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This synopsis covers the main results and conclusions from the platform presentations during the International Gap Junction Conference. More detailed information is provided in the mini reviews on controversial scientific issues, short reports of research results and conference abstracts published in this issue of Cell Communication and Adhesion. [ABSTRACT FROM AUTHOR]

In mouse heart, four connexins (Cxs), Cx30.2, Cx40, Cx43, and Cx45, form gap junction (GJ) channels for electric and metabolic cell-to-cell signaling. Extent and pattern of Cx isoform expression together with cytoarchitecture and excitability of cells determine the velocity of excitation spread in different regions of the heart. In the SA node, cell–cell coupling is mediated by Cx30.2 and Cx45, which form low-conductance (approximately 9 and 32 pS, respectively) GJ channels. In contrast, the working cardiomyocytes of atria and ventricles express mainly Cx40 and Cx43, which form GJ channels of high conductance (approximately 180 and 115 pS, respectively) that facilitate the fast conduction necessary for efficient mechanical contraction. In the AV node, cell–cell coupling is mediated by abundantly expressed Cx30.2 and Cx45 and Cx40, which is expressed to a lesser extent. Cx30.2 and Cx45 may determine higher intercellular resistance and slower conduction in the SA- and AV-nodal regions than in the ventricular conduction system or the atrial and ventricular working myocardium. Cx30.2 and its putative human ortholog, Cx31.9, under physiologic conditions form unapposed hemichannels in nonjunctional plasma membrane; these hemichannels have a conductance of approximately 20 pS and are permeable to cationic dyes up to approximately 400 Da in molecular mass. Genetic ablation of Cxs confirmed that Cx40 and Cx43 are important in determining the high conduction velocities in atria and ventricles, whereas the deletion of the Cx30.2 complementary DNA led to accelerated conduction in the AV node and reduced the Wenckebach period. We suggest that these effects are caused by (1) a dominant-negative effect of Cx30.2 on junctional conductance via formation of low-conductance homotypic and heterotypic GJ channels, and (2) open Cx30.2 hemichannels in non-junctional membranes, which shorten the space constant and depolarize the excitable membrane. [Copyright &y& Elsevier]

Gap junctions (Gj) form conduits between adjacent cells that are composed of connexin (Cx) protein subunits and allow direct intercellular communication. To date, the connexin gene family comprises 20 members in the mouse and 21 members in the human genome, 19 of which can be grouped as sequence-orthologous pairs. The structure of connexin genes is relatively simple. An untranslated exon 1 is separated by an intron of different length from exon 2, containing the uninterrupted coding region and the 3′-untranslated region (3′-UTR). However, in some connexin genes, the untranslated regions and the reading frame are spliced. Among the known “cardiovascular” connexins, Cx37 and Cx40 were demonstrated to be functionally expressed in mouse and human endothelial cells and Cx40, Cx43 as well as Cx45 in cardiomyocytes of both species. Functional properties, like permeabilities, charge selectivity and unitary conductivity were investigated after directed expression of these connexins in cultured cell lines or paired Xenopus oocytes. Targeted deletion of their coding sequence in the mouse genome allowed study of the biological relevance of Cx37, Cx40, Cx43 and Cx45 with regard to cardiovascular morphology and function. After ablation of Cx37 or Cx40, mice were viable and could be used to study defects in the adult cardiovascular system but loss of Cx43 or Cx45 led to neonatal or embryonic lethality, respectively. Conditional and cell-type specific deletion of both connexins in the heart or blood vessels can help to overcome this obstacle. As yet only little is known about mutations in the human genes for Cx37, Cx40, Cx43 and Cx45. Thus, a profound comparison between human and mouse phenotypes is not yet possible. [Copyright &y& Elsevier]

