Taking pain out of NGF: a "painless" NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrophic activity.
Bottom Line: NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8-10 mice/group).We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF.Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.
Affiliation: European Brain Research Institute, Rome, Italy.
During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8-10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.
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Mentions: The activity of the hNGF and hproNGF mutants (hNGFX and hproNGFX) was further tested in cultures from mouse dorsal root ganglia (DRG) and superior cervical ganglia (SCG). Cell cultures were first exposed for 4 days to 100 ng/ml or 200 ng/ml of NGF or proNGF, respectively (Fig. 4A). This assay allows to measure both induction of dependency and survival activity of NGF. On the fifth day, DRG and SCG neurons were deprived of hNGFX or hproNGFX for 24 hours, before cell counting (Fig. 4A). In DRG cultures, hNGF and hNGFR100E were similarly effective in determining mouse DRG survival or dependency from NGF (compare blue versus green bars in Fig. 4B). hproNGF was as effective as hNGF in determining neuronal survival (blue versus red bars in Fig. 4B) and dependency (red versus pink bars in Fig. 4B) while hproNGFR100E was less effective than hNGFR100E in inducing DRG survival and dependency (Fig. 4B). Parallel experiments on chick embryo DRG neurons confirmed that survival curves for chick DRG neurons obtained after incubation with different doses of hNGF or hNGