Ischemic stroke elicits a strong neuroinflammatory response characterized by massive microglia activation. However, microglia do not only cause damage by releasing pro-inflammatory cytokines and reactive oxygen species, they can also exert beneficial functions. Similar to macrophages, microglia can assume a classically activated (M1-like) or alternatively activated (M2-like) phenotype. While M2-like microglia are presumably neuroprotective and anti-inflammatory and have been described to peak relatively early in rodent models of ischemic stroke, M1-polarized microglia begin to appear later in the infarct area, especially in the border zone, and expand neuronal injury. An effective anti-inflammatory treatment for stroke should therefore not be a general immunosuppressant but instead suppress microglia in a subtype specific manner by preferentially targeting pro-inflammatory microglia. Our group has a long history of studying K+ channels in the immune system and previously developed small molecule inhibitors for the voltage-gated KV1.3 and the Ca2+-activated KCa3.1 channel as immunomodulators. We recently obtained exciting new data showing that M1 and M2 microglia significantly differ in their K+ channel expression profiles and here propose to test whether KV1.3 blockers can preferentially inhibit M1-like microglia functions and preserve beneficial M2-like functions. We propose to test this therapeutic hypothesis with three interrelated Specific Aims:
Under Aim -1 we will investigate the expression profile and the functional role of K+ channels in cultured M1 and M2 microglia and macrophages.
In Aim -2 we will study microglia in a more ?natural environment? and use organotypic slices exposed to hypoxia/aglycemia or acute slices from Cx3cr1GFP/+ mice subjected to reversible middle cerebral artery occlusion (MCAO) to determine K+ channel expression and function using whole-cell patch-clamp, immunohistochemistry, qPCR and flow cytometry. As part of these experiments we will characterize the time courses of K+ channel and M1 and M2 marker expression and correlate them with brain cytokine profiles and pathology. Parallel immunohistochemical experiments will be performed on brain sections from stroke patients to evaluate K+ channel expression in the context of M1 and M2 markers in humans. Finally, in Aim-3 we are proposing to test our hypothesis that selective targeting of M1-like microglia with KV1.3 blockers is beneficial in ischemic stroke by evaluating the effect of KV1.3 knockout and pharmacological blockade with our KV1.3 blocker PAP-1 in MCAO. These experiments will include studies where PAP-1 administration will match the time-course of the presence of KV1.3 on microglia in the infarct. Overall, we expect that KV1.3 blockade will spare beneficial microglia functions such as phagocytosis of debris and production of neurotrophic factors and preferentially target detrimental pro-inflammatory microglia functions. This strategy could be very beneficial for ischemic stroke but could also be applied to other neuroinflammatory brain disorders, where neuroinflammation is pathologically significant.
Microglia are cells of the immune system, that reside in the brain. Microglia exert both beneficial and detrimental effects following ischemic stroke. With the help of this grant we are going to test the hypothesis that small molecule inhibitors of the voltage-gated potassium channel Kv1.3 preferentially target detrimental inflammatory microglia functions. If our experiments are successful, our work could lead to the development of novel therapeutics for the treatment of stroke.
