Stuart J. Rabin

GM1 Ganglioside Activates the High‐Affinity Nerve Growth Factor Receptor trkA

Abstract: The monosialoganglioside GM1 has been shown to possess neurotrophic activity in vitro and in vivo and is now used as an experimental treatment for a variety of neurological disorders and trauma. Little is known about the mechanism of action used by GM1. Because GM1 appears to enhance nerve growth factor (NGF) activity, we have used C6trk+ cells, a derivative of C6‐2B glioma cells that express the high‐affinity receptor for NGF trkA, to determine whether the neurotrophic effects of GM1 occurs through induction of trkA activity. Exposure of C6trk+ cells to NGF (10–50 ng/ml) resulted in a five‐ to 10‐fold increase in trkA tyrosine phosphorylation within 5 min. Incubation of cells with GM1 resulted in a threefold increase in trkA phosphorylation beginning within 1 h and peaking between 3 and 6 h. Optimal responses to GM1 were obtained using 80–100 µM concentrations. Moreover, tyrosine phosphorylation of known trkA target proteins, such as extracellular signal‐regulated kinases, and suc‐associated neurotrophic factor‐induced tyrosine‐phosphorylated target, were activated upon stimulation of C6trk+ cells with GM1. In addition, GM1 potentiated the NGF‐mediated activation of tyrosine phosphorylation of trkA. GM1 failed to induce phosphorylation of trkA and target proteins in mock transfected cells. Thus, our data demonstrate that GM1 mimics some of the effects of NGF and suggest that the neurotrophic properties of GM1 may be attributed to its activation of trkA signal transduction.

K‐252b Selectively Potentiates Cellular Actions and trk Tyrosine Phosphorylation Mediated by Neurotrophin‐3

Abstract: K‐252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K‐252b was found to prevent trophic actions of two other neurotrophins, brain‐derived neurotrophic factor, and neurotrophin‐3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC 12 pheochromocytoma cells, when used at >2 μM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1‐100 nM, K‐252b selectively enhanced the trophic action of neurotrophin‐3 on central cholinergic neurons, peripheral sensory neurons, and PC 12 cells. In PC 12 cells, K‐252b potentiated the neurotrophin‐3‐induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K‐252a, K‐252b, and staurosporine, only the first two also mediated neurotrophin‐3 potentiation. These findings indicate that K‐252b generally and selectively potentiates the neurotrophic action of neurotrophin‐3 and suggest that this action involves trk‐type neurotrophin receptors.

Rapid Phosphorylation of Phospholipase Cγ 1 by Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 in Cultures of Embryonic Rat Cortical Neurons

Abstract: Phospholipase Cγ1 (PLC‐γ1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5‐bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron‐like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC‐γ1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) on phosphorylation of PLC‐γ1 in primary cultures of embryonic rat brain cells. BDNF and NT‐3 stimulated the phosphorylation of PLC‐γ1, followed by hydrolysis of PI. The stimulation of PLC‐γ1 phosphorylation occurred within 20 s after addition of BDNF or NT‐3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT‐3, with τ25 ng/ml. Phosphorylation of PLC‐γ1 by BDNF and NT‐3 was found in cultures from all major brain areas. K‐252b, a compound known to inhibit selectively neurotrophin actions by interfering with the phosphorylation of trk‐type neurotrophin receptors, prevented the BDNF‐ and NT‐3‐stimulated phosphorylation of PLC‐γ1. Receptors of the trk type were coprecipitated with anti‐PLC‐γ1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT‐3 seems to be neuron specific because no phosphorylation of PLC‐γ1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT‐3, and they indicate that the transduction mechanism of BDNF and NT‐3 in the brain involves rapid phosphorylation of PLC‐γ1 followed by PI hydrolysis.

Increased basic fibroblast growth factor expression following contusive spinal cord injury.