Gap junctions, composed of connexin protein subunits, allow direct communication through conduits between neighboring cells. Twenty and twenty-one members of the connexin gene family are likely to be expressed in the mouse and human genome, respectively, 19 of which can be grouped into sequence-orthologous pairs. Their gene structure appears to be relatively simple. In most cases, an untranslated exon1 is separated by an intron of different lengh from exon2 that includes the uninterrupted coding region and the 3′-untranslated region. However, there are several exceptions to this scheme, since some mouse connexin genes contain different 5′-untranslated regions spliced either in an alternative and/or consecutive manner. Additionally, in at least 3 mouse and human connexin genes (mCx36, mCx39, mCx57 and hCx31.3, hCx36, as well as hCx40.1) the reading frame is spliced together from 2 different exons. So far, there are two nomenclatures to classify the known connexin genes: The "Gja/Gjb" nomenclature, as it is currently adopted by the NCBI data base, contains some inconsistencies compared to the "Cx" nomenclature. Here we suggest some minor corrections to co-ordinate the "Gja/Gjb" nomenclature with the "Cx" nomenclature. Furthermore, this short review contains an update on phenotypic correlations between connexin deficient mice and patients bearing mutations in their orthologous connexin 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]

Background: The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium. Methods: Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin- 43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function. Results: Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin- 43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci. Conclusion: Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis. [ABSTRACT FROM AUTHOR]

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. [ABSTRACT FROM AUTHOR]

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C 2 C 12 mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C 2 C 12 cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo. [ABSTRACT FROM AUTHOR]

Gap junctions are channel-forming structures in contacting plasma membranes that allow direct metabolic and electrical communication between almost all cell types in the mammalian brain. At least 20 connexin genes and 3 pannexin genes probably code for gap junction proteins in mice and humans. Gap junctions between murine neurons (also known as electrical synapses) can be composed of connexin 36, connexin 45 or connexin 57 proteins, depending on the type of neuron. Furthermore, pannexin 1 and 2 are likely to form electrical synapses. Here, we discuss the roles of connexin and pannexin genes in the formation of neuronal gap junctions, and evaluate recent functional analyses of electrical synapses that became possible through the characterization of mouse mutants that show targeted defects in connexin genes. [ABSTRACT FROM AUTHOR]

Summary: Eight different connexins are expressed in mouse epidermis with overlapping expression patterns in different epidermal layers. Analyses of mice with deficiency or modifications of distinct connexins yielded insights into the large variety of connexins in the epidermis. Connexin43 (Cx43) deficiency in mouse epidermis resulted in a significant acceleration of wound closure. Truncation by 125 amino acid residues of the Cx43 C-terminal region led to an altered epidermal expression pattern of Cx43 and defective development of the epidermal water barrier in transgenic mice, although the truncated Cx43 protein could still form open gap junctional channels in transfected HeLa cells. Thus, the phenotypic abnormalities observed in mice with truncated Cx43 protein (Cx43K258Stop) are more likely due to defective regulation of this protein rather than the closed Cx43 channel. Our studies of connexin-deficient mice revealed an extensive redundancy of connexins expressed in mouse epidermis. Epidermal connexins seem to form two functional groups in which deficiency of one connexin isoform can be compensated by other connexin isoforms of the same group. [Copyright &y& Elsevier]

The recently identified mouse connexin39 (mCx39) gene encodes a peptide of 364 amino acids that shows only 61% sequence similarity to its putative human orthologue connexin40.1 (hCx40.1). The coding regions of mCx39 and hCx40.1 are located on two different exons as described for murine and human connexin36. Northern blot and RT-PCR analyses revealed that mCx39 is expressed after embryonic day (ED) 13.5 up to birth and is absent from the adult stage. Polyclonal antibodies raised to a peptide corresponding to the 16 C-terminal amino acid residues detected a protein band of about 40 kDa apparent molecular mass in lysates of several embryonic tissues. In sections of ED14.5, ED16.5 and neonatal (P0) tissues, immunofluorescent signals were prominent between myotubes in the developing diaphragm, within the intercostal muscle, in the region around the occipital bone, as well as in muscles of the limb, tongue and connective tissue around the eye. These antibodies yielded punctate signals on apposed plasma membranes of HeLa cells transfected with Cx39 cDNA but did not react with wild-type cells. Furthermore, no intercellular permeation of microinjected neurobiotin and other tracers could be detected in Cx39 transfected HeLa cells. However, after microinjection of Alexa488 into myotubes of dissected neonatal diaphragm, we found spreading of this dye into neighbouring cells. As expression of no other known connexin could be verified in these cells, intercellular dye transfer might result from functional expression of Cx39 in developing striated muscle fibers. [ABSTRACT FROM AUTHOR]

