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

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 (Sip1fl/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 Sip1fl/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 Sip1fl/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 Sip1fl/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.

Mutation in the Sip1 transcription factor leads to a disturbance of the preconditioning of AMPA receptors by episodes of hypoxia in neurons of the cerebral cortex due to changes in their activity and subunit composition. The protective effects of interleukin-10.

The Sip1 mutation plays the main role in pathogenesis of the Mowat-Wilson syndrome, which is characterized by the pronounced epileptic symptoms. Cortical neurons of homozygous mice with Sip1 mutation are resistant to AMPA receptor activators. Disturbances of the excitatory signaling components are also observed on such a phenomenon of neuroplasticity as hypoxic preconditioning. In this work, the mechanisms of loss of the AMPA receptor's ability to precondition by episodes of short-term hypoxia were investigated on cortical neurons derived from the Sip1 homozygous mice. The preconditioning effect was estimated by the level of suppression of the AMPA receptors activity with hypoxia episodes. Using fluorescence microscopy, we have shown that cortical neurons from the Sip1fl/fl mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca2+-responses to the application of the AMPA receptor agonist, 5-Fluorowillardiine, in neurons from the Sip1 mice brainstem is suppressed by brief episodes of hypoxia. The mechanism responsible for this process is hypoxia-induced desensitization of the AMPA receptors, which is absent in the cortex neurons possessing the Sip1 mutation. However, the appearance of preconditioning in these neurons can be induced by phosphoinositide-3-kinase activation with a selective activator or an anti-inflammatory cytokine interleukin-10.

Sip-1 mutations cause disturbances in the activity of NMDA- and AMPA-, but not kainate receptors of neurons in the cerebral cortex.

Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b), Zeb2] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome (MWS) in humans. MWS is a rare genetic autosomal dominant disease caused by a mutation in the Sip1 gene (aka Zeb2 or Zfhx1b) mapped to 2q22.3 locus. MWS affects 1 in every 50-100 newborns worldwide. It is characterized by mental retardation, small stature, typical facial abnormalities as well as disturbances in the development of the cardio-vascular and renal systems as well as some other organs. Sip1 mutations cause abnormal neurogenesis in the brain during development as well as susceptibility to epileptic seizures. In the current study we investigated the role of the Sip1 gene in the activity of NMDA-, AMPA- and KA- receptors. We showed that a particular Sip1 mutation in the mouse causes changes in the activity of both NMDA- and AMPA- receptors in the neocortical neurons in vitro. We demonstrate that neocortical neurons that have only one copy of Sip1 (heterozygous, Sip1fI/wt), are more sensitive to both NMDA- and AMPA- receptors agonists as compared to wild type neurons (Sip1wt/wt). This is reflected in higher amplitudes of agonist induced Ca2+ signals as well as a lower half maximal effective concentration (ЕC50). In contrast, neurons from homozygous Sip1 mice (Sip1fI/fI), demonstrate higher resistance to these respective receptor agonists. This is reflected in lower amplitudes of Ca2+-responses and so a higher concentration of receptor activators is required for activation.