Biomedical subjects

H W Müller

Publications and source records attributed to H W Müller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Alternative exon usage of rat septins.

Septins represent a family of phylogenetically conserved proteins required for cytokinesis. Their presence in pre- and postsynaptic neuronal membranes suggests a general function as scaffolds for membrane reorganization. The transcriptional regulation of all septins examined so far is complex, resulting in alternatively spliced variants. We focus here on the rat homologue of the gene for the human septin MSF, a truncated form of which, designated eseptin, had been described previously. It will be shown here that there is an alternative usage of the first exon by two forms, named exon r1a and r1b, respectively. Exon r1a, but not exon r1b, contains a part of the coding sequence while the start of translation for the remaining coding sequence resides in the second exon. The complete genomic organization was resolved and data on the temporal and spatial expression of this septins are presented.

Alternative Splicing↗

PubMed2000

Glycosaminoglycan-binding properties and secondary structure of the C-terminus of netrin-1.

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the "deleted in colorectal carcinoma" gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an alpha-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (K(D) = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, K(D) = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an alpha-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.

Amino Acid Sequence↗

PubMed2000

Chondroitin sulfates expressed on oligodendrocyte-derived tenascin-R are involved in neural cell recognition. Functional implications during CNS development and regeneration.

Tenascin-R (TN-R), an extracellular matrix constituent of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions underlying axon growth inhibition/guidance, myelination and neural cell migration during development and regeneration. Although most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide first evidence that TN-R derived from whole rat brain or cultured oligodendrocytes expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e., C-4S and C-6S, that are recognized by CS-56, a CS/dermatan sulfate-specific monoclonal antibody. Based on different in vitro approaches utilizing substrate-bound glycoprotein, we found that TN-R-linked CS GAGs (1) promote oligodendrocyte migration from white matter microexplants and increase the motility of oligodendrocyte lineage cells; (2) similar to soluble CS GAGs, induce the formation of glial scar-like structures by cultured cerebral astrocytes; and (3) contribute to the antiadhesive properties of TN-R for neuronal cell adhesion in an F3/F11-independent manner, but not to neurite outgrowth inhibition, by mechanism(s) sensitive to chondroitinase or CS-56 treatments. Furthermore, after transection of the postcommissural fornix in adult rat, CS-bearing TN-R was found to be stably upregulated at the lesion site. Our findings suggest the functional impact of TN-R-linked CS on neural cell adhesion and migration during brain morphogenesis and the contribution of TN-R to astroglial scar formation (CS-dependent) and axon growth inhibition (CS-independent), i.e., suppression of axon regeneration after CNS injury.

Animals↗

PubMed2000

Tetraspan myelin protein PMP22 and demyelinating peripheral neuropathies: new facts and hypotheses.

It has been demonstrated that abnormal levels of PMP22 expression due to altered gene dosage in CMT1A neuropathy alters Schwann cell growth and differentiation. On the other hand, disease-related missense mutations within transmembrane domains of PMP22 disturb intracellular protein trafficking leading to accumulation of the mutant protein in the endoplasmic reticulum/Golgi compartment. Further, the recently reported association of PMP22 and P0 in peripheral myelin sheds new light on the almost identical phenotypes of CMT1A and CMT1B giving rise to a unifying hypothesis on disease mechanism.

Animals↗

PubMed2000

Cellular localization of the disintegrin CRII-7/rMDC15 mRNA in rat PNS and CNS and regulated expression in postnatal development and after nerve injury.

Disintegrins perform putative functions in cell adhesion, signaling and fusion. We have isolated a 2815-bp rat cDNA (CRII-7) representing a transcript that is differentially expressed during sciatic nerve regeneration. Nucleotide sequence comparison indicates that CRII-7 is the rat homologue to the recently cloned cDNAs MDC15 (ADAM 15) and metargidin (hMDC15) of mouse and human, respectively. The CRII-7 cDNA (rMDC15) encodes a membrane-anchored glycoprotein of approximately 85 kDa containing a disintegrin and a metalloprotease domain. Cellular metalloprotease disintegrins are a family of proteins (ADAMs or MDC proteins) with important roles, e.g., in cell-cell interactions during fertilization, muscle and nerve development, or tumor necrosis factor-alpha (TNF-alpha) cleavage. Northern blot analysis demonstrated a predominant expression of CRII-7/rMDC15 in the nervous system (PNS and CNS) and lung. Analysis of the CRII-7/rMDC15 transcript levels following peripheral nerve lesions demonstrated regulated mRNA expression during Wallerian degeneration and nerve regeneration. The steady-state levels of CRII-7/rMDC15 transcripts markedly increased within the first day after lesion and then steadily decreased for at least 4 weeks. CRII-7/rMDC15 mRNA expression was further examined during postnatal development and maturation of rat sciatic nerve and brain, as well as in cultured Schwann cells, meningeal fibroblasts, and astrocytes. In situ hybridization on paraffin sections showed the cellular localization of CRII-7/rMDC15 mRNA in Schwann cells and endothelial cells of peripheral nerve and in various neuronal populations in brain and spinal cord.

