Characterization of the internal IRES element of the zebrafish connexin55.5 reveals functional implication of the polypyrimidine tract binding protein

<p>Abstract</p> <p>Background</p> <p>Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides evidence that this gap junction protein may participate in a putative cytoplasmic to nuclear signal transfer.</p> <p>Results</p> <p>We characterized the IRES element of Cx55.5 in terms of sequence elements necessary for its activity and protein factor(s), which may play a role for its function. Two stretches of polypyrimidine tracts designated PPT1 and PPT2 which influence the IRES activity of this neuronal gap junction protein were identified. Selective deletion of PPT1 results in an appreciable decrease of the IRES activity, while the deletion of PPT2 results in a complete loss. RNA-EMSA and UV-cross linking experiments showed that protein complexes bind to this IRES element, of which the polypyrimidine tract binding protein (PTB) was identified as one of the interacting partners with influence on IRES activity. These results indicate that PTB conveys a role in the regulation of the IRES activity of Cx55.5.</p> <p>Conclusion</p> <p>Our findings indicate that the activity of the IRES element of the neuronal gap junction protein Cx55.5 is subject of regulation through flanking polypyrimidine tracts, and that the non-canonical trans-activation factor PTB plays an essential role in this process. This observation is of considerable importance and may provide initial insight into molecular-functional relationships of electrical coupling in horizontal cells.</p

Exploring the Role of PANNEXIN1A in an Acute Experimental Model of Parkinson's Disease

In the nervous system Pannexin1 channels are major ATP and glutamate release sites. The channels are implicated in neurodegenerative disorders including Parkinsons disease, but the underlying mechanisms remain unclear. Here, an interdisciplinary approach tested roles of the mammalian Pannexin1 ortholog Pannexin1a in a zebrafish model of MPTP-induced early stages of Parkinsons disease at molecular, systems, and behavioral levels. The short-term treatment of wild-type TL and gene-edited Panx1a-KO larvae caused metabolic stress, regulated inflammatory pathways, and reduced ATP production. Local field potentials recorded from three regions of the ascending visual pathway showed complex changes in the beta- and gamma-band power and in the coherence between these regions. MPTP treatment produced significantly impaired movements which were partially rescued by targeting the NLRP3 inflammasome. The main findings of this research provide evidence that Panx1a serves a neuroprotective role in an acute MPTP model of Parkinson's disease

Exploring the Role of PANNEXIN1A in an Acute Experimental Model of Parkinson's Disease

In the nervous system Pannexin1 channels are major ATP and glutamate release sites. The channels are implicated in neurodegenerative disorders including Parkinson’s disease, but the underlying mechanisms remain unclear. Here, an interdisciplinary approach tested roles of the mammalian Pannexin1 ortholog Pannexin1a in a zebrafish model of MPTP-induced early stages of Parkinson’s disease at molecular, systems, and behavioral levels. The short-term treatment of wild-type TL and gene-edited Panx1a-KO larvae caused metabolic stress, regulated inflammatory pathways, and reduced ATP production. Local field potentials recorded from three regions of the ascending visual pathway showed complex changes in the beta- and gamma-band power and in the coherence between these regions. MPTP treatment produced significantly impaired movements which were partially rescued by targeting the NLRP3 inflammasome. The main findings of this research provide evidence that Panx1a serves a neuroprotective role in an acute MPTP model of Parkinson's disease

Elevated intracellular chloride level in albino visual cortex neurons is mediated by Na-K-Cl co-transporter

<p>Abstract</p> <p>Background</p> <p>During development the switch from a depolarizing to a hyperpolarizing action of GABA is a consequence of a decrease of the Na⁺-K⁺-2Cl^-co-transporter (NKCC1, Cl^--uptake) and increase of the K⁺-Cl^-co-transporter (KCC2, Cl^--extrusion) expression. However albino visual cortex neurons don't show a corresponding decrease in intracellular chloride concentration during development of the visual system as compared to pigmented animals.</p> <p>Results</p> <p>Our study revealed that more cells express NKCC1 in albinos compared to pigmented rat visual cortex neurons whereas KCC2 is expressed in all cells in both strains. We determined a positive relationship between the presence of NKCC1 and an inhibitory deficit in single neurons of the albino visual cortex. After pharmacological blockade of NKCC1 function with its specific inhibitor, bumetanide, the reversal potential of electrically evoked GABAA receptor-mediated postsynaptic currents and, as a consequence, [Cl-]_iin albino visual cortex neurons shifted to the pigmented rat brain value. In conclusion, our pharmacological experiments and subsequent single cell real time PCR analysis of the co-transporter mRNA demonstrated that the inhibitory deficit present in the albino visual cortical network is almost exclusively mediated by NKCC1.</p> <p>Conclusion</p> <p>Our findings suggest that blocking of NKCC1 in albino visual cortex neurons could improve processing in visual cortex and therefore might be beneficial for vision in albinos.</p

EUV Emission and Scattered Light Diagnostics of Equatorial Coronal Holes as Seen by Hinode/EIS

Spectroscopic diagnostics of solar coronal plasmas critically depends on the uncertainty in the measured line intensities. One of the main sources of uncertainty is instrumental scattered light, which is potentially most important in low-brightness areas. In the solar corona, such areas include polar and equatorial coronal holes, which are the source regions of the solar wind; instrument-scattered light must thus pose a significant obstacle to studies of the source regions of the solar wind. In this paper we investigate the importance of instrument-scattered light on observations of equatorial coronal holes made by the Hinode/EIS spectrometer in two different phases of the solar cycle. We find that the instrument-scattered light is significant at all temperatures, and in both regions it amounts to approximately 10% of the average intensity of the neighboring quiet Sun regions. Such contribution dominates the measured intensity for spectral lines formed at temperatures larger than Log T = 6.15 K, and has deep implications for spectroscopic diagnostics of equatorial coronal hole plasmas and studies of the source regions of a large portion of the solar wind which reaches Earth. Our results suggest that the high temperature tail of in the coronal hole plasma distribution with temperature, however small, is an artifact due to the presence of scattered light.Comment: 11 pages, 6 figure

IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

<p>Abstract</p> <p>Background</p> <p>Changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.</p> <p>Results</p> <p>Connexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus <it>in vivo </it>and <it>in vitro</it>.</p> <p>Conclusion</p> <p>Our results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.</p

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-β (CaMKII-β) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-β Antibody With Connexin36

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-β at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-β. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-β-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-β. However, a monoclonal antibody (CB-β-1) recognized CaMKII-β but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-β expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-β also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-β influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-β-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-β is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-β with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-β may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs

Synaptic Transmission from Horizontal Cells to Cones Is Impaired by Loss of Connexin Hemichannels

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina