Nicolas Heck

Regulation of RPTPβ/phosphacan expression and glycosaminoglycan epitopes in injured brain and cytokine-treated glia

Several chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS injury and are thought to limit axonal regeneration in the adult mammalian CNS. Therefore, we examined the expression of the CSPG, receptor protein tyrosine phosphatase beta (RPTPbeta)/phosphacan, after a knife lesion to the cerebral cortex and after treatment of glial cultures with regulatory factors. The three splice variants of this CSPG gene, the secreted isoform, phosphacan, and the two transmembrane isoforms, the long and short RPTPbeta, were examined. Western blot and immunostaining analysis of injured and uninjured tissue revealed a transient decrease of phosphacan protein levels, but not of short RPTPbeta, in the injured tissue from 1 to 7 days postlesion (dpl). By real time RT-PCR, we show that phosphacan and long RPTPbeta mRNA levels are transiently down-regulated at 2 dpl, unlike those of short RPTPbeta which increased after 4 dpl. In contrast to the core glycoprotein, the phosphacan chondroitin sulfate (CS) glycosaminoglycan epitope DSD-1 was up-regulated after 7 dpl. Phosphacan was expressed by cultivated astrocytes and oligodendrocyte precursors but was more glycanated in oligodendrocyte precursors, which produce more of DSD-1 epitope than astrocytes. Epidermal growth factor/transforming growth factor alpha strongly increased the astrocytic expression of long RPTPbeta and phosphacan and slightly the short RPTPbeta protein levels, while interferon gamma and tumor necrosis factor alpha reduced astrocytic levels of phosphacan, but not of the receptor forms. Examining the effects of phosphacan on axon growth from rat E17 cortical neurons, we found that phosphacan stimulates outgrowth in a largely CS dependent manner, while it blocks the outgrowth-promoting effects of laminin through an interaction that is not affected by removal of the CS chains. These results demonstrate complex injury-induced modifications in phosphacan expression and glycanation that may well influence axonal regeneration and repair processes in the damaged CNS.

The extracellular matrix glycoprotein Tenascin-C is expressed by oligodendrocyte precursor cells and required for the regulation of maturation rate, survival and responsiveness to platelet-derived growth factor

Analysis of Tenascin‐C (TN‐C) knockout mice revealed novel roles for this extracellular matrix (ECM) protein in regulation of the developmental programme of oligodendrocyte precursor cells (OPCs), their maturation into myelinating oligodendrocytes and sensitivity to growth factors. A major component of the ECM of developing nervous tissue, TN‐C was expressed in zones of proliferation, migration and morphogenesis. Examination of TN‐C knockout mice showed roles for TN‐C in control of OPC proliferation and migration towards zones of myelination [E. Garcion et al. (2001) Development, 128, 2485–2496]. Extending our studies of TN‐C effects on OPC development we found that OPCs can endogenously express TN‐C protein. This expression covered the whole range of possible TN‐C isoforms and could be strongly up‐regulated by leukaemia inhibitory factor and ciliary neurotrophic factor, cytokines known to modulate OPC proliferation and survival. Comparative analysis of TN‐C knockout OPCs with wild‐type OPCs reveals an accelerated rate of maturation in the absence of TN‐C, with earlier morphological differentiation and precocious expression of myelin basic protein. TN‐C knockout OPCs plated on poly‐lysine displayed higher levels of apoptosis than wild‐type OPCs and there was also an earlier loss of responsiveness to the protective effects of platelet‐derived growth factor (PDGF), indicating that TN‐C has anti‐apoptotic effects that may be associated with PDGF signalling. The existence of mechanisms to compensate for the absence of TN‐C in the knockout is indicated by the development of oligodendrocytes derived from TN‐C knockout neurospheres. These were present in equivalent proportions to those found in wild‐type neurospheres but displayed enhanced myelin membrane formation.

DSD-1-Proteoglycan/Phosphacan and Receptor Protein Tyrosine Phosphatase-Beta Isoforms during Development and Regeneration of Neural Tissues

Interactions between neurons and glial cells play important roles in regulating key events of development and regeneration of the CNS. Thus, migrating neurons are partly guided by radial glia to their target, and glial scaffolds direct the growth and directional choice of advancing axons, e.g., at the midline. In the adult, reactive astrocytes and myelin components play a pivotal role in the inhibition of regeneration. The past years have shown that astrocytic functions are mediated on the molecular level by extracellular matrix components, which include various glycoproteins and proteoglycans. One important, developmentally regulated chondroitin sulfate proteoglycan is DSD-1-PG/phosphacan, a glial derived proteoglycan which represents a splice variant of the receptor protein tyrosine phosphatase (RPTP)-beta (also known as PTP-zeta). Current evidence suggests that this proteoglycan influences axon growth in development and regeneration, displaying inhibitory or stimulatory effects dependent on the mode of presentation, and the neuronal lineage. These effects seem to be mediated by neuronal receptors of the Ig-CAM superfamily.

Differential upregulation of extracellular matrix molecules associated with the appearance of granule cell dispersion and mossy fiber sprouting during epileptogenesis in a murine model of temporal lobe epilepsy.

We have investigated changes in the extracellular matrix of the hippocampus associated with the early progression of epileptogenesis in a murine model of temporal lobe epilepsy using immunohistochemistry. In the first week following intrahippocampal injection of the glutamate agonist, domoate, there is a latent period at the end of which begins a sequential upregulation of extracellular matrix (ECM) molecules in the granule cell layer of the dentate gyrus, beginning with neurocan and tenascin-C. This expression precedes the characteristic dispersion of the granule cell layer which is evident at 14 days post-injection when the first recurrent seizures can be recorded. At this stage, an upregulation of the chondroitin sulfate proteoglycan, phosphacan, the DSD-1 chondroitin sulfate motif, and the HNK-1 oligosaccharide are also observed. The expression of these molecules is localized differentially in the epileptogenic dentate gyrus, especially in the sprouting molecular layer, where a strong upregulation of phosphacan, tenascin-C, and HNK-1 is observed but there is no expression of the proteoglycan, neurocan, nor of the DSD-1 chondroitin sulfate motif. Hence, it appears that granule cell layer dispersion is accompanied by a general increase in the ECM, while mossy fiber sprouting in the molecular layer is associated with a more restricted repertoire. In contrast to these changes, the expression of the ECM glycoproteins, laminin and fibronectin, both of which are frequently implicated in tissue remodelling events, showed no changes associated with either granule cell dispersion or mossy fiber sprouting, indicating that the epileptogenic plasticity of the hippocampus is accompanied by ECM interactions that are characteristic of the CNS.

GABAC receptors are functionally expressed in the intermediate zone and regulate radial migration in the embryonic mouse neocortex

Radial neuronal migration in the cerebral cortex depends on trophic factors and the activation of different voltage- and ligand-gated channels. To examine the functional role of GABA(C) receptors in radial migration we analyzed the effects of specific GABA(A) and GABA(C) receptor antagonists on the migration of BrdU-labeled neurons in vitro using organotypic neocortical slice cultures. These experiments revealed that the GABA(A) specific inhibitor bicuculline methiodide facilitated neuronal migration, while the GABA(C) specific inhibitor (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic-acid (TPMPA) impeded migration. Co-application of TPMPA and bicuculline methiodide or the unspecific ionotropic GABA receptor antagonist picrotoxin both impeded migration, suggesting that the GABA(C) receptor mediated effects dominate. Addition of the specific GABA(C) receptor agonist cis-4-aminocrotonic acid (CACA) also hampered migration, indicating that a physiological GABAergic stimulation is required for appropriate function. RT-PCR experiments using specific probes for GABA(C) receptor mRNA and Western blot assays using an antibody directed against rho subunits revealed the expression of GABA(C) receptor mRNA and translated GABA(C) receptor protein in the immature cortex. Microfluorimetric Ca(2+) imaging in neurons of identified cortical layers using Calcium Green revealed the functional expression of GABA(A) and GABA(C) receptors in the intermediate zone, while only GABA(A) receptor mediated responses were observed in the upper cortical plate. In summary, these results demonstrate that activation of GABA(C) receptors is a prerequisite for accurate migration and that GABA(C) receptors are functionally expressed in the intermediate zone.

Oxygen and glucose deprivation induces major dysfunction in the somatosensory cortex of the newborn rat

The mechanisms and functional consequences of ischemia‐induced injury during perinatal development are poorly understood. Subplate neurons (SPn) play a central role in early cortical development and a pathophysiological impairment of these neurons may have long‐term detrimental effects on cortical function. The acute and long‐term consequences of combined oxygen and glucose deprivation (OGD) were investigated in SPn and compared with OGD‐induced dysfunction of immature layer V pyramidal cortical neurons (PCn) in somatosensory cortical slices from postnatal day (P)0–4 rats. OGD for 50 min followed by a 10–24‐h period of normal oxygenation and glucose supply in vitro or in culture led to pronounced caspase‐3‐dependent apoptotic cell death in all cortical layers. Whole‐cell patch‐clamp recordings revealed that the majority of SPn and PCn responded to OGD with an initial long‐lasting ischemic hyperpolarization accompanied by a decrease in input resistance (Rin), followed by an ischemic depolarization (ID). Upon reoxygenation and glucose supply, the recovery of the membrane potential and Rin was followed by a Na+/K+‐ATPase‐dependent postischemic hyperpolarization, and in almost half of the investigated SPn and PCn by a postischemic depolarization. Whereas neither a moderate (2.5 mm) nor a high (4.8 mm) increase in extracellular magnesium concentration protected the SPn from OGD‐induced dysfunction, blockade of NMDA receptors with MK‐801 led to a significant delay and decrease of the ID. Our data demonstrate that OGD induces apoptosis and a profound dysfunction in SPn and PCn, and underline the critical role of NMDA receptors in early ischemia‐induced neuronal damage.