Connexins are the protein subunits of gap junction channels and are expressed in a highly regulated temporal and spatial pattern in embryonic development and adult life, with most cell types expressing more than one isoform. Connexin31 (Cx31) is encoded by the gene Gjb3 and expressed throughout mouse development n a complex pattern; in adult mice it becomes restricted to the granular layer of epidermis, testis, and placenta. In placenta, lack of Cx31 leads to transient dysmorphogenesis affecting embryonic survival. Here we have analyzed the structure of mouse Gjb3 as well as its transcriptional regulation by transient transfection of reporter gene constructs in HM1 mouse embryonic stem cells and a mouse keratinocytederived cell line, Hel37, as model systems for early development and skin, respectively. Like most connexin genes, Gjb3 is composed of two exons, the second of which contains the whole coding region and is separated from the first exon by an intron of 2.3 kb. Expression in keratinocytes is regulated by a basal promoter extending to 561 bp upstream of exon 1 in conjunction with a regulatory region between upstream positions 561 and 841. In contrast, expression of Gjb3 in embryonic stem cells depended on the basal promoter together with the intron. The enhancing effect of the ntron was found only in embryonic stem cells and depended on its native position and the integrity of the splice sites. Thus, expression of Gjb3 in keratinocytes and embryonic stem cells is regulated by different cis-regulatory elements and differs in its requirements for the intron in situ. [Copyright &y& Elsevier]

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life. [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]

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart-brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters. [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]

Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2. [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]

Abstract: Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and a major cause of stroke. In the mammalian heart the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) are strongly expressed in the atrial myocardium mediating effective propagation of electrical impulses. Different heterozygous mutations in the coding region for Cx40 were identified in patients with AF. We have generated transgenic Cx40A96S mice harboring one of these mutations, the loss-of-function Cx40A96S mutation, as a model for atrial fibrillation. Cx40A96S mice were characterized by immunochemical and electrophysiological analyses. Significantly reduced atrial conduction velocities and strongly prolonged episodes of atrial fibrillation were found after induction in Cx40A96S mice. Analyses of the gating properties of Cx40A96S channels in cultured HeLa cells also revealed significantly lower junctional conductance and enhanced sensitivity voltage gating of Cx40A96S in comparison to Cx40 wild-type gap junctions. This is caused by reduced open probabilities of Cx40A96S gap junction channels, while single channel conductance remained the same. Similar to the corresponding patient, heterozygous Cx40A96S mice revealed normal expression levels and localization of the Cx40 protein. We conclude that heterozygous Cx40A96S mice exhibit prolonged episodes of induced atrial fibrillation and severely reduced atrial conduction velocities similar to the corresponding human patient. [Copyright &y& Elsevier]

Abstract: During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled ‘Current Pharmacology of Gap Junction Channels and Hemichannels’. [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 N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain. [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]

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]

Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelinassociated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1- derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior [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]

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]

Abstract: Functional gap junction channels composed of certain connexin proteins are essential for the function of the cochlea. Homozygous deficiency in the Gjb2 (mice) or GJB2 (human) gene coding for connexin26 (Cx26) in the cochlea leads to hearing impairment in mice and humans, respectively. Here we have studied the functional equivalence of Cx26 and connexin32 (Cx32) isoforms in the cochlea. We analyzed a conditional mouse mutant in which the Gjb2 coding DNA was exchanged by LacZ DNA coding for the reporter protein beta-galactosidase. This allowed us to follow the unrestricted and cell type specific expression of Gjb2 promoter activity. After inner ear specific, Otogelin-Cre recombinase mediated deletion of the loxP-site-flanked LacZ coding DNA, transcription of the Gjb1 gene, coding for Cx32 was activated by the Gjb2 promoter. Interbreeding of these mice with conditional Gjb2 null mice resulted in animals in which Cx32 instead of Cx26 protein is expressed in the non-sensory epithelial network of the cochlea. When we analyzed the auditory function in these mice, we found that the expression of Cx32 protein is sufficient to support hearing in the absence of Cx26. Thus Cx32 can functionally replace Cx26 in the mouse cochlea resulting in almost normal hearing. [Copyright &y& Elsevier]

