Biomedical subjects

R Dermietzel

Publications and source records attributed to R Dermietzel.

At least 37 records · Page 2Linked to original sources

PubMed1998

Gap junction wiring: a 'new' principle in cell-to-cell communication in the nervous system?

This review gives an updated excerpt of recent advances in our understanding of brain gap junctions. It starts with a brief description of the principle molecular composition of gap junctions before specific issues concerning brain tissues are addressed. The following questions and matters are subjected to a detailed analysis: First, why are there so many gap junctions in the brain? Second, what is the functional significance of the cellular diversity of brain gap junctions? Third, how do astrocytic gap junctions mediate intercellular volume transmission (IVT), and what does IVT mean for glial-neuronal interaction? Fourth, how frequent are interneuronal gap junctions; and what is their functional significance in brain development and in interrelated chemical-electrotonic transmission at mixed synapses.

Animals↗

PubMed1998

Large conductance channel in plasma membranes of astrocytic cells is functionally related to mitochondrial VDAC-channels.

Large conductance anion channels with similar electrophysiological characteristics were found in plasma membranes and in outer mitochondrial membranes of various cell types. Although their large conductance and their peculiar voltage dependence point to a close relation, it was questioned whether they belong to the same family. We therefore compared some biochemical features of a plasmalemmal channel with those known from the mitochondrial channel. Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN). The underlying channels exhibited a conductance of 401 +/- 50 pS. Open probability was highest between +/- 10 mV and gradually approached zero beyond +/- 25 mV. Activity as induced by voltage ramps between +/- 40 mV appeared after a delay of up to several min. The delay could be reduced by bathing either side of the patch in an acidic Ringer solution (pH 6.2). 1 mM Al3+ increased the open time at potentials more positive than 20 mV. 10 mM dextran sulfate (MW 8000) caused reversible flickering, increasing the closed probability. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) also caused a reversible flickering into the closed state, reducing the apparent single channel amplitude by up to 70% at 0.5 mM DIDS. Application of 5 mM ATP resulted in reversible blockade; ATP was more effective from the outside than from the inside (blocking activity 65% vs. 16% of the patches). We conclude that the large conductance anion channel from astrocytic cells displays electrophysiological and pharmacological characteristics that resemble those of VDAC (Voltage Dependent Anion Channel) from the outer mitochondrial membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Regulation of a blood-brain barrier-specific enzyme expressed by cerebral pericytes (pericytic aminopeptidase N/pAPN) under cell culture conditions.

In this study we show that the aminopeptidase N of cerebral pericytes (pAPN) associated with the blood-brain barrier (BBB) is downregulated in pericytic cell cultures. This observation is in accordance with previous data describing comparable in vitro effects for BBB-specific enzymes of endothelial or pericytic origin, such as gamma-glutamyl transpeptidase or alkaline phosphatase. By polymerase chain reaction and in situ hybridization we were able to determine that the down-regulation of pAPN occurs at the posttranscriptional level. The mRNA of pAPN was found to be constitutively expressed even when the protein is no longer detectable. Culturing the pericytes in an endothelial cell-conditioned medium allowed pAPN to be reexpressed. However, the reexpression effect depended largely on the culturing conditions of the pericytes. Although purified pericytes deprived of endothelial cells did not reveal a reexpression effect, pericytes that were kept in contact with endothelial cells were able to acquire a pAPN-positive phenotype, indicating that endothelial cells constitute an essential requirement for the in vitro reexpression of pAPN. Astrocytes, however, were insufficient in exerting any reexpression effect.

Animals↗

PubMed1998

Fibroblast growth factor 2 (FGF-2) differentially regulates connexin (cx) 43 expression and function in astroglial cells from distinct brain regions.

