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1‐O‐Acylceramides (1‐OACs) have a fatty acid esterified to the 1‐hydroxyl of the sphingosine head group of the ceramide, and recently we identified these lipids as natural components of human and mouse epidermis. Here we show epidermal 1‐OACs arise shortly before birth during the establishment of the water permeability barrier in mice. Fractionation of human epidermis indicates 1‐OACs concentrate in the stratum corneum. During in vitro maturation into reconstructed human epidermis, human keratinocytes dramatically increase 1‐OAC levels indicating they are one source of epidermal 1‐OACs. In search of potential enzymes responsible for 1‐OAC synthesis in vivo, we analyzed mutant mice with deficiencies of ceramide synthases (Cers2, Cers3, or Cers4), diacylglycerol acyltransferases (Dgat1 or Dgat2), elongase of very long fatty acids 3 (Elovl3), lecithin cholesterol acyltransferase (Lcat), stearoyl‐CoA desaturase 1 (Scd1), or acidic ceramidase (Asah1). Overall levels of 1‐OACs did not decrease in any mouse model. In Cers3 and Dgat2‐deficient epidermis they even increased in correlation with deficient skin barrier function. Dagt2 deficiency reshapes 1‐OAC synthesis with an increase in 1‐OACs with N‐linked non‐hydroxylated fatty acids and a 60% decrease compared to control in levels of 1‐OACs with N‐linked hydroxylated palmitate. As none of the single enzyme deficiencies we examined resulted in a lack of 1‐OACs, we conclude that either there is functional redundancy in forming 1‐OAC and more than one enzyme is involved, and/or an unknown acyltransferase of the epidermis performs the final step of 1‐OAC synthesis, the implications of which are discussed. [ABSTRACT FROM AUTHOR]

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however,howhorizontal cell signals also affect the temporal, spatial,andcontrast tuning in retinal output neurons, the ganglion cells.Tostudy this,wegenerated a genetically modifiedmouseline inwhichweeliminated the lightdependencyof feedbackbydeleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actualmodeof feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-+ retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. [ABSTRACT FROM AUTHOR]

Well-defined gradients of the lipid mediator sphingosine-1-phosphate (S1P) direct chemotactic egress of mature thymocytes from the thymus into the circulation. Although it is known that these gradients result from low S1P levels in the thymic parenchyma and high S1P concentrations at the exit sites and in the plasma, the biochemical mechanisms that regulate these differential S1P levels remain unclear. Several studies demonstrated that ceramide synthase 2 (Cers2) regulates the levels of the S1P precursor sphingosine. We, therefore, investigated whether Cers2 is involved in the regulation of S1P gradients and S1P-dependent egress into the circulation. By analyzing Cers2-deficient mice, we demonstrate that Cers2 limits the levels of S1P in thymus and blood to maintain functional S1P gradients that mediate thymocyte emigration into the circulation. This function is specific for Cers2, as we also show that Cers4 is not involved in the regulation of thymic egress. Our study identified Cers2 as an important regulator of S1P-dependent thymic egress, and thus contributes to the understanding of how S1P gradients are maintained in vivo. [ABSTRACT FROM AUTHOR]

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. S tem C ells 2017;35:859-871 [ABSTRACT FROM AUTHOR]

Complex sphingolipids are strongly expressed in neuronal tissue and contain ceramides in their backbone. Ceramides are synthesized by six ceramide synthases (CerS1-6). Although it is known that each tissue has a unique profile of ceramide synthase expression and ceramide synthases are implicated in several neurodegenerative disorders, the expression of ceramide synthase isoforms has not been investigated in the retina. Here we demonstrate CerS1, CerS2 and CerS4 expression in mouse retina and cornea, with CerS4 ubiquitously expressed in all retinal neurons and M€uller cells. To test whether ceramide synthase deficiency affects retinal function, we compared electroretinograms and retina morphology between wild-type and CerS1-, CerS2- and CerS4-deficient mice. Electroretinograms were strongly reduced in amplitude in ceramide synthase-deficient mice, suggesting that signalling in the outer retina is affected. However, the number of photoreceptors and cone outer segment length were unaltered and no changes in retinal layer thickness or synaptic structures were found. Mass spectrometric analyses of ceramides, hexosylceramides and sphingomyelins showed that C20 to C24 acyl-containing species were decreased whereas C16-containing species were increased in the retina of ceramide synthase-deficient mice. Similar but smaller changes were also found in the cornea. Thus, we hypothesize that the replacement of very long-chain fatty acyl residues by shorter C16 residues may affect the electrical properties of retina and cornea, and alter receptor-mediated signal transduction, vesicle-mediated synaptic transmission or corneal light transmission. Future studies need to identify the molecular targets of ceramides or derived sphingolipids in light signal transduction and transmission in the eye. [ABSTRACT FROM AUTHOR]

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43Cx45:Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43Cx45:Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43Cx45:Myo-Cre mice was significantly better than that of control mdx Cx43Cx45 mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD. [ABSTRACT FROM AUTHOR]

Background: Synthesis of clonal IgG is a consistent feature of patients with multiple sclerosis (MS). Whether oligoclonal bands (OCBs) represent unspecific disease bystanders or active components in MS pathology is an open question. The aim of this study was to develop a method to quantify and compare the reactivity of cerebrospinal fluid (CSF) antibodies from patients with and without MS. Methods: We collected CSF from 262 patients from two different cohorts, which included 148 patients with MS and 114 with other neurological diseases (OND). We established a highly sensitive electrochemiluminescence (ECL)-based assay to measure CSF antibody reactivity against purified myelin particles and biotin anchored liposomes. The diagnostic value of the ECL score against myelin particles was assessed with receiver operating characteristic curves. Results: CSF from patients with MS have higher reactivity toward purified myelin particles as compared to those with OND with OCBs. Using liposomes with defined lipid compositions and myelin particles from ceramide synthase 2 (CerS2) knockout mice, we find that some of the CSF antibody reactivity is directed against cerebrosides. Conclusion: The ECL-based assay system expands the currently available toolbox for the detection of autoantibodies in MS and related diseases. [ABSTRACT FROM AUTHOR]

Ceramides are mediators of inflammatory processes. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that CerS6 mRNA expression was upregulated 15‐fold in peripheral blood leukocytes before the onset of EAE symptoms. In peripheral blood leukocytes from MS patients, a 3.9‐fold upregulation was found. Total genetic deletion of CerS6 and the selective deletion of CerS6 in peripheral blood leucocytes exacerbated the progression of clinical symptoms in EAE mice. This was associated with enhanced leukocyte, predominantly neutrophil infiltration and enhanced demyelination in the lumbar spinal cord of EAE mice. Interferon‐gamma/tumor necrosis factor alpha (IFN‐γ/TNF‐α) and granulocyte colony‐stimulating factor (G‐CSF) both drive EAE development and induce expression of the integrin CD11b and the chemokine receptor C‐X‐C motif chemokine receptor 2 (CXCR2), and we found they also induce CerS6 expression. In vivo, the genetic deletion of CerS6 enhanced the activation/migration of neutrophils, as reflected by an enhanced upregulation of CD11b and CXCR2. In vitro, the genetic deletion of CerS6 enhanced the activation status of IFN‐γ/TNF‐α‐stimulated neutrophils, as shown by increased expression of nitric oxide and CD11b and an increased adhesion capacity. In G‐CSF‐stimulated neutrophils, the migration status was enhanced, as reflected by an elevated level of CXCR2 and an increased migration capacity. These data suggest that CerS6/C16‐Cer mediates feedback regulation by inhibiting the formation of CD11b and CXCR2, which are induced either by IFN‐γ/TNF‐α or by G‐CSF, respectively. We conclude that CerS6/C16‐Cer mediates anti‐inflammatory effects during the development of EAE and MS possibly by suppressing the migration and deactivation of neutrophils. [ABSTRACT FROM AUTHOR]

The keratitis–ichthyosis–deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome. [ABSTRACT FROM AUTHOR]

