Immune regulatory roles for Kv1.3 channels in Microglia in Alzheimer’s Disease

Background In Alzheimer’s Disease (AD), a sub‐population of disease associated microglia present increased Kv1.3 channel abundance. Pharmacological blockade of Kv1.3 reduces Aβ pathology and dampens pro‐inflammatory function of microglia. To identify Kv1.3‐regulated neuroinflammatory mechanisms in microglia, we examined the interactome of Kv1.3 channels in vitro, and Kv1.3 deletion and inhibition in vivo. Methods TurboID, a proximity‐dependent biotin ligase, was fused to the N‐term or C‐term of Kv1.3 and transduced in BV2 microglia. Kv1.3 and TurboID presence and activity were confirmed via qPCR, electrophysiology, flow cytometry, and western blot. Mass spectrometry(MS) of biotinylated proteins, under resting and lipopolysaccharide(LPS)‐treated conditions, identified Kv1.3 interactomes. Kcna3(Kv1.3)‐floxed mice were generated and crossed to CMV‐Cre mice for global Kv1.3 deletion. Three and six month old 5xFAD mice were treated with PAP1, a small molecule intracellular blockade of Kv1.3, for three months. At nine months of age behavioral tests were performed. Aβ pathology was evaluated using ELISA and Immunofluorescence. Brain nuclei were isolated for snRNAseq. CSF, plasma, and brain were processed for MS. Results Proteomics of BV2‐Kv1.3‐TurboID microglia identified distinct N‐term (n = 991) and C‐term Kv1.3 (n = 849) interactors. Kv1.3 N‐term interacts with plasma membrane proteins (e.g. Cars1, Psma2) and mitochondrial trafficking proteins (Timm23). C‐term interactors were modified by LPS‐stimulation (C3, STAT1, Oasl1) (n = 36), and partly dependent on a PDZ‐binding motif (Snx3, ND3, n = 70). Predicted by MS, western blot confirmed mitochondrial Kv1.3 presence. In vivo global Kv1.3 deletion was verified by qPCR. 9month 5xFAD mice with the Kv1.3 blockade show a reduction in Aβ pathology and a rescue of fear conditioning. Brain cell type‐specific and tissue‐level effects of Kv1.3 blockade will be evaluated by snRNAseq and MS. Conclusions We identified N and C‐term domain‐specific and context‐dependent interactors of Kv1.3 channels in microglia, including several pro‐inflammatory C‐term interactors. We developed and validated a novel Kv1.3‐floxed line for global, and conditional, Kv1.3 deletion studies in mouse models of AD pathology. Kv1.3 blockers reduce Aβ pathology and correct behavioral deficits in 5xFAD mice. The transcriptomic and proteomic studies of brain and biofluids from this cohort will provide mechanistic insights and identify novel biomarkers o