Fibroblast growth factor (FGF)-2 is a peptide growth factor that promotes the generation, differentiation, and survival of neurons and glial cells. In the CNS, astroglial cells are coupled in a region-specific manner by gap junctions consisting of connexin 43 (cx43). In the present study we have investigated effects of FGF-2 and of other growth factors on the expression and function of cx43 in astroglial cells cultured from telencephalic cortex, striatum, and mesencephalon of newborn rats. Confluent cultures were maintained for two days in low serum, and then exposed to FGF-2 (10 ng/ml) for 48 h. FGF-2 caused a reduction of cx43-protein, -mRNA, and intercellular communication revealed by dye spreading. These changes occurred in cortical and striatal cells, but not in mesencephalic astroglial cells. Effects of FGF-2 were time- and concentration-dependent, with a minimal effective dose of 1 ng/ml FGF-2, and an onset of effects after 6 h of incubation. The reduction of coupling by FGF-2 was transient, since in cortical and striatal cultures coupling recovered to control levels 48 h after removal of the growth factor. Like FGF-2, transforming growth factor-beta3 (TGF-beta3) decreased coupling of cortical and striatal, but not mesencephalic astroglial cells. Astroglial cells from all brain regions showed a slight FGF-mediated increase in 5-bromo-2'-desoxy-uridine (BrdU) incorporation, which was abolished upon co-treatment with TGF-beta3. However, TGF-beta3 did not interfere with the repression of cx43-function by FGF-2. Epidermal growth factor (EGF) that has been demonstrated to influence coupling in other cell types had no effect on dye spreading but significantly increased BrdU incorporation. Our results reveal a novel function of FGF-2 on cultured astroglial cells which may be relevant to the regulation of astroglial cell connectivity in vivo.

Animals↗

PubMed1998

Dye coupling between spinal cord oligodendrocytes: differences in coupling efficiency between gray and white matter.

Oligodendrocytes express two gap junction proteins, connexin32 (Cx32) and Cx45. To test for functional coupling between oligodendrocytes, cells were filled with the (Cx32-permeable) dyes Lucifer Yellow (LY) and Neurobiotin. Cells in slices from rat spinal cord were dialyzed via the patch pipette containing the dye while recording with the patch-clamp technique. The dye-labeled cells were identified as oligodendrocytes by their characteristic pattern of membrane currents and by morphology. In gray matter, 18% of the injected cells (N = 94) were coupled to more than three adjacent cells (slices from postnatal day 1 to 19). In contrast, in white matter, the dye was restricted to the injected cell (N = 63 for Lucifer Yellow injection only; N = 11 for LY and Neurobiotin) indicating a lack of functional coupling. Immunolabeling of Cx32 in mature oligodendrocytes of white matter revealed that the gap junction protein is localized on the cell bodies and abaxonal processes which occupy non-overlapping territories. In immature white matter and gray matter, Cx32 is mostly concentrated in the somatic region of the cells. In addition to Cx32, we have obtained immunocytochemical data that oligodendrocytes can express Cx45 with a labeling pattern different from the Cx32 expression. Two alternative interpretations of the coupling data are discussed: 1) that the presence of Cx32 in mature white matter oligodendrocytes does not serve for communication between cells, but rather for communication within oligodendrocytes in the sense of autocellular coupling, or 2) that the glial syncytium is furnished with a high degree of functional rectification at the oligodendrocytic side.

Animals↗

PubMed1997

Advances in Charcot-Marie-Tooth disease research: cellular function of CMT-related proteins, transgenic animal models, and pathomechanisms. The European CMT Consortium.

The First Workshop of the European Consortium on Charcot-Marie-Tooth (CMT) disease brought together neuroscientists, molecular and cell biologists, neuropathologists, neurologists, and geneticists with a common interest in the understanding of the fundamental mechanisms that underlie the pathogenesis of CMT. The interdisciplinary group of 25 expert scientists discussed recent advances in (i) molecular genetics and histopathology of CMT, (ii) development of suitable animal models, (iii) understanding of the cellular function of CMT-related proteins, and (iv) studies using nerve biopsies from CMT patients. In this minireview, we summarize the key findings presented and discuss their impact on CMT research.

Animals↗

PubMed1997

Expression of connexin43 mRNA in adult rodent brain.

The expression of connexin43 mRNA was detected in adult rat brains by in situ hybridization methods. Specific digoxigenin riboprobes were generated by in vitro transcription of two PCR-amplified fragments of connexin43 cDNA which lack homology with any other published connexin. Following immunohistochemical detection, the digoxigenin cRNA was found to occur in various neuronal populations including Purkinje cells of the cerebellum, pyramidal cells of the neocortex and the hippocampal formation, as well as granule cells of the dentate gyrus and various neurons of diverse hindbrain nuclei. This ubiquituous expression of connexin43 mRNA in adult neurons, in particular in neocortical pyramidal cells, is surprising insofar as gap junction communication in adult bains has been considered to be confined to specific subpopulations of neurons revealing a high incidence of synchronized electrical activities in contrast to the postnatal brain where interneuronal coupling via gap junctions precedes the formation of chemical transmission. In addition, connexin43 is regarded as being preferentially expressed in astrocytes, although its presence in adult neurons has not definitely been excluded. We propose that adult neurons preserve their capability of expressing functional gap junctions more frequently than presently considered and that connexin43 is a most likely neuronal gap junction protein candidate.