Aims It is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant ‘Cx43D378stop’) led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell–cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43–INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein ‘end-binding 1’ (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. Methods and results Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules. Conclusions NaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism. [ABSTRACT FROM AUTHOR]

Five ceramide synthases (CerS2-CerS6) are expressed in mouse \skin. Although CerS3 has been shown to fulfill an essential \function during skin development, neither CerS6- nor CerS2- \deficient mice show an obvious skin phenotype. In order to study \the role of CerS4, we generated CerS4-deficient mice (Cers4-/- ) \and CerS4-specific antibodies. With these biological tools we \analysed the tissue distribution and determined the cell-type \specific expression of CerS4 in suprabasal epidermal layers of \footpads as well as in sebaceous glands of the dorsal skin. Loss of \CerS4 protein leads to an altered lipid composition of the sebum, \which is more solidified and therefore might cause progressive hair loss due to physical blocking of the hair canal. We also noticed a strong decrease in C20 1,2-alkane diols consistent with the decrease of wax diesters in the sebum of Cers4-/- mice. Cers4-/- mice at 12 months old display additional epidermal tissue destruction due to dilated and obstructed pilary canals. Mass spectrometric analyses additionally show a strong decrease in C20-containing sphingolipids. [ABSTRACT FROM AUTHOR]

Highlights: [•] First transgenic mouse line expressing a human Clouston syndrome mutation. [•] The mutation Cx30A88V induces hyperproliferation and hearing impairments in homozygous mice. [•] A high expression level of Cx30A88V is required to cause abnormalities in mice. [Copyright &y& Elsevier]

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing. [ABSTRACT FROM AUTHOR]

Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22-C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein. [ABSTRACT FROM AUTHOR]

Denervation of skeletal muscles induces atrophy, preceded by changes in sarcolemma permeability of causes not yet completely understood. Here, we show that denervation-induced Evans blue dye uptake in vivo of fast, but not slow, myofibers was acutely inhibited by connexin (Cx) hemichannel/pannexin1 (Panx1) channel and purinergic ionotropic P2X7 receptor (P2X7R) blockers. Denervated myofibers showed up-regulation of Panx1 and de novo expression of Cx39, Cx43, and Cx45 hemichannels as well as P2X7Rs and transient receptor potential subfamily V, member 2, channels, all of which are permeable to small molecules. The sarcolemma of freshly isolated WT myofibers from denervated muscles also showed high hemichannel-mediated permeability that was slightly reduced by blockade of Panx1 channels or the lack of Panx1 expression, but was completely inhibited by Cx hemichannel or P2X7R blockers, as well as by degradation of extracellular ATP. However, inhibition of transient receptor potential subfamily V, member 2, channels had no significant effect on membrane permeability. Moreover, activation of the transcription factor NFκB and higher mRNA levels of proinflammatory cytokines (TNF-α and IL-1β) were found in denervated WT but not Cx43/Cx45-deficient muscles. The atrophy observed after 7 d of denervation was drastically reduced in Cx43/Cx45-deficient but not Panx1-deficient muscles. Therefore, expression of Cx hemichannels and P2X7R promotes a feed-forward mechanism activated by extracellular ATP, most likely released through hemichannels, that activates the inflammasome. Consequently, Cx hemichannels are potential targets for new therapeutic agents to prevent or reduce muscle atrophy induced by denervation of diverse etiologies. [ABSTRACT FROM AUTHOR]

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Na1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function. [ABSTRACT FROM AUTHOR]

