NG2 positive cells of rat spinal cord activated during experimental autoimmune encephalomyelitis are spatially associated with radially oriented astroglia and express p75 receptor: a role for nerve growth factor in oligodendrocyte progenitor migration?

Data have been provided from several studies that support the proposal that the adult oligodendrocyte progenitors migrate into the lesioned areas under conditions of experimental autoimmune encephalomyelitis (EAE). However, the routes of migration of these cells and the governing mechanisms are not clear. In the present studies, we have examined the effect of EAE upon activation of endogenous oligodendroglia progenitors and their spatial distribution in the spinal cord of Lewis rats using immunocytochemical procedures. Antibodies against the marker chondroitin sulfate proteoglycan NG2, are used for identification of oligodendroglia progenitors. We find that the activated elongated subpopulation of NG2 positive oligodendroglia progenitors of white matter is spatially associated with the radially-oriented astroglia during the acute phase of EAE. The latter re-expressed the phenotypic embryonic marker nestin while still expressing the mature astroglial marker GFAP. The elongated oligodendroglia progenitors express p75 receptor. In addition, colocalization of NG2 and p75 is observed also in ependymal neural cells of the central canal and the subventricular zone. This raises the possibility that the activated NG2+/p75+ parenchymal cell pool may also be recruited from multipotent neural cells of the germination areas. Our data suggest that, under EAE conditions, the radially oriented astroglia of juvenile phenotype may serve as scaffolding for migrating activated endogenous oligodendroglia progenitors just like radial glia provide a path for neuronal and oligodendroglia progenitor cells in embryonic stage. The expression of p75 receptor in oligodendroglia progenitors associated with radially oriented astroglia during EAE may implicate a role for NGF in the regulation of migration of oligodendroglia progenitors.

High-affinity NGF receptor in the rat spinal cord during acute and chronic phases of experimental autoimmune encephalomyelitis: a possible functional significance.

The biological effects of Nerve Growth Factor (NGF) are primarily mediated via its high affinity receptor-TrkA. In the present study, we examined the effect of experimental autoimmune encephalomyelitis (EAE) upon the expression of TrkA in neuronal and non-neuronal cells of the spinal cord of Lewis rats during the acute (14 days postimmunization) and chronic (12 months postimmunization) phases of the disease. In the normal spinal cord, both of mature and aged rats, we found TrkA immunoreaction (TrkA-IR) in the motoneurons of the Rexed lamina IX and in both oligo- and astroglia cells. In the acute phase of the disease, we found a reduction of TrkA immunoreactivity in motoneurons and its up-regulation in oligodendroglia, mainly in the white matter. We also confirmed our previous findings concerning the up-regulation of TrkA-IR in astroglia. Both neuronal and non-neuronal changes of TrkA immunoreactivity had a transient character: they were not seen in the chronic phase of the disease. Our results suggest that both neuronal and glial TrkA expression changes depend on inflammation. Moreover, our data indicate that, during the acute phase of EAE, the glial cells become more receptive to NGF, pointing to glia as an important target for pharmacological manipulations, particularly for exogenously administered NGF.

Circulating NGF antibody alters the distribution of NG2 and CD56 positive cells in the brain of an animal model of inflammatory disorder.

Using an animal model of endogenous Nerve Growth Factor (NGF) deprivation, we have investigated the effect of this molecule on the distribution of NG2 and CD56-positive cells in the brain of normal and of Experimental Allergic Encephalomyelitis (EAE) rats. We found that the number of these cells is significantly altered in the SubVentricular Zone (SVZ) and in other brain regions. These findings indicate that NGF might be implicated in the regulation of the antigen expressed by oligodendrocyte progenitors (NG2) and of the neural cell adhesion molecule (CD56/NCAM). Both antigens are associated with mechanisms of brain repair thus providing additional evidence for a major role of NGF in the brain response to pathological conditions such as EAE. Acknowledgements.--We thank Prof. R. Levi-Montalcini for her stimulating discussions. We also thank Dr. L. Aloe for critical reading of the manuscript. This study was supported by Proj. Strategico CNR, by AISM and by Fondazione CARISBO, Bologna. Dr. V. Triaca is a recipient of a fellowship from the AISM.

Nerve growth factor antibody exacerbates neuropathological signs of experimental allergic encephalomyelitis in adult lewis rats.

In this study, experimental allergic encephalomyelitis (EAE) rats and rats exhibiting EAE expressing high circulating anti-nerve growth factor antibody were daily monitored for clinical signs and chronic relapses. Eighty-five days after EAE induction, blood, spinal cord and brain stem were used for histological examination, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) evaluation. The results showed that NGF-deprived rats display more severe clinical signs of disease. These effects were associated with a significant reduction of NGF in the brain stem and spinal cord but not of BDNF, which decreased only in spinal cord. These observations provide additional support to the hypothesis of a protective NGF role in rats exhibiting EAE.