Animals↗

PubMed1997

Expression of the gap junction protein connexin43 in the subependymal layer and the rostral migratory stream of the mouse: evidence for an inverse correlation between intensity of connexin43 expression and cell proliferation activity.

Connexins constitute the channel-forming proteins of gap junctions. Gap junctions are considered to be involved in the regulation of cell proliferation. To verify this hypothesis for connexin43, the most abundant connexin in brain tissue, we have analyzed the expression of this gap junction protein in the subependymal layer and the rostral migratory stream of the murine telencephalon. These regions reveal high proliferative activity, even during postnatal stages and in adulthood. Proliferating cells were labeled in vivo by means of the bromodeoxyuridine method and were later processed for double immunocytochemistry by using an antibody to connexin43. The relationship between connexin43 expression and cell proliferation was also determined in primary cell cultures of olfactory bulbs from newborn mice. The intercellular coupling efficiency of cultured bulbar cells was also analyzed by dye-transfer experiments in combination with the bromodeoxyuridine technique. In the rostral migratory stream, connexin43 was upregulated during postnatal development, coinciding with a decrease of BrdU incorporation. Comparative quantification of the intensity of connexin43 immunoreactivity by confocal laser microscopy and of BrdU-labeled cells showed a clear reverse correlation between connexin43 expression and cell proliferation in the rostral migratory stream during postnatal development. A marked reverse correlation of both parameters was also observed in primary cell cultures from olfactory bulbs at day 6 after seeding.

Animals↗

PubMed1997

Coexpression of connexin45 and -32 in oligodendrocytes of rat brain.

Connexin proteins are the subunits of gap junction channels, and are encoded by a gene family. Although several connexin mRNAs were detected in brain, only a few connexin-proteins have been localized to specific cell types in this tissue. Here we describe expression of connexin45 protein in oligodendrocytes in rat hippocampus. Double immunofluorescent staining using specific antibodies to connexin45 and connexin32 paired with cell-type specific marker proteins revealed that connexin45 and connexin32 were co-expressed and colocalized in oligodendrocytes. Each of the connexin antibodies gave rise to the same pattern of punctate fluorescence in the plasma membrane of cell bodies and proximal processes of oligodendrocytes. Connexins in the plasma membrane of oligodendrocytes may form gap junctions between oligodendrocytes, or between oligodendrocytes and astrocytes. Expression of connexin45 in oligodendrocytes may prevent dysmyelinating effects of connexin32 mutations in the central nervous system of Charcot-Marie-Tooth (X-type) patients.

Animals↗

PubMed1997

Oligodendrocytes express gap junction proteins connexin32 and connexin45.

Oligodendrocytes, the myelin-forming glia of brain, are connected by gap junctions in situ and in culture. Cultured oligodendrocytes from adult bovine and porcine brains were studied using immunocytochemical, molecular, and electrophysiological techniques in order to characterize the gap junction types. The expression of connexin32 was substantiated by the detection of low, but significant, signals using connexin-specific probes in Northern and Western blot analyses. Connexin43, which comprises gap junctions in astrocytes, was not detectable in pure oligodendrocytic cultures; mRNAs of connexin40 and connexin37 and connexin26 were also not detected. By means of two specific antibodies directed to the recently cloned connexin45 and by RT-PCR we were able to identify this connexin as a second oligodendrocytic gap junction protein. Whole cell voltage clamp recording provided evidence for electrical coupling between pairs of cultured oligodendrocytes (mean junctional conductance 3.9 nS, n = 38 pairs) and intracellular Lucifer Yellow injection indicated that oligodendrocytes were usually only weakly dye coupled, with spread generally being restricted to nearest neighbors. Unitary conductances ranged from > 20 to < 150 pS with modes of distribution at about 100 to 120pS and 40 to 20 pS, respectively. These unitary conductances are consistent with the channel events expected for connexin32 and connexin45. The low degree of functional coupling between oligodendrocytes in vitro corresponds with the low levels of connexin32 and connexin45 messenger RNAs and protein expression.

