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Microglial SK3 and SK4 currents and activation state are modulated by the neuroprotective drug, riluzole
B-S Liu1, R Ferreira, S Lively
1Toronto Western Research Institute, University Health Network, MC9-417, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada.
Abstract:
Microglia monitor the CNS for 'danger' signals after acute injury, such as stroke and trauma, and then undergo complex activation processes. Classical activation of microglia can produce neurotoxic levels of glutamate and immune mediators (e.g., pro-inflammatory cytokines, reactive oxygen and nitrogen species), while alternative activation up-regulates anti-inflammatory molecules and is thought to resolve inflammation and protect the brain. Thus, pharmacological strategies to decrease classical- and/or promote alternative activation are of interest. Here, we assessed actions of the neuroprotective drug, riluzole, on two Ca(2+)- activated K channels in microglia - SK3 (KCa2.3, KCNN3) and SK4 (KCa3.1, KCNN4) - and on classical versus alternative microglial activation. Riluzole is used to treat amyotrophic lateral sclerosis, and is in clinical trials for several other CNS disorders, where it has been presumed to target neurons and reduce glutamate-mediated toxicity. We show that simply elevating intracellular Ca(2+) to micromolar levels in whole-cell recordings does not activate SK channels in a cell line derived from primary rat microglia (MLS-9). In intact cells, riluzole raised cytoplasmic Ca(2+), but it was marginal (~200 nM) and transient (2 min). Surprisingly then, in whole cell recordings, riluzole rapidly activated SK3 and SK4 channels for as long as it was present, and did not require elevated intracellular Ca(2+). We then used primary rat microglia to analyze expression of several activation markers and inflammatory mediators. Riluzole decreased classical LPS-induced activation, and increased some aspects of IL-4-induced alternative activation. These actions on microglia suggest an additional mechanism underlying the neuroprotective actions of riluzole.
Insights
The neuroprotective drug riluzole directly activates microglial SK3 and SK4 channels, independent of calcium levels. This action shifts microglial activation away from harmful classical responses towards beneficial alternative pathways, suggesting a novel mechanism for riluzole
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglia are key immune cells in the central nervous system (CNS) that respond to injury through classical or alternative activation states.
- Classical microglial activation can lead to neurotoxicity, while alternative activation is associated with resolving inflammation and brain protection.
- Pharmacological modulation of microglial activation is a therapeutic target for CNS disorders.
Purpose of the Study:
- To investigate the effects of the neuroprotective drug riluzole on microglial SK3 and SK4 calcium-activated potassium channels.
- To determine if riluzole modulates classical versus alternative microglial activation pathways.
Main Methods:
- Whole-cell patch-clamp recordings were used to assess SK3 and SK4 channel activity in a microglial cell line (MLS-9) and primary rat microglia.
- Riluzole's effects on intracellular calcium levels were measured in intact cells.
- Expression of microglial activation markers and inflammatory mediators was analyzed following riluzole treatment in the presence of classical (LPS) or alternative (IL-4) stimuli.
Main Results:
- Riluzole rapidly activated SK3 and SK4 channels in microglia without requiring elevated intracellular calcium.
- Riluzole caused a marginal and transient increase in intracellular calcium in intact cells.
- Riluzole decreased lipopolysaccharide (LPS)-induced classical microglial activation and enhanced certain aspects of interleukin-4 (IL-4)-induced alternative activation.
Conclusions:
- Riluzole directly targets and activates microglial SK3 and SK4 channels, representing a novel mechanism of action.
- Riluzole's ability to modulate microglial activation states may contribute to its neuroprotective effects.
- These findings suggest riluzole as a potential therapeutic agent for CNS conditions by influencing microglial immune responses.
