DIFFERENTIAL EXPRESSION OF PAIN-RELATED NEUROTROPHIC FACTOR (NGF, GDNF AND IL-6) mRNAs IN HUMAN PERIPHERAL NEUROPATHY
Yamamoto M., Mitsuma N., Ito Y., Li M., Sobue G. Department of Neurology, Nagoya University School of Medicine, Nagoya, Japan
Nociception is regulated by a large number of endogenous molecules including neurotrophic factors and neuropoietic cytokines. NGF and GDNF have been found to promote the survival of small diameter sensory neurons with myelinated and unmyelinated axons. IL-6, a cytokine, is known to lower the threshold of nociception. In the present study, we investigated mRNA levels of NGF, GDNF and IL-6, and cognate receptors (p75 and TrkA, GFRa-1 and Ret, IL-6Ra and gp 130) in 81 various human peripheral neuropathies by the methods of RT-PCR and in situ hybridization. The mRNA expression was assessed in relation to myelinated and unmyelinated nerve fiber pathology, and T cell and macrophage invasions in the diseased nerves. NGF, GDNF and IL-6, and their receptor mRNA levels were elevated to variable extent in the diseased nerves, particularly angiopathic and inflammatory neuropathies. The p75 and GFRa-1 mRNA levels were correlated with the extent of the nerves with axonal pathology. TrkA and Ret signaling receptor mRNAs were not detected in normal nor diseased nerves. The increase of IL-6 mRNA was proportional to the extent of invasion of the nerves by macrophages. The levels of NGF, GDNF and IL-6, and p75, GFRa-1 and IL-6Ra mRNAs were not related each other. These results indicate that the expression of pain-related neurotrophic factor (NGF, GDNF and IL-6) and cognate receptors is differentially regulated by an underlying pathology-related process, suggesting multiple therapeutic approaches to nociception in human peripheral neuropathies.
DOWNREGULATION OF NGF RECEPTORS (TRK A) ON THE DRG CELL SURFACE IS RESPONSIBLE FOR INHIBITION OF AXONAL GROWTH BY HIGH LEVELS OF NGF
Conti A.M., Windebank A.J. Mayo Molecular Neuroscience Program, Rochester, MN 55905.
We have demonstrated that supraphysiological levels of NGF (50-200 ng/ml) rapidly and reversibly inhibit neurite outgrowth from embryonic rat DRG neurons (Conti et al: Ann Neurol 42:838-846, 1997). These levels have been achieved in human trials when NGF is intrathecally administered. NGF binds to two cell surface receptors; TrkA with high affinity (Kd =10(exp-11) and p75 with low affinity. (Kd = 10(exp-9). We now identify which receptor is involved in this inhibition. Receptor binding studies were performed on enriched cultures of dissociated DRG neurons using (125)I-NGF. Cells were pre-treated with either 200 ng/ml or 10 ng/ml NGF for 1h for binding studies. In high NGF concentrations, there was a shift to the right of the binding displacement curve, compared to control. Using LIGAND, a computer program for the interpretation of multiple binding sites, there was one-site binding (Kd = 10(exp-9). Under control conditions, binding compatible with two sites corresponding to the low and high affinity receptors was observed. Semiquantitative RT-PCR showed that TrkA mRNA level was not reduced with high NGF. Co-incubation with REX antibody (a gift from Dr. Louis Reichardt, UCSF) blocked binding of NGF to p75 but did not block the inhibitory effect of 200 ng/ml NGF on axonal growth. Axonal growth inhibition by 200 ng/ml NGF was also observed in DRG from p75 knockout mice. These results demonstrated that TrkA was not detectable on the plasma membrane of DRG following treatment with 200 ng/ml NGF for 1h. This was not due to a reduction in transcription. Inhibition of axonal growth by 200 ng/ml NGF still occurred when p75 was blocked or absent. Decreased expression of TrkA on the cell surface provides the first evidence of a mechanism by which high dose NGF inhibits axonal growth. This may have important implications for the use of NGF in clinical trials for the treatment of neuropathy.
