Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4⁺CCR7⁻CD45RA⁻effector memory T cells (T_EMcells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (T_CM) cells. In T_EMcells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP, p56^lck, and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca²⁺-signaling, cytokine production, and proliferation of autoantigen-specific T_EMcells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

Disease-associated-microglia (DAM) represent transcriptionally-distinct and neurodegeneration-specific microglial profiles with unclear significance in Alzheimer’s disease (AD). An understanding of heterogeneity within DAM and their key regulators may guide pre-clinical experimentation and drug discovery. Weighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to identify modules of highly co-expressed genes. These modules were contrasted with known signatures of homeostatic microglia and DAM to reveal novel molecular heterogeneity within DAM. Flow cytometric validation studies were performed to confirm existence of distinct DAM sub-populations in AD mouse models predicted by WGCNA. Gene ontology analyses coupled with bioinformatics approaches revealed drug targets and transcriptional regulators of microglial modules predicted to favorably modulate neuroinflammation in AD. These guided in-vivo and in-vitro studies in mouse models of neuroinflammation and neurodegeneration (5xFAD) to determine whether inhibition of pro-inflammatory gene expression and promotion of amyloid clearance was feasible. We determined the human relevance of these findings by integrating our results with AD genome-wide association studies and human AD and non-disease post-mortem brain proteomes. WGCNA applied to microglial gene expression data revealed a transcriptomic framework of microglial activation that predicted distinct pro-inflammatory and anti-inflammatory phenotypes within DAM, which we confirmed in AD and aging models by flow cytometry. Pro-inflammatory DAM emerged earlier in mouse models of AD and were characterized by pro-inflammatory genes (Tlr2, Ptgs2, Il12b, Il1b), surface marker CD44, potassium channel Kv1.3 and regulators (NFkb, Stat1, RelA) while anti-inflammatory DAM expressed phagocytic genes (Igf1, Apoe, Myo1e), surface marker CXCR4 with distinct regulators (LXRα/β, Atf1). As neuro-immunomodulatory strategies, we validated LXRα/β agonism and Kv1.3 blockade by ShK-223 peptide that promoted anti-inflammatory DAM, inhibited pro-inflammatory DAM and augmented Aβ clearance in AD models. Human AD-risk genes were highly represented within homeostatic microglia suggesting causal roles for early microglial dysregulation in AD. Pro-inflammatory DAM proteins were positively associated with neuropathology and preceded cognitive decline confirming the therapeutic relevance of inhibiting pro-inflammatory DAM in AD. We provide a predictive transcriptomic framework of microglial activation in neurodegeneration that can guide pre-clinical studies to characterize and therapeutically modulate neuroinflammation in AD.

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4⁺T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedlyin vitroexpressed a unique channel phenotype (“chronically activated”) with large numbers of Kv1.3 voltage-gated channels (≈1500 per cell) and small numbers of IKCa1 Ca²⁺-activated K⁺channels (≈50–120 per cell). In contrast, resting T cells displayed 0–10 Kv1.3 and 10–20 IKCa1 channels per cell (“quiescent” phenotype), whereas T cells stimulated once or twice expressed ≈200 Kv1.3 and ≈350 IKCa1 channels per cell (“acutely activated” phenotype). Consistent with their channel phenotype, [³H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap²²) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap²²and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on thesein vitroresults, we assessed the efficacy of K⁺channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap²²plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap²², but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap²²plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.

Peptides are recognized as being highly selective, potent and relatively safe as potential therapeutics. Peptides isolated from the venom of different animals satisfy most of these criteria with the possible exception of safety, but when isolated as single compounds and used at appropriate concentrations, venom-derived peptides can become useful drugs. Although the number of venom-derived peptides that have successfully progressed to the clinic is currently limited, the prospects for venom-derived peptides look very optimistic. As proteomic and transcriptomic approaches continue to identify new sequences, the potential of venom-derived peptides to find applications as therapeutics, cosmetics and insecticides grows accordingly.

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthusinhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys²² in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap²²) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap²², at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [³H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of ∼200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap²² in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap²² or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.