CC BY
15
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
ОМ&Р
Section MOLECULAR NEUROSCIENCE
It is known that a mutation of Sipl gene leads to disruption of normal development and as consequence to distort functioning of cortex [1] and hippocampus [1] of the brain. Sip1 mutation physiologically expresses in dysregulation of neurogenesis [2], an increases the risk of developing of epilepsy [3], and mental retardation [4]. The aim of this research is to analyze effect of the mutation in transcription factor Sipl on receptor-mediated component of calcium signaling in cortical neurons.
Cells were loaded by calcium-sensitive probe Fura-2 for registration of cytosolic calcium levels. Fluorescence detection was performed with help of an image analysis system «Cell observer» (Carl Zeiss, Germany), on the basis of inverted motorized microscope Axiovert 200M. As the object of research we used primary cultures of cells from cortex and brainstem of SIP1 mutant mice (p1-p2) [5,6]. Experiments were performed on 8th day of culturing of neurons, when culture has well-developed neural network. Neurons distinguished from astroglia by presence of Ca2+ response on depolarization by KCl application. The main measuring parameter was amplitude of Ca2+ -response of neurons on short-term application of agonists of ionotropic glutamate receptors.
Sequential application of activators of NMDA- (fig. la) and AMPA-receptors (fig. lb) causes a transient increase in cytosolic Ca2+ in both neurons from wild-type mice and in cells of homo- and heterozygous mutant Sipl mice. In this case, the amplitude of the Ca2+-signal on NMDA and FW is significantly lower in neurons of Siplfl/fl mice than in neurons of Sip1fl/wt and wild type mice (Sip1wt/wt). In neurons derived from brainstem of homozygous mutant Sipl mice (control for the cortex) amplitude of Ca2+-response to NMDA and FW are almost the same (not shown) as in neurons of wild type mice and heterozygote because this part of the brain is not damage by directed mutagenesis.
Diagrams of dependence of dose that was built for activators of NMDA- and AMPA-receptors shown that Sip1£l/£l-neurons characterize by considerable resistance (fig. lc and d), and the EC 50 value is 425 and l88 nM, respectively. Whereas for heterozygous mutant neurons (Siplfl/wt) characterize on the contrary by increase of sensitivity to NMDA activators (83 nM) and AMPA receptors (8,4 nM), relative to wild-type neurons whose EC50 for NMDA and FW is 96 and l8 nM, respectively.
Fig.1. Characteristics of NMDA- и AMPA-receptors activity in cortical neurons Sipl homozygotes (Siplfl/fl), heterozygotes (Siplfl/wt) and wild type mice (Siplwt/wt). a - Ca2+- response of neurons on sequential increasing of concentration of NMDA-receptors (N-methyl-D-aspartate) activator in surrounding without magnesium. b. - Ca2+- response of neurons on sequential increasing of concentration of AMPA-receptors (5-fluorow-illardiine, FW) activator in a sign of inhibitor of agonist dependent desensitization of receptors (Cyclothiazide, CTZ, 5^M). c - Dependence of the amplitude of Ca2+- response of neurons from concentration of NMDA. d - Dependence of the amplitude of Ca2+- response of neurons from concentration ofFW. NMDA concentrations: 1 - 0.001 рМ, 2 - 0.01 рМ, 3 - 0.1 рМ, 4 - 0.3 рМ, 5 - 05 рМ, 6 -1рМ, 7-3 рМ, 8 -5 рМ, 9 -10 рМ,10 -30 рМ,11 -50 рМ,12 -100 рМ. Концентрации FW: 1 - 0.001 рМ, 2 - 0.005 рМ, 3 - 0.01 рМ, 4 - 0.03 рМ, 5 - 0.05 рМ, 6 - 0.1 рМ, 7 - 0.5 рМ, 8 -1 рМ, 9 - 3 рМ, 10 - 5 рМ, 11 -10 рМ.
Thereby Sip1 mutation changes the activity of basic ionotropic glutamate receptors of cortical neurons but does not change in brainstem. Besides in neurons of homozygotes these changes express in common signal insufficiency, and contrariwise in neurons of heterozygotes was shown the increasing sensitivity to NMDA- and AMPA-receptors activators. These changes in receptors activity can lead to faster activation of neuronal pathways, excitotoxicity and induction of cell death processes under moderate activity (short-time hypoxia) on physiological level [6].
