The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro.
BACKGROUND
Small-conductance Ca2+ activated K+ channels are expressed in the CNS, where KCNN2/SK2/KCa2.2 and KCNN3/SK3/KCa2.3 help shape the electrical activity of some neurons. The SK3 channel is considered a potential therapeutic target for diseases and disorders involving neuron hyper-excitability but little is known about its expression and roles in non-neuronal cells in either the healthy or damaged CNS. The purpose of this study was to examine expression of KCNN3/SK3 in CNS microglia in vivo and in vitro, and to use an established in vitro model to determine if this channel contributes to the neurotoxic capacity of activated microglia.


METHODS
KCNN3 mRNA (real-time RT-PCR) and SK3 immunoreactivity were examined in rat microglia. Lipopolysaccharide was then used to activate microglia (monitored by iNOS, nitric oxide, activation of NF-kappaB and p38 MAPK) and transform them to a neurotoxic state. Microglia-mediated neuron damage (TUNEL, activated caspase 3) and nitrotyrosine levels were quantified using a two-chamber system that allowed microglia to be treated with channel blockers, washed and then added to neuron/astrocyte cultures. Contributions of SK3 to these processes were discriminated using a subtractive pharmacological approach with apamin and tamapin. ANOVA and post-hoc tests were used to assess the statistical significance of differences between treatment groups. SK3 immunoreactivity was then compared in the normal and damaged adult rat striatum, by injecting collagenase (a hemorrhagic stroke) or endothelin-1 (a transient ischemic stroke).


RESULTS
KCNN3 mRNA was prevalent in cultured microglia and increased after lipopolysaccharide-induced activation; SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons. SK3 immunoreactivity was prevalent in cultured microglia and throughout the adult rat striatum (except white matter tracts). After strokes, SK3 was highly expressed in activated microglia/macrophages within the lesions, but reduced in other cells.


CONCLUSIONS
SK3 is expressed in microglia in both the healthy and damaged adult striatum, and mechanistic in vitro studies show it contributes to transformation of microglia to an activated neurotoxic phenotype. Thus, SK3 might be a therapeutic target for reducing inflammation-mediated acute CNS damage. Moreover, its roles in microglia must be considered when targeting this channel for CNS diseases, disorders and reducing neuron hyper-excitability.
Schlichter
Lyanne C
LC
Genes and Development Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario, M5T 2S8, Canada. schlicht@uhnres.utoronto.ca
Kaushal
Vikas
V
Moxon-Emre
Iska
I
Sivagnanam
Vishanthan
V
Vincent
Catherine
C
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001030:Apamin / Q000494:pharmacology; D001921:Brain / Q000166:cytology; D039481:CD11b Antigen / Q000378:metabolism; D002478:Cells, Cultured; D002543:Cerebral Hemorrhage / Q000378:metabolism / Q000473:pathology; D018920:Coculture Techniques / Q000379:methods; D003342:Corpus Striatum / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D020521:Stroke / Q000378:metabolism / Q000473:pathology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D039481:CD11b Antigen; C496901:Kcnn3 protein, rat; D008070:Lipopolysaccharides; D016328:NF-kappa B; D026902:Potassium Channel Blockers; D012333:RNA, Messenger; D051640:Small-Conductance Calcium-Activated Potassium Channels; D001030:Apamin; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D048051:p38 Mitogen-Activated Protein Kinases