PubMed Health⌕ Search

Biomedical subjects

H W Müller

Publications and source records attributed to H W Müller.

At least 73 records · Page 4Linked to original sources
PubMed1997

Chondroitin/dermatan sulphate promotes the survival of neurons from rat embryonic neocortex.

Recently we have shown that biglycan, a small chondroitin sulphate proteoglycan of the extracellular matrix, supports the survival of cultured neurons from the developing neocortex of embryonic day 15 rats. Here we investigate the structure-function relationship of this neurotrophic proteoglycan and show that chondroitin/dermatan sulphate chains are the active moieties supporting survival. Heparin, a highly sulphated glucosaminoglycan, is less active than the galactosaminoglycans (chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate), whereas hyaluronic acid, an unsulphated glucosaminoglycan, does not support neuron survival. Galactosaminoglycans must be in direct contact with neurons to cause survival. Experiments with elevated potassium concentrations and antagonists of voltage-gated calcium channels exclude the involvement of membrane depolarization. However, genistein and an erbstatin analogue, which are inhibitors of tyrosine kinases with low specificity, abolished neuron survival in the presence of chondroitin/dermatan sulphate, whereas a selective inhibitor of neurotrophin receptor kinases (K252a) had no suppressive effect. Thus, yet unidentified tyrosine kinases are involved in the chondroitin/dermatan sulphate-dependent survival of neocortical neurons. In the embryonic stages of rat neocortical development chondroitin sulphate is mainly located in layers I, V and VI and the subplate. Chondroitin sulphate expression is maintained after birth, extends up to cortical layer IV on postnatal day 7, and is down-regulated until postnatal day 21 concomitant with the period of naturally occurring cell death. The latter observation is consistent with a putative role of chondroitin sulphate in the control of neuron survival during cortical histogenesis.

Animals↗
PubMed1997

Lack of immune responses to immediate or delayed implanted allogeneic and xenogeneic Schwann cell suspensions.

Previous studies have shown that Schwann cell implantation offers a potential therapeutic approach to a variety of neurodegenerative disorders and traumatic injuries. In a clinically relevant paradigm, however, the implantation of autologous Schwann cells is problematic and the use of heterogenetic Schwann cells will be required. In the present study we addressed this important issue and analysed the immunogenicity and survival of allogeneic and xenogeneic Schwann cell suspension grafts in a prelesioned CNS fiber tract, the transected postcommissural fornix of the adult Wistar rat. Cultured Schwann cells from Wistar rat or human peripheral nerve were injected either immediately or after a delay into the transection site and the spatio-temporal pattern of leukocyte infiltration and of major histocompatibility antigen expression was characterized and semiquantified with immunocytochemical methods. Our main findings are that (1) invasive cerebral lesions induce the expression of MHC class I and II antigens, but only sparse infiltration of T-lymphocytes, (2) both allogeneic and xenogeneic discordant Schwann cell suspension grafts, from either neonatal or adult peripheral nerve, survive without any overt signs of rejection for up to 10 weeks after implantation; and (3) delayed implantation procedures have no effect on immune responses to allogeneic Schwann cell grafts. These results demonstrate that there is no marked ongoing immune reactions to heterogenetic Schwann cell suspension grafts and that long-term survival of cross-species Schwann cell grafts can be achieved in the absence of any immunsuppressive treatment. Thus the conditions for functional transplantation of Schwann cells across immunological barriers seem to be favourable and will have implications for future cross-species studies, and possibly also for clinical application.

Animals↗
PubMed1996

Cultured astrocytes express biglycan, a chondroitin/dermatan sulfate proteoglycan supporting the survival of neocortical neurons.

Astrocyte-conditioned medium (ACM) supports the survival of rat E15 neocortical neurons. Using a microtiter assay for neuronal survival, we demonstrated that part of the survival activity is associated with a proteoglycan fraction obtained after two chromatographic steps: (1) preparative Q-Sepharose anion-exchange chromatography under non-denaturating conditions and (2) MonoQ chromatography in the presence of 8 M urea. Analytical SDS-polyacrylamide gradient gel electrophoresis of pooled active MonoQ-fractions (MQ-pool) revealed a broad proteoglycan band migrating with an apparent M(r) in the range of 150-400 kDa. Digestion of the MQ-pool with chondroitin-ABC-lyase yielded a major core protein of 50 kDa. In Western blots the high molecular weight (150-400 kDa) material as well as the 50 kDa core protein band were immunoreactive to chicken polyclonal antibodies raised against purified biglycan from rat meningeal fibroblasts. Northern blot analysis of total RNA prepared from highly enriched astrocyte cultures revealed a single 2.9 kb biglycan transcript. By using in situ hybridization we demonstrated that essentially all cells in these cultures expressed biglycan mRNA. Furthermore, highly purified biglycan from bovine cartilage was shown to markedly enhance survival of rat neocortical neurons. In conclusion, we have shown that astrocytes synthesize and release the small chondroitin/dermatan sulfate proteoglycan (CS/DSPG) biglycan, a molecule that was found to support survival of neocortical neurons in vitro.