Acknowledgements
This work was supported by the Russian Science Foundation (project no. l5-l4-l002l).
Section MOLECULAR NEUROSCIENCE References
1. A. Miquelajauregui, T. Van de Putte, A. Polyakov et al., PNAS, 2007, 104(31): 12919-12924
2. E. Seuntjens, A. Nityanandam, A. Miquelajauregui et al., Nature Neuroscience, 2009, 12, 1373 - 1380
3. G. L. McKinsey, S. Lindtner, B. Trzcinski, et al., Neuron, 2013, 77(1), 83-98
4. D. Mowat, M. Wilson, and M. Goossens, J. Med. Genet., 2003, 40(5): 305-310
5. S. Srivatsa, S. Parthasarathy, Z. Molnâr et al., Neuron, 2015, 85(5):998-1012
6. E. A. Turovsky, M. V. Turovskaya, A. V. Kononov et al., Exp. Neurol., 2013, 250:1-7
Multiplex Approach in Depressive Disorders Research
AS. Boiko*, LS. Losenkov, L.A. Levchuk, G.G. Simutkin, S.A. Ivanova
Mental Health Research Institute, Tomsk, Russia. * Presenting e-mail: [email protected]
Aims
Depressive disorders, with a lifetime prevalence of over 15%, is a great burden for both patients and the society. Depressive disorders is also increasingly implicated in a wide range of other medical conditions. As yet a reliable (biological) diagnostic marker for depressive disorders does not exist. Nevertheless, an increasing body of evidence indicates that the underlying neurobiology of depression likely involves a complex interplay of various factors. Thus far, research into single biomarkers for depression was not very successful. Arguably, a combination of biomarkers reflecting the various neurobiological disturbances in depression might enable a more specific and selective diagnosis of depressive disorder.
Aim of the study was to investigate levels of the following substances in the serum of patients with depressive disorders and healthy subjects: ß-endorphin, orexin A, cortisol, melatonin, insulin-like growth factor 1 (IGF-1), interferon y (INF-y), interleukin 1ß (IL-1ß), interleukin 3 (IL-3), interleukin 6 (IL-6), tumor necrosis factor a (TNF-a).
Materials and methods
78 patients (46 patients with depressive episode (ICD-10: F32), 32 patients with recurrent depressive disorder (ICD-10: F33)) were included in the study. Control group consisted of 71 physically and mental healthy persons. ß-endorphin, orexin A, cortisol, melatonin, IGF-1, INFy, IL-1ß, IL-3, IL-6, TNFa were measured in the serum using Luminex xMAP® technology. Concentrations of investigated substances were measured by MAGPIX apparatus (Merck Millipore). Statistical analyses were performed using the SPSS software, release 20.0, for Windows. Results were expressed as median (25% quartile — 75% quartile). Between-group differences were evaluated using the Mann - Whitney U-test. p values less than 0.05 were considered as significant.
Results
Our study showed significantly higher concentration of cortisol in the group of patients with depressive disorders (214.17 (155.51 - 315.19) ng/ml) compare to control group (168.48 (121.61 - 263.34) ng/ml), (p=0.037). We found no difference between groups in concentration of neuropeptides and hormones, such as ß-endorphin, orexin A, me-latonin and IGF-1. Concentration of ß-endorphin in control group was 252.28 (136.90 - 366.84) pg/ml, in group of depressive patients concentration was 259.26 (204.06 - 314.47) pg/ml., (p=0.436); concentration of orexin A in control group was 614.13 (379.25 - 891.34) pg/ml, in group of depressive patients concentration was 687.42 (504.65 - 837.34) pg/ml., (p=0.394); concentration of IGF-1 in control group was 8.75 (5.90 - 20.75) ng/ml, in group of depressive patients concentration was 6.26 (4.52 - 13.57) ng/ml., (p=0.101); concentration of melatonin in control group was 46.15 (27.55 - 77.86) pg/ml, in group of depressive patients concentration was 66.30 (32.59 - 132.59) pg/ml., (p=0.071). No difference between groups in concentration of cytokines INF-y, IL-1b, IL-3 and TNF-a except level of IL-6 was observed. Concentration of IL-6 in the group of depressive patients was significantly higher (3.79 (2.90 - 5.63) pg/ml) compare to control group (3.22 (2.67 - 4.02) pg/ml) (p=0.002).
OM&P