Neurotrophic factors appear to be crucial for the survival and potential regeneration of injured neurons. We have previously demonstrated that contusive spinal cord injury (SCI) increases the levels of mRNA for basic fibroblast growth factor (FGF2). To determine whether FGF2 protein levels also increase, Western blot analysis was performed on extracts of spinal cord tissue after a standardized SCI and compared to laminectomy controls. In spinal cord extracts, a monoclonal antibody to FGF2 recognized various molecular forms of FGF2 (18-24 kDa) and some characteristic proteolytic fragments. Extracts of spinal cords 1 day after SCI showed a slight increase in the levels of these polypeptides. By 4 days, a significant increase (two-fold) was detected in the levels of the 18-kDa and higher molecular weight forms as well as the proteolytic fragments. Immunohistochemical analyses on spinal cord tissue sections confirmed an increased cellular (glial) FGF2 as well as interstitial immunoreactivity surrounding neurons and along blood vessels. Heparinpurified spinal cord extracts from tissue 4 days after SCI showed increased biological activity as indicated by their ability to (i) increase [3H]thymidine incorporation in cultures of Balb/c 3T3 cells and (ii) induce phosphorylation of suc-associated neurotrophic factor-induced tyrosine-phosphorylated target, a FGF2 target protein. These data suggest that SCI induces increased FGF2 expression and support the hypothesis that FGF2 may play a role in the partial recovery of function seen following SCI.

Gangliosides Prevent Excitotoxicity Through Activation of TrkB Receptor

Gangliosides protect cerebellar granule cells from excitotoxicity; however, their mechanism of action remains to be fully characterized. GM1 ganglioside has been shown to activate Trk, the tyrosine kinase receptor implicated in the neuroprotective properties of the neurotrophins. In these studies, we used primary cultures of cerebellar granule cells to determine whether gangliosides exert neuroprotective effect via the activation of Trk receptors. We first examined the relative potency of the neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin-3 and nerve growth factor to prevent glutamate-mediated apoptosis. BDNF was the only neurotrophin that elicited a complete neuronal protection against glutamate. GM1 and its semisynthetic compound LIGA20 also prevented glutamate toxicity, however, LIGA20 was more potent than GM1. Both LIGA20 and BDNF blocked glutamate-mediated activation of caspase-3 and consequently apoptosis; however, the anticaspase-3 activity was seen only when these compounds were added to the cultures several hours before glutamate, suggesting that LIGA20 and BDNF share an identical molecular mechanism. To test this hypothesis, we compared the ability of LIGA20 and BDNF to activate TrkB. Both compounds elicited a similar time-dependent increase in Trk tyrosine phosphorylation. Moreover, the neuroprotective effect of BDNF and LIGA20 was abolished in neurons exposed to the Trk tyrosine kinase inhibitor k252a, demonstrating a relationship between neuroprotection and activation of Trk receptors. Our data suggest that by activating the Trk neurotrophin receptors, gangliosides may be used as neuroprotective agents.

BIOLOGICAL ACTIVITY OF INTERLEUKIN-10 IN THE CENTRAL NERVOUS SYSTEM

Cytokines play a crucial role as mediators of inflammation. Astrocytes and microglia are the two major glial cells involved in the central nervous system immune responses. In this study we examined the effects of interleukin-10 (IL-10), one of the naturally occurring inhibitory cytokines, on different types of glial cells in culture such as rat astrocytes, hamster microglia and C6-2B glioma cells. Phosphorylation of signal transducers and activators of transcription (STAT) proteins was used as a marker for IL-10 activity. Within minutes, IL-10 elicited a strong and weak increase in STAT3 and STAT1 phosphorylation, respectively, in human T lymphocytes, suggesting that STAT3 is a main IL-10 signaling event in these cells. In contrast, IL-10 failed to induce STAT3 in glial cells, but elicited a weak increase in STAT1 phosphorylation in microglia and C6-2B glioma cells only, suggesting that in some glial cell population(s) IL-10 may produce cellular responses via activation of the STAT1 pathway. Moreover, in C6-2B cells, IL-10 elicited a decrease in the level of basic fibroblast growth factor mRNA. A similar decrease was observed in adult rat hypothalamus, indicating that this cytokine may regulate glial production of trophic factors. Our data suggest that IL-10 may play a role in glial cell differentiation and proliferation.

TrkA receptors delay C6-2B glioma cell growth in rat striatum.

Nerve growth factor (NGF) acts as an anti-mitogenic factor in C6-2B glioma cells stably expressing TrkA (C6trk+). To study the effect of TrkA on cell growth in vivo, we grafted mock and C6trk+ cells into the striatum of ACI nude rats. Thy 1.1 and p75NTR immunohistochemistry revealed that wild type C6-2B cells formed a tumor mass in the striatum by 14 days. In contrast, C6trk+ transplanted rats did not show the presence of a significant tumor mass until 71 days. Analysis of this tumor showed that expression of TrkA was retained, but the synthesis of NGF was abolished. Our data encourage the speculation that expression of TrkA in glioblastoma in vivo will attenuate tumor progression.