Hans-Werner Müller

Restricted appearance of tenascin and chondroitin sulphate proteoglycans after transection and sprouting of adult rat postcommissural fornix

SummaryTransected fibres of the adult rat postcommissural fornix sprout over short distances but fail to traverse the lesion site and terminate in close vicinity to the wound. As a step in defining the molecular environment responsible for regeneration failure at the lesion site, we have used immunocytochemistry to analyse the spatio-temporal expression pattern of two putative growth-inhibitory extracellular matrix components, tenascin and chondroitin sulphate proteoglycans and their topographical relationship to the sprouting axons. Both tenascin and chondroitin sulphate proteoglycan labelling appeared after fornix transection and were confined to the immediate vicinity of the lesion site. While tenascin-labelling was associated with astrocytes and microglia/macrophages, which accumulate preferentially at the tract borders, chondroitin sulphate proteoglycan labelling appeared as a homogeneous meshwork around the wound. Tenascin-like immunoreactivity disappeared between 17 days and 4 weeks, but chondroitin sulphate proteoglycan staining persisted at least up to 14 months after transection. Regrowing fornix fibres invaded and elongated within the chondroitin sulphate proteoglycanimmunopositive region up to the lesion site, where they terminated. This zone of axonal growth inhibition was neither characterized by an increase of chondroitin sulphate proteoglycan immunoreactivity nor by the presence of tenascinimmunopositive structures. The spatio-temporal distribution patterns of tenascin and chondroitin sulphate proteoglycan and the permeability of the chondroitin sulphate proteoglycan-immunopositive region for sprouting axons do not support the hypothesis that chondroitin sulphate proteoglycan alone and/or tenascin inhibit the advance of sprouting fornix fibres.

Improved culture methods to expand schwann cells with altered growth behaviour from CMT1A patients

A duplication of the gene for myelin protein PMP22 is by far the most common cause of the hereditary demyelinating neuropathy CMT1A. A role for PMP22 in cell growth in addition to its function as a myelin protein has been suggested because PMP22 is homologous to a gene specifically upregulated during growth arrest. Furthermore, transfected rat Schwann cells overexpressing PMP22 show reduced growth. In addition, abnormal Schwann cell differentiation has been described in nerve biopsies from CMT1A patients. To analyse whether the duplication of the PMP22 gene in CMT1A neuropathy primarily alters Schwann cell differentiation and to exclude nonspecific secondary responses, we improved human Schwann cell culturing. This allowed us long‐term passaging of human Schwann cells with unchanged phenotype, assessed by expression of different Schwann cell markers. Subsequently we established Schwann cell cultures from CMT1A nerve biopsies. We find decreased proliferation of Schwann cells from different CMT1A patients in all passages. We also demonstrate PMP22 mRNA overexpression in cultured CMT1A Schwann cells. We conclude that decreased proliferation in cultured Schwann cells that carry the CMT1A duplication indicates abnormal differentiation of CMT1A Schwann cells. The identification of an abnormal phenotype of CMT1A Schwann cells in culture could possibly lead to an in vitro disease model. GLIA 23:89–98, 1998.

Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts

Abstract.The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth-arrest-specific gene gas3. The biological function of PMP22 is unknown, but recent progress in the analysis of rat Schwann cells expressing altered levels of PMP22 revealed that one role of PMP22 is as a negative growth modulator. We have investigated the influence of rat PMP22 (rPMP22) and a mutant of PMP22 (rPMP22Tr) resembling the murine trembler mutation on cell growth of retrovirus-vector-infected mouse NIH3T3 cells. Transduced cells carrying the two different sense constructs expressed rPMP22 and rPMP22Tr mRNAs and proteins. Elevated levels of rPMP22 and rPMP22Tr significantly reduced fibroblast growth as judged by proliferation assays. Despite a negative modulatory influence of rPMP22 and rPMP22Tr on cell proliferation, cell cycle analyses by flow cytometry did not reveal an influence of rPMP22 or rPMP22Tr on the synchronous progression of resting NIH3T3 cells from G0 into S phase. However, cell cycle analyses by flow cytometry of asynchronously dividing cultures demonstrated that the expression of rPMP22 and rPMP22Tr increased the fraction of cells in the G1 phase of the cell cycle. Furthermore, cell death analyses revealed that, in contrast to control cells and cells carrying the rPMP22Tr construct, a significantly increased fraction of NIH3T3 cells expressing rPMP22 exit the proliferation compartment showing hallmarks of programmed cell death. These results indicate that (i) rPMP22 and rPMP22Tr act as negative modulators of proliferation in murine fibroblasts probably through extension of the G1 phase of the cell cycle and (ii) rPMP22 but not rPMP22Tr promotes programmed death of these cells.

