Blockade of Microglial Kv1.3 Potassium Channels by the Peptide HsTX1[R14A] Attenuates Lipopolysaccharide-mediated Neuroinflammation

Nicolazzo, Joseph A.; Pan, Yijun; Stefano, Ilenia Di; Choy, Kwok Ho Christopher; Reddiar, Sanjeevini Babu; Low, Yi Ling; Wai, Dorothy C.C.; Norton, Raymond S.; Jin, Liang

doi:10.1016/j.xphs.2021.11.003

Cited by 13 publications

(12 citation statements)

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“…We next investigated whether Cy5–HsTX1[R14A] could be used to visualize K V 1.3 at more physiologically relevant expression levels. Microglia are known to upregulate K V 1.3 in neuroinflammatory disorders such as Alzheimer’s and Parkinson’s diseases, , as well as upon exposure to pro-inflammatory stimuli such as lipopolysaccharide (LPS) derived from Gram-negative bacterial membranes . We therefore examined the staining of BV-2 cells (immortalized murine microglia) by Cy5–HsTX1[R14A] in the absence or presence of LPS stimulation.…”

Section: Resultsmentioning

confidence: 99%

“…Microglia are known to upregulate K V 1.3 in neuroinflammatory disorders such as Alzheimer's and Parkinson's diseases, 7,8 as well as upon exposure to pro-inflammatory stimuli such as lipopolysaccharide (LPS) derived from Gram-negative bacterial membranes. 51 We therefore examined the staining of BV-2 cells (immortalized murine microglia) by Cy5−HsTX1[R14A] in the absence or presence of LPS stimulation. The amoeboid morphology of the cells in the presence of LPS (Figure 8B, top left panel) compared with rounder unstimulated cells (Figure 8A, top left panel) indicated successful microglial activation.…”

Section: Synthesis and Biophysical Characterization Of Cy5− Hstx1[r14a]mentioning

confidence: 99%

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A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3

et al. 2022

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Upregulation of the voltage-gated potassium channel K V 1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of K V 1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds K V 1.3 with high affinity (IC 50 of 45 pM) and selectivity (2000-fold for K V 1.3 over K V 1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5−HsTX1[R14A] retains activity against K V 1.3 (IC 50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (K.1 and K Ca 3.1), as well as selectivity against heteromeric channels assembled from K V 1.3/K V 1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged K V 1.3 shows co-localization of Cy5−HsTX1[R14A] and K V 1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5−HsTX1[R14A] can detect K V 1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of K V 1.3, was associated with increased staining by Cy5−HsTX1[R14A], demonstrating that it can be used to identify K V 1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5−HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5−HsTX1[R14A] as a tool for visualizing K V 1.3, with broad applicability in fundamental investigations of K V 1.3 biology, and the validation of novel disease indications where K V 1.3 inhibition may be of therapeutic value.

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Section: Resultsmentioning

confidence: 99%

Section: Synthesis and Biophysical Characterization Of Cy5− Hstx1[r14a]mentioning

confidence: 99%

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3

et al. 2022

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“…We performed these studies in mice to complement previous studies by our group that showed that subcutaneous administration of HsTX1[R14A] can attenuate the LPSmediated increase in plasma and brain levels of proinflammatory mediators following either immediate or delayed subcutaneous administration of HsTX1[R14A]. 21 In this study, we compared the biodistribution of the [ 64 Cu]Cu-labeled conjugate of DOTA-HsTX1[R14A] (IC 50 ∼ 397 pM, Figure 2A) with that of [ 64 Cu]Cu-DOTA-HsTX1-[R14A,Y21A,K23A] (IC 50 ∼ 6 μM, Figure 2C) and [ 64 Cu]Cuacetate in healthy mice up to 48 h. Of the available positronemitting radionuclides, 64 Cu was the most practical isotope ] was significantly higher than that of [ 64 Cu]Cu-acetate at 6 h postinjection, reflecting the rapid accumulation and subsequent clearance of these cationic peptides in the kidney (Figure 4). The mechanism of cortical retention has been most thoroughly described for octreotide.…”

Section: Discussionmentioning

confidence: 95%

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation

et al. 2022

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The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer’s and Parkinson’s diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 μM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood–brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

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“…It is pertinent that lipopolysaccharide(LPS)-induced microglial activation, which transforms into classical ("M1") phenotype, has been shown to require the participation of microglial Kv1.3 (12,26,27,33). In mice genetically devoid of Kv1.3, LPS failed to activate microglia, or produce neuroin ammation (34). Progranulin could inhibit LPS-induced macrophage M1 polarization via NF-кB and MAPK signaling pathways (35).…”

Section: Discussionmentioning

confidence: 99%

Spinal voltage‐gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome

Yuan

Han

Manyande

et al. 2022

European Journal of Pain

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Background: Studies have shown that activation of microglia is the main mechanism of neuropathic pain. Kv1.3 channel is a novel therapeutic target for treating neuroin ammatory disorders due to its crucial role in subsets of microglial cells. As such, it may be involved in the processes of neuropathic pain, however, whether Kv1.3 plays a role in neuroin ammation following peripheral nerve injury is unclear.Methods: The spared nerve injury model (SNI) was used to establish neuropathic pain. Western blot and immuno uorescence were used to examine the effect of Kv1.3 in the SNI rats. PAP-1, a Kv1.3 speci c blocker was administered to alleviate neuropathic pain in the SNI rats.Results: Neuropathic pain and allodynia occurred after SNI, the levels of M1 (CD68, iNos) and M2 (CD206, Arg-1) phenotypes were up-regulated in the spinal cord, and the protein levels of NLRP3, caspase-1 and IL-1β were also increased. Pharmacological blocking of Kv1.3 with PAP-1 alleviated hyperpathia induced by SNI. Meanwhile, intrathecal injection of PAP-1 reduced M1 polarization and decreased NLRP3, caspase-1, and IL-1β expressions of protein levels.Conclusion: Our research indicates that the Kv1.3 channel in the spinal cord contributes to neuropathic pain by promoting microglial M1 polarization and activating the NLRP3 in ammasome.

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Blockade of Microglial Kv1.3 Potassium Channels by the Peptide HsTX1[R14A] Attenuates Lipopolysaccharide-mediated Neuroinflammation

Cited by 13 publications

References 56 publications

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation

Spinal voltage‐gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome

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Blockade of Microglial Kv1.3 Potassium Channels by the Peptide HsTX1[R14A] Attenuates Lipopolysaccharide-mediated Neuroinflammation

Cited by 13 publications

References 56 publications

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation

Spinal voltage‐gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome

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A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3