Andreas Faissner

The time course of loss of dopaminergic neurons and the gliotic reaction surrounding grafts of embryonic mesencephalon to the striatum.

Grafts of embryonic ventral mesencephalic tissue placed in the striatum of 6-hydroxydopamine-lesioned rats survive, and make and receive connections to and from the host brain. The dopaminergic neurons of the graft can grow processes into the host brain, and thereby alleviate many of the behavioral deficits of this form of experimental Parkinsons disease. However, when examined some weeks after implantation, grafted substantia nigra only contains about 5% of the expected complement of dopaminergic neurons. We have examined the time course of loss of grafted neurons. We find that the majority die during the first 7 days after transplantation. However, we have shown previously that three-dimensional cultures with the same dimensions as a graft, made of identical cell suspensions, have much better dopaminergic neuronal survival. There must, therefore, be features in the environment surrounding a graft that are toxic to dopaminergic neurons. A limiting factor in the efficacy of dopaminergic grafts is the small distance over which the neurons are able to grow neurites and form connections in the host brain. We find that the growth of neurites from dopaminergic neurons into the host striatum occurs in two phases. Neurites reach their maximum length within 7 days of transplantation, and this is followed by a much slower process of branch and terminal formation. Since axon growth in the adult brain may be inhibited by a number of factors associated with reactive gliosis, we have immunostained various ages of graft for vimentin, tenascin, chondroitin sulfate proteoglycan (CS-PG) using the CS56 antibody, the DSD-1 proteoglycan, and microglia using the OX-42 antibody. We have compared this staining with that surrounding a simple stab wound. Vimentin staining was initially seen in the graft and in astrocytes immediately surrounding it. By 7 weeks staining was restricted to a ring of astrocytes surrounding the graft. Tenascin, DSD-1, and CS-PG were initially seen in and around the grafts. By 7 weeks they had disappeared from grafts, but CS-PG and tenascin persisted in small amounts around stab wounds. In general, immunostaining of these molecules persisted longer around a stab lesion than around a graft. There was also an intense local microglial reaction surrounding both grafts and stab wounds which had largely resolved by 7 weeks.

The DSD-1 Carbohydrate Epitope Depends on Sulfation, Correlates with Chondroitin Sulfate D Motifs, and Is Sufficient to Promote Neurite Outgrowth

The neural chondroitin sulfate (CS) proteoglycan (PG) DSD-1-PG was originally identified with the monoclonal antibody (mAb) 473HD. It promotes neurite outgrowth of hippocampal neurons when coated as a substrate in the presence of polycations. This effect is inhibited by mAb 473HD that specifically recognizes the DSD-1 epitope. The DSD-1 epitope is also detectable in CS-C and CS-D preparations from shark cartilage but not in other chondroitin sulfates that are structurally related and differ in their sulfation patterns. Non-sulfated DSD-1-PG and chemically desulfated CS-D were not recognized by mAb 473HD, suggesting that the DSD-1 epitope depends on sulfation. It was possible to enrich DSD-1 epitope-bearing carbohydrates and D disaccharide units from CS-C and CS-D preparations on a mAb 473HD affinity matrix. This indicates that the DSD-1 epitope represents a distinct glycosaminoglycan structure containing D units. The analysis of glycosaminoglycan digestion products by high pressure liquid chromatography revealed that DSD-1-PG preparations contain a unique D disaccharide unit as well as an A, a C, and a non-sulfated disaccharide unit. In neurite outgrowth assays with hippocampal neurons, substrate-bound CS-D promoted neurite outgrowth, whereas CS-A, CS-B, or CS-C did not. This effect of CS-D was inhibited by mAb 473HD. DSD-1 epitope-enriched fractions obtained from CS-D and CS-C promoted neurite outgrowth, whereas CS-C had no such effect prior to enrichment on the mAb 473HD matrix. Based on these findings we conclude that the DSD-1 epitope by itself is sufficient to promote neurite outgrowth and that this activity is possibly associated with D motifs.