Relationship between injury-induced astrogliosis, laminin expression and axonal sprouting in the adult rat brain

SummaryLesion-induced regenerative sprouting of CNS axons is accompanied by structural and metabolic changes of astrocytes. In order to evaluate the effects of these astrocytic changes on axonal regeneration, we investigated the spatio-temporal relationship of gliosis, laminin expression and axonal sprouting in the postcommissural fornix of the adult rat. Using immunocytochemical methods we observed (1) a perilesional area with a transient lack of astrocytes and axons, (2) the reappearance of reactive astrocytes followed by the ingrowth of sprouting fibres and finally an increase in laminin-immunoreactivity, (3) the absence of lesion-induced laminin-expression in reactive astrocytes and (4) the formation and long-lasting (at least 28 months) persistence of a dense plexus of laminin-immunopositive blood vessels at the site of transection and in the proximal and distal stumps. These data indicate that astrogliosis is permeable for regrowing axons and that injury-induced axonal sprouting in the transected postcommissural fornix may be mediated by laminin-independent mechanisms.

Upregulation of GABAA-receptor α1- and α2-subunit mRNAs following ischemic cortical lesions in rats

Abstract 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 α-subunits of the GABA A -receptor (α1- and α2-subunits) change following ischemic cortical lesioning. The results show that 7 days after lesion induction mRNA-levels for both the α1- and α2-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 α1-subunit-protein and no change for the α2-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.

Proteins of peripheral myelin are associated with glycosphingolipid/cholesterol-enriched membranes

A characteristic feature of the vertebrate nervous system is the ensheathment of axons by myelin, a multilamellar membrane specialization produced by polarized glial cells. Although the main protein and lipid components of the myelin sheath are well characterized, relatively little is known about the mechanisms of their intracellular distribution to the respective sites of assembly within the myelin sheath. To analyze whether peripheral myelin protein trafficking is mediated by glycosphingolipid/cholesterol‐enriched membranes (GEMs), we studied the association of established myelin proteins, peripheral myelin protein 22 (PMP22), protein zero (P0), plasmolipin, and myelin basic protein (MBP), with these membrane microdomains. To examine the association of the selected peripheral myelin proteins with detergent‐insoluble GEMs, purified myelin from sciatic nerve of adult rat was extracted with Triton X‐100 at 4°C and 37°C and, in additional experiments, was pretreated with the cholesterol chelator methyl‐β‐cyclodextrin. The material was then centrifuged to equilibrium in sucrose gradients, and fractions were analyzed by Western blotting. Here we demonstrate for the first time that PMP22, P0, and plasmolipin prepared from purified peripheral myelin are associated with GEMs. To characterize whether the association of these proteins is a specialized feature of myelinating Schwann cells, we studied the distribution of PMP22, P0, and plasmolipin in transiently transfected HeLa cells. These experiments confirm the specific association of these proteins with GEMs in both neural and nonneural cell types.

Combinatorial code of growth factors and neuropeptides define neuroendocrine differentiation in PC12 cells.

