Changes in Calcium Homeostasis and Gene Expression Implicated in Epilepsy in Hippocampi of Mice Overexpressing <i>ORAI1</i>

• Main Authors: Lukasz Majewski, Bartosz Wojtas, Filip Maciąg, Jacek Kuznicki
• Format: Article
• Language: English
• Published: MDPI AG 2019-11-01
• Series: International Journal of Molecular Sciences
• Subjects: epileptic seizures; orai1; stim1; stim2; nsoce; rnaseq; rt-pcr; cdkl5; arx; dclk1
• Online Access: https://www.mdpi.com/1422-0067/20/22/5539

Previously, we showed that the overexpression of <i>ORAI</i><i>1</i> calcium channel in neurons of murine brain led to spontaneous occurrence of seizure-like events in aged animals of transgenic line FVB/NJ-Tg(ORAI1)Ibd (Nencki Institute of Experimental Biology). We aimed to identify the mechanism that is responsible for this phenomenon. Using a modified Ca²⁺-addback assay in the CA1 region of acute hippocampal slices and FURA-2 acetomethyl ester (AM) Ca²⁺ indicator, we found that overexpression of <i>ORAI1</i> in neurons led to altered Ca²⁺ response. Next, by RNA sequencing (RNAseq) we identified a set of genes, whose expression was changed in our transgenic animals. These data were validated using customized real-time PCR assays and digital droplet PCR (ddPCR) ddPCR. Using real-time PCR, up-regulation of hairy and enhancer of split-5 (<i>Hes-5</i><i>)</i><i> </i>gene and down-regulation of aristaless related homeobox<i> (</i><i>Arx</i><i>)</i>, doublecortin-like kinase 1 (<i>Dclk1</i><i>),</i> and cyclin-dependent kinase-like 5 (<i>Cdkl5</i>, also known as serine/threonine kinase 9 (Stk9)) genes were found. Digital droplet PCR (ddPCR) analysis revealed down-regulation of <i>Arx</i>. In humans, <i>ARX</i>, <i>DCLK1</i><i>,</i> and <i>CDLK5</i> were shown to be mutated in some rare epilepsy-associated disorders. We conclude that the occurrence of seizure-like events in aged mice overexpressing <i>ORAI1</i> might be due to the down-regulation of<i> </i><i>Arx</i><i>, </i>and possibly of <i>Cdkl</i>5 and <i>Dclk1</i> genes.