Panglial networks are essential for normal physiology in the CNS, and the function of distinct connexins participating in these networks is not well understood. We generated Connexin32 (Cx32)-deficient mice with additional deletion of astrocytic Cx43 to explore the role of both connexins in panglial networks. Cx43/Cx32 double knock-out (dKO) mice revealed strong microglial activation in corpus callosum and cingulum along with severe astrogliosis and scar formation. In addition, most of the fine myelinated fibers projecting from the corpus callosum into the cortex were lost. Myelin loss was caused by a strong decrease of oligodendrocytes in the cingulum of Cx43/Cx32dKO mice. Immunoblot analyses using newly generated specific Cx47 antibodies revealed that oligodendrocytic Cx47 is phosphorylated in vivo depending on astrocytic Cx43 expression. In Cx43-deficient mice, Cx47 protein levels were strongly decreased, whereas Cx47 mRNA levels were not altered. Using Cx43G 138R/Cx30KC) mice, we show that Cx47 expression depends on the presence of astrocytic Cx43 protein and that its gap junctional channel function is not necessary for Cx47 stabilization. In consequence, Cx43/Cx32dKO mice additionally lack Cx47 expression and therefore cannot form oligodendrocytic gap junctions, which explains the phenotypic similarities to Cx32/Cx47dKO mice. Our findings provide strong evidence that phosphorylation and stability of oligodendrocytic Cx47 proteins is dependent on astrocytic Cx43 expression. These results further unravel the complexity of panglial networks and show that results of previous studies using astrocytic Cx43-deficient mice have to be reconsidered. [ABSTRACT FROM AUTHOR]

Connexins have been implicated in the regulation of precursor cell migration and proliferation during embryonic development of the mammalian brain. However, their function in postnatal neurogenesis is unclear. Here we demonstrate that connexin (Cx) 45 is expressed in transit-amplifying cells and neuroblasts in the postnatal subventricular zone (SVZ) and modulated the proliferation of SVZ-derived precursor cells in vivo. Thus, overexpression of Cx45 by retroviral injections increased the proliferation of Mash-1-positive transit-amplifying precursor cells in the SVZ. Conversely, conditional deletion of Cx45 in precursor cells decreased proliferation. Finally, we established that Cx45 positively influences cell cycle reentry via ATP signaling that involves intracellular calcium stores and ERK1/2 signaling. [ABSTRACT FROM AUTHOR]

Gene inactivation reporters are powerful tools to circumvent limitations of the widely used Cre/loxP system of conditional mutagenesis. With new conditional transgenic mouse lines expressing the enhanced cyan fluorescent protein (ECFP) instead of connexin43 (Cx43) after Cre-mediated recombination, we demonstrate dual reporter approaches to simultaneously examine astrocyte subpopulations expressing different connexins, identify compensatory up-regulation within gene families, and quantify Cre-mediated deletion at the allelic level. Analysis of a newly generated Cx43 knock-in ECFP mouse revealed an unexpected heterogeneity of Cx43-expressing astrocytes across brain areas. [ABSTRACT FROM AUTHOR]

Pelizaeus-Merzbacher-like disease is characterized by impaired psychomotor development, ataxia, progressive spasticity and mental retardation. It is induced by mutations in the gap junction gene GJC2 that encodes for the gap junction protein connexin 47. Mice bearing a human Cx47M283T missense mutation have been generated as a transgenic mouse model of Pelizaeus-Merzbacher-like disease. Homozygous expression of the mutant connexin 47 gene in oligodendrocytes resulted in a complex and variable neuropathologic phenotype, which was associated with impairments in motor coordination in juvenile, but not adult mice. In the present study, we have investigated anxiety-like behaviour and spatial working memory in juvenile (P23) and adult (3-month-old) Cx47M282T mutant mice. Adult Cx47M282T mice were also evaluated in terms of neuromotor functions and in the novel object recognition test. Juvenile Cx47M282T mutant mice exhibited an increase in anxiety-like behaviour in the open field test, but no changes in spatial working memory performance. No significant changes in anxiety-like behaviour, spatial working memory or neuromotor functions were observed in the adult Cx47M282T mutant mice. However, novel object recognition was significantly impaired in adult Cx47M282T mice. Our results suggest that the expression of the human Cx47M282T mutation in the mouse causes changes in anxiety-like behaviour in juvenile and novel object recognition impairments in adult mice. It appears that the distortion of panglial gapjunction coupling in white and grey matter tissue in the Cx47M282T mice is associated with a complex age-dependent behavioural phenotype including changes in psychomotor, emotional and memory functions. [ABSTRACT FROM AUTHOR]

