TSQ fluorochrome incubation increase autometallographic zinc detection in primary cell cultures of astrocytes

In this work, we have analyzed the combination of direct (TSQ) and indirect (AMG) zinc detection methods in order to increase the sensitivity to a better study of the zinc uptake. For that purpose, rat primary cell cultured astrocytes were used. Intracellular Zn levels were visualized by using the TSQ zinc fluorochrome, either in normal conditions or after supplementary addition of 50 µM ZnSO4 to the culture. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Monolayers were fixed and ionic zinc precipitated by sodium sulphide or sodium selenite. Autometallographic zinc detection was the last step to detect zinc in cell cultures. Zincosome formation were visualized meanwhile the zinc fluorochrome TSQ in light microscopy, with or without adding zinc to cell monolayers. Additionally, we tested the better zinc precipitation and fixative method in order to preserve zinc ions and make them suitable for AMG development, selecting 0.1% sodium sulphide and 4% PFA. TSQ incubation demonstrated that are crucial for AMG zinc detection in both, prior and after the cell fixation. We found the setting parameters by which the zinc ion content of zincosomes can be maintained and visualized in optimal conditions and circumstances, either for optical and electron microscopy zinc detection studies

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency

Generation of a disease-specific iPS cell line derived from a patient with Charcot-Marie-Tooth type 2K lacking functional GDAP1 gene

Human CMT2-FiPS4F1 cell line was generated from fibroblasts of a patient with Charcot-Marie-Tooth disease harbouring the following mutations in the GDAP1 gene in heterozygosis: p.Q163X/p.T288NfsX3. This patient did not present mutations in the PM22, MPZ or GJB genes. Human reprogramming factors OCT3/4, KLF4, SOX2 and C-MYC were delivered using a non-integrative methodology that involves the use of Sendai virus

c‑MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency

Searching free zinc at the ultrastructural level in cultured astrocytes

Zinc is an element that is necessary for many physiological functions in the body but may play an important role in diseases affecting most systems in the body if its balance is altered by environmental, toxicological or idiosyncrasy of subjects. We have centred our investigation in central nervous system, using cultured astrocytes since they are involved in clearance of zinc exocytated to the extracellular medium during synaptic transmission. In previous works we have used a zinc fluorochrome, i.e., the TSQ (6-Methoxy-(8-p-toluenesulfonamido)quinoline) to in vivo zinc uptake in cultured astrocytes and its accumulation in organelles named zincosomes. However, the precise location of these zinc-enriched structures (zincosomes) at the ultrastructural level is a very hard task. In a previous attempt at the electron microscopy level, only topographical approximation by combining light and electron microscopy allowed us to identify selected zincosomes previously marked with TSQ. Now, our objective is to adapt zinc autometallography (Timm’s method) to TSQ labelled cultured astrocytes. For the electron microscopic detection of zincosomes, the first important step is to achieve a good zinc precipitation during or previous to glutaraldehyde fixation. Surprisingly, neither ditizone nor selenite were successful as zinc precipitating agents; only sodium sulphide gave us good results. We also found that while glutaraldehyde is the best option for animal experimentation, paraformaldehyde prefixation gave us best results. Paraformaldehyde prefixation allowed both ultrastructure preservation as well as zinc-precipitated-detection with Timm autometallography in semithin sections. These semithin sections were included again and zincosomes become clearly visible in ultrathin sections