Adrenal chromaffin cells constitute one of the first cell types to have been defined as a neuroendocrine cell type. Since they produce dopamine, these cells have been proposed for the treatment of neuronal deficits in human Parkinsons disease. However, the factors involved in the development of chromaffin cells are still poorly understood. Based on recent insights from stem cell research, we decided to study the role of extracellular matrices, growth factors and neuropeptides on the neuroendocrine differentiation in a serum-free medium of PC12 cells. Employing immunohistochemistry, quantitative PCR and HPLC analysis, neuroendocrine differentiation was determined by evaluating neurite outgrowth, catecholamine biosynthesis and release as well as neuropeptide and vesicular protein mRNA expression. The combination of bFGF, NGF and PACAP could prevent the inhibition of neurite process development induced by dexamethasone in PC12 cells cultured on ECM. Whereas glucocorticoids were essential in the regulation of enzymes of catecholamine biosynthesis and metabolism, growth factors and PACAP were more efficient in inducing neuropeptide and chromogranin B expression as well as release of dopamine and 3-methoxytyramine. Therefore, in addition to glucocorticoids, chromaffin cells need a gradient of matrix, growth factors, and neuropeptides to develop the full functional phenotype of a neuroendocrine cell.

Osteopontin: A novel axon-regulated Schwann cell gene

Osteopontin (OPN) is a RGD‐containing glycoprotein with cytokine‐like, chemotactic, and pro‐adhesive properties. During wound healing, OPN is abundantly expressed by infiltrating macrophages and has been implicated in posttraumatic tissue repair. To delineate a role in the regenerative response to axotomy we examined the expression of OPN in Wallerian degeneration of the sciatic nerve in rats. Unexpectedly, we found high constitutive expression of OPN by myelinating Schwann cells (SCs) in uninjured control nerves. OPN mRNA expression was confirmed in primary cultures of rat SCs. Upon axotomy, SC‐expressed OPN in the degenerating distal nerve stump transiently increased during the first days after injury, but was continuously downregulated thereafter, reaching its minimum at Day 14. Macrophages invading axotomized nerves were OPN‐negative. During late stages after axotomy, SC‐OPN was reexpressed in regenerating but not permanently transected nerves. We also found OPN expression by myelinating SCs in human sural nerves with a dramatic reduction in severe axonal polyneuropathies. Taken together, our study identifies OPN as a novel Schwann cell gene regulated by axon‐derived signals. The lack of OPN induction in infiltrating macrophages indicates fundamental differences in tissue repair between axonal injury in the peripheral nervous system and structural lesions in other organ systems.

Full‐length 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 differentially 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 the N‐terminus. The open reading frame of the NTII11 transcript encodes a 19.4 kDa protein with four putative transmembrane domains. Northern blot analyses revealed a tissue‐specific expression of the plasmolipin transcript that is restricted to sciatic nerve, brain and kidney. The neural 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 the sciatic nerve and brain, (ii) after sciatic nerve injury, and (iii) in cultured 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.

Proteolipid plasmolipin: localization in polarized cells, regulated expression and lipid raft association in CNS and PNS myelin

The proteolipid plasmolipin is member of the expanding group of tetraspan (4TM) myelin proteins. Initially, plasmolipin was isolated from kidney plasma membranes, but subsequent northern blot analysis revealed highest expression in the nervous system. To gain more insight into the functional roles of plasmolipin, we have generated a plasmolipin‐specific polyclonal antibody. Immunohistochemical staining confirms our previous observation of glial plasmolipin expression and proves plasmolipin localization in the compact myelin of rat peripheral nerve and myelinated tracts of the CNS. Western blot analysis indicates a strong temporal correlation of plasmolipin expression and (re‐) myelination in the PNS and CNS. However, following axotomy plasmolipin expression is also recovered in non‐regenerating distal nerve stumps. In addition, we detected plasmolipin expression in distinct neuronal subpopulations of the CNS. The observed asymmetric distribution of plasmolipin in compact myelin, as well as in epithelial cells of kidney and stomach, indicates a polarized cellular localization. Therefore, we purified myelin from the CNS and PNS and demonstrated an enrichement of phosphorylated plasmolipin protein in detergent‐insoluble lipid raft fractions, suggesting selective targeting of plasmolipin to the myelin membranes. The present data indicate that the proteolipid plasmolipin is a structural component of apical membranes of polarized cells and provides the basis for further functional analysis.