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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.

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.

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.

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 Müller 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, hexosyl-ceramides 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.

Correction to:NPG Asia Materials (2018) https://doi.org/10.1038/s41427-018-0014-9 published online on 16 April 2018

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer-amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound's function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS(2) (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS(2) analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.

The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1–6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.

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'.

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.

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 phosphorylatedin vivodepending on astrocytic Cx43 expression. In Cx43-deficient mice, Cx47 protein levels were strongly decreased, whereas Cx47 mRNA levels were not altered. Using Cx43G138R/Cx30KO 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.

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 myelin-associated 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.

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.

Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I–III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.

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.

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.

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 co-expressed 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.

Arterial PCO2, a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of PCO2 in the blood and brain, yet the mechanisms by which the brain senses CO(2) 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 CO(2)-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO(2)-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of PCO2. ATP release is independent of extracellular Ca(2+) 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 CO(2)-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 CO(2)-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO(2)-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 PCO2 is an important mechanism contributing to central respiratory chemosensitivity.

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30(T5M/T5M) mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca(2+) signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

Connexin43 plays an important role in neuroprotection in experimental stroke models; reducing the expression of this gap junction protein in astrocytes enhances injury upon middle cerebral artery occlusion (MCAO). Because the C-terminal region of connexin43 isimportant for channel activity, we carried out MCAO stroke experiments in mice expressing a truncated form of connexin43 (Cx43DeltaCT mice). Brain sections were analyzed for infarct volume, astrogliosis, and inflammatory cell invasion 4 days after MCAO. Adult cortices and astrocyte cultures were examined for connexin43 (Cx43) expression by immunohistochemistry and Western blot. Cultured astrocytes were also examined for dye coupling, channel conductance, hemichannel activity, and Ca wave propagation. The Cx43DeltaCT mice exhibit enhanced cerebral injury after stroke. Astrogliosis was reduced and inflammatory cell invasion was increased inthe peri-infarct region in these mice compared with controls; Cx43 expression was also altered. Lastly, cultured astrocytes from Cx43DeltaCT mice were less coupled and displayed alterations in channel gating, hemichannel activity, and Ca wave properties. These results suggest that astrocytic Cx43 contributed to the regulation of cell death after stroke and support the view that the Cx43 C-terminal region is important in protection in cerebral ischemia.

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

(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 (or=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.

In the adult dentate gyrus, radial glia-like cells represent putative stem cells generating neurons and glial cells. Here, we combined patch-clamp recordings, biocytin filling, immunohistochemistry, single-cell transcript analysis, and mouse transgenics to test for connexin expression and gap junctional coupling of radial glia-like cells and its impact on neurogenesis. Radial glia-like cells were identified in mice expressing EGFP under control of the nestin and gfap promoters. We show that a majority of Radial glia-like cells are coupled and express Cx43. Neuronal precursors were not coupled. Mice lacking Cx30 and Cx43 in GFAP-positive cells displayed almost complete inhibition of proliferation and a significant decline in numbers of radial glia-like cells and granule neurons. Inducible virus-mediated ablation of connexins in the adult hippocampus also reduced neurogenesis. These findings strongly suggest the requirement of connexin expression by radial glia-like cells for intact neurogenesis in the adult brain and point to possible communication pathways of these cells.

Oligodendrocytes in CNS are linked to astrocytes by heterotypic gap junctions composed of Cx32 and Cx47 in oligodendrocytes and Cx30 and Cx43 in astrocytes. These gap junctions also harbour regulatory proteins, including ZO-1 and ZONAB. Here, we investigated the localization of multi-PDZ domain protein 1 (MUPP1) at these gap junctions and examined accessory proteins and connexins associated with oligodendrocytes in Cx47-knockout mice. In every CNS region tested, punctate immunolabelling for MUPP1 was found on all oligodendrocyte somata in wild-type mice. These MUPP1-positive puncta were colocalized with punctate labelling for oligodendrocytic Cx32 or Cx47, and with astrocytic Cx30 or Cx43 at oligodendrocyte-astrocyte (O/A) gap junctions, but were not found at astrocyte-astrocyte gap junctions. In Cx47-knockout mice, immunolabelling of MUPP1 and ZONAB was absent on oligodendrocytes, whereas some ZO-1-positive puncta remained. In Cx32-knockout mice, MUPP1 and ZONAB persisted at O/A gap junctions. The absence of Cx47 in Cx47-knockout mice was accompanied by a total loss of punctate labelling for Cx30, Cx32 and Cx43 on oligodendrocyte somata, and by a dramatic increase in immunolabelling for Cx32 along myelinated fibers. These results demonstrate MUPP1 at O/A gap junctions and Cx47-dependent targeting of connexins to the plasma membranes of oligodendrocyte somata. Further, it appears that deficits in myelination reported in Cx47-knockout mice may arise not only from a loss of Cx47 but also from the accompanied loss of gap junctions and their regulatory proteins at oligodendrocyte somata, and that loss of Cx47 may be partly compensated for by elevated levels of Cx32 along myelinated fibers.

