Interleukin-10 restores glutamate receptor-mediated Ca2+-signaling in brain circuits under loss of Sip1 transcription factor

Abstract

Objective: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca²⁺-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro.

Methods: We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used.

Results: It was found that cortical neurons isolated from homozygous (Sip1^fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca²⁺ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca²⁺ oscillations occurring in all neurons of the network in response to Mg²⁺-free medium and bicuculline. But cortical Sip1^fl/fl neurons only single Ca²⁺ pulses or attenuated Ca²⁺ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1^fl/fl neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1^fl/fl neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca²⁺ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca²⁺ oscillations to a Mg²⁺-free medium and bicuculline increases.

Conclusion: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.

Keywords: Sip1; epileptiform activity; glutamate receptors; interleukin-10; neurons; protective genes.