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Section MOLECULAR NEUROSCIENCE Methods
To study effects of calcineurin inhibition on the KCC2 activity cyclosporine was applied in vivo or in rat brain slices. The evaluations were performed using immunofluorescence, immunoblotting and co-immunoprecipitation, quantitative PCR, and electrophysiological measurements with sharp microelectrode.
Results
Calcineurin and KCC2 co-localized in rat neocortical neurons by immunofluorescence and interacted by co-immuno-precipitation. Short term administration of cyclosporine to rats (25 mg/kg for 1 to 4h) resulted in increased tyrosine phosphorylation of KCC2 suggesting inhibition of its activity. In line with this, intracellular recordings of chloride homeostasis after iontophoretic Cl- loading revealed strong cyclosporine-induced prolongation of the Cl- extrusion time (+3.4s, p<0.05) which was compatible with KCC2 blockade. Chronic administration of cyclosporine to rats (5 to 25 mg/kg for 14 days) drastically reduced the level of activating KCC2 phosphorylation at S940 (-65%, p<0.05). In addition, expression of the KCC2-inhibiting SPAK kinase was significantly increased upon chronic cyclosporine treatment (+59%, p<0.05).
Conclusions
In summary, our data suggest that calcineurin inhibition using cyclosporine attenuates KCC2 function in acute and chronic settings. Our data have clinical implications for immunosuppressive therapy using calcineurin inhibitors.
Lack of Diap3 Relaxes the Spindle Checkpoint Causing the Loss of Neural Progenitors and Microcephaly
Fadel Tissir*
University of Louvain, Belgium. * Presenting e-mail: [email protected]
The diaphanous homologue Diap3 (also referred to as mDia2, DIAPH3 in humans) is a major regulator of actin dynamics. Loss of Diap3 has been constantly associated with cytokinesis failure ascribed to impaired accumulation of F-actin in the cleavage furrow. We provide evdence that Diap3 is required prior to cell fission to ensure the accurate segregation of chromosomes. In mice, inactivation of the Diap3 gene causes a massive loss of neural progenitor cells with subsequent reduction in the number of intermediate progenitors and neurons, and ultimately results in microcephaly. Mechanistically, we show that Diap3 co-localises with chromosomal passenger complex (CPC) proteins at the kinetochore-mitotic spindle interface, and interacts physically with BubRl and Survivin, components of the spindle assembly checkpoint (SAC) and CPC respectively. Diap3-deficient neural progenitor cells have decreased levels of BubRl, and fail to properly distribute CPC proteins, or activate the SAC. Hence, they bypass the mitotic arrest and embark on anaphase in spite of incorrect chromosome segregation which causes their apoptotic death. These findings identify Diap3 as an important guard of cortical progenitors, shed new light on the mechanisms of action of Diaphanous formins during cell division, and add insights into the pathobiology of primary microcephaly
Sip 1-Mutahon Causes a Disturbance in Activity of NMDA- and AMPA-Receptors of Neurons in Cerebral Cortex
M.V. Turovskaya*, A.A. Babaev, E.A. Turovsky, V.S. Tarabykin
Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod. * Presenting e-mail: [email protected]
OM&P
Opera Med Physiol 2016 Vol. 2 (S1) 31