Animals↗

PubMed1996

Regulation of connexin-43, GFAP, and FGF-2 is not accompanied by changes in astroglial coupling in MPTP-lesioned, FGF-2-treated parkinsonian mice.

Basic fibroblast growth factor (bFGF; FGF-2) has potent trophic effects on developing and toxically impaired midbrain dopaminergic (DAergic) neurons which are crucially affected in Parkinson's disease. The trophic effects of FGF-2 are largely indirect, both in vitro and in vivo, and possibly involve intermediate actions of astrocytes and other glial cells. To further investigate the cellular and molecular mechanisms underlying the restorative actions of FGF-2, and to analyse in more detail the changes within astroglial cells in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned striatum, we have studied striatal expression and regulation of connexin-43 (cx43), the principal gap junction protein of astroglial cells, along with the expression of glial fibrillary acidic protein (GFAP), FGF-2, and functional coupling. Our results show an immediate, yet transient increase in cx43 mRNA, and a sustained increase in FGF-2 mRNA, GFAP-positive cells, and cx43-immunoreactive punctata following the MPTP lesion, without any induction of functional coupling between astrocytes and other glial cells as revealed by dye coupling of patched cells. Unilateral administration of FGF-2 in a piece of gelfoam caused a further increase in cx43-positive punctata immediately adjacent to the implant, which was more pronounced than after application of a gelfoam containing the nontrophic control protein cytochrome C. These changes were parallelled by a small increase in cx43 protein determined by Western blot, but not by alterations in the coupling state of cells in the vicinity of the gelfoam implant. Although our data indicate that MPTP and exogenous FGF-2 may alter expression and protein levels of cx43, they do not support the notion that increases in cellular coupling may underly the trophic and widespread actions of FGF-2 in the MPTP-model of Parkinson's disease.

Animals↗

PubMed1996

Mitral and tufted cells of the mouse olfactory bulb possess gap junctions and express connexin43 mRNA.

Analyses of freeze-fracture replicas of mouse olfactory bulb reveal the presence of gap junctions in the plasma membranes of the cell bodies of mitral cells. Due to their localization and morphology we presume that they interconnect mitral and granule cells. Since the quality of electrical transmission between neurons is considered to be determined by the biochemical nature of the gap junction channel forming proteins (connexins) we performed immunohistochemistry and in situ hybridization using probes for connexin43 (Cx43), the most abundant connexin in brain tissue. Attribution of Cx43 immunolabel to specific neurons could not definitely be assessed by means of immunohistochemistry. In situ hybridization, however, using a specific cRNA probe for Cx43 revealed a label confined to cell bodies of mitral and tufted cells of the olfactory bulb. These data indicate that Cx43 is expressed by bulbar neurons and suggest that Cx43 is a molecular constituent of gap junction channels in neurons.

Animals↗

PubMed1996

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Animals↗

PubMed1996

C-erbB2/neu transfection induces gap junctional communication incompetence in glial cells.

Astrocytes form functional networks that participate in active signaling in which external stimuli are generated and amplified in many of the same ways as in neurons. Gap junctions between astrocytes offer the structural avenue by which the electrical and metabolic signals are propagated from one cell to another. Little is known about the trafficking, assembly, and degradation mechanisms of the major astrocytic gap junction protein connexin43. We have studied a glial cell line transfected with the C-erbB2/neu oncogene (neu+), finding severe interruption of gap junctional communication after stable transfection. Evidence from Western blotting and phosphorylation studies showed that the processing of connexin43 to its higher phosphorylated isoforms is disturbed. Confocal laser imaging indicates that the major deficit in the neu+ cells is attributable to a lack in plaque assembly of connexin43. Because the neu+ cells also lack N-CAM proteins and because work from others has indicated a close relationship between communication competence and constitutive CAM expression, our data suggest that expression of C-erbB2/neu oncogene alters cell-cell association via CAM proteins, which thereby affects gap junction plaque assembly and appropriate phosphorylation of connexin43.

Animals↗

PubMed1996

TNF alpha inhibits Schwann cell proliferation, connexin46 expression, and gap junctional communication.

Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-alpha or the mitogen neu differentiation factor (NDF)-beta. TNF alpha inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNF alpha also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both Po and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDF beta initially had little effect on cell division although it reduced junctional coupling within 8 h. However, by 48 h, NDF beta stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU+) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNF alpha may be a signal which terminates proliferation as well as junctional communication.