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]

Abstract: During muscle development and regeneration of skeletal muscle in mice connexin43 (Cx43) and connexin39 (Cx39) are specifically expressed: Cx43 in satellite cells and myoblasts, whereas Cx39 is exclusively expressed in myogenin-positive cells. We generated Cx39 deficient mice by replacing the coding region of the Gjd4 gene by DNA coding for the enhanced green fluorescent protein eGFP. Adult Cx39 deficient mice exhibit no obvious phenotypic alterations of skeletal muscle compared to wild type mice in the resting state. However, myogenesis in Cx39 deficient embryos is accelerated as indicated by increased myogenin expression on ED13.5 and ED16.5 and increased expression of Cx43 in developing skeletal muscle. In addition, the regeneration process of skeletal muscle in Cx39 deficient mice is accelerated as shown by a 2day earlier onset of MyoD and myogenin expression, relative to wild type littermates. Interestingly, Cx43 expression was also upregulated in Cx39 deficient mice during regeneration of skeletal muscle. We hypothesize that Cx43 may compensate for the loss of Cx39 during myogenesis and regeneration. [Copyright &y& Elsevier]

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]

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]

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]

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]

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]

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22-C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulting mice lack ceramide synthase activity toward C24:1 in the brain as well as the liver and show only very low activity toward C18:0-C22:0 in liver and reduced activity toward C22:0 residues in the brain. In addition, these mice exhibit strongly reduced levels of ceramide species with very long fatty acid residues (⩾C22) in the liver, kidney, and brain. From early adulthood on, myelin stainability is progressively lost, biochemically accompanied by about 50% loss of compacted myelin and 80% loss of myelin basic protein. Starting around 9 months, both the medullary tree and the internal granular layer of the cerebellum show significant signs of degeneration associated with the formation of microcysts. Predominantly in the peripheral nervous system, we observed vesiculation and multifocal detachment of the inner myelin lamellae in about 20% of the axons. Beyond 7 months, the CERS2-deficient mice developed hepatocarcinomas with local destruction of tissue architecture and discrete gaps in renal parenchyma. Our results indicate that CERS2 activity supports different biological functions: maintenance of myelin, stabilization of the cerebellar as well as renal histological architecture, and protection against hepatocarcinomas. [ABSTRACT FROM AUTHOR]

Abstract: Connexin45 (Cx45) is a member of the connexin family which can form gap junction channels and is known to be expressed in several cell types in the embryonic as well as adult mouse including working cardiomyocytes and certain types of neurons. Until now its subcellular localization could not be unequivocally determined in certain tissues due to the lack of sensitive and specific antibodies. In order to investigate the localization of Cx45, we have generated a transgenic mouse expressing a fusion protein composed of Cx45 and eGFP under control of the endogenous Cx45 promoter using a bacterial artificial chromosome (BAC). In previous studies it had been shown that a C-terminal tag of connexin proteins only slightly altered the properties of gap junction channels in cultured cells and allowed direct visualization of the fusion protein. In the adult brain the expression of the Cx45eGFP protein was found in the subventricular zone in transient amplifying cells as well as in neuroblasts and ependymal cells. In addition Cx45eGFP is expressed in the atrial and ventricular working myocardium, i.e. regions of the heart where divergent results regarding Cx45 expression had previously been published. In the lung we identified Cx45eGFP in the smooth muscle cell layer of bronchioles. The Cx45eGFP transgene could not rescue embryonic lethality of Cx45-deficient mice, i.e. Cx45eGFP//Cx45−/− mice die around ED10.5 presumably due to altered properties of gap junction channels as a result of C-terminal tagging of Cx45. [Copyright &y& Elsevier]

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]