Animals↗
PubMed1996

Reconstruction of transected postcommissural fornix in adult rat by Schwann cell suspension grafts.

Schwann cell (SC) transplantation has emerged as a powerful tool to promote regeneration in the lesioned central nervous system (CNS). Most studies focused on the use of guidance channels to introduce the SCs into CNS neuropil; a technique that itself causes extensive damage to host tissue and is only applicable in superficial brain areas. The present study examines the efficacy of microinjected SC suspensions to promote structural reconstruction of the transected postcommissural fornix in the adult rat. Stereotactic, unilateral fornix transection was performed using a tungsten wire knife. Immediately after transection lesion cavities received either a Dulbecco's modified Eagle's medium injection or a pure SC suspension graft derived from a highly purified SC culture that was prepared from syngeneic rat (P1) sciatic nerves. After 4 days to 8 months, the implant characteristics as well as the structural reconstruction of the tract were analyzed using immunocytochemical methods and anterograde tracing techniques. Numerous SCs of the graft could be identified for up to 8 months. They rapidly dispersed from the injection site and migrated freely for considerable distances into the host tissue. The SCs exhibited a low proliferation activity that ceased within 2 weeks after transplantation. They did not prevent retrograde axonal degeneration of the fornix tract for a short distance (600 micron) but promoted structural reconstruction of the transected fornix tract. Regenerating fibers traversed the lesion site and extended along their former pathway up to the mammillary body, their proper target. Moreover, the applied transplantation technique allowed remyelination of the regenerating fibers by host oligodendrocytes. In conclusion, microtransplantation of SC suspensions represents a promising strategy for promoting structural reconstruction of lesioned CNS projections.

Animals↗
PubMed1996

Low affinity NGF receptor expression in CMT1A nerve biopsies of different disease stages.

Duplication of the gene for the peripheral myelin protein 22 (PMP22) is the most common cause for Charcot-Marie-Tooth neuropathy type 1a (CMT1A) neuropathy. In early stages of the disease PMP22 is overexpressed in nerve biopsies from CMT1A patients. Recent studies with genetically modified Schwann cells have demonstrated that the altered expression of PMP22 modulates cell growth. Thus we hypothesized that elevated expression of PMP22 at the beginning of the disease might alter Schwann cell differentiation and phenotype. In this study we investigated Schwann cell phenotype in different stages of CMT1A neuropathy using antibodies to established Schwann cell markers. We found a pathological expression of low affinity nerve growth factor receptor (LNGF-R-also referred to in the literature as p75) in numerous Schwann cells of young CMT1A patients with almost normal myelin thickness and very few onion bulbs. During further progression of the disease, when Schwann cells began to form onion bulbs, we observed an intense LNGF-R immunoreactivity in all layers of the onion bulbs. In the most advanced stages of the disease, characterized by massive onion bulb formation, no LNGF-R immunoreactivity was shown. In age-matched control nerves LNGF-R staining was barely detectable. Furthermore, onion bulbs seen in patients with chronic idiopathic polyneuropathy (CIDP) were always negative for LNGF-R. In addition, at all CMT1A disease stages analysed, LNGF-R-positive Schwann cells were glial fibrillary acidic protein (GFAP) negative. Immunostaining with an antibody to the proliferation marker, proliferating cellular nuclear antigen (PCNA) indicated Schwann cell proliferation when onion bulb formation was well developed. In conclusion, we describe a disease stage-dependent altered Schwann cell phenotype in CMT1A neuropathy, which could be a direct consequence of the PMP22 overexpression on Schwann cell growth behaviour or, less likely, a secondary phenomenon related to myelin loss.

Adolescent↗
PubMed1996

Full-lenth cloning, expression and cellular localization of rat plasmolipin mRNA, a proteolipid of PNS and CNS.

