No abstract

The studies of signaling mechanisms involved in the disruption of the cytoskeleton homeostasis were performed in a model of quinolinic acid (QUIN) neurotoxicity in vitro. This investigation focused on the phosphorylation level of intermediate filament (IF) subunits of astrocytes (glial fibrillary acidic protein - GFAP) and neurons (low, medium and high molecular weight neurofilament subunits - NFL, NFM and NFH, respectively). The activity of the phosphorylating system associated with the IFs was investigated in striatal slices of rat exposed to QUIN or treated simultaneously with QUIN plus glutamate receptor antagonists, calcium channel blockers or kinase inhibitors. Results showed that in astrocytes, the action of 100 μM QUIN was mainly due to increased Ca(2+) influx through NMDA and L-type voltage-dependent Ca(2+) channels (L-VDCC). In neuronal cells QUIN acted through metabotropic glutamate receptor (mGluR) activation and influx of Ca(2+) through NMDA receptors and L-VDCC, as well as Ca(2+) release from intracellular stores. These mechanisms then set off a cascade of events including activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Also, Cdk5 was activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. mGluR1 was upstream of phospholipase C (PLC) which, in turn, produced diacylglycerol (DAG) and inositol 3,4,5 triphosphate (IP3). DAG is important to activate PKC and phosphorylate NFL, while IP(3) contributed to Ca(2+) release from internal stores promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH. The present study supports the concept of glutamate and Ca(2+) contribution in excitotoxic neuronal damage provoked by QUIN associated to dysfunction of the cytoskeleton homeostasis and highlights the differential signaling mechanisms elicited in striatal astrocytes and neurons.

The maintenance of viable and functional pancreatic islets is crucial for successful islet transplantation from brain-dead donors. To overcome islet quality loss during culture, some studies have co-cultured islets with mesenchymal stem/stromal cells (MSC). However, it is still uncertain if MSC-secreted factors are enough to improve islet quality or if a physical contact between MSCs and islets is needed. Therefore, we performed a systematic review and meta-analysis to clarify the effect of different culture contact systems of islets with MSCs on viability and insulin secretion outcomes. Pubmed and Embase were searched. Twenty studies fulfilled the eligibility criteria and were included in the qualitative synthesis and/or meta-analysis. For both outcomes, pooled weighted mean differences (WMD) between islet cultured alone (control group) and the co-culture condition were calculated. Viability mean was higher in islets co-cultured with MSCs compared with islet cultured alone [WMD = 18.08 (95% CI 12.59-23.57)]. The improvement in viability was higher in islets co-cultured in indirect or mixed contact with MSCs than in direct physical contact (P <0.001). Moreover, the mean of insulin stimulation index (ISI) was higher in islets from co-culture condition compared with islet cultured alone [WMD = 0.83 (95% CI 0.54-1.13)], independently of contact system. Results from the studies that were analyzed only qualitatively are in accordance with meta-analysis data. Co-culture of islets with MSCs has the potential for protecting islets from injury during culture period. Moreover, culture time appears to influence the beneficial effect of different methods of co-culture on viability and function of islets.

Engineering neural tissue by combining biodegradable materials, cells and growth factors is a promising strategy for the treatment of central and peripheral nervous system injuries. In this study, neural differentiation of mouse embryonic stem cells (mESCs) was investigated in combination with three dimensional (3D) electrospun nanofibers as a substitute for the extracellular matrix (ECM). Nano/microfibrous poly(lactic-co-glycolic acid) (PLGA) 3D scaffolds were fabricated through electrospinning and characterized. The scaffolds consisted of either a randomly oriented or an aligned structure of PLGA fibers. The mESCs were induced to differentiate into neuronal lineage and the effect of the polymer and fiber orientation on cell survival, morphology and differentiation efficiency was studied. The neural progenitors derived from the mESCs could survive and migrate onto the fibrous scaffolds. Aligned fibers provided more contact guidance with the neurites preferentially extending along the long axis of fiber. The mESCs differentiated into neural lineages expressing neural markers as seen by the immunocytochemistry. The nestin and beta3-tubulin expression was enhanced on the PLGA aligned fibers in comparison with the other groups, as seen by the quantitative analysis. Taken together, a combination of electrospun fiber scaffolds and mESC derived neural progenitor cells could provide valuable information about the effects of topology on neural differentiation and axonal regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1333-1345, 2017.

