Keratin 18 provides resistance to Fas-mediated liver failure in mice.

BACKGROUND: Keratins are intermediate filament proteins of epithelial cells with pivotal functions for cell integrity. They comprise keratins 18 [K18] and 8 [K8] in hepatocytes. Keratins are of major importance for an intact cellular microarchitecture and have protective functions in human liver diseases. In mice, K8 has been demonstrated to protect against Fas-antibody-induced liver failure by direct interaction with apoptotic regulators, while the role of K18 remains unresolved. MATERIALS AND METHODS: We analysed effects of K18 deficiency on Fas-induced liver failure in mice. We determined survival and analysed induction of apoptosis after injection of the agonistic Fas antibody Jo2 into K18(-/-) and wild-type control mice by TUNEL assay and fluorometrically analysed caspase-3, -8 and -9 activities 1, 2 and 3 h after Jo2 injection. RESULTS: In K18(-/-) mice, survival of Fas-antibody treated mice was significantly shorter than that of wild-type controls (P = 0.02). However, shortened survival of K18(-/-) mice was caused by increased hepatic damage but was not correlated to enhanced induction of apoptotic pathways, as neither numbers of TUNEL positive apoptotic cells nor activities of caspases-3, -8 and -9 differed between K18(-/-) and K18(+/+) mice at any point of time. CONCLUSION: K18(-/-) mice are significantly more susceptible to Fas-antibody-induced liver failure. The cytoprotective effect of K18 is not explained by a differential activation of caspases-3, -8 and -9, suggesting that K18 does not directly interfere with apoptotic regulators. Importantly, however, K18 exerts significant protective functions by other mechanisms.

Visual transmission deficits in mice with targeted disruption of the gap junction gene connexin36.

In the mammalian retina, rods feed into the cone pathway through electrotonic coupling, and recent histological data suggest the involvement of connexin36 (Cx36) in this pathway. We therefore generated Cx36 null mice and monitored the functional consequences of this deficiency on early visual transmission. The homozygous mutant mice had a normally developed retina and showed no changes in the cellular organization of the rod pathway. In contrast, the functional coupling between AII amacrine cells and bipolar cells was impaired. Recordings of electroretinograms revealed a significant decrease of the scotopic b-wave in mutant animals and an increased cone threshold that is compatible with a distorted, gap junctional transmission between AII amacrine cells and cone bipolar cells. Recordings of visual evoked potentials showed extended latency in mutant mice but unaffected ON and OFF components. Our results demonstrate that Cx36-containing gap junctions are essential for normal synaptic transmission within the rod pathway.

Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons.

A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.

Stimulus complexity dependent memory impairment and changes in motor performance after deletion of the neuronal gap junction protein connexin36 in mice.

Gap junction channels, composed of connexin (Cx) proteins, are conduits for intercellular communication and metabolic exchange in the central nervous system. Connexin36 (Cx36) is expressed in distinct subpopulations of neurons throughout the mammalian brain. Deletion of the Cx36 gene in the mouse affected power and frequency of gamma and sharp wave-ripple oscillations, putative correlates of memory engram inscription. Here, we present a behavioral analysis of Cx36-deficient mice. Activity patterns, exploratory- and anxiety-related responses were largely unaffected by elimination of Cx36, while sensorimotor capacities and learning and memory processes were impaired. Repeated testing on the rotarod suggested that the Cx36-deficient mice showed slower motor-coordination learning. After a retention interval of 24 h the Cx36-deficient mice showed habituation to an open-field, but failed to habituate to a more complex spatial environment (Y-maze). A more pronounced memory impairment was found when Cx36 knockout mice had to remember recently explored objects. Cx36-deficient mice were unable to recognize objects after short delays of 15 and 45 min. These data suggest that lack of Cx36 induces memory impairments that vary in dependence of the complexity of the stimuli presented. Our results suggest that neuronal gap junctions incorporating Cx36 play a role in learning and memory.

A second alternative transcript of the gap junction gene connexin32 is expressed in murine Schwann cells and modulated in injured sciatic nerve.