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed β-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.

The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using β-galactosidase staining, we have shown that lacZ/Connexin31.1 was expressed in the spinous and granular layers of the epidermis, in cells of olfactory epithelium and in the vomeronasal organ. During embryogenesis, Connexin31.1 was co-expressed with another isoform, Connexin31, in the post-implantation trophoblast cell lineage and, later in gestation, in placental glycogen cells. Although homozygous Connexin31.1-deficient mice were fertile and showed no morphological or functional defects in adult organs expressing this gene, 30% of the offspring expected according to Mendelian inheritance were lost between embryonic days 11.5 and 14.5 and surviving embryos were significantly reduced in weight near the end of pregnancy. Placentas of Connexin31.1-deficient embryos were reduced in weight and showed altered morphology of the spongiotrophoblast and labyrinth layer. The spongiotrophoblast formed a compact barrier at the decidual border that might restrict the maternal blood supply. We conclude that Connexin31.1 is critical for normal placental development but appears to be functionally compensated by other connexin isoforms in the embryo proper and adult mouse.

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linked to approximately half of all cases of human autosomal nonsyndromic prelingual deafness. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs). This molecular arrangement implies that homomeric GJs would remain in the cochlea if one of the coassembly partners were mutated resulting in null expression. We generated mice in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome in a Cx30 −/− background. In the absence of the Cx30 gene, Cx26 expressed from extra alleles completely restored hearing sensitivity and prevented hair cell death in deaf Cx30 −/− mice. The results indicated that hybrid GJs consisting of Cx26 and Cx30 were not essential for normal hearing in mice and suggested that up-regulation of Cx26 or slowing down its protein degradation might be a therapeutic strategy to prevent and treat deafness caused by Cx30 mutations.

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.

Mutations in connexin (Cx) genes are responsible for a large proportion of human inherited prelingual deafness cases. The most commonly found human Cx mutations are either Cx26 or Cx30 deletions. Histological observations made in the organ of Corti of homozygous Cx26 and Cx30 gene knockout mice show that cochlear hair cells degenerate after the onset of hearing. However, it is unclear whether vestibular hair cells undergo similar degeneration in connexin knockout mice. To address this question, we first examined expression patterns of Cx26 and Cx30 in the saccule, utricle, and ampulla by immunolabeling. In wild-type mice, Cx26 and Cx30 immunoreactivity was found extensively in vestibular supporting cells and connective tissue cells, and the two Cxs were co-localized in most gap junction (GJ) plaques. Targeted deletion of the Cx30 gene, which caused little change in Cx26 expression pattern, resulted in a significant and age-related loss of vestibular hair cells only in the saccule. dUTP nick end labeling (TUNEL) staining also revealed on-going apoptosis specifically in saccular hair cells of Cx30(-/-) mice. These results indicated that hair cell survival in the utricle and ampulae does not require Cx30. Importantly, over-expressing the Cx26 gene from a modified bacterial artificial chromosome in the Cx30(-/-) background rescued the saccular hair cells. These results suggest that it is the reduction in the total amount of GJs rather than the specific loss of Cx30 that underlies saccular hair cell death in Cx30(-/-) mice. Hybrid GJs co-assembled from Cx26 and Cx30 were not essential for the survival of saccular hair cells.

The keratitis-ichthyosis-deafness (KID) syndrome is characterized by corneal, skin, and hearing abnormalities. KID has been linked to heterozygous dominant missense mutations in the GJB2 and GJB6 genes, encoding connexin26 and 30, respectively. In vitro evidence indicates that KID mutations lead to hyperactive (open) hemichannels, which in some cases is accompanied by abnormal function of gap junction channels. Transgenic mouse models expressing connexin26 KID mutations reproduce human phenotypes and present impaired epidermal calcium homeostasis and abnormal lipid composition of the stratum corneum affecting the water barrier. Here we have compiled relevant data regarding the KID syndrome and propose a mechanism for the epidermal aspects of the disease.

