Inhibition of Kir4.1 channel elevates BDNF expression in primary cultures of astrocytes

[Speaker] Yukihiro Ohno:1
[Co-author] Masato Kinboshi:1,2,3, Saki Shimizu:1, Takahiro Mukai:1, Yuki Nagao:1, Hidefumi Ito:3, Akio Ikeda:2
1:Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan, 2:Department of Epilepsy, Movement Disorders and Physiology, Graduate School of Medicine, Kyoto University, Japan, 3:Department of Neurology, Wakayama Medical University, Japan

Background: Inwardly rectifying potassium (Kir) 4.1 channels are specifically expressed in astrocytes and regulate neuronal excitability by modulating the spatial potassium buffering activity. It is now known that loss-of-function mutations of the gene encoding Kir4.1 or down-regulation of Kir4.1 expression causes neural hyperexcitation and generalized tonic-clonic seizures both in patients and animal models, which probably results from the elevated extracellular potassium and glutamate levels due to the disruption of spatial potassium buffering. However, pathophysiological role and mechanism of astrocytic Kir4.1 dysfunction in the development of chronic seizures (epileptogenensis) are still unknown. In this study, we evaluated the effects of Kir4.1 dysfunction (blockade and knockdown of Kir4.1 channels) on the expression of BDNF, a key molecule mediating epileptogenensis, in astrocyte primary cultures.
Methods: Primary cultures of cerebrocortical astrocytes were prepared from 1 or 2 day-old new born ICR mice. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner (Brain Res., 1178, 44-51, 2007; Mol. Pharmacol., 75, 1287-1295, 2009). For knock-down of Kir4.1 channels, small interfering RNA (siRNA) targeting Kir4.1 was transfected into astrocytics.
Results: Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of Kir4.1 siRNA significantly increased the mRNA and protein levels of BDNF. The BDNF induction by the Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but only slightly by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125.
Conclusions: Our results show that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may lead to the development of epilepsy and other neuropsychiatric disorders.
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