Ethanol impairs extracellular zinc intake in cultured astrocytes

Zinc (Zn) deficiency is present in many physiological and health problems. Among circumstances involved in Zn deficiency, ethanol consumption appears as a prominent cause. In the CNS substantial amounts of Zn appear accumulated in synaptic vesicles of a particular class of neurons: the Zn enriched neurons very abundant in the telencephalon and cerebral cortex. This is the so called synaptic Zn which is simultaneously released with the neurotransmitter thus exerting a neuromodulator role during synaptic transmission. Neighbour astrocytic processes have to capture the excess of both extracellular Zn and neurotransmitter in order to maintain efficient synaptic transmission between neurons. In this work we analyze the effect of exposure to 30 mM ethanol for 7 days in the ability of cultured rat astrocytes to capture and manage extracellular Zn. Intracellular Zn levels were visualized by using the TSQ Zn fluorochrome, either in normal culture conditions or after supplementary addition of 50 μM ZnSO4 to the culture. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Basal Zn levels in cultured astrocytes was greatly and significantly lower in ethanol treated cells (about 30% of control cultures). These differences were consistently maintained after addition of extracellular Zn to cell monolayers, resulting in a lower ability to uptake or retain Zn. The Zn was uptaked by the endocytic pathway, as demonstrated by the marker FM1-43 and was mainly confined to bright organelles that were more abundant in control cells. In conclusion, ethanol impairs astrocyte Zn management resulting in a lower capacity for extracellular Zn intake in resting conditions and after extracellular addition. It has been proposed that an efficient method to palliate Zn deficiency it could be a dietary supplement. Nevertheless, this study suggests that a dietary Zn supplementation may not be enough for recovery of cellular normal function in alcoholic cultured astrocytes

Extracellular zinc intake in cultured astrocytes is altered by ethanol exposure

Ethanol reduces the amount of intracellular zinc detectable with TSQ. Ethanol impairs astrocyte Zn management. It results in a lower capacity for exogenous Zn intake and delivering to zincosomes. Thus, Zn supplementation (dietary?) may not be enough for recovery of cellular normal function; as it happens in alcohol treated astrocytes. Zincosomes are a kind of low density primary endosomes

Ethanol impairs extracellular zinc intake in cultured astrocytes

Zinc is an ion that participates in increasing described cellular and tissular functions. On the other hand, zinc deficiency is also present in many physiological and health problems affecting most organs along the body, including teratological problems. Among circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. In the central nervous system zinc is also present and it is especially important in neurons that include zinc in the transmission synaptic vesicles. This zinc is delivered as the neurotransmitter exerting a neuromodulator role in the synaptic transmission. Neighbour astrocytes have to maintain the extracellular zinc homeostasis in order to maintain neurons continuously able to perform synaptic transmission. It has been proposed that an efficient method to palliate zinc deficiency it could be a dietary supplement. In this work we analyze the ability of cultured astrocytes in the management of extracellular zinc. Cultured rat astrocytes were used in this work, incubated in presence or not of 30 mM Ethanol for 7 days. Intracellular zinc levels were visualized by using the TSQ zinc fluorochrome, after addition or not of 50 µM ZnSO4. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Basal zinc levels in cultured astrocytes was greatly and significantly lower in ethanol treated cells (about 30% of control cultures). These differences were consistently maintained after addition of extracellular zinc to cell monolayers, resulting in a lower ability to uptake or retain zinc. The zinc was uptaked by the endocitic pathway, as demonstrated with the endosome marker FM 1-43 and was mainly confined to bright organelles that were more abundant in control cells. In conclusion, ethanol impairs astrocyte zinc management resulting in a lower capacity for extracellular zinc intake in resting conditions and after extracellular addtion. Consequently the efficiency of a dietary zinc supplementation may not be enough for recovery of cellular normal function

Endocytosis is altered by chronic alcohol exposure in cultured neurons

Endocytosis is required for many cellular pivotal processes including membrane recycling, nutrient uptake and signal transduction. This process is particularly relevant in polarized cells such as neurons. Exposure to ethanol causes a variety of anomalies in the developing brain and previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin (BSA) and transferrin (Tf) in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. The present study shows that the exposure of neurons in primary culture to chronic ethanol (0 to 14 DIV, 30 mM) affects both clathrindependent and clathrinindependent endocytosis by probably acting not only on the several proteins which are directly involved in the different steps of these processes, but also on the proteins that participate in the organization and dynamics of the actin cytoskeleton as well as on cholesterol cellular levels. Therefore, a toxic effect of alcohol on endocytosis could affect some of the important neuronal activities which depend on the endocytic process, including synaptic vesicle recycling, trafficking of postsynaptic receptors, polarized axon elongation, growth cone navigation and neuronal migration