of minocycline treatment in other models of brain injury.

Neuron death and inflammation in a rat model of intracerebral hemorrhage: Effects of delayed minocycline treatment

After intracerebral hemorrhage (ICH), blood entry is followed by neuron death and an inflammatory response, but development of pharmacological therapies has been hampered by an inadequate understanding of the spatial and temporal relationship between neuron death and inflammation. Using a rat model of ICH, we first investigated these relationships at 6 h, and 1, 3 and 7 days. At the edge of the hematoma, no degenerating neurons were observed at 6 h; however, dying neurons were present between 1 and 3 days, with peak neuron death occurring at 1 day. This is apparently the first report of ongoing neuron death at the edge of the hematoma during a time window that is appropriate for human therapy. Neuron death was limited to the edge of the hematoma, with no degenerating neurons in the striatum surrounding the hematoma, despite robust and prolonged microglia activation. Importantly, neuron loss at the edge of the hematoma was spatially and temporally associated with accumulation and activation of microglia/macrophages. We then tested the hypothesis that treatment with the tetracycline derivative, minocycline, after the hematoma had reached a maximal size, will reduce inflammation and neuron damage. Minocycline injection (45 mg/kg i.v. at 6 h, and i.p. at 24, 48 and 72 h) failed to reduce neuron loss outside the hematoma or striatal tissue loss (assessed at 7 days), despite reducing the number of neutrophils and activated microglia/macrophages. Thus, minocycline does not appear to target the mechanisms responsible for cell death in this model of ICH.

Ca2+-activated K+ channels in human B lymphocytes and rat thymocytes

1. Previous evidence for the existence of Ca2(+)‐activated K+ channels in lymphocytes comes from measurements using voltage‐sensitive dyes and from tracer flux studies. We have now directly measured these channels in human tonsillar B lymphocytes and rat thymocytes in single‐channel recordings from cell‐attached and excised patches. 2. In cell‐attached recordings, intracellular Ca2+ was raised by either ionomycin or replacement of external Ca2+ following incubation in Ca2(+)‐free medium. Indo‐1 measurements during the Ca2(+)‐replacement technique showed that [Ca2+]i rose from approximately 90 to 260 nM. Both techniques activated two channels of approximately 25 and 8 pS (slope conductance at 0 mV applied, with 140 mM‐K+ in the pipette). Over 90% of patches displayed this activity, indicating a high density of these channels in the membrane. 3. Both channels reversed near the K+ equilibrium potential with either KCl or potassium aspartate in the pipette, when the cells were bathed in normal or high‐K+ saline. Therefore, these channels are selective for K+. 4. The larger channel was studied in more detail. It displayed inward rectification in symmetrical K+ solutions. The open‐channel probability was weakly dependent on membrane potential. 5. Ca2(+)‐dependent K+ channels were also recorded from excised, inside‐out membrane patches. The threshold for activation was 200‐300 nM [Ca2+i]. 6. Patch excision altered some characteristics of IK(Ca). Channels were activated in fewer than 50% of patches and the main conductance level was approximately 34 pS (at ‐80 mV). The duration of single‐channel events was shorter than in cell‐attached patches; kinetic analysis suggested that this was due to the loss of an open state in excised patches. 7. We conclude that B and T lymphocytes have K(+)‐selective channels which are activated by internal [Ca2+] in the physiological range and which will influence the membrane potential during cell activation.