Animals↗

PubMed1996

Altered connexin expression after peripheral nerve injury.

The identification of connexin32 (Cx32) in myelinating Schwann cells and the association of Cx32 mutations with peripheral neuropathies suggest a functional role for gap junction proteins in the nerve. However, after nerve crush injury, Cx32 expression dramatically decreases in Schwann cells in the degenerating region, returning to control levels at newly formed nodes of Ranvier and Schmidt-Lantermann incisures by 30 days. The present study examined increases in expression of other connexins that occur after peripheral nerve injury. A 56/58-kDa connexin46 (Cx46) protein species was detected in adult rat sciatic nerve, along with very low levels of Cx46 mRNA. However, by 3 days after crush injury, coincident with changes in Schwann cell phenotype, Cx46 mRNA rapidly increased in the degenerating regions. Additionally, the 56/58-kDa Cx46 protein species present in adult nerve decreased and a 53-kDa Cx46 species, which was also present in cultured Schwann cells, became apparent. Connexin43 (Cx43) mRNA and protein, which was localized to perineurial cells in adult nerve, dramatically increased in endoneurial fibroblasts in the crush and distal regions by 3 days, coincident with macrophage infiltration. By 12 days after injury, Cx43 decreased and was comparable to normal nerve. These results suggest that enhanced expression of Cx46 and Cx43, by nonneuronal cells, may be important for the injury and regenerative responses of peripheral nerves.

Animals↗

PubMed1996

Connexin expression and intercellular communication in two- and three-dimensional in vitro cultures of human bladder carcinoma.

The identification of gap-junctional proteins (connexins) and the preparation of related antibodies provides new tools to study patterns of intercellular communication in tumors. Focusing on the biology of human bladder carcinoma, we compared the expression of gap-junctional proteins (connexins Cx26, Cx32, and Cx43) with a dye-coupling assay for gap-junctional intercellular communication in three cell lines with different urothelial differentiation. The cell lines HCV-29, RT4, and J82 were initially grown as monolayers of different ages. Connexin expression was found mostly positive over the time of culture and found constantly negative only in J82 cells for Cx26 and HCV-29 cells for Cx32. In HCV-29 cells, Cx26 increased in positivity over the time of culture. Western blotting with the antibodies confirmed the findings. Comparisons of dye transfer using Lucifer Yellow showed an increase of coupling in the normal urothelial cell line HCV-29 in contrast to a decrease of coupling in the tumor cell lines. Data were extended by multicellular spheroid (MCS) co-cultures with the stromal fibroblast line N1. In three-dimensional cultures as MCSs, Cx26 was increased in proximity of RT4 tumor cells to fibroblasts, and positivity was maintained in J82 cells. E-cadherin expression in cell lines showed no change in dependence of growth state. The data suggest that Cx26 plays a role in negative growth control or differentiation of urothelial cells. Preliminary comparative data on normal and neoplastic urothelium show all three connexins in normal urothelium, in contrast to varying amounts of Cx43 and low amounts of Cx32 in tumors and evident loss of Cx26 in low-grade tumors. Discrepancies between monolayer and MCS cultures are most likely due to higher differentiation in MCSs, and the continuation of systematic work with heterologous MCSs is indicated for more information on the role of gap-junctional proteins in human tumors.

Blotting, Western↗

PubMed1995

Morphological study in the early stages of complement C5a fragment-induced experimental meningitis: activation of macrophages and astrocytes.

Subarachnoidal application of the complement C5a fragment was used to induce acute experimental meningitis in rabbits and rats within 30-60 min. The early stages of the cellular inflammatory response were studied by means of flow cytometry, transmission electron microscopy and immunofluorescence microscopy. Infiltration of polymorphonuclear leukocytes (PMN) into the subarachnoidal space was the earliest event of the inflammatory reaction. By morphological criteria we found that PMN interacted with cells of the mononuclear-macrophage lineage (MML) and the marginal astrocytes via pseudopodia, whereas the pial cells were not involved in early stages of the inflammatory response. The number of invaded MML that were positive with the ED2 marker increased, indicating the hematogenous origin of the immigrating cell population. PMN were found to infiltrate the perivascular space of the marginal arterial vessel segments. This perivascular infiltration was assumed to be the first manifestation of cerebral vasculitis. The intimate association of resident cerebral cells (astrocytes) with invading PMN and MML is suggestive of a transient interaction of these cell types.

Animals↗