ADAM Proteins↗

PubMed2000

Enhanced proliferation and potassium conductance of Schwann cells isolated from NF2 schwannomas can be reduced by quinidine.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that is characterized mainly by schwannomas, as well as menigiomas and gliomas. The NF2 gene product merlin/schwannomin acts as a tumor suppressor. Schwann cells derived from NF2 schwannomas showed an enhanced proliferation rate, and electrophysological studies revealed larger K(+) outward currents as compared with controls. Schwann cells isolated from schwannomas of NF2 patients or multiorgan donors were treated with different concentrations of the K(+) current blockers quinidine, tetraethylammonium chloride, and 4-aminopyridine and K(+) outward currents and proliferation rates of these cells were compared. K(+) outward currents of both cell types can be blocked by quinidine. Importantly, treatment with quinidine reduces proliferation of NF2 Schwann cells in a concentration dependent manner but did not reduce proliferation of normal Schwann cells. Therefore, the use of quinidine or quinidine-like components would possibly provide a novel adjuvant therapeutic option for NF2 patients to slow down or freeze growth of schwannomas.

Antineoplastic Agents↗

PubMed2000

Facilitation of learning and long-term ventral pallidal-cortical cholinergic activation by proteoglycan biglycan and chondroitin sulfate C.

We have shown previously in the rat that biglycan, a recently discovered chondroitin sulfate proteoglycan, has neurotrophic effects which are mediated by its chondroitin/dermatan sulfate chains. Here we report that biglycan has neurochemical effects when injected into the nucleus basalis magnocellularis of the ventral pallidum, a site of dense cholinergic cell bodies. The effects on the cholinergic output in the frontal cortex are long lasting, indicating profound neuroactive function akin to that expected of a long-acting hormone. Injected into the same area of the brain, as well as into the ventricles in behaviorally impaired old animals, we found that biglycan can improve learning and memory in several behavioral paradigms. Furthermore, we show that both the neurochemical effectiveness as well as the promotion of learning is carried not by the proteoglycan per se, but rather by its chondroitin sulfate moiety, thus, demonstrating for the first time memory-promoting and neuroactive effects of a glycosaminoglycan, namely, chondroitin sulfate C. The results suggest that biglycan and other extracellular matrix molecules can have neurobehavioral and pharmacological functions for beyond those traditionally attributed to this class of molecules.

Acetylcholine↗

PubMed2000

Cloning and characterization of SDF-1gamma, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system.

The cytokines SDF-1alpha and -1beta are two alternatively spliced variants of the CXC (alpha) chemokines that are highly conserved among species. SDF-1alpha was shown to function as a B-cell maturation factor, a ligand for the CXCR4 (LESTR/fusin) chemokine receptor, thereby inhibiting replication of T cell-tropic HIV-1 strains and inducing cell death in human neuronal cell lines. In this report the cloning of the rat SDF-1beta cDNA and a new SDF-1 isoform, SDF-1gamma, are presented. Using Northern blot analysis, the expression pattern of both isoforms was studied in different tissues and it is shown that during postnatal development of the central and peripheral nervous system SDF-1beta- and SDF-1gamma-mRNA expression is inversely regulated. Whilst SDF-1beta-mRNA is the predominant isoform in embryonic and early postnatal nerve tissue, SDF-1gamma-mRNA is expressed at higher levels in adulthood. After peripheral nerve lesion a transient increase in SDF-1beta-mRNA expression is observed. As revealed by in situ hybridization, neurons and Schwann cells are the main cellular sources of both SDF-1beta and SDF-1gamma mRNAs in the nervous system. Computer-assisted analysis revealed that both transcripts encode secreted peptides with putative proteolytic cleavage sites which might generate novel neuropeptides.

Alternative Splicing↗

PubMed2000

Mutation-dependent alteration in cellular distribution of peripheral myelin protein 22 in nerve biopsies from Charcot-Marie-Tooth type 1A.

The hereditary demyelinating neuropathy Charcot-Marie-Tooth type 1A is caused by duplication or by point mutations of the PMP22 gene. Histopathological differences in these genotypes suggest distinct disease mechanisms. In the present investigation we demonstrate a pathologically altered cellular distribution of PMP22 in sural nerve biopsies of patients with PMP22 point mutations. In these patients, in contrast to findings in patients with PMP22 duplication, PMP22 partially accumulates in the Schwann cells instead of being inserted in the myelin sheath. These findings may explain the different histopathology and may suggest different mechanisms of pathogenesis in these genotypes.