In th e brain, including the retina, interneurons show an enormous structural and functional diversity. Retinal horizontal cells represent a class of interneurons that form triad synapses with photoreceptors and ON bipolar cells. At this first retinal synapse, horizontal cells modulate signal transmission from photoreceptors to bipolar cells by feedback and feedforward inhibition. To test how the fully developed retina reacts to the specific loss of horizontal cells, these interneurons were specifically ablated from adult mice using the diphtheria toxin (DT)/DT-receptor system and the connexin57 promoter. Following ablation, the retinal network responded with extensive remodeling: rods retracted their axons from the outer plexiform layer and partially degenerated, whereas cones survived. Cone pedicles remained in the outer plexiform layer and preserved synaptic contacts with OFF but not with ON bipolar cells. Consistently, the retinal ON pathway was impaired, leading to reduced amplitudes and prolonged latencies in electroretinograms. However, ganglion cell responses showed only slight changes in time course, presumably because ON bipolar cells formed multiple ectopic synapses with photoreceptors, and visual performance, assessed with an optomotor system, was only mildly affected. Thus, the loss of an entire interneuron class can be largely compensated even by the adult retinal network. [ABSTRACT FROM AUTHOR]

Connexins (Cx) form gap junctions and allow the coordination of cellular behaviour. In vessels, expression of Cx40, Cx37, and Cx43 is well established and specifically Cx40 serves important functions in endothelial cells. In contrast, expression and physiological functions of Cx45 is unclear although its expression has been suggested in vascular smooth muscle (VSM). Therefore, we studied expression and function of Cx45 in vessels using different mice models allowing to identify and delete Cx45. Smooth muscle cell (SMC)-specific deletion was achieved by the Cre/loxP system using Cre-recombinase driven by a Nestin promoter. Deletion of Cx45 leads concomitantly to the expression of enhanced green fluorescence protein (EGFP) in these mice. Conduction of vasomotor responses was studied in cremasteric arterioles using intravital microscopy and arterial pressure was measured telemetrically. Cx45 is transcriptionally expressed in VSM as detected by EGFP expression in SMC-specific Cx45-deficient mice (Cx45fl/fl:Nestin-Cre) but not in endothelial cells (Cx45fl/fl:TIE2-Cre). Moreover, EGFP was located at VSM cell borders in arterioles of transgenic mice carrying an EGFP-tagged Cx45. Expectedly, arteriolar conduction of dilations evoked by the endothelium-dependent agonist acetylcholine were not different between Cx45fl/fl:Nestin-Cre mice and controls carrying homozygously a floxed Cx45 gene (Cx45fl/fl). Surprisingly, the amplitude of locally initiated endothelium-independent constrictions (K+) and dilations (adenosine) declined similarly with distance in both genotypes indicating an intact VSM conduction pathway also in mice being deficient for Cx45 in VSM. Arterial pressure was not different between freely moving Cx45fl/fl and Cx45fl/fl:Nestin-Cre mice during day or night. We conclude that Cx45 is physiologically expressed in VSM, but not in EC in murine arterioles. However, Cx45 is dispensable for the conduction of vasomotor responses along these arterioles. Possibly, other Cx functionally replace the lack of Cx45 in VSM. The reported role of Cx45 in renin secretion does not seem to alter arterial pressure in freely moving mice. [ABSTRACT FROM AUTHOR]

In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and ∼40% of Cx30/Cx47 doubledeficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin. [ABSTRACT FROM AUTHOR]