Four connexin32 (Cx32) cDNA clones isolated from a rat sciatic nerve cDNA library differ in the nucleotide sequence of their 5' untranslated region (UTR) from the corresponding Cx32 cDNA clones previously characterized from liver. The new Cx32 5'UTR sequence detected in the sciatic nerve cDNA clones is identical to one previously found in the 6.5 kb intron of the murine Cx32 gene. Using primer extension and S1 nuclease protection analysis, we determined the transcriptional starting point of this new alternative Cx32 transcript expressed in the sciatic nerve. This starting point is located 444 bp (409 bp) upstream of exon2 in a region previously described as an intron of the Cx32 gene in the rat (and mouse) genome, respectively. The alternative exon1B comprises 99 bp in rat, but 97 bp in the mouse genome, and is spliced to the same exon2 acceptor site also used for splicing of exon1 in liver. Both transcripts are likely to code for the same Cx32 protein whose reading frame is located in exon2. The putative promoter region, upstream of the alternative exon1B, contains a TATAAA motif and has been sequenced and noticed before by Miller et al. (Biosci. Rep. 8, 455-464, (1988)). The alternative exon1B transcript is highly expressed in the sciatic nerve, (i.e. Schwann cells) and very low in liver (i.e. hepatocytes). Its expression is regulated after sciatic nerve injury. The time course of expression was similar to previously established myelin genes and, therefore, we suggest that the expression of the alternative exon1B Cx32 transcript is related to the process of myelination. Very recently, we have characterized another alternative Cx32 exon1A which is transcribed in mouse embryonic stem cells but not in the sciatic nerve (Dahl et al., submitted for publication, 1995). Thus, the murine Cx32 gene is likely to be regulated by three alternative promoters that appear to be activated in a cell type-specific manner.

The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development.

A new gap junction gene isolated from rat brain cDNA, mouse retina cDNA and mouse genomic DNA is called connexin36, since it codes for a connexin protein of 321 amino acids corresponding to the theoretical molecular mass of 36045 kDa (rat) and 36084 kDa (mouse). Only one amino acid residue differs between rat and mouse connexin36. In the single murine connexin36 gene, an 1.14-kb intron interrupts the coding region, similar as in the homologous skate connexin35 gene. Because of this unique feature, mouse connexin36 differs from the other 13 murine connexin genes and is suggested to form a new delta subclass of connexins. Connexin36 mRNA (2.9 kb) is highly expressed in adult retina and less abundant in brain where it gradually increased during fetal development until day 7 post partum, and decreased thereafter.

Expression patterns of connexin genes in mouse retina.

To analyze the molecular basis of gap junctional communication in mouse retina, we examined the expression pattern of the following 13 connexin (Cx) genes: Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50. By using reverse transcriptase-polymerase chain reactions with primer oligonucleotides to murine connexin genes, we detected mRNAs of Cx26, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Retinae from heterozygous mice with targeted replacement of most of the Cx45 open reading frame by a lacZ reporter gene showed Cx45 promoter activity in somata of the ganglion cell layer and the inner nuclear layer. Immunoblot and immunofluorescence analyses with antibodies generated to murine connexin epitopes revealed the presence of Cx36, Cx37, Cx43, and Cx45 proteins: The outer and inner plexiform layer were immunopositive for Cx36 and Cx45. Cx37 immunoreactivity was found in blood vessels of the inner retina. Cx43 immunolabeling was detected in the ganglion cell layer and nerve fiber layer where it was largely colocalized with immunostaining of glial fibrillary acidic protein suggesting that Cx43-positive cells could be of glial origin. No Cx26 protein was detected in retina by using Cx26 antibodies for immunoblot analyses or confocal microscopy. Furthermore, comparative immunofluorescence analyses of retinae from mice deficient for Cx31, Cx32, or Cx40 with retinae of wild-type mice revealed no specific immunostaining. Our results demonstrate regional specificity in expression of connexin genes in mouse retina and, thus, provide a basis for future assignments of functional defects in connexin-deficient mice to cells in different regions of the retina.

Connexin genes in the mouse and human genome.

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.

Cx36 and the function of endocrine pancreas.