Adolescent↗

PubMed1999

Biohybride nerve guide for regeneration: degradable polylactide fibers coated with rat Schwann cells.

The restricted capacity of the nervous system to regenerate calls for novel therapeutic concepts. We have tested biocompatible polylactide fibers as potential nerve guides that could bridge proximal nerve stumps and synaptic target regions after nerve lesion. Polylactides have the great advantage that they degrade and resorb after completion of regeneration. Material surface properties were optimized three-fold by oxygen plasma treatment, polyanion coating and the seeding of Schwann cells from rat sciatic nerve. Immunocytochemistry and scanning electron microscopy revealed that in vitro axonal outgrowth of dorsal root ganglia on two specifically synthesized lactide polymers can be greatly improved by these surface treatments. The approach aims to develop an 'intelligent neuroprosthesis' that in vivo facilitates directed axonal regrowth in the first place and disappears thereafter.

Animals↗

PubMed1999

New vistas on the pathomechanism of Charcot-Marie-Tooth and related peripheral neuropathies.

A gene-dosage mechanism in CMT1A and HNPP has been postulated previously. Here, recent findings are discussed concerning (i) the functional consequences of altered PMP22 expression on Schwann cell growth regulation and on the capacity of genetically modified Schwann cells to myelinate peripheral axons, (ii) the cell physiological effects caused by the expression of certain disease-related missense mutations of PMP22 that are known to alter the Schwann cell phenotype and impair myelination in vivo, and (iii) the pathomechanism of CMT1 in light of findings on a novel association between PMP22 and P0 in PNS myelin.

Animals↗

PubMed1999

Peripheral myelin protein 22 and protein zero: a novel association in peripheral nervous system myelin.

Mutations found in the two major glycosylated transmembrane proteins of the PNS myelin, the peripheral myelin protein zero (P0) and peripheral myelin protein 22 (PMP22), have been independently associated with the most common hereditary demyelinating peripheral neuropathies. Genotype-phenotype correlations in humans and transgenic animals have provided functional evidence that P0 and PMP22 are involved in formation and maintenance of compact myelin. Here, we demonstrate for the first time that P0 and PMP22 proteins form complexes in the myelin membrane, as shown by coimmunoprecipitation experiments, and that glycosylation is not involved in mediating these interactions. Complex formation was also detected when the two proteins were coexpressed in heterologous cells. In transfected cells, P0 and PMP22 are recruited and colocalize at the apposed plasma membranes of expressors as shown by confocal microscopy. These findings provide a new basis for a better understanding of myelin assembly and of the pathomechanisms involved in demyelinating peripheral neuropathies. Furthermore, these results propose a possible explanation why alterations in either of these molecules are sufficient to destabilize the myelin structure and cause a similar disease phenotype.

Animals↗

PubMed1999

Upregulation of GABAA-receptor alpha1- and alpha2-subunit mRNAs following ischemic cortical lesions in rats.

Focal cortical lesions are associated with a functional downregulation of the GABAergic system in perilesional tissue lasting (at least) several weeks. The molecular mechanisms underlying this phenomenon are still poorly understood. Here we used RT-PCR to investigate whether mRNA-levels of two alpha-subunits of the GABAA-receptor (alpha1- and alpha2-subunits) change following ischemic cortical lesioning. The results show that 7 days after lesion induction mRNA-levels for both the alpha1- and alpha2-subunits are increased threefold in perilesional tissue ipsilateral, but not contralateral to the lesion. Taken together with the results of a previous immunohistochemical study in which a moderate decrease of the alpha1-subunit-protein and no change for the alpha2-subunit [T. Neumann-Haefelin, J.F. Staiger, C. Redecker, K. Zilles, J.M. Fritschy, H. Mohler, O.W. Witte, Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats. Neuroscience (Oxford) 87 (4) (1998) 871-879] was observed, this is interpreted as a partial block of translation in the perilesional tissue surrounding cortical ischemic lesions.

Animals↗

PubMed1999

Post-transcriptional regulation of the peripheral myelin protein gene PMP22/gas3.

The peripheral myelin protein PMP22 gene has been described as a growth arrest-specific gene gas3 and has been identified as disease gene of various demyelinating neuropathies. The gene consists of two highly conserved alternative noncoding 5'-exons la (CD25) and 1b (SR13), respectively. Differential expression patterns of these transcripts in vivo and in vitro suggest a very complex mode of PMP22 gene regulation, which cannot be explained merely by transcriptional control. In fact, the PMP22 gene is regulated on different post-transcriptional levels. While reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no alterations in stability for both PMP22 transcripts in randomly growing Schwann cell cultures of rat sciatic nerve for at least 8 hours, in serum-induced synchronized cultures of resting cells we observed a specific cell cycle-regulated degradation of both transcripts. We further prepared diverse PMP22/CAT fusion genes to study the influence of the alternative 5'UTRs on PMP22 translation. Transient transfection of NIH3T3-fibroblasts and rat Schwann cells demonstrated that the alternative 5'UTRs (CD25 and SR13) and the 3'UTR exert differential regulatory influences on the translation efficiency.