Gap junction coupling synchronizes activity among neurons in adult neural circuits, but its role in coordinating activity during development is less known. The developing retina exhibits retinal waves-spontaneous depolarizations that propagate among retinal interneurons and drive retinal ganglion cells (RGCs) to fire correlated bursts of action potentials. During development, two connexin isoforms, connexin36 (Cx36) and Cx45, are expressed in bipolar cells and RGCs, and therefore provide a potential substrate for coordinating network activity. To determine whether gap junctions contribute to retinal waves, we compared spontaneous activity patterns using calcium imaging, whole-cell recording, and multielectrode array recording in control, single-knock-out (ko) mice lacking Cx45 and double-knock-out (dko) mice lacking both isoforms. Wave frequency, propagation speed, and bias in propagation direction were similar in control, Cx36ko, Cx45ko, and Cx36/45dko retinas. However, the spontaneous firing rate of individual retinal ganglion cells was elevated in Cx45ko retinas, similar to Cx36ko retinas (Hansen et al., 2005; Torborg and Feller, 2005), a phenotype that was more pronounced in Cx36/45dko retinas. As a result, spatial correlations, as assayed by nearest-neighbor correlation and functional connectivity maps, were significantly altered. In addition, Cx36/45dko mice had reduced eye-specific segregation of retinogeniculate afferents. Together, these findings suggest that although Cx36 and Cx45 do not play a role in gross spatial and temporal propagation properties of retinal waves, they strongly modulate the firing pattern of individual RGCs, ensuring strongly correlated firing between nearby RGCs and normal patterning of retinogeniculate projections. [ABSTRACT FROM AUTHOR]

Gap junction channels are intercellular conduits that allow diffusional exchange of ions, second messengers, and metabolites. Human oligodendrocytes express the gap junction protein connexin47 (Cx47), which is encoded by the GJC2 gene. The autosomal recessive mutation hCx47M283T causes Pelizaeus-Merzbacher-like disease 1 (PMLD1), a progressive leukodystrophy characterized by hypomyelination, retarded motor development, nystagmus, and spasticity. We introduced the human missense mutation into the orthologous position of the mouse Gjc2 gene and inserted the mCx47M282T coding sequence into the mouse genome via homologous recombination in embryonic stem cells. Three-week-old homozygous Cx47M282T mice displayed impaired rotarod performance but unchanged open-field behavior. 10-15-day-old homozygous Cx47M282T and Cx47 null mice revealed a more than 80% reduction in the number of cells participating in glial networks after biocytin injections into oligodendrocytes in sections of corpus callosum. Homozygous expression of mCx47M282T resulted in reduced MBP expression and astrogliosis in the cerebellum of ten-day-old mice which could also be detected in Cx47 null mice of the same age. Three-month-old homozygous Cx47M282T mice exhibited neither altered open-field behavior nor impaired rotarod performance anymore. Adult mCx47M282T expressing mice did not show substantial myelin alterations, but homozygous Cx47M282T mice, additionally deprived of connexin32, which is also expressed in oligodendrocytes, died within six weeks after birth and displayed severe myelin defects accompanied by astrogliosis and activated microglia. These results strongly suggest that PMLD1 is caused by the loss of Cx47 channel function that results in impaired panglial coupling in white matter tissue. [ABSTRACT FROM AUTHOR]

Gap junctional coupling of renin-producing cells is of major functional importance for the control of renin synthesis and release. This study was designed to determine the relevance of the vascular gap junction protein connexin 45 (Cx45) for the control of renin expression and secretion. By crossbreeding mice which drive Cre recombinase under the control of the endogenous renin promoter with mice harboring floxed Cx45 gene alleles, we generated viable mice with a deletion of Cx45 in the renin cell lineage. These mice were normotensive, and renin cells in their kidneys were normal with regard to localization and number. Sodium deficiency induced typical recruitment of renin-producing cells along afferent arterioles, whereas sodium overload resulted in a decrease in the number of cells expressing renin. Regulation of renin secretion by perfusion pressure, catecholamines, and angiotensin II from isolated kidneys of mice with renin cell-specific deletion of Cx45 was normal. Analyzing Cx45 promoter activity in cells of the preglomerular arteriolar tree by using mice driving the reporter gene LacZ under the control of the Cx45 promoter revealed strong staining in smooth muscle cells of the media, whereas renin-expressing cells were almost devoid of LacZ staining. Conversely, renin-producing cells, but not vascular smooth muscle cells expressed the gap junction protein Cx40. These findings suggest that Cx45 plays no major functional role in renin-producing cells, probably because the expression of Cx45 is downregulated in these cells. Since renin-producing cells in the adult kidney can reversibly transform into vascular smooth muscle cells and vice versa, our findings on connexin expression indicate that these phenotype switches are paralleled by characteristic reciprocal changes in the transcriptional activity of Cx40 and Cx45 genes. [ABSTRACT FROM AUTHOR]