CISPLATIN INDUCED NEURONAL APOPTOSIS IS PREVENTED BY NERVE GROWTH FACTOR THROUGH THE HIGH AFFINITY RECEPTOR
Fischer S.J., McDonald E.S., Gill J.S., Windebank A.J. Mayo Molecular Neuroscience Program, Rochester, MN 55905.
Apoptosis occurs in neurodegenerative, metabolic, and toxic diseases of the nervous system. Cisplatin (CP) induced death of sensory neurons is the dose-limiting side effect for this effective chemotherapeutic agent. Dorsal root ganglion neurons undergo apoptosis when exposed to CP (Gill and Windebank JCI 101:2842-2850, 1998). Death was preceded by upregulation of elements indicative of GO to G1 transition in the cell cycle. Cyclin D1 mRNA and protein levels increased in CP treated cultures. This was accompanied by a decrease in expression of the endogenous cdk inhibitor pl6(INK4a). Levels of the hyperphosphorylated form of the retinoblastoma gene product increased. These changes were followed by activation of caspase-3 demonstrated by the appearance of the active form of CPP 32 and increased cleavage of the fluorescent substrate (Ac-DEVD-AMC). Use of caspase inhibitors prevented neuronal death. Inhibition of cdk activity demonstrated that the cell cycle changes were occurring upstream of caspase-3. Preincubation of cells with 50 ng/ml NGF prevented apoptosis by increasing endogenous levels of p16. Use of molecular chimeras between the TNF receptor and either the low (p75) or high (Trk A) affinity NGF receptor demonstrated that this protective effect was mediated through Trk A. The protective effect was abolished by K252a. This data demonstrated that NGF protected neurons against CP induced death by preventing progression from GO to G1. We are now determining whether similar changes occur in vivo in rats treated with CP. This has significant therapeutic implications for the prevention of CP induced chemotherapeutic neurotoxicity.
INSULIN-LIKE GROWTH FACTOR I ENHANCES SH-SY5Y NEUROBLASTOMA CELL MOTILITY
Meyer G.E., Shelden E.(1), Feldman E.L. University of Michigan, Neuroscience Program Departments of Neurology, (1)Anatomy & Cell biology, Ann Arbor, MI
Insulin-like growth factor I (IGF-1) plays a key role in neuronal development as well as protection from environmental stressors. In vitro, IGF-I enhances neuronal mitogenesis and survival. SH-SY5Y neuroblastoma cells express the type I IGF receptor (IGF-IR) and provide a model system to examine the effects of IGF-I on neuronal function and recovery from insult. In the current study, we examined the ability of IGF-I to enhance neuronal motility. We have employed two assays to investigate the effect of' IGF-I on SH-SY5Y motility. First, cells are plated on gold-coated coverslips in serum-free media and stimulated with IGF-I. After incubation for 6 or 12 h, the coverslips are fixed, mounted, and imaged with a computer. The areas of the tracks etched into the gold by the cells are quantitated using a computer imaging program. At 6 and 12 h post stimulation, 1 nM IGF-I causes SH-SY5Y motility to almost double. Higher doses of IGF-I (10 nM) increase motility over baseline, but not as effectively as lower doses. In a second approach, cell motility and morphological responses to IGF-I are observed with time lapse microscopy. Changes in cell shape are quantitated by measuring he area that changed between two sequential time lapse images taken 1 min apart. 1 nM IGF-I induces an instantaneous increase in the amount of shape change occurring in cells plated on plastic and subjected to overnight serum starvation. 10 nM IGF-I induces a lesser effect. On laminin 1 nM IGF-I causes dramatic production of lamellipodia an subsequent motility at 40 min post stimulation. These observations suggest that IGF-I is a potent stimulator of SH-SY5Y motility. Currently, studies are being done to understand which aspects of the IGF-I intracellular signaling mechanisms are involved in promoting cell motility. Supported by NIH R01 NS38849 (ELF) and 5 T32 GM07863 (GM).