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.

中文翻译：

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白细胞介素 10 在 Sip1 转录因子缺失的情况下恢复脑回路中谷氨酸受体介导的 Ca2+ 信号传导

摘要

客观的

本研究旨在探讨Sip1转录因子的突变与受损的 Ca ^{2+信号传导之间的联系，这反映}了体外大脑皮层神经传递的变化。

方法

我们使用来自Sip1突变小鼠的混合神经胶质皮质细胞培养物。细胞装有荧光比例钙敏感探针 Fura-2 AM，并通过从外部介质中排除镁离子或添加 GABA(A) 受体拮抗剂荷包牡丹碱来模拟癫痫样活动。使用荧光显微镜记录细胞内钙动力学。为了确定基因表达水平，使用了实时 PCR 方法。

结果

发现从具有Sip1突变的纯合子 ( Sip1 ^fl/fl ) 小鼠中分离出的皮质神经元在体外癫痫样活动模型中表现出抑制的 Ca ²⁺信号。野生型皮层神经元的特点是响应于无 Mg ²⁺培养基和荷包牡丹碱，在网络的所有神经元中发生同步的高频和高振幅 Ca ^2+振荡。但皮质Sip1 ^fl/fl神经元只有单个 Ca ²⁺脉冲或减弱的 Ca ²⁺振荡只记录在单个神经元中，而大多数细胞网络对这些刺激没有反应。Sip1 ^fl/fl神经元的这种信号缺陷与编码 NMDA、AMPA 和 KA 受体亚基的基因表达水平受到抑制有关。蛋白激酶 PKA、JNK、CaMKII；以及转录因子 Hif1α。当Sip1 ^fl/fl神经元在具有抗炎细胞因子 IL-10 的培养基中生长时，这些负面影响被部分消除。IL-10 增加上述基因的表达，但不增加野生型的表达水平。同时，Ca ^2+的幅值信号响应于 NMDA、AMPA 和 KA 受体的选择性激动剂而增加，并且响应于 Ca ²⁺振荡对无 Mg ²⁺培养基和荷包牡丹碱的神经元的比例增加。

结论

IL-10通过调节编码信号蛋白的基因的表达来恢复具有Sip1突变的神经元网络中的神经传递。