Carbonyl compounds such as methylglyoxal (MGO) seem to play an important role in complications resulting from diabetes mellitus, in aging and neurodegenerative disorders. In this study, we are showing, that MGO is able to suppress cell viability and induce apoptosis in the cerebral cortex and hippocampus of neonatal rats ex-vivo. These effects are partially related with ROS production, evaluated by DCFH-DA assay. Coincubation of MGO and reduced glutathione (GSH) or Trolox (vitamin E) totally prevented ROS production but only partially prevented the MGO-induced decreased cell viability in the two brain structures, as evaluated by the MTT assay. Otherwise, L-NAME, a nitric oxide (NO) inhibitor, partially prevented ROS production in the two structures but partially prevented cytotoxicity in the hippocampus. Pharmacological inhibition of Erk, has totally attenuated MGO-induced ROS production and cytotoxicity, suggesting that MEK/Erk pathway could be upstream of ROS generation and cell survival. Otherwise, p38MAPK and JNK failed to prevent ROS generation but induced decreased cell survival consistent with ROS-independent mechanisms. We can propose that Erk, p38MAPK and JNK are involved in the cytotoxicity induced by MGO through different signaling pathways. While Erk could be an upstream effector of ROS generation, p38MAPK and JNK seem to be associated with ROS-independent cytotoxicity in neonatal rat brain. The cytotoxic damage progressed to apoptotic cell death at MGO concentration higher than those described for adult brain, suggesting that the neonatal brain is resistant to MGO-induced cell death. The consequences of MGO-induced brain damage early in life, remains to be clarified. However, it is feasible that high MGO levels during cortical and hippocampal development could be, at least in part, responsible for the impairment of cognitive functions in adulthood.

Aim: Scaffolds are a promising approach for spinal cord injury (SCI) treatment. FGF-2 is involved in tissue repair but is easily degradable and presents collateral effects in systemic administration. In order to address the stability issue and avoid the systemic effects, FGF-2 was encapsulated into core–shell microfibers by coaxial electrospinning and its in vitro and in vivo potential were studied. Materials & methods: The fibers were characterized by physicochemical and biological parameters. The scaffolds were implanted in a hemisection SCI rat model. Locomotor test was performed weekly for 6 weeks. After this time, histological analyses were performed and expression of nestin and GFAP was quantified by flow cytometry. Results: Electrospinning resulted in uniform microfibers with a core–shell structure, with a sustained liberation of FGF-2 from the fibers. The fibers supported PC12 cells adhesion and proliferation. Implanted scaffolds into SCI promoted locomotor recovery at 28 days after injury and reduced GFAP expression. Conclusion: These results indicate the potential of these microfibers in SCI tissue engineering. [Formula: see text]

We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 μM DL-AP5 and 50 μM MK801, 100 μM CNQX and 100 μM DNQX. Also, 10 μM verapamil and 10 μM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 μM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 μM) totally prevented this effect. Results obtained with 0.2 μM calyculin A (PP1 and PP2A inhibitor), 1 μM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 μM FK-506 or 100 μM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.

Central nervous system dysfunctions are among the most significant effects of exposure to ethanol and the glial cells that play an important role in maintaining neuronal function, are extremely involved with these effects. The actin cytoskeleton plays a crucial role in a wide variety of cellular functions, especially when there is some injury. Therefore the aim of the present study was to analyze the short-term effects of ethanol (50, 100 and 200 mM) on the cytoskeleton of C6 glioma cells. Here we report that acute ethanol exposure profoundly disrupts the actin cytoskeleton in C6 cells decreasing stress fiber formation and downregulating RhoA and vinculin immunocontent. In contrast, microtubule and GFAP networks were not altered. We further demonstrate that anti-oxidants prevent ethanol-induced actin alterations, suggesting that the actions of ethanol on the actin cytoskeleton are related with generation of reactive oxygen species (ROS) in these cells. Our results show that ethanol at concentrations described to be toxic to the central nervous system was able to target the cytoskeleton of C6 cells and this effect could be related with increased ROS generation. Therefore, we propose that the dynamic restructuring of the cytoskeleton of glial cells might contribute to the response to the injury provoked by binge-like ethanol exposure in brain.