MicroRNA-92 modulates K(+) Cl(-) co-transporter KCC2 expression in cerebellar granule neurons.
MicroRNAs have been associated to fine-tuning spatial and temporal control of gene expression during neuronal development. The neuronal Cl(-) extruding, K(+)Cl(-) co-transporter 2 (KCC2) is known to play an important role in neuronal Cl(-) homeostasis and in determining the physiological response to activation of anion selective GABA receptors. Here we show that microRNA-92 is developmentally down-regulated during maturation of rat cerebellar granule neurons (CGNs) in vitro. Computational predictions suggest several high-ranking targets for microRNA-92 including the KCC2 gene. Consistently, the KCC2 protein levels were up-regulated in mature CGN in vitro and a functional association between microRNA-92 and KCC2 3' untranslated region was established using luciferase assays. The generation of an inward directed Cl(-) electrochemical gradient, necessary for the hyperpolarizing effect of GABA, requires robust KCC2 expression in several neuronal types. Here we show that lentiviral-mediated microRNA-92 over-expression reduced KCC2 protein levels and positively shifted reversal potential of GABA induced Cl(-) currents in CGNs. In addition KCC2 re-expression reversed microRNA-92 electrophysiological phenotype. Consistently microRNA-92 inhibition induced both an increase of the level of KCC2 and a negative shift in GABA reversal potential. These findings introduce a new player in the developmental change of GABA from depolarization to hyperpolarization.
Barbato
Christian
C
EBRI - European Brain Research Institute - Fondazione EBRI - Rita Levi-Montalcini, Rome, Italy.
Ruberti
Francesca
F
Pieri
Massimo
M
Vilardo
Elisa
E
Costanzo
Manuela
M
Ciotti
Maria Teresa
MT
Zona
Cristina
C
Cogoni
Carlo
C
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism; D004594:Electrophysiology; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D027981:Symporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D005680:gamma-Aminobutyric Acid / Q000502:physiology; D000096922:K Cl- Cotransporters
D020413:3' Untranslated Regions; D008156:Luciferases; D035683:MicroRNAs; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters