Relationship between rat retinal degeneration and potassium channel KCNQ5 expression.

KCNQ5/Kv7.5 is a low-threshold non-inactivating voltage-gated potassium channel preferentially targeted to excitatory endings in brain neurons. The M-type current is mediated by KCNQ5 channel subunits in monkey retinal pigment epithelium cells and in brain neurons. This study was undertaken to analyze KCNQ5 expression and the interaction signals of KCNQ5 with other proteins in normal rat retina and during photoreceptor degeneration. The KCNQ5 expression pattern was studied by immunocytochemistry and Western blot in normal rat retinas (Sprague-Dawley, SD) and P23H-1 rats as a retinitis pigmentosa model. The physical interactions of KCNQ5 with calmodulin (CaM), vesicular glutamate transporter 1 (VGluT1) and glial fibrillary acidic protein (GFAP) were analyzed by in situ proximity ligation assays and were supported by calcium recording. KCNQ5 expression was found in the plexiform layers, ganglion cell layer and basal membrane of the retinal pigment epithelium. The physical interactions among KCNQ5 and CaM, VGluT1 and GFAP changed with age and during retinal degeneration. The maximal level of KCNQ5/CaM interaction was found when photoreceptors had almost completely disappeared; the KCNQ5/VGluT1 interaction signal decreased and the KCNQ5/GFAP interaction increased in the inner retina, while degeneration progressed. The basal calcium levels in the astrocytes and neurons of P23H-1 were higher than in the control SD retinas. This study demonstrates that KCNQ5 is present in the rat retina where its activity may be moderated by CaM. Retinal degeneration progression in P23H-1 rats can be followed by an interaction between KCNQ5 with CaM in an in situ system. The relationship between KCNQ5 and VGluT1 or GFAP needs to be more cautiously interpreted.

Response of transcription factor NFATc3 to excitotoxic and traumatic brain insults: identification of a subpopulation of reactive astrocytes.

Astrocytes react to brain injury triggering neuroinflammatory processes that determine the degree of neuronal damage. However, the signaling events associated to astrocyte activation remain largely undefined. The nuclear factor of activated T-cells (NFAT) is a transcription factor family implicated in activation of immune cells. We previously characterized the expression of NFAT isoforms in cultured astrocytes, and NFAT activation in response to mechanical lesion. Here we analyze NFATc3 in two mouse models of inflammatory brain damage: hippocampal excitotoxicity induced by intracerebral kainic acid (KA) injection and cortical mechanical lesion. Immunofluorescence results demonstrated that NFATc3 is specifically induced in a subset of reactive astrocytes, and not in microglia or neurons. In KA-treated brains, NFATc3 expression is transient and NFATc3-positive astrocytes concentrate around damaged neurons in areas CA3 and CA1. Complementary Western blot and RT-PCR analysis revealed an NFAT-dependent induction of RCAN1-4 and COX-2 in hippocampus as soon as 6 h after KA exposure, indicating that NFAT activation precedes NFATc3 over-expression. Moreover, activation of NFAT by ATP increased NFATc3 mRNA levels in astrocyte cultures, suggesting that NFATc3 expression is controlled through an auto-regulatory loop. Meanwhile, stab wound enhanced NFATc3 expression specifically in a subclass of reactive astrocytes confined within the proximal layer of the glial scar, and GFAP immunoreactivity was attenuated in NFATc3-expressing astrocytes. In conclusion, our work establishes NFATc3 as a marker of activation for a specific population of astrocytes in response to brain damage, which may have consequences for neuronal survival.

Fluorescent Protein-photoprotein Fusions and Their Applications in Calcium Imaging.

Calcium-activated photoproteins, such as aequorin, have been used as luminescent Ca2+ indicators since 1967. After the cloning of aequorin in 1985, microinjection was substituted by its heterologous expression, which opened the way for a widespread use. Molecular fusion of green fluorescent protein (GFP) to aequorin recapitulated the nonradiative energy transfer process that occurs in the jellyfish Aequorea victoria, from which these two proteins were obtained, resulting in an increase of light emission and a shift to longer wavelength. The abundance and location of the chimera are seen by fluorescence, whereas its luminescence reports Ca2+ levels. GFP-aequorin is broadly used in an increasing number of studies, from organelles and cells to intact organisms. By fusing other fluorescent proteins to aequorin, the available luminescence color palette has been expanded for multiplexing assays and for in vivo measurements. In this report, we will attempt to review the various photoproteins available, their reported fusions with fluorescent proteins and their biological applications to image Ca2+ dynamics in organelles, cells, tissue explants and in live organisms.

'Green mice' display limitations in enhanced green fluorescent protein expression in retina and optic nerve cells.

Characterization of retinal cells, cell transplants and gene therapies may be helped by pre-labeled retinal cells, such as those transfected with vectors for green fluorescent protein expression. The aim of this study was to analyze retinal cells and optic nerve components from transgenic green mice (GM) with the 'enhanced' green fluorescent protein (EGFP) gene under the control of the CAG promoter (a chicken ß-actin promoter and a cytomegalovirus enhancer). The structural analysis and electroretinography recordings showed a normal, healthy retina. Surprisingly, EGFP expression was not ubiquitously located in the retina and optic nerve. Epithelial cells, photoreceptors and bipolar cells presented high green fluorescence levels. In contrast, horizontal cells, specific amacrine cells and ganglion cells exhibited a null EGFP expression level. The synaptic terminals of rod bipolar cells displayed a high green fluorescence level when animals were kept in the dark. Immature retinas exhibited different EGFP expression patterns to those noted in adults. Axons and glial cells in the optic nerve revealed a specific regional EGFP expression pattern, which correlated with the presence of myelin. These results suggest that EGFP expression might be related to the activity of both the CAG promoter and ß-actin in mature retinal neurons and oligodendrocytes. Moreover, EGFP expression might be regulated by light in both immature and adult animals. Since GM are used in numerous retina bioassays, it is essential to know the differential EGFP expression in order to select cells of interest for each study.

Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

Bioluminescence recording of Ca(2+) signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+) in various cell compartments. In addition, they would also serve to monitor Ca(2+) in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R(0)) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+) oscillations in single HeLa cells expressing tdTA. Ca(2+) rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+) activity of HeLa cells injected subcutaneously into mice, and Ca(2+) signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+) sensor reported to date and is, therefore, a promising probe to study Ca(2+) dynamics in whole organisms or tissues expressing the transgene.

Glitazones induce astroglioma cell death by releasing reactive oxygen species from mitochondria: modulation of cytotoxicity by nitric oxide.

The glitazones (or thiazolidinediones) are synthetic compounds used in type-2 diabetes, but they also have broad antiproliferative and anti-inflammatory properties still not well understood. We described previously the apoptotic effects of glitazones on astroglioma cells ( J Biol Chem 279: 8976-8985, 2004 ). At certain concentrations, we found a selective lethality on glioma cells versus astrocytes that was dependent on a rapid production of reactive oxygen species (ROS) and seemed unrelated to the receptor peroxisome proliferator activated receptor-gamma. The present study was aimed at characterizing the oxygen derivatives induced by ciglitazone, rosiglitazone, and pioglitazone in C6 glioma cells and to investigate their intracellular source. We examined the interaction of ROS with nitric oxide (NO) and its consequences for glioma cell survival. Fluorescence microscopy and flow cytometry showed that glitazones induced superoxide anion, peroxynitrite, and hydrogen peroxide, with ciglitazone being the most active. ROS production was completely prevented by uncoupling of the electron transport chain and by removal of glucose as an energy substrate, whereas it was unaffected by inhibition of NADPH-oxidase and xanthine-oxidase. Moreover, glitazones inhibited state 3 respiration in permeabilized cells, and experiments with mitochondrial inhibitors suggested that complex I was the likely target of glitazones. Therefore, these results point to the mitochondrial electron transport chain as the source of glitazone-induced ROS in C6 cells. Glitazones also depolarized mitochondria and reduced mitochondrial pH. NO synthase inhibitors revealed that superoxide anion combines with NO to yield peroxynitrite and that the latter contributes to the cytotoxicity of glitazones in astroglioma cells. Future antitumoral strategies may take advantage of these findings.

PKCepsilon induces astrocyte stellation by modulating multiple cytoskeletal proteins and interacting with Rho A signalling pathways: implications for neuroinflammation.

Despite the importance of stellation to maintain astrocyte functionality, the intracellular signals controlling morphology in these cells are poorly characterized. Our goal was to examine the implication of protein kinase C epsilon (PKCepsilon) in astrocyte stellation. We found that the morphological transformation of astrocytes induced by exposure to the pro-inflammatory agent lipopolysaccharide is enhanced by adenoviral expression of wild-type PKCepsilon, and that activation of PKCepsilon is sufficient to trigger a dramatic stellation. Such an effect is mediated by the rearrangement of microtubules and filaments of glial fibrillary acidic protein, disorganization of stress fibres, and formation of new actin filaments within growing cellular processes. Furthermore, PKCepsilon regulates actin-interacting elements such as non-muscle myosin and proteins of the ezrin/radixin/moesin family. We also observed that at least part of the actions of PKCepsilon depend on its catalytic activity. Finally, stellation by PKCepsilon could be blocked by the expression of a constitutively active form of Rho A implicated in the stability of the flat astrocytic morphology. In summary, PKCepsilon stands out as a key intracellular regulator of morphological plasticity in astrocytes, affecting a large range of cytoskeletal elements and inactivating Rho A-dependent pathways. These morphological effects of PKCepsilon may play essential roles during the course of neuroinflammation.

Glitazones differentially regulate primary astrocyte and glioma cell survival. Involvement of reactive oxygen species and peroxisome proliferator-activated receptor-gamma.

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.