3' Untranslated Regions↗

PubMed1999

Basal membrane-depleted scar in lesioned CNS: characteristics and relationships with regenerating axons.

The lesion scar formed after CNS injury is an impediment to axonal regeneration and leads to growth arrest or misrouting of sprouting axons. Our previous study showed that pharmacological reduction of basal membrane formation within the scar can overcome this scar impermeability [Stichel C. C. et al. (1999) Eur. J. Neurosci. 11, 632-646]. The aim of the present study was to characterize the basal membrane-depleted scar and to analyse its relationships with penetrating axons. The experiments comprised two groups of animals in which the left postcommissural fornix was transected; in addition, one group received a local immediate injection of the collagen IV-reducing agent dipyridyl, while the other group received an injection of phosphate-buffered saline. Immunohistochemical methods were used to characterize scar formation and scar-axon relationships. Animals receiving dipyridyl showed reduction of collagen IV-immunopositive basal membrane in the lesion center, which was accompanied by: (i) a decrease in laminin, as well as chondroitin and heparan sulfate proteoglycan, deposition in the lesion center; (ii) an increase in chondroitin and keratan sulfate proteoglycan expression in the perilesional area; (iii) a typical activation pattern of astrocytes and microglia/macrophages; (iv) axons regenerating through this modified scar were closely associated with various glial cell types and crossed a prominent chondroitin/keratan sulfate proteoglycan matrix. Our results suggest that neither the formation of a reactive astroglial network nor the accumulation of microglia/macrophages or the deposition of chondroitin and keratan sulfate proteoglycans in the perilesional area represent a barrier to regrowing axons. The present approach demonstrates that the lesion-induced basal membrane itself is the primary determinant of scar impermeability.

2,2'-Dipyridyl↗

PubMed1999

Inhibition of collagen IV deposition promotes regeneration of injured CNS axons.

Scarring impedes axon regrowth across the lesion site and is one major extrinsic constraint to effective regeneration in the adult mammalian central nervous system. In the present study we determined whether specific biochemical or immunochemical modulation of one major component of the scar, the basal membrane (BM), would provide a means to stimulate axon regeneration in the mechanically transected postcommissural fornix of the adult rat. Basal membrane developed within the first 2 weeks after transection in spatiotemporal coincidence with the abrupt growth arrest of spontaneously regrowing axons. Local injection of anticollagen IV antibodies or alpha, alpha'-dipyridyl, an inhibitor of collagen triple helix formation and synthesis, significantly reduced lesion-induced BM deposition. This treatment allowed massive axon elongation across the lesion site. Anterograde tracing provided unequivocal evidence that regenerating axons follow their original pathway, reinnervate the appropriate target, the mammillary body, and become remyelinated with compact myelin. Presynaptic electrophysiological recordings of regenerated fibre tracts showed recovery to nearly normal conduction properties. Our results indicate that lesion-induced BM is an impediment for successful axonal regeneration and its reduction is a prerequisite and sufficient condition for regrowing axons to cross the lesion site.

2,2'-Dipyridyl↗

PubMed1999

Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

3T3 Cells↗

PubMed1999

Dissipation in ferrofluids: mesoscopic versus hydrodynamic theory.

Part of the field dependent dissipation in ferrofluids occurs due to the rotational motion of the ferromagnetic grains relative to the viscous flow of the carrier fluid. The classical theoretical description due to Shliomis (Zh. Eksp. Teor. Fiz. 61, 2411 (1971) [Sov. Phy JETP 34, 1291 (1972)]) uses a mesoscopic treatment of the particle motion to derive a relaxation equation for the nonequilibrium part of the magnetization. Complementary, the hydrodynamic approach of Liu [Phys. Rev. Lett. 70, 3580 (1993)] involves only macroscopic quantities and results in dissipative Maxwell equations for the magnetic fields in the ferrofluid. Different stress tensors and constitutive equations lead to deviating theoretical predictions in those situations, where the magnetic relaxation processes cannot be considered instantaneous on the hydrodynamic time scale. We quantify these differences for two situations of experimental relevance, namely, a resting fluid in an oscillating oblique field and the damping of parametrically excited surface waves. The possibilities of an experimental differentiation between the two theoretical approaches is discussed.

Journal Article↗