Mechano growth factor interacts with nucleolin to protect against cisplatin-induced neurotoxicity.

Mechano growth factor (MGF) is an alternatively spliced form of insulin-like growth factor-1 (IGF-1) that has shown to be neuroprotective against 6-hydroxydopamine toxicity and ischemic injury in the brain. MGF also induces neural stem cell proliferation in the hippocampus and preserves olfactory function in aging mice. Cisplatin is a chemotherapy drug that induces peripheral neuropathy in 30-40% of treated patients. Our studies were designed to see if MGF would protect dorsal root ganglion (DRG) neurons from cisplatin-induced neurotoxicity and to identify potential mechanisms that may be involved. Expression of endogenous MGF in adult DRG neurons in vivo ameliorated cisplatin-induced thermal hyperalgesia. Exogenous MGF and MGF with a cysteine added to the N-terminus (CMGF) also protected embryonic DRG neurons from cisplatin-induced cell death in vitro. Mass spectroscopy analysis of proteins bound to MGF showed that nucleolin is a key-binding partner. Antibodies against nucleolin prevented the neuroprotective effect of MGF and CMGF in culture. Both nucleolin and MGF are located in the nucleolus of DRG neurons. RNAseq of RNA associated with MGF indicated that MGF may be involved in RNA processing, protein targeting and transcription/translation. Nucleolin is an RNA binding protein that is readily shuttled between the nucleus, cytoplasm and plasma membrane. Nucleolin and MGF may work together to prevent cisplatin-induced neurotoxicity. Exploring the known mechanisms of nucleolin may help us better understand the mechanisms of cisplatin toxicity and how MGF protects DRG neurons.

Autocrine regulation of neurite outgrowth from PC12 cells by nerve growth factor.

The PC12 cell line may be used as a model of NGF-induced neuronal differentiation. Exposure to NGF is accompanied by extension of neurites, cessation of growth and differentiation into cells resembling sympathetic neurons. In this study neurite outgrowth from PC12 cells was induced in serum-free, NGF-free medium conditions. Neurite outgrowth in serum-free conditions was abolished by exposure to anti-NGF antisera. Reverse transcription combined with polymerase chain reaction (RT-PCR) and in situ hybridization of PC12 cells in serum-free medium conditions revealed NGF transcripts. Western blot analysis of these cells revealed tyrosine phosphorylation of the high affinity NGF receptor (TrkA/gp140) and activation of a downstream signal cascade element, ERK-1/MAP kinase. NGF was also detected by a specific enzyme-linked immunoabsorbant assay (ELISA) revealing picogram levels of protein in conditioned medium and cell lysates. Survival of embryonic rat dorsal root ganglion neurons was maintained in cultures grown in this serum-free conditioned medium. This demonstrated that NGF may act as an autocrine or paracrine growth factor for PC12 cell differentiation.

Nerve growth factor rescue of cisplatin neurotoxicity is mediated through the high affinity receptor: studies in PC12 cells and p75 null mouse dorsal root ganglia.

Nerve growth factor (NGF) rescues dorsal root ganglion neurons and PC12 cells from cisplatin-induced cell death. Two model systems were used to demonstrate that rescue is mediated through the high affinity NGF receptor. In dorsal root ganglion (DRG) neurons isolated from p75(-/-) and control mice, 20 ng/ml NGF completely prevented cisplatin-induced death. In PC12 cells, we overexpressed receptor chimeras between the tumor necrosis factor and NGF receptors. We demonstrated that activation of the intracellular domain of Trk A is responsible for the NGF rescue effect.

Acidity is involved in the development of neuropathy caused by oxidized cellulose.

Recently we demonstrated that oxidized cellulose (OC), a surgical topical hemostatic agent, induces subjacent nerve fiber degeneration by a diffusible chemical mechanism. Since OC is highly acidic, we examined the role of acidity in the development of neuropathy by OC in this study. Fifteen minutes' exposure to culture media containing OC (2 mg/ml, pH 3.47 or 10 mg/ml, pH 2.57) suppressed the subsequent neurite outgrowth of precultured rat DRG neurons in vitro. However, the neurotoxicity of OC disappeared when the pH of the media was restored to 7.42. Topical application of 20 mg OC lowered the pH in the subperineurium of the adjacent rat sciatic nerve to around 3, and kept it below 4 for 2 h in vivo. Application of 0.1 ml neutralized physiological saline containing 40 mg OC did not produce pathological changes in the adjacent rat sciatic nerve in vivo, in contrast to the marked subperineurial nerve damage by direct application of 20 or 40 mg OC observed in our previous study. These results strongly indicate that local neurotoxicity of OC is due to its high acidity. Further care is needed to avoid direct application of large amounts of OC to peripheral nerve.

Role of nerve growth factor in suramin neurotoxicity studied in vitro.

We determined whether suramin neurotoxicity can be prevented by nerve growth factor (NGF) and if this interaction occurs at the level of the NGF receptor. Neurite outgrowth from rat dorsal root ganglia in vitro was measured serially in the presence of suramin (100-600 microM) alone or with beta-NGF (50-1,000 ng/ml). Competitive NGF receptor-binding studies were done with 125I-labeled NGF in the presence or absence of suramin. Neurite growth was inhibited in a dose-dependent manner, but at usual neurotoxic levels this inhibition could be overcome completely by increasing the concentration of NGF. Receptor-binding assays showed similar dose-dependent inhibition of 125I-labeled NGF binding. In the presence of suramin, the dissociation constant for high-affinity binding was decreased from 1.2 x 10(-11) to 3.9 x 10(-10) and low-affinity binding from 2.7 x 10(-9) to 1.2 x 10(-8). Increasing doses of suramin inhibited 125I-labeled NGF specific binding in a dose-dependent fashion, and doses of suramin > or = 1,000 microM were able to completely inhibit 125I-labeled NGF specific binding. Suramin-induced dorsal root ganglia damage can be ameliorated by high-dose NGF. This effect is most likely due to competition between suramin and NGF at the high-affinity NGF receptor.

Role of the extracellular matrix in myelination of peripheral nerve.

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination.

Myelination by Schwann cells in the absence of extracellular matrix assembly.

Assembly of extracellular collagen fibrils and Schwann cell basal lamina has previously been identified as a prerequisite for compact myelin formation in the peripheral nervous system. Synthesis of this extracellular matrix (ECM) in vitro required the presence of serum and ascorbic acid. Using rat embryonic dorsal root ganglion neurons and Schwann cells, we have developed a fully defined medium in which myelination occurs. In the absence of ascorbic acid, normal myelin was formed without ECM assembly. This demonstrates that although myelination and ECM assembly are usually closely linked, ECM formation is not a prerequisite for myelination in vitro.