Astrocytes in culture express fibrillar collagen

The use of monoclonal antibodies has led to much progress in the characterization of extracellular matrix components of the CNS. F1C3 is a monoclonal antibody raised against the astrocytic cell line, Neu7. Analysis by immunoprecipitation and Western blots of the F1C3 antigen in Neu7 cell lysates and conditioned medium reveals a recognition of several protein bands around 140–230 kD. Internal peptide sequence data from these bands indicate that they are highly homologous to fibrillar collagens, and the F1C3 antigen is specifically digested by the collagenase I protease. Other glial cell lines show F1C3 antigen expression including A7, C6, and U373. Cultures of neonatal primary astrocytes also express F1C3 antigen, and Western blot analysis of rat brain extracts from different ages and parts of the brain confirm an in vivo expression of F1C3 protein. The significance of the expression of fibrillar collagen‐like proteins by astrocytes is discussed together with its possible implication during developmental processes and in the context of CNS lesions and regeneration. GLIA 41:382–392, 2003.

Cortical neurons express PSI, a novel isoform of phosphacan/RPTPbeta

Phosphacan is a chondroitin sulfate proteoglycan representing the secreted extracellular part of a transmembrane receptor protein tyrosine phosphatase (RPTP-β). These isoforms have been implicated in cell-extracellular matrix signaling events associated with myelination, axon growth, and cell migration in the developing central nervous system and may play critical roles in the context of brain pathologies. Recently, we have reported the identification of a new isoform of phosphacan, the phosphacan short isoform (PSI), the expression of which peaks in the second postnatal week. PSI interacts with the neuronal receptors L1 and F3/contactin and can promote neurite growth of cortical neurons. In this study, we have assessed, by in situ hybridization, the expression profile of PSI in the rat brain at postnatal day 7. PSI is largely expressed in the gray matter of the developing cerebral cortex in which it colocalizes with phosphacan, whereas the expression of RPTPbeta receptor forms is restricted to the ventricular area in which PSI has not been observed. Neurons from all layers of the cortex express PSI. In the cerebellum, on the other hand, no expression of PSI has been detected, although the other phosphacan/RPTP-beta isoforms show strong PSI expression here. Overall, our study suggests that PSI is expressed during the postnatal period in differentiated neurons of the cortex but is absent from structures in which proliferation and migration occur. The significance of these observations is discussed in the context of previous models of phosphacan/RPTP-beta functions.

Evidence for distinct leptomeningeal cell-dependent paracrine and EGF-linked autocrine regulatory pathways for suppression of fibrillar collagens in astrocytes.

A unique and unresolved property of the central nervous system is that its extracellular matrix lacks fibrillar elements. In the present report, we show that astrocytes secrete triple helices of fibrillar collagens type I, III and V in culture, while no astroglial collagen expression could be detected in vivo. We discovered two inhibitory mechanisms that could underlie this apparent discrepancy. Thus, we uncover a strong inhibitory effect of meningeal cells on astrocytic collagen expression in coculture assays. Furthermore, we present evidence that EGF-receptor activation downregulates collagen expression in astrocytes via an autocrine loop. These investigations provide a rational framework to explain why the brain is devoid of collagen fibers, which is a unique feature that characterizes the structure of the neural extracellular matrix. Moreover, fibrillar collagens were found transiently upregulated in a laser-induced cortical lesion, suggesting that these could contribute to the glial scar that inhibits axonal regeneration.

A new technique for real-time analysis of caspase-3 dependent neuronal cell death

Several markers are available to identify cells undergoing programmed cell death, but so far they are only applicable on fixed material. Therefore, no information on the kinetics of apoptosis can be obtained, although apoptosis is a dynamic cell process. Here, we describe a new technique that allows the real-time observation of the onset of apoptosis in primary neurons. Neurons are transfected with a plasmid that codes for a fluorescent protein localized in the soma. Upon activation of caspase-3, which represents the point-of-no-return in the apoptosis process, the fusion protein is cleaved and as a consequence translocates into the nucleus. The onset of apoptosis is thus visualized by translocation of the fluorescent signal from the soma to the nucleus. The translocation process was found to be specific for the apoptosis process as it correlates with the activation of caspase-3 and TUNEL staining. This tool does not require complex detection systems and allows for the first time the analysis of the kinetics of apoptosis in a simple and efficient manner.