Presentation Title

The Role of KV1.3 Channels in Neuroinflammation

Faculty Mentor

Dr. Maria Lioudyno, Dr. Jorge Busciglio

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 10:15 AM

Location

9-255

Session

Bio Sciences 1

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

The formation of Noll-like Receptor Protein 3 (NLRP3) inflammasomes, key protein complexes involved in the innate immune response, triggers a signaling cascade that produces mature forms of pro-inflammatory cytokines interleukin IL-1β and IL-18. It has been shown that permeability of the cell membrane to K+ is required for NLRP3-mediated interleukin production, although the type of channel(s) that support potassium efflux in response to lipopolysaccharide (LPS) is (are) still not identified. Given that Kv1.3 channels have long been implicated in immune function, we investigated their role in inflammasome formation using the mouse microglial cell line BV2. The selective peptide blocker of Kv1.3 channels HsTx1 (1) blocked redistribution of one of the key inflammasome proteins, apoptosis-associated speck-like protein containing a CARD (ASC), from the nucleus to the para-nuclear region and the cytoplasm of the cells and (2) significantly inhibited LPS-induced increase in the level of intracellular reactive oxygen species (ROS). These results implicate Kv1.3 channels as candidates for supporting potassium efflux essential for the formation of inflammasome complexes and suggest a role for Kv1.3 channels in LPS-induced ROS production in microglia. The data also provide a rationale for potential therapeutic targeting of the Kv1.3 channels with selective inhibitors (e.g. HsTx1) or their analogs in patients with neurodegenerative diseases that involve neuroinflammation.

Summary of research results to be presented

To investigate the response of the BV2 microglial cells to a pro-inflammatory stimulus, we measured intracellular reactive oxidative species (ROS) levels in cells incubated with or without LPS. Intracellular ROS levels decreased at 1h time point in cells treated with LPS compared to that in untreated cells. In contrast, the ROS level is significantly increased after 6 hours of LPS addition to the media and remained elevated for at least 24 hours compared to untreated control cells. These data indicate that cellular response to LPS is time-dependent and may involve feedback mechanisms that underlie cytokine-dependent sustained ROS production for as long as 24 h.

We next tested whether LPS-induced ROS increase is dependent on Kv1.3 channels activity using a potent and selective inhibitor of the Kv1.3 channels, HsTx-1[R14A]. When HsTx1 was added to cultured cells at 10nM, 100nM, 300nM, 600nM, and 900nM simultaneously with LPS, the LPS effect was significantly suppressed.

We decided to test if HsTx1 affects one of the initial steps in the mechanism of inflammasome formation, the recruitment of nuclear ASC protein to the cytoplasm, by testing whether inhibition of Kv1.3 channels will have an effect on ASC protein level or sub-cellular localization.

ASC immunoreactivity re-distributed from the nucleus towards the para-nuclear region and the cytoplasm upon LPS treatment. The LPS-induced re-distribution of ASC was not observed when the cells were simultaneously treated with both LPS and selective Kv1.3 inhibitor, HsTx1, suggesting a role for Kv1.3 channels in the early events of the formation of inflammasome complexes.

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Nov 18th, 10:00 AM Nov 18th, 10:15 AM

The Role of KV1.3 Channels in Neuroinflammation

9-255

The formation of Noll-like Receptor Protein 3 (NLRP3) inflammasomes, key protein complexes involved in the innate immune response, triggers a signaling cascade that produces mature forms of pro-inflammatory cytokines interleukin IL-1β and IL-18. It has been shown that permeability of the cell membrane to K+ is required for NLRP3-mediated interleukin production, although the type of channel(s) that support potassium efflux in response to lipopolysaccharide (LPS) is (are) still not identified. Given that Kv1.3 channels have long been implicated in immune function, we investigated their role in inflammasome formation using the mouse microglial cell line BV2. The selective peptide blocker of Kv1.3 channels HsTx1 (1) blocked redistribution of one of the key inflammasome proteins, apoptosis-associated speck-like protein containing a CARD (ASC), from the nucleus to the para-nuclear region and the cytoplasm of the cells and (2) significantly inhibited LPS-induced increase in the level of intracellular reactive oxygen species (ROS). These results implicate Kv1.3 channels as candidates for supporting potassium efflux essential for the formation of inflammasome complexes and suggest a role for Kv1.3 channels in LPS-induced ROS production in microglia. The data also provide a rationale for potential therapeutic targeting of the Kv1.3 channels with selective inhibitors (e.g. HsTx1) or their analogs in patients with neurodegenerative diseases that involve neuroinflammation.