Patients treated with antitumor drugs displaying neurological deficits are characterized by a low circulating level of nerve growth factor.

The aim of our study was to explore whether nerve growth factor (NGF) plays any role in the development of peripheral neuropathy induced by anticancer treatment. We measured the circulating NGF levels in 23 cancer patients before and after chemotherapy. We evaluated whether the development of peripheral neurotoxicity was associated with changes in basal NGF concentrations in patients studied with a comprehensive neurological and neurophysiological examination. The results of these studies showed that the circulating levels of NGF, which are about 20 pg/ml in plasma of controls, decrease during chemotherapy and in some cases completely disappeared after prolonged treatment with antitumor agents. The decrease in NGF levels seems to be correlated with the severity of neurotoxicity. These results clearly suggest that NGF might become a useful agent to prevent neuropathies induced by antineoplastic drugs and restore peripheral nerve dysfunction induced by these pharmacological compounds.

Role of superoxide in apoptosis induced by growth factor withdrawal.

We have examined the role of reactive oxygen species (ROS) in apoptosis induced by growth factor deprivation in primary cultures of mouse proximal tubular (MPT) cells. When confluent monolayers of MPT cells are deprived of all growth factors, the cells die by apoptosis over a 10- and 14-day period. Both epidermal growth factor (EGF) and high-dose insulin directly inhibit apoptosis of MPT cells deprived of growth factors. Growth factor deprivation results in an increase in the cellular levels of superoxide anion while apoptosis of MPT cells induced by growth factor withdrawal is inhibited by a number of antioxidants and scavengers of ROS. Growth factor deprivation also results in activation of caspase activity, which is inhibited by EGF and high-dose insulin as well as by the ROS scavengers and antioxidants that inhibit apoptosis. The cell-permeant caspase inhibitor, z-Val-Ala-Asp-CH2F (zVAD-fmk), prevents the increase in caspase activity and markedly inhibits apoptosis induced by growth factor deprivation. However, zVAD-fmk had no effect on the increased levels of superoxide associated with growth factor deprivation. Thus we provide novel evidence that ROS play an important role in mediating apoptosis associated with growth factor deprivation. ROS appear to act upstream of caspases in the apoptotic pathway. We hypothesize that oxidant stress, induced by growth factor withdrawal, represents a signaling mechanism for the default pathway of apoptosis.

Lysophosphatidic acid: a novel growth and survival factor for renal proximal tubular cells.

Lysophosphatidic acid (LPA) is the smallest and structurally simplest of all glycerophospholipids. LPA is a normal constituent of serum and binds with high affinity to albumin while retaining its biological activity. The effects of LPA are pleiotropic and range from mitogenesis to stress fiber formation. In this report, we demonstrate two novel functions for LPA. LPA acts as a survival factor to inhibit apoptosis of primary cultures of mouse renal proximal tubular (MPT) cells. LPA also acts as a potent mitogen for MPT cells. The ability of LPA to act as both a survival factor and a mitogen is mediated by the lipid kinase phosphatidylinositol 3-kinase (PI3K), since these activities were completely blocked by wortmannin or LY-294002, two structurally dissimilar inhibitors of PI3K. The identification of LPA as a proliferative and anti-apoptotic factor suggests a potential role for this lipid mediator during the injury and/or recovery phases following tubular damage.

Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis.

We have examined the mechanisms of cell death induced by cisplatin in primary cultures of mouse proximal tubular cells. High concentrations of cisplatin (800 microM) led to necrotic cell death over a few hours. Much lower concentrations of cisplatin (8 microM) led to apoptosis, which caused loss of the cell monolayer over several days. Necrosis was characterized by a cytosolic swelling and early loss of plasma membrane integrity. In contrast, early features of cells undergoing apoptosis included cell shrinkage and loss of attachment to the monolayers. Nuclear chromatin became condensed and fragmented in apoptosing cells. These features were absent in necrotic cells. DNA electrophoresis of cells exposed to 800 microM cisplatin yielded a "smear" pattern, due to random DNA degradation. In contrast, the DNA of apoptosing cells demonstrated a "ladder" pattern resulting from internucleosomal DNA cleavage. Antioxidants delayed cisplatin-induced apoptosis but not necrosis. Thus the mechanism of cell death induced by cisplatin is concentration dependent. Reactive oxygen species play a role in mediating apoptosis but not necrosis induced by cisplatin.