Enhanced expression of the extracellular matrix molecule J1/tenascin in the regenerating adult mouse sciatic nerve

SummaryWe have investigated the expression of J1/tenascin in the sciatic nerve of the adult mouse under normal and regenerating conditions by immunocy tological and immunochemical methods. In the normal nerve, J1/tenascin expression was confined to the extracellular matrix at the node of Ranvier and in the perineurium. At 2 days after nerve transection, J1/tenascin was detectable in the fibroblast-containing caps of the distal and proximal nerve stumps, in the distal nerve stump along its entire length and in the distal end of the proximal nerve stump. In the nerve stumps immunoreactivity was predominantly associated with extracellular matrix consisting of collagen fibrils and Schwann cell basal laminae. Approximately 7 days after transection, the caps of the nerve stumps had usually grown together forming a bridge. This bridge consisted of a J1/tenascin-negative perineurium-like structure and an inner part of predominantly fibroblasts, endothelial cells and macrophages. All cell types in this inner part were embedded in a J1/tenascin-positive matrix of collagen fibrils indicating the prospective direction of growth of neural elements. A few days later, J1/tenascin in the bridge was confined to the extracellular matrix around small Schwann cell-containing nerve fascicles. In nerves chronically denervated for 19 days, J1/tenascin was poorly detectable in the cap of the distal stump, although Schwann cells had infiltrated this cap. Approximately 19 days after the lesion, J1/tenascin expression returned to control levels in the proximal nerve stump. In the distal nerve stump, J1/tenascin immunoreactivity reached a peak at approximately 14 days after nerve transection and vanished only at approximately 35 days, thus correlating with the time of active regrowth of axons into the distal nerve stump. This reduction was prevented by chronic denervation, suggesting that reinnervation of target structures may be related to the down-regulation of J1/tenascin. These combined observations suggest that J1/tenascin is differentially regulated in the individual parts of the regenerating nerve, possibly triggered by different cellular and molecular signals.

DSD-1-Proteoglycan Is the Mouse Homolog of Phosphacan and Displays Opposing Effects on Neurite Outgrowth Dependent on Neuronal Lineage

DSD-1-PG is a chondroitin sulfate proteoglycan (CSPG) expressed by glial cells that can promote neurite outgrowth from rat embryonic mesencephalic (E14) and hippocampal (E18) neurons, an activity that is associated with the CS glycosaminoglycans (GAGs). Further characterization of DSD-1-PG has included sequencing of peptides from the core protein and the cloning of the corresponding cDNA using polyclonal antisera against DSD-1-PG to screen phage expression libraries. On the basis of these studies we have identified DSD-1-PG as the mouse homolog of phosphacan, a neural rat CSPG. Monoclonal antibodies 3H1 and 3F8 against carbohydrate residues on rat phosphacan recognize these epitopes on DSD-1-PG. The epitopes of the antibodies, L2/HNK-1 and L5/Lewis-X, which have been implicated in functional interactions, are also found on DSD-1-PG. Although DSD-1-PG has previously been shown to promote neurite outgrowth, its upregulation after stab wounding of the CNS and its localization in regions that are considered boundaries to axonal extension suggested that it may also have inhibitory functions. Neonatal dorsal root ganglion (DRG) explants grown on a rich supportive substrate (laminin) with and without DSD-1-PG were strikingly inhibited by the proteoglycan. The inhibitory effects of DSD-1-PG on the DRG explants were not relieved by removal of the CS GAGs, indicating that this activity is associated with the core glycoprotein. The neurite outgrowth from embryonic hippocampal neurons on laminin was not affected by the addition of DSD-1-PG. This indicates that DSD-1-PG/mouse phosphacan can have opposing effects on the process of neurite outgrowth dependent on neuronal lineage.

Biosynthesis and membrane topography of the neural cell adhesion molecule L1.

The biosynthesis and membrane topography of the neural cell adhesion molecule L1 have been studied in cerebellar cell cultures by metabolic labeling and immunoprecipitation. Pulse and pulse‐chase experiments with [35S]methionine show that L1 is synthesized in its high mol. wt. form, the 200 kd component. The lower mol. wt. components with 40, 80 and 140 K apparent mol. wts. can be generated by proteolysis in intact cellular membranes. Peptide maps generated by protease treatment of L1 isolated from adult mouse brain show that the 80 and 140 kd components are related to the 200 kd component, but not to each other. The 200, 80 and 40 kd components can be biosynthetically phosphorylated. The 140 kd component is not phosphorylated and not released from the surface membrane during tryspinization. The phosphorylated amino acid is serine. In the presence of tunicamycin the 200 kd component is synthesized as a 150 kd protein. Pulse‐chase experiments in the presence of tunicamycin indicate that the carbohydrate moieties are predominantly N‐glycosidically linked and that the contribution of O‐glycosylation is minimal. The carbohydrate moieties are of the complex type as shown by treatment with endoglycosidase H. Since monensin inhibits processing of the carbohydrate moieties, the 200 kd component appears to be transported to the surface membrane via the Golgi apparatus.