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 Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca(2+) 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 Cx43(fl/fl)Cx45(fl/fl):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 Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice was significantly better than that of control mdx Cx43(fl/fl)Cx45(fl/fl) 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.

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.

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS) 2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.

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-gamma/TNF-alpha) 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-gamma/TNF-alpha-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-gamma/TNF-alpha 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.

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.

Connexin43 (Cx43) is involved in bone development, but its role in adult bone homeostasis remains unknown. To overcome the postnatal lethality of Cx43 null mutation, we generated mice with selective osteoblast ablation of Cx43, obtained using a Cx43fl allele and a 2.3-kb fragment of the α1(I) collagen promoter to drive Cre in osteoblasts (ColCre). Conditionally osteoblast-deleted ColCre;Cx43–/fl mice show no malformations at birth, but develop low peak bone mass and remain osteopenic with age, exhibiting reduced bone formation and defective osteoblast function. By both radiodensitometry and histology, bone mineral content increased rapidly and progressively in adult Cx43+/fl mice after subcutaneous injection of parathyroid hormone (PTH), an effect significantly attenuated in ColCre;Cx43–/fl mice, with Cx43–/fl exhibiting an intermediate response. Attenuation of PTH anabolic action was associated with failure to increase mineral apposition rate in response to PTH in ColCre;Cx43–/fl, despite an increased osteoblast number, suggesting a functional defect in Cx43-deficient bone-forming cells. In conclusion, lack of Cx43 in osteoblasts leads to suboptimal acquisition of peak bone mass, and hinders the bone anabolic effect of PTH. Cx43 represents a potential target for modulation of bone anabolism.

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.

Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of uterine contractions during labour. Expression of connexin43 (Cx43), the major myometrial gap junction protein, is increased during delivery. We have generated a mouse mutant (Cx43fl/fl:SM-CreERT2), in which the coding region of Cx43 can be specifically deleted in smooth muscle cells at any given time point by application of tamoxifen. By this approach, we were able to study long-term effects on myometrial functions that are necessary for parturition as well as gap junction intercellular communication in primary myometrial cell cultures. We found a prolongation of the pregnancy in 82% of tamoxifen-treated Cx43fl/fl:SM-CreERT2 mice as well as decreased dye coupling in cultured primary myocytes of these animals. Other parturition-specific parameters such as the regulation of oxytocin receptor, prostaglandin F receptor or progesterone remained unchanged. Our results indicate the important function of Cx43 during parturition in the living animal and suggest further strategies to investigate the role of connexins in uterine contractility in transgenic mice.

In the gastrulating mouse embryo, the gap junction protein connexin43 is expressed exclusively in cells derived from the inner cell mass, whereas connexin31 is expressed in cells of the trophoblast lineage. Since connexin43 and connexin31 do not form heterotypic gap junction channels in exogenous expression systems, such as HeLa cells and Xenopus oocytes, previous studies have suggested that the incompatibility of these two connexins could contribute to the separation of connexin43-expressing and connexin31-expressing compartments between embryo and extraembryonic tissues at gastrulation, respectively. Thus, we have generated connexin43 knock-in connexin31 mice, in which the coding region of the connexin43 gene was replaced by that of connexin31. Interbreeding of heterozygous connexin43 knock-in connexin31 mice resulted in homozygous connexin43 knock-in connexin31 mice, but none of them survived to adulthood. As these mice were born at the expected Mendelian frequency, we conclude that the reported incompatibility of connexin43 and connexin31 to form heterotypic gap junction channels does not interfere with normal embryonic development. Neonatal homozygous connexin43 knock-in connexin31 hearts showed malformation in the subpulmonary outlet of the right ventricle, similar to general connexin43-deficient mice. Electrocardiograms of neonatal hearts in homozygous connexin43 knock-in connexin31 mice revealed significantly low voltage of the QRS complex. This is in contrast to previous results from our laboratory which showed that replacement of connexin43 by connexin40 resulted in morphologically and functionally normal hearts. We conclude that connexin31 cannot functionally replace connexin43 during cardiac morphogenesis.