We have isolated a 1.476 bp cDNA (NTII11) representing a transcript that is differntially expressed during sciatic nerve development and regeneration in the rat. Nucleotide sequence comparison indicates partial identity with a recently isolated plasmolipin cDNA. However, our clone extends the published sequence by 234 bp at the 5' end and predicts a protein that contains an additional 25 amino acids at th N-terminus. The open reading frame of th NTII11 transcript encodes a 19.4 kDa protein with four putative transmembrane domains. Northern blot analyses revealed a tissue-specific expression was confirmed by in situ hybridization, and cellular localization of plasmolipin mRNA was demonstrated in Schwann cells of the sciatic nerve and in glial cells of myelinated brain structures. The steady-state levels of plasmolipin mRNA were markedly altered (i) during development of sciatic nerve and brain. (ii) after sciatic nerve injury, and (ii) in cured Schwann cells maintained under different conditions of cell growth and arrest. Our data indicate a function of plasmolipin during myelination in the central as well as in the peripheral nervous system.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed1995

Differential expression of the small chondroitin/dermatan sulfate proteoglycans decorin and biglycan after injury of the adult rat brain.

Chondroitin sulfate proteoglycans are widespread extracellular matrix proteins and are specifically upregulated after CNS injury at the lesion site. Many proteoglycan core proteins have been described in the rat brain, but detailed analysis of individual proteoglycans expressed after injury are missing. The present study represents an initial attempt to assess the diversity and timing of lesion-induced expression of proteoglycans in order to elucidate their functional role in CNS injury and repair. Using immunocytochemical methods we analysed the expression of decorin and biglycan in the transected postcommissural fornix of the adult rat. Transection of the fornix induced the upregulation of both decorin and biglycan. However, their expression differed with respect to time course, regional extent and cellular localization. The rapid upregulation of decorin within a wide area around the lesion was followed by a massive appearance of biglycan that remained restricted to the transection site. Three months after lesion, differences of the area size of decorin- and biglycan-immunoreactivities were no longer detectable. Both proteoglycans were restricted to the lesion site and the fornix stumps. While decorin was primarily expressed by astrocytes, biglycan was deposited extracellularly in sheet-like structures. The upregulation of both proteoglycans persisted for at least up to 6 months after lesion. These strong but divergent lesion-induced expression patterns indicate important but different roles of decorin and biglycan in CNS injury.

Animals↗
PubMed1995

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan.

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.

Animals↗
PubMed1995

Differentially expressed genes after peripheral nerve injury.

In an attempt to identify genes associated with Wallerian degeneration and peripheral nerve regeneration we have performed differential hybridization screening of a cDNA library from crushed rat sciatic nerve (7 days postlesion) using radioactively labeled cDNA prepared from poly(A)+ RNA of normal vs. crushed nerve. Screening of 5,000 randomly selected colonies yielded 24 distinct clones that were regulated following nerve injury. Fifteen of the differentially expressed sequences could be classified as induced, whereas 9 sequences appeared to be repressed at 1 week postcrush. Sequencing and computer-assisted sequence comparison revealed 3 classes of regulated cDNA clones representing 1) novel gene sequences (8 clones) including 3 transcripts containing a repetitive "brain identifier" (ID) element; 2) identified genes (7 clones) with previously undetected expression in the peripheral nervous system (PNS), such as apolipoprotein D, peripheral myelin protein 22kD (PMP22), SPARC (secreted protein, acidic and rich in cysteine), sulfated glycoprotein SGP-1, apoferritin, decorin, and X16/SRp20; and 3) identified genes (9 clones) with known expression in the PNS including, e.g., the myelin protein P0, gamma-actin, vimentin, alpha-tubulin, chargerin II, and cytochrome c-oxidase subunit I. Northern blot and polymerase chain reaction analyses with RNA from crushed and transected nerve demonstrated that sequences with related function, like the group of myelin genes, cytoskeleton genes, genes involved in RNA processing and translation, in lipid transport or energy metabolism showed closely related temporal patterns of expression during nerve degeneration and regeneration. Finally, we compared the differentially expressed genes identified at 7 days after crush injury (this investigation) with the regulated sequences isolated previously by De Leon et al. (J Neurosci Res 29:437-488, 1991) from a 3 day postcrush sciatic nerve cDNA library.

Animals↗
PubMed1995

Peripheral myelin protein-22 is expressed in rat and mouse brain and spinal cord motoneurons.

