Upregulation of pleiotrophin gene expression in developing microvasculature, macrophages, and astrocytes after acute ischemic brain injury

Pleiotrophin (PTN) is a heparin-binding, 18 kDa secretory protein that functions to induce mitogenesis, angiogenesis, differentiation, and transformation in vitro. PTN gene (Ptn) expression is highly regulated during development and is highest at sites in which mitogenesis, angiogenesis, and differentiation are active. In striking contrast, with the exception of the neuron, the Ptn gene is only minimally expressed in adults. We now demonstrate that Ptn gene expression is strikingly upregulated within 3 d in OX42-positive macrophages, astrocytes, and endothelial cells in areas of developing neovasculature after focal cerebral ischemia in adult rat. Ptn gene expression remains upregulated in these same cells and sites 7 and 14 d after ischemic injury. However, expression of the Ptn gene is significantly decreased in cortical neurons 6 and 24 hr after injury and is undetectable in degenerating neurons at day 3. Neurons in contralateral cortex continue to express Ptn in levels equal to control, uninjured brain. It is suggested that PTN may have a vital role in neovascular formation in postischemic brain and that postischemic brain is an important model in which to analyze sequential gene expression in developing neovasculature. In contrast, Ptn gene expression in injured neurons destined not to recover is strikingly reduced, and potentially its absence may contribute to the failure of the neuron to survive.     

Different cell surface areas of polarized radial glia having opposite effects on axonal outgrowth

During neuronal development neurites are likely to be specifically guided to their targets. Within the chicken retina, ganglion cell axons are extended exclusively into the optic fibre layer, but not into the outer retina. We investigated, whether radial glial cells having endfeet at the optic fibre layer and somata in the outer retina, might be involved in neurite guidance. In order to analyse distinct cell surface areas, endfeet and somata of these glial cells were purified. Glial endfeet were isolated from flat mounted retina by a specific detachment procedure. Glial somata were purified by negative selection using a monoclonal antibody/complement mediated cytolysis of all non-glial cells. Retinal tissue strips were explanted either onto pure glial endfeet or onto glial somata. As revealed by scanning and fluorescence microscopy, essentially no ganglion cell axons were evident on glial somata, whereas axonal outgrowth was abundant on glial endfeet. However, when glial somata were heat treated and employed thereafter as the substratum, axon extension was significantly increased. Time-lapse video recording studies indicated that purified cell membranes of glial somata but not of endfeet induced collapse of growth cones. Collapsing activity was destroyed by heat treatment of glial membranes. The collapsing activity of retinal glia was found to be specific for retinal ganglion cell neurites, because growth cones from dorsal root ganglia remained unaffected. Employing four different kinase inhibitors revealed that the investigated protein kinase types were unlikely to be involved in the collapse reaction. The data show for the first time that radial glial cells are functionally polarized having permissive endfeet and inhibitory somata with regard to outgrowing axons. This finding underscores the pivotal role of radial glia in structuring developing nervous systems.     

Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene

Mutations of the proteolipid protein (Plp) gene cause a generalized central nervous system (CNS) myelin deficit in Pelizaeus-Merzbacher disease of man and various tremor syndromes in animal models. X-linked spastic paraplegia is also due to Plp gene mutations but has a different clinical profile and more restricted pathology involving specific tracts and regions. We have shown previously that PLP overexpression in mice homozygous for a Plp transgene results in premature arrest of CNS myelination and premature death. Here, we demonstrate that a low-level increase in Plp gene expression in transgenic mice causes significant axonal degeneration and demyelination with predilection for specific tracts. Following normal motor development, aged mice develop progressive myelin loss, axonal swellings with resultant Wallerian degeneration, and marked vacuolation of the neuropil associated with ataxia, tremor, and seizures. The age of onset and severity of the phenotype is a function of Plp gene dosage. The corticospinal tracts, optic nerve, fasciculus gracilis cerebellum, and brainstem are particularly involved. Although oligodendrocyte cell bodies show little abnormality, their inner adaxonal tongue is often abnormal, suggesting a perturbation of the axon/glial interface that may underlie the axonal changes. We conclude that abnormal expression of an oligodendrocyte-specific gene can cause axonal damage, a finding that is relevant to the pathogenesis of PLP-associated disorders and probably to other myelin-related diseases.     

Commissure formation in the embryonic CNS of Drosophila

Most of the neurons of the ventral nerve cord send out long projecting axons which cross the midline. In the Drosophila central nervous system (CNS) cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. Here we present evidence that beside the previously known NETRIN/FRAZZLED (DCC) signalling system an additional attractive system(s) is operating in the developing embryonic nervous system of Drosophila. Attractive cues appear to be provided by the midline neurons. We show that the glial cells present repulsive signals to the previously described ROUNDABOUT receptor in addition to a permissive contact-dependent signal helping commissural growth cones across the midline. A novel repulsive component is encoded by the karussell gene. Furthermore the midline glial cells separate anterior and posterior commissures. By genetic criteria we demonstrate that some of the genes we have identified are acting in the midline glia whereas other genes are required in the midline neurons. The results lead to a detailed model relating different cellular functions to axonal patterning at the midline.     

