In addition to directly causing neuronal damage ischemic stroke elicits a delayed neuroinflammatory response that is characterized by lymphocyte infiltration, hyperthermia and robust microglia activation. """"""""Reactive"""""""" microglia in particular contribute to this """"""""secondary damage"""""""" by producing inflammatory cytokines, reactive oxygen species, NO, and cyclooxygenase-2 reaction products. However, activated microglia might also be neuroprotective by releasing neurotrophic factors and phagocytosing cellular debris. The goal of microgliatargeted therapies therefore should be to reduce the neurotoxic effects of activated microglia while at the same time maintaining their beneficial functions. We here hypothesize, that blockers of the microglial K+ channels Kv1.3 and KCa3.1 might be able to do exactly this based on the preliminary data presented in this application. We previously designed potent and selective small molecule inhibitors for both channels, PAP-1 for Kv1.3 and TRAM-34 for KCa3.1, and demonstrated that these compounds can prevent or treat various autoimmune diseases and inflammatory conditions in rodents such as contact dermatitis, type-1 diabetes, inflammatory bowel disease, atherosclerosis and EAE. More recently we made the exciting observation that our KCa3.1 blocker TRAM-34 reduces infarct area and neurological deficit scores following ischemic stroke in rats even if treatment is commenced 12 hours after reperfusion. Another strong rationale for our study is a report that TRAM-34 does not prevent microglia from phagocytosing damaged neurons but increases the number of surviving retinal ganglion cells following optic nerve transection in rats by reducing the production and/or secretion of neurotoxic molecules in the retina. Taken together with previous work from our laboratory and other groups implicating both Kv1.3 and KCa3.1 in microglia mediated neuronal killing, these results suggest Kv1.3 and KCa3.1 as novel targets for CNS pathologies involving inflammation. With the help of this grant we therefore intend to test the hypothesis that both channels constitute novel targets for the treatment of stroke.
Under Aim -1 we will more rigorously evaluate Kv1.3 and KCa3.1 as targets for stroke by testing the effect of both pharmacological blockade and genetic deletion in reperfusion MCAO and by performing parallel in vitro studies to investigate the role Kv1.3 and KCa3.1 in microglia functions.
Under Aim -2 we will use our expertise in medicinal chemistry to design a less lipophilic and more brain penetrant small molecule Kv1.3 inhibitor than our existing lead compound PAP-1 (IC50 2 nM). We further will resynthesize a brain-penetrant KCa3.1 inhibitor, which was abandoned by Bayer, when the company pulled out of stroke research.
Under Aim -3 we will directly compare the new Kv1.3 and KCa3.1 blockers to minocycline in a 4-week trial by assessing in vivo cytokine production, neurogenesis and functional recovery. As a first step towards translating our findings to humans, we will further obtain brain sections from stroke patients and controls and perform immunohistochemistry for KCa3.1, Kv1.3, and microglia activation markers.
Microglia are cells of the immune system, that reside in the brain, and have been shown to significantly contribute to the delayed inflammatory damage following ischemic stroke. With the help of this grant we will test whether small molecule inhibitors of two potassium channels that are involved in microglia activation constitute therapeutic targets for the treatment of stroke.
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