Dieter R. Zimmermann

Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell–like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest–derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.

Extracellular matrix of the central nervous system: from neglect to challenge

The basic concept, that specialized extracellular matrices rich in hyaluronan, chondroitin sulfate proteoglycans (aggrecan, versican, neurocan, brevican, phosphacan), link proteins and tenascins (Tn-R, Tn-C) can regulate cellular migration and axonal growth and thus, actively participate in the development and maturation of the nervous system, has in recent years gained rapidly expanding experimental support. The swift assembly and remodeling of these matrices have been associated with axonal guidance functions in the periphery and with the structural stabilization of myelinated fiber tracts and synaptic contacts in the maturating central nervous system. Particular interest has been focused on the putative role of chondroitin sulfate proteoglycans in suppressing central nervous system regeneration after lesions. The axon growth inhibitory properties of several of these chondroitin sulfate proteoglycans in vitro, and the partial recovery of structural plasticity in lesioned animals treated with chondroitin sulfate degrading enzymes in vivo have significantly contributed to the increased awareness of this long time neglected structure.

Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult, mouse central nervous systems

Bral1, a brain-specific hyaluronan-binding protein, has been cloned recently. To gain insight into the role of Bral1, we generated a specific antibody against this protein. We have examined the detailed localization pattern of Bral1 protein and compared it with that of other members of the lectican proteoglycan family, such as brevican and versican, with which Bral1 is predicted to interact. The immunoreactivity of Bral1 antibody was predominantly observed in myelinated fiber tracts in the adult brain and could be detected at P20 in the white matter of the developing cerebellum, suggesting that expression starts when axonal myelination takes place. Furthermore, immunostaining demonstrated that Bral1 colocalized with the versican V2 isoform at the nodes of Ranvier. The present data suggest that Bral1 may play a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes.

Differential expression of versican isoforms in brain tumors

Versican is a member of the family of large aggregating chondroitin sulfate proteoglycans which are one of the major constituents of brain extracellular matrix (ECM). We examined the expression of versican splice variants at mRNA and protein levels in normal human brain and in gliomas, medulloblastomas, schwannomas, neurofibromas, and meningiomas. RT-PCR revealed transcripts for V0 and V1 in all tissues. V2 mRNA was restricted to gliomas and normal brain, while V3 mRNA was detected in all tissues except for medulloblastomas. Immunohistochemistry using antibodies to the glycosaminoglycan (GAG)-α attachment domain of versican (present in V0 and V2) revealed decreased staining of most glioma ECMs compared to normal neuropil, while some abnormal tumor vessels, but not normal cerebral vessels, were GAG-α-positive. Expression of the GAG-β attachment domain (present in V0 and V1) was faint in normal neuropil and cerebral vessels, but increased in tumor vessels and was absent in most glioma ECMs. Both GAG-α and GAG-β were expressed in connective tissue of all nonglial tumors. Our data suggest that V2 is the major versican isoform of normal neuropil and glioma ECM. Furthermore, increased expression in tumor vessels and decreased expression in glioma ECM of the anti-adhesive versican may be related to marked local invasivity and rarity of extracranial metastasis of gliomas.

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Three Mechanisms Assemble Central Nervous System Nodes of Ranvier

Rapid action potential propagation in myelinated axons requires Na⁺ channel clustering at nodes of Ranvier. However, the mechanism of clustering at CNS nodes remains poorly understood. Here, we show that the assembly of nodes of Ranvier in the CNS involves three mechanisms: a glia-derived extracellular matrix (ECM) complex containing proteoglycans and adhesion molecules that cluster NF186, paranodal axoglial junctions that function as barriers to restrict the position of nodal proteins, and axonal cytoskeletal scaffolds (CSs) that stabilize nodal Na⁺ channels. We show that while mice with a single disrupted mechanism had mostly normal nodes, disruptions of the ECM and paranodal barrier, the ECM and CS, or the paranodal barrier and CS all lead to juvenile lethality, profound motor dysfunction, and significantly reduced Na⁺ channel clustering. Our results demonstrate that ECM, paranodal, and axonal cytoskeletal mechanisms ensure robust CNS nodal Na⁺ channel clustering.

Versican V0 and V1 Guide Migratory Neural Crest Cells

We previously showed the selective expression of the chondroitin sulfate proteoglycans versican V0 and V1 in barrier tissues that impede the migration of neural crest cells during embryonic trunk development (Landolt, R. M., Vaughan, L., Winterhalter, K. H., and Zimmermann, D. R. (1995) Development 212, 2303-2312). To test for an active involvement of these isoforms in the guidance process, we have now established protocols to isolate intact versican V0 and V1 in quantities sufficient for functional experiments. Using stripe choice assays, we demonstrate that pure preparations of either a mixture of versican V0/V1 or V1 alone strongly inhibit the migration of multipotent Sox10/p75NTR double-positive early neural crest stem cells on fibronectin by interfering with cell-substrate adhesion. We show that this inhibition is largely core glycoprotein-dependent, as the complete removal of the glycosaminoglycan chains has only a minor effect on the inhibitory capacity. Our findings support the notion that versican variants V0 and V1 act, possibly in concert with other inhibitory molecules such as aggrecan and ephrins, in directing the migratory streams of neural crest cells to their appropriate target tissues.

Versican V2 Assembles the Extracellular Matrix Surrounding the Nodes of Ranvier in the CNS

The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix surrounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2 during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality of the complete knock-out and preventing compensation by the remaining versican splice variants. These mice are viable and fertile; however, they display major molecular alterations at the nodes of Ranvier. While the clustering of nodal sodium channels and paranodal structures appear in versican V2-deficient mice unaffected, the formation of the extracellular matrix surrounding the nodes is largely impaired. The conjoint loss of tenascin-R and phosphacan from the perinodal matrix provide strong evidence that versican V2, possibly controlled by a nodal receptor, organizes the extracellular matrix assembly in vivo.

A new emerging genotype subgroup within PCV-2b dominates the PMWS epizooty in Switzerland

Postweaning multisystemic wasting syndrome (PMWS) is among the most important emerging pig diseases worldwide. Initially, the insidious nature of the disease made it difficult to pinpoint the pathogen. The presence of porcine circovirus type 2 (PCV2) in all PMWS diseased animals led to its acceptance, possibly together with an unknown factor, as the causative agent for PMWS. Also, presence of PCV2 in healthy individuals did not facilitate the understanding of the disease. Phylogenetic classification separates PCV2 viruses into at least two major groups. With the aid of a signature motif, a short amino acid motif encoded within the capsid protein, the viruses are determined as belonging to PCV-2a or PCV-2b. Recently, this classification received more attention, as it seemed to define PCV-2b to be more virulent. This simplification, however, could not be confirmed experimentally. Hence, we investigated whether virus genetic shift was an initiator for the PMWS epizooty in Switzerland. Piglet lymphoid tissues from 1973 to 2005 were investigated by histology, immunohistochemistry (IHC) and PCR. For genotype classification, a sequence amplificate of 137bp was used encompassing the signature motif. The onset of Swiss PMWS epizooty exhibited a marked shift in PMWS diseased and subclinically infected piglets to PCV-2b and specifically to one genotype subgroup. Complementary to these observations, healthy piglets also defined by IHC as negative are positive in the PCR reaction and are void of any PCV-2b virus during epizooty. Consequently, our data support PCV2 genome plasticity as a major contributing factor for PMWS disease manifestation.

Chlamydia-related abortions in cattle from Graubunden, Switzerland

In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.