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.     

Axonal versus dendritic outgrowth is differentially affected by radial glia in discrete layers of the retina

Formation of neural cell polarity defined by oriented extension of axons and dendrites is a crucial event during the development of the nervous system. Ganglion cells of the chicken retina extend axons exclusively into the inner retina, whereas their dendrites grow into the outer retina. To analyze guidance cues for specific neurite extension, novel in vitro systems were established. Ganglion cells were purified by enzymatically facilitated detachment of the ganglion cell layer. A newly developed retrograde labeling technique and the expression analysis of the cell type-specific 2A1 antigen were used to monitor ganglion cell purification. In highly purified ganglion cells explanted onto retinal cryosections (cryoculture), axon formation was induced when the cells were positioned on the inner retina. In contrast, on outer layers of the developing retina dendritic outgrowth was prevalent. Because radial glia have been demonstrated to be instructive in neuritogenesis, distinct glial cell compartments located in inner and outer retina, respectively, were isolated for functional assays. Glial end feet were purified by a physical detachment technique. Glial somata were purified by complement mediated cytolysis of all nonglial cells. When ganglion cells were cultured on different glial compartments, axon formation occurred on end feet but not on glial somata. In striking contrast, on glial somata dendrites were formed. The data support the notion that ganglion cell polarity is affected by the retinal microenvironment, which in turn is possibly influenced by radial glia, being themselves polarized.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system.     

N-syndecan and HB-GAM (heparin-binding growth-associated molecule) associate with early axonal tracts in the rat brain

Heparin-Binding Growth-Associated Molecule (HB-GAM)/pleiotrophin is an 18 kDa extracellular matrix- and cell-surface-associated protein shown to enhance neurite outgrowth of perinatal forebrain neurones in vitro. The heparan sulphate proteoglycan N-syndecan (Raulo et al., 1994) has been isolated as a receptor/coreceptor for the HB-GAM. We have investigated, whether HB-GAM and N-syndecan could have a similar role in neurite outgrowth and axon guidance in early axonal tracts of brain. In the present study N-syndecan was found to be spatiotemporally associated with the developing axonal tracts already on embryonic day 9 in rat, as revealed by coexpression with class III beta-tubulin, which is one of the earliest neuronal markers (Easter et al., 1993; Brittis et al., 1995). Later, N-syndecan and HB-GAM were detected in the first afferent serotonergic projections arising from the pontine raphe nuclei. The expression pattern of HB-GAM peaked in the developing rhombencephalon at embryonic stage (E) 13-14. At the same time, N-syndecan was expressed in the developing raphe neurones growing neurites towards the diencephalon along HB-GAM immunoreactive pathways. When rhombencephalic neurones were cultured on decreasing concentrations of substrate-bound HB-GAM, E13 neurones showed a significantly better neurite outgrowth response than E11, E16 or E18 neurones. The neurite outgrowth of raphe neurones in vitro was inhibited by adding soluble heparin or N-syndecan into the culture medium, whereas addition of chondroitin sulphate had no effect. In a simple pathway assay, E13 raphe neurones selectively preferred attaching and growing neurites on pathways containing HB-GAM as compared with regions containing either laminin or fibronectin alone. Our results suggest that HB-GAM may function as a developmentally regulated cue for rhombencephalic neurones that possess N-syndecan on their cell membrane.     

HIV-1 gene expression and replication in neuronal and glial cell lines with immature phenotype: effects of nerve growth factor

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.     

Decreased axosomatic input to motoneurons and astrogliosis in the spinal cord of aged rats

An increasing body of evidence indicates that aging-related impairments of nervous functions are caused by damage to neuron integrity rather than by loss of neurons. By using electron microscopy, we have examined axosomatic boutons on spinal cord motoneurons derived from aged and young adult Sprague-Dawley rats. The main finding was that about half of the examined motoneuron somata from aged rats had a reduced (50%) bouton coverage, which seemed to be caused by a smaller number of axosomatic bouton profiles. Long stretches of the cell body plasma membrane were apposed by pale processes, and immunolabeling for glial fibrillary acidic protein (GFAP) disclosed that a number of the aged motoneurons appeared embedded in GFAP immunopositive processes. Lumbar motoneurons seemed to be more severely affected than cervical motoneurons. At the ultrastructural level, affected motoneurons disclosed plasma membrane irregularities with appendages/sprout-like extensions that in some cases were sites for axosomatic contacts.