The structure and function of tenascins in the nervous system

The tenascins are a family of large extracellular matrix glycoproteins that comprise five known members. Three of these, tenascin-C (TN-C) tenascin-R (TN-R) and tenascin-Y (TN-Y) are expressed in specific patterns during nervous system development and are down-regulated after maturation. The expression of TN-C, the best studied member of the family, persists in restricted areas of the nervous system that exhibit neuronal plasticity and is reexpressed after lesion. Numerous studies in vitro suggest specific roles for tenascins in the nervous system involving precursor cell migration, axon growth and guidance. TN-C has been shown to occur in a large number of isoform variants generated by combinatorial variation of alternatively spliced fibronectin type III (FNIII) repeats. This finding indicates that TN-C might specify neural microenvironments, a hypothesis supported by recent analysis of TN-C knockout animals, which has begun to reveal subtle nervous system dysfunctions.

The tenascin gene family in axon growth and guidance

Abstract. Glial cells are thought to play an important role in the regulation of neural pattern formation, e.g. by guiding migrating neuroblasts and growth cones to their target regions. In addition to these supportive roles, astro- and oligodendroglia have also been attributed inhibitory functions. Thus, these lineages are believed to constrain the pathways of migrating neurons and growth cones. Recent studies have led to the current view that the inhibitory roles of the glia of the central nervous system (CNS) may be important for neural pattern formation. Furthermore, inhibitory effects of glia may play an essential role in the failure of CNS regeneration, e.g. in the astrocytic scar. Advances have been made in deciphering the molecular basis of glia-mediated inhibitory influences in the CNS. The present review focuses on the tenascin gene family of extracellular matrix glycoproteins. Of these, tenascin-C and -R are expressed in developing and lesioned neural tissue and embody both stimulatory and anti-adhesive or inhibitory properties for axon growth.

Contributions of astrocytes to synapse formation and maturation — Potential functions of the perisynaptic extracellular matrix

The concept of the tripartite synapse proposes that in addition to the presynapse and the postsynaptic membrane closely apposed processes of astrocytes constitute an integral part of the synapse. Accordingly, astrocytes may influence synaptic activity by various ways. Thus glia- and neuron-derived neurotrophins, cytokines and metabolites influence neuronal survival, synaptic activity and plasticity. Beyond these facts, the past years have shown that astrocytes are required for synaptogenesis, the structural maintenance and proper functioning of synapses. In particular, astrocytes seem to play a key role in the organization of the brains extracellular matrix (ECM) - most prominently the so-called perineuronal nets (PNNs), complex macromolecular assemblies of ECM components. Due to progress in cellular and molecular neurosciences, it has been possible to decipher the composition of ECM structures and to obtain insight into their function(s) and underlying mechanisms. It appears that PNN-related structures are involved in regulating the sprouting and pruning of synapses, which represents an important morphological correlate of synaptic plasticity in the adult nervous system. Perturbation assays and gene elimination by recombinant techniques have provided clear indications that astrocyte-derived ECM components, e.g. the tenascins and chondroitinsulfate proteoglycans (CSPGs) of the lectican family participate in these biological functions. The present review will discuss the glia-derived glycoproteins and CSPGs of the perisynaptic ECM, their neuronal and glial receptors, and in vitro assays to test their physiological functions in the framework of the synapse, the pivotal element of communication in the central nervous system.

Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.

Long‐term changes in the molecular composition of the glial scar and progressive increase of serotoninergic fibre sprouting after hemisection of the mouse spinal cord

The scarring process occurring after adult central nervous system injury and the subsequent increase in the expression of certain extracellular matrix molecules are known to contribute to the failure of axon regeneration. This study provides an immunohistochemical analysis of temporal changes (8 days to 1 year) in the cellular and molecular response of the Swiss mouse spinal cord to a dorsal hemisection and its correlation with the axonal growth properties of a descending pathway, the serotoninergic axons. In this lesion model, no cavity forms at the centre of the lesion. Instead, a dense fibronectin‐positive tissue matrix occupies the centre of the lesion, surrounded by a glial scar mainly constituted by reactive astrocytes. NG2 proteoglycan and tenascin‐C, potential axon growth inhibitors, are constantly associated with the central region. In the glial scar, tenascin‐C is never observed and the expression of chondroitin sulphate proteoglycans (revealed with CS‐56 and anti‐NG2 antibodies) highly increases in the week following injury to progressively return to their control level. In parallel, there is an increasing expression of the polysialilated neural cell adhesion molecule by reactive astrocytes. These molecular changes are correlated with a sprouting process of serotoninergic axons in the glial scar, except in a small area in contact with the central region. All these observations suggest that while a part of the glial scar progressively becomes permissive to axon regeneration after mouse spinal cord injury, the border of the glial scar, in contact with the fibronectin‐positive tissue matrix, is the real barrier to prevent axon regeneration.