The secretory, duct, connective and vascular cells of pancreas are connected by gap junctions, made of different connexins. The insulin-producing beta-cells, which form the bulk of endocrine pancreatic islets, express predominantly Cx36. To assess the function of this connexin, we have first studied its expression in rats, during sequential changes of pancreatic function which were induced by the implantation of a secreting insulinoma. We observed that changes in beta-cell function were paralleled by changes in Cx36 expression. We have also begun to investigate mutant mice lacking Cx36. The absence of this protein did not affect the development and differentiation of beta-cells but appeared to alter their secretion. We have studied this effect in MIN6 cells which spontaneously express Cx36. After stable transfection of a construct that markedly reduced the expression of this connexin, we observed that MIN6 cells were no more able to secrete insulin, in contrast to wild type controls, and differentially displayed a series of still unknown genes. The data provide evidence that Cx36-dependent signaling contributes to regulate the function of native and tumoral insulin-producing cells.

Expression of connexin genes in hippocampus of kainate-treated and kindled rats under conditions of experimental epilepsy.

We have analyzed whether the expression of connexin genes is altered in the hippocampus of kindled and kainate-treated rats, i.e., animal models of human temporal lobe epilepsy. We have tested this hypothesis by analyzing mRNA, protein abundance and cellular location of connexins (Cx) 43, 36, 32 and 30. The expression of glial fibrillary acid protein and mRNA was also monitored both in kainate-treated and kindled rats, in order to take into account reactive gliosis under these conditions. We found significantly increased expression of GFAP mRNA (100%) and protein (178%) in kainate-treated rats 4 weeks after kainate application, whereas in kindled rats only moderate increases of GFAP mRNA and protein were detected 2-3 weeks (group 2) or 4-6 weeks (group 1) after the last stage 5 induced seizure. Under gliotic conditions, connexins 43 and 30 mRNA or protein expression in astrocytes of kainate-treated rats were nearly unaffected. Cx36 mRNA expression (presumably in neurons) was significantly reduced (44%), whereas abundance of Cx36 protein was only slightly reduced. In both groups of kindled rats, Cx30 and Cx43 mRNA or protein expression were either slightly decreased or unchanged. Again, Cx36 mRNA and protein expression were reduced by about half in group 2. Immunofluorescence analysis of Cx43, Cx36 and Cx30 expression revealed that 4 weeks after the last kainate administration or kindling, cellular localization of these connexins was indistinguishable from control animals.

Analysis of Cx36 knockout does not support tenet that olivary gap junctions are required for complex spike synchronization and normal motor performance.

Electrotonic coupling by gap junctions between neurons in the inferior olive has been claimed to underly complex spike (CS) synchrony of Purkinje cells in the cerebellar cortex and thereby to play a role in the coordination of movements. Here, we investigated the motor performance of mice that lack connexin36 (Cx36), which appears necessary for functional olivary gap junctions. Cx36 null-mutants are not ataxic, they show a normal performance on the accelerating rotorod, and they have a regular walking pattern. In addition, they show normal compensatory eye movements during sinusoidal visual and/or vestibular stimulation. To find out whether the normal motor performance in mutants reflects normal CS activity or some compensatory mechanism downstream of the cerebellar cortex, we determined the CS firing rate, climbing-fiber pause, and degree of CS synchrony. None of these parameters in the mutants differed from those in wildtype littermates. Finally, we investigated whether the role of coupling becomes apparent under challenging conditions, such as during application of the tremorgenic drug harmaline, which specifically turns olivary neurons into an oscillatory state at a high frequency. In both the mutants and wildtypes this application induced tremors of a similar duration with similar peak frequencies and amplitudes. Thus surprisingly, the present data does not support the notion that electrotonic coupling by gap junctions underlies synchronization of olivary spike activity and that these gap junctions are essential for normal motor performance.

Vasoactive hormones in children with chronic renal failure.

Plasma renin activity (PRA), aldosterone, vasopressin and catecholamines were measured in 15 children (ages 7.3 to 16.2 years) with chronic renal failure (CRF) before and after one session of hemodialysis and in 15 control children. Basal levels of PRA and aldosterone in children with CRF did not differ significantly from control values, but showed a wider range. Uremic patients with nephronophthisis showed the highest basal PRA and aldosterone levels. In children with CRF, basal vasopressin levels were significantly higher (9.7 +/- [SEM] 2.0 ng/liter) than control values (3.2 +/- 0.8 ng/liter). Plasma noradrenalin and adrenalin concentrations were similar in children with CRF and controls. During hemodialysis, a fall in blood pressure and a rise in heart rate was observed in all children. PRA and catecholamines increased twofold to fivefold during dialysis while aldosterone and vasopressin showed a variable response. In contrast to reports in adults, there is no evidence for an insufficiency of vasoactive hormones or of the sympathetic nervous system in children on hemodialysis.

Treatment of cerebral Langerhans cell histiocytosis.

Cerebral Langerhans cell histiocytosis (LCH) is a rare granulomatous disorder which may be primary or secondary or solitary or multiple. Brain structures outside the hypothalamic-pituitary axis are only scarcely involved, even in multisystem varieties. Since there are neither controlled therapeutic trials nor systematic analyses of hitherto reported cases, optimal treatment strategies are not known. To evaluate the effect of different treatment modalities, we analyzed previous reports of extrahypothalamic LCH back to 1980 in which the diagnosis was made on the basis of examination of cerebral tissues. Thirty-five histologically examined cases were identified, including 10 patients presenting with multiple cerebral lesions. Adding one own case followed up for 10 years, 16 patients had cerebral involvement secondary to multisystem LCH, while another 20 patients had primary cerebral LCH. The peak incidence was far beyond the pediatric range for both primary and secondary cerebral LCH. Localized lesions can be treated successfully by surgery or radiation following biopsy. Chemotherapy may be an additional option. Multiple lesions can tentatively be controlled by chemotherapy and, possibly, radiation. The ultimate outcome is determined by whether or not recurrencies or de-novo lesions will appear and the course of the systemic disease. Studies addressing the effects of therapy in cerebral LCH are urgently needed.

Connexin expression in the retina.

Here, we review recent results from our laboratory on connexin expression in mouse retina in the context of previous results with other vertebrate species. In mouse retina, four different connexin proteins were detected by immunoblot and immunofluorescence: connexin (Cx)-36, -37, -43 and -45. Cx36 and Cx45 immunoreactive signals were found in the inner and outer plexiform layer, both of which are known to show interneuronal gap junctions. Cx43 was detected in the ganglion cell layer, presumably in astrocytes, where it appeared to be colocalized with glial fibrillary acid protein. Cx37 was expressed in retinal endothelial cells. Additionally, Cx26, -31, -32 and -40 mRNAs were detected in retina by RT-PCR but none of the corresponding proteins were found. In order to exclude cross reactions of the corresponding antibodies, retinae from targeted connexin-deficient mice (Cx31 -/-, Cx32 -/- and Cx40 -/-) were used as negative controls for immunoblot and immunofluorescence analyses of wild-type retina. Further detailed investigation of cell type specific connexin expression in the mouse retina will be necessary for functional analyses of targeted mouse mutants with defects in connexins expressed in retinal neuronal cells.

The mouse gap junction gene connexin29 is highly expressed in sciatic nerve and regulated during brain development.

A novel mouse gap junction gene, coding for a presumptive protein of 258 amino acids (molecular mass: 28 981 Da), has been designated connexin29. This single copy gene was mapped to distal mouse chromosome 5 and shows 75% sequence identity to a human connexin30.2 sequence in the database. Connexin29 mRNA (4.4 kb) is highly expressed in mouse sciatic nerve and less abundant in spinal cord as well as in adult brain, where it increased 12-fold between day 7 and 14 post partum. Our expression data suggest that the new connexin gene is active in myelin-forming glial cells.

A new alternatively spliced transcript of the mouse connexin32 gene is expressed in embryonic stem cells, oocytes, and liver.