Using newly generated transgenic mice in which the coding region of the connexin29 (Cx29) gene was replaced by the lacZ reporter gene, we confirmed previous immunochemical results that Cx29 is expressed in Schwann cells, oligodendrocytes and Bergmann glia cells. In addition, we detected lacZ/Cx29 in Schwann cells of the sciatic nerve and in particular of the spiral ganglion in the inner ear, as well as at low abundance in the stria vascularis. Furthermore, we found lacZ/Cx29 expression in nonmyelinating Schwann cells of the adrenal gland, in chondrocytes of intervertebral discs and the epiphysis of developing bones. Electron microscopic analyses of myelin sheaths in the central and peripheral nervous system of Cx29-deficient mice detected no abnormalities. The nerve conduction in the sciatic nerve of adult Cx29-deficient mice and the auditory brain stem response as well as visually evoked potentials in 4- to 10-week-old Cx29-deficient mice were not different from wild-type littermate controls. Thus, in contrast to connexin32 and connexin47, which are also expressed in myelinating cells, Cx29 does not contribute to the function of myelin in adult mice.

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: a) the morphology of clusters formed by the microtubule plus-end tracking protein “end binding 1” (EB1), b) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin rich sites, c) 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 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.

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein αolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Gαolf in odorant-specific signaling out of OR. The employment of the non-typical G protein α15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Gα15 or Gαolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.

Connexin45 (Cx45) is known to be expressed in the retina, but its functional analysis was problematic because general deletion of Cx45 coding DNA resulted in cardiovascular defects and embryonic lethality at embryonic day 10.5. We generated mice with neuron-directed deletion of Cx45 and concomitant activation of the enhanced green fluorescent protein (EGFP). EGFP labeling was observed in bipolar, amacrine, and ganglion cell populations. Intracellular microinjection of fluorescent dyes in EGFP-labeled somata combined with immunohistological markers revealed Cx45 expression in both ON and OFF cone bipolar cells. The scotopic electroretinogram of mutant mice revealed a normal a-wave but a 40% reduction in the b-wave amplitude, similar to that found in Cx36-deficient animals, suggesting a possible defect in the rod pathway of visual transmission. Indeed, neurotransmitter coupling between AII amacrine cells and Cx45-expressing cone bipolar cells was disrupted in Cx45-deficient mice. These data suggest that both Cx45 and Cx36 participate in the formation of functional heterotypic electrical synapses between these two types of retinal neurons that make up the major rod pathway.

The molecular mechanisms regulating skeletal muscle regeneration and differentiation are not well understood. We analyzed the expression of connexins (Cxs) 40, 43 and 45 in normal and regenerating tibialis anterior muscle and in primary cultures of differentiating myoblasts in adult and newborn mice, respectively. Cxs 45 and 43, but not 40, were strongly expressed in normal muscle and their expression was upregulated during regeneration. Furthermore, the functional role of Cx43 during differentiation and regeneration was examined after induced deletion of Cx43 in transgenic mice. In vivo, the inducible deletion of Cx43 delayed the formation of myofibers and prolonged the expression of myogenin during regeneration. In primary cultures of satellite cell-derived myoblasts, induced deletion of Cx43 led to decreased expression of myogenin and MyoD, dye coupling, creatine kinase activity and myoblast fusion. Thus, the expression of Cx45 and Cx43 is upregulated during skeletal muscle regeneration and Cx43 is required for normal myogenesis in vitro and adult muscle regeneration in vivo.

Alpha-ganglion cells are present in all vertebrate retinae and are subdivided into ON and OFF types according to their level of dendritic ramification within the inner plexiform layer. They have large dendritic fields and usually a good responsiveness to moving stimuli. They were the first ganglion cells in which tracer coupling was observed, suggesting the presence of gap junctions composed of unknown connexins. Here we show that ON-alpha-ganglion cells in the mouse retina are coupled to amacrine cells, whereas OFF-alpha-ganglion cells are coupled to other OFF-alpha-ganglion cells and to amacrine cells. These tracer coupling patterns were completely absent in mice deficient in connexin36 (Cx36). The expression of Cx36 protein in alpha-ganglion cells but not in coupled amacrine cells was confirmed in mice in which the Cx36 coding DNA was replaced by the lacZ reporter gene. The dendritic localization and the distribution pattern of Cx36 patches, analyzed in mice in which the enhanced green fluorescent protein (EGFP) was linked to the C-terminal region of the Cx36 protein, revealed a rather small number of fluorescent plaques and different patterns for ON- and OFF-alpha-ganglion cells. Furthermore, tracer coupling between OFF-alpha-ganglion cells could be inhibited by quinine, a gap junctional blocker with a slight preference for gap junctions formed by Cx36. These data strongly suggest that Cx36 gap junction channels are functional not only in interneurons but also in output neurons of the retina and are responsible for distinct coupling patterns of ganglion cells.