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons. J. Comp. Neurol. 519:433-450, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region ("entry site") to increasingly complex pat- terns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTEC5 followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance. [ABSTRACT FROM AUTHOR]

The gap junction protein connexin-45 (Cx45) is expressed in the conduction system of the heart and in certain neurons of the retina and brain. General and cardiomyocyte-directed deficiencies of Cx45 in mice lead to lethality on embryonic day 10.5 as a result of cardiovascular defects. Neuron-directed deletion of Cx45 leads to defects in transmission of visual signals. Connexin-36 (Cx36) is coexpressed with Cx45 in certain types of retinal interneurons. To determine whether these two connexins have similar functions and whether Cx36 can compensate for Cx45, we generated knock-in mice in which DNA encoding Cx45 was replaced with that encoding Cx36. Neuron-directed replacement of Cx45 with Cx36 resulted in viable animals. Electroretinographic and neurotransmitter coupling analyses demonstrated functional compensation in the retina. By contrast, general and cardiomyocyte-directed gene replacement led to lethality on embryonic day 11.5. Mutant embryos displayed defects in cardiac morphogenesis and conduction. Thus, functional compensation of Cx45 by Cx36 did not occur during embryonic heart development. These data suggest that Cx45 and Cx36 have similar functions in the retina, whereas Cx45 fulfills special functions in the developing heart that cannot be compensated by Cx36. [ABSTRACT FROM AUTHOR]

Arterial , a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of in the blood and brain, yet the mechanisms by which the brain senses CO2 remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO2-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO2-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of . ATP release is independent of extracellular Ca2+ and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO2-dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO2-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO2-dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in is an important mechanism contributing to central respiratory chemosensitivity. [ABSTRACT FROM AUTHOR]

Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin36, connexin45, connexin50, and connexin57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin30.2 (Cx30.2), and its regulation in the mouse retina. To analyze the expression of Cx30.2, we used a transgenic mouse line in which the coding region of Cx30.2 was replaced by lacZ reporter DNA. We detected the lacZ signal in the nuclei of neurons located in the inner nuclear layer and the ganglion cell layer (GCL). In this study, we focused on the GCL and characterized the morphology of the Cx30.2- expressing cells. Using immunocytochemistry and intracellular dye injections, we found six different types of Cx30.2-expressing ganglion cells: one type of ON-OFF, three types of OFF, and two types of ON ganglion cells; among the latter was the RGA1 type. We show that RGA1 cells were heterologously coupled to numerous displaced amacrine cells. Our results suggest that these gap junction channels may be heterotypic, involving Cx30.2 and a connexin yet unidentified in the mouse retina. Gap junction coupling can be modulated by protein kinases, a process that plays a major role in retinal adaptation. Therefore, we studied the protein kinase-induced modulation of coupling between RGA1 and displaced amacrine cells. Our data provide evidence that coupling of RGA1 cells to displaced amacrine cells is mediated by Cx30.2 and that the extent of this coupling is modulated by protein kinase C. [ABSTRACT FROM AUTHOR]

Background: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown. Methods: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina. Results: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina. Conclusion: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublaminaspecific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies. [ABSTRACT FROM AUTHOR]

Aims: This study aimed at characterizing expression and the functional role of the Gjb6 gene, encoding for connexin 30 (Cx30) protein, in the adult mouse heart. [ABSTRACT FROM PUBLISHER]

Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA-S4/5 cells. They belong to the group of medium-field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA-S4/5 cells are GABAergic and express GAT-1, a plasma-membrane-bound GABA transporter possibly involved in non-vesicular GABA release. To characterize the light responses of IPA-S4/5 cells, patch-clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M- and S-cones, possibly with reduced S-cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter. [ABSTRACT FROM AUTHOR]