The rodent gap junction protein connexin32 (Cx32) is highly expressed in hepatocytes, less abundantly in Schwann cells and oligodendrocytes, and at low levels in the early mouse embryo. In both hepatocytes and Schwann cells, Cx32 expression is directed by alternative promoter regions (P1 and P2) which activate differently spliced transcript isoforms. Here we describe a third Cx32 transcript isoform expressed in embryonic cells, oocytes, and liver. Using competitive polymerase chain reaction, we have found that this new Cx32 transcript containing exon 1A is 200-fold less abundant in liver than the Cx32 isoform with exon 1. In mouse oocytes, the exon 1A-containing Cx32 transcript is exclusively expressed. Immunoblot analyses revealed no Cx32 protein expression in embryonic stem cells, whereas it has previously been demonstrated in oocytes. When the putative Cx32 promoter region upstream of exon 1A was cloned before the lacZ reporter gene, transient transfection yielded weak expression in embryonic stem cells. Our results suggest that the exon 1A-containing Cx32 isoform is likely to be inherited as an oogenetic product but not translated during early embryogenesis.

Connexin30-deficient mice show increased emotionality and decreased rearing activity in the open-field along with neurochemical changes.

Gap-junction channels in the brain, formed by connexin (Cx) proteins with a distinct regional/cell-type distribution, allow intercellular electrical and metabolic communication. In astrocytes, mainly the connexins 43, 26 and 30 are expressed. In addition, connexin30 is expressed in ependymal and leptomeningeal cells, as well as in skin and cochlea. The functional implications of the astrocytic gap-junctional network are not well understood and evidence regarding their behavioural relevance is lacking. Thus, we have tested groups of Cx30-/-, Cx30+/-, and Cx30+/+ mice in the open-field, an object exploration task, in the graded anxiety test and on the rotarod. The Cx30-/- mice showed reduced exploratory activity in terms of rearings but not locomotion in the open-field and object exploration task. Furthermore, Cx30-/- mice exhibited anxiogenic behaviour as shown by higher open-field centre avoidance and corner preference. Graded anxiety test and rotarod performance was similar across groups. The Cx30-/- mice had elevated choline levels in the ventral striatum, possibly related to their aberrant behavioural phenotypes. The Cx30+/- mice had lower dopamine and metabolite levels in the amygdala and ventral striatum and lower hippocampal 5-hydroxyindole acid (5-HIAA) concentrations relative to Cx30+/+ mice. Furthermore, the Cx30+/- mice had lower acetylcholine concentrations in the ventral striatum and higher choline levels in the neostriatum, relative to Cx30+/+ mice. Our data suggest that the elimination of connexin30 can alter the reactivity to novel environments, pointing to the importance of gap-junctional signalling in behavioural processes.

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.

Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.

Functional expression of the murine connexin 36 gene coding for a neuron-specific gap junctional protein.

The mouse connexin 36 (Cx36) gene was mapped on chromosome 2 and an identical transcriptional start site was determined in brain and retina on exon I. Rabbit polyclonal antibodies to the presumptive cytoplasmic loop of the Cx36 protein recognized in immunohistochemical analyses Cx36 expression in the retina, olfactory bulb, hippocampus, inferior olive and cerebellum. In olivary neurons strong punctate labeling at dendritic cell contacts and weaker labeling in the cytoplasm of dendrites were shown by immuno electron microscopy. After expression of mouse Cx36 cDNA in human HeLa cells, neurobiotin transfer was increased 1.8-fold and electrical conductance at least 15-fold compared to untransfected HeLa cells. No Lucifer Yellow transfer was detected in either untransfected or Cx36 transfected HeLa cells. Single Cx36 channels in transfected HeLa cells showed a unitary conductance of 14.3 + or - 0. 8 pS. The sensitivity of Cx36 channels to transjunctional voltage was low in both HeLa-Cx36 cells and Xenopus oocytes expressing mouse Cx36. No increased transfer of neurobiotin was detected in heterotypic gap junctions formed by Cx36 and 9 other connexins expressed in HeLa cells. Our results suggest that Cx36 channels function as electrical synapses for transmission of electrical and metabolic signals between neurons in the central nervous system.