Peripheral myelin protein PMP-22 is expressed by Schwann cells and is a constituent of peripheral nervous system (PNS) myelin. Two PMP-22 transcripts, SR13 and CD25, differing in their 5' non-coding sequences have been described. SR13 is present both in the PNS and in non-neural tissue, whereas CD25 mRNA is almost exclusively expressed in Schwann cells. PMP-22 mRNA is also present in the central nervous system (CNS), but at much lower levels than in the PNS. We have investigated the regional distribution of PMP-22 mRNA in the rat and mouse CNS by the reverse transcriptase-polymerase chain reaction method, using oligonucleotide primers specific for the SR13 or CD25 transcripts. SR13 mRNA was detected in all the CNS regions analysed, whereas the CD25 message was present only in the brainstem and the spinal cord. Localization of the PMP-22 transcripts, determined by in situ hybridization in 21 day-old animals, showed selective expression in the motor nuclei. The PMP-22 signal was very weak in the nuclei of the oculomotor and trochlear nerves and absent in the nucleus of the abducens nerve. A strong PMP-22 signal was observed in the motor nuclei of the trigeminal, facial, ambigus, vagus, hypoglossal and accessory spinal nerves and in the ventral horn of the spinal cord. The PMP-22-positive cells were identified as motoneurons on the basis of topographic and morphological criteria, as well as immunolabelling with neuron-specific antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth arrest-specific gene gas3. Besides a putative role of PMP22 in myelination, a regulatory function in cell growth has been suspected. Here we have investigated both the expression of PMP22 during cell cycle progression of cultured rat Schwann cells and the influence of altered levels of PMP22 on Schwann cell growth. When resting cells were stimulated to begin the cell cycle, the regulation of PMP22 mRNA resembled the growth arrest-specific pattern of gas3 expression observed previously in NIH3T3 fibroblasts. To prove a growth regulatory function of PMP22, we generated Schwann cell cultures by infection with retroviral PMP22 expression vectors that constitutively expressed PMP22 cDNA sequences, in either the sense or antisense orientation. Transduced cells carrying the sense construct overexpressed PMP22 mRNA and protein, whereas in cells infected with an antisense PMP22 expression vector PMP22 mRNA levels were reduced markedly. Altered levels of PMP22 significantly modulated Schwann cell proliferation, as judged by 5-bromo-2'-deoxy-uridine incorporation into replicated DNA. In asynchronously dividing cultures enhanced expression of PMP22 decreased DNA synthesis to 60% of the control level. Conversely, reduced levels of PMP22 mRNA led to enhanced DNA synthesis of approximately 150%. Further cell cycle analyses by flow cytometry revealed that overexpression of PMP22 delayed serum- and forskolin-stimulated entry of resting Schwann cells from G0/G1 into the S + G2/M phases by approximately 8 h, whereas underexpression of PMP22 mRNA slightly increased the proportion of cells that entered the S + G2/M phases.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Clearance of myelin constituents and axonal sprouting in the transected postcommissural fornix of the adult rat.

Following transection of the postcommissural fornix in the adult rat, fibres retract from the lesion zone but then start to regrow within their former pathway up to the lesion site, where they terminate. The fibres neither penetrate nor bypass this region. In order to define the molecular mechanisms that cause regenerative failure at the lesion site, we analysed the spatiotemporal relationship between clearance/re-expression of myelin constituents and axon sprouting. Using immunocytochemical methods, we investigated the distribution of myelin-associated growth inhibitor (NI-35/250) and myelin basic protein after transection of the postcommissural fornix. In the studies described here we demonstrate the sequential removal of neurofilaments and myelin constituents in a perilesion zone and at the lesion site. The removal of myelin constituents was followed by the extensive regrowth of fornix fibres in the proximal segment. However, these fibres stopped at the lesion site, an area that lacked immunostaining for NI-35/250 and. In the distal stump we observed the disappearance of neurofilament along the entire fornix segment but spatial differences in the removal of myelin constituents. While both NI-35/250 and myelin basic protein disappeared in the perilesion zone, they persisted in the more distal segment for at least 28 months after lesion. In conclusion, our study indicates that the onset of axon sprouting is correlated with the removal of myelin basic protein and NI-35/250. Furthermore, we suggest that it seems unlikely that the myelin growth inhibitor NI-35/250 constitutes the stop signal of the axon growth barrier in the transected formix.

Animals↗
PubMed1995

Amplification of the MET gene in glioma.

We have previously reported the finding of MET amplification linked to double minutes (dmins) in a human glioblastoma (TX3095). Because dmins are found in approximately 50% of glioblastomas, 18 gliomas were analyzed for MET amplification. Three grade IV glioblastomas and one grade II astrocytoma showed amplification. We could also localize the MET amplicon to dmins in glioblastoma TX3095 by fluorescence in situ hybridization.

Base Sequence↗