The gap junction protein connexin43 (Cx43) is thought to be involved in growth control in several tissues. Using the doxycycline inducible tet-on system, we generated human malignant trophoblast Jeg3 cells transfected with either Cx40, Cx43, or C-terminal truncated Cx43 (trCx43). Cx43, but not Cx40 or trCx43, displayed a reduced cell growth of Jeg3 cells in vitro and tumor growth in nude mice, suggesting a role of the C terminus of Cx43 in growth regulation. Using gene array analysis, the growth regulator NOV (CCN3), a member of the CCN gene family, was found to be up-regulated only in the Cx43-transfected cells. Validation by reverse transcriptase-PCR confirmed an up-regulation of the NOV transcript exclusively upon Cx43 induction. In contrast to Cx40 or trCx43, induction of Cx43 led to a switch in localization of NOV from the nucleus to the cell membrane, where it is colocalized with Cx43. Coimmunoprecipitation showed a binding of NOV to the C terminus of Cx43 in vitro as well as in transfected cells. Jeg3 cells transfected only with NOV revealed that NOV itself acts as a growth regulator. We suggest that Cx43 is able to regulate cell growth via an up-regulation of NOV transcription, a change in localization of the NOV protein and a binding of NOV to the C terminus of Cx43.

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morpho- logical and functional characteristics, specifically a nonoverlapping expression of either AMPA-type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter-controlled EGFP expression and patch-clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junc- tional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing -Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individ- ual cells. This study supports the notion that the two subpopulations of hGFAP-EGFP- positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes. © 2004 Wiley-Liss, Inc.

Targeted deletion of the connexin36 (Cx36) gene in the mouse genome leads to visual transmission defects, weakened synchrony of rhythmic inhibitory potentials in the neocortex, and disruption of -frequency network oscillations. We have generated transgenic mice in which a reporter protein consisting of the exon1 coded N-terminal part of Cx36 fused to -galactosidase (N36--gal) is expressed instead of Cx36. Here, we have used these mice for a detailed analysis of the reporter gene expression. By -gal staining of adult retina, we found expression of the lacZ reporter gene in the ganglion cell layer, in two rows of the inner nuclear layer, and in the photoreceptor layer. In the brain, -gal staining was present in -aminobutyric acid (GABA)ergic neurons of the cerebellar nuclei, in non-GABAergic neurons of the inferior olive, in mitral cells of the olfactory bulb, and in parvalbumin-positive cells of the cerebral cortex. Outside the central nervous system, N36--gal signals were detected in insulin producing -cells of the pancreas and in the medulla of the adrenal gland of adult Cx36 /del[LacZ] mice. This expression pattern suggests that Cx36 fulfills functional roles not only in several types of neurons in the retina and central nervous system but also in excitable cells of the pancreas and adrenal gland. J. Comp. Neurol. 473:511–525, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: retina; inferior olive; cerebellum; cortex; pancreas; adrenal gland

Conditional gene targeting using the Cre/loxP technology generally includes integration of a selection marker cassette flanked by loxP recognition sites (floxed) in the target gene locus. Subsequent marker removal avoids possible impairment of gene expression or mosaicism due to partial and total deletions after Cre-mediated recombination in vivo. The use of deleter Cre mice for in vivo marker removal in floxed connexin43 mice revealed considerable mosaicism, but no selective marker removal. In addition, we noted that several Cre transgenic lines displayed spontaneous ectopic activity, reminiscent of deleter Cre mice, and required the confirmation of cell type-specific deletion in every individual mouse. When we used myosin heavy chain promoter Cre (alphaMyHC-Cre) mice for cardiomyocyte specific deletion, we observed, in addition to cardiomyocyte-restricted or complete excision, selective marker removal in a subgroup of mice as well. Thus, selective marker removal can be achieved as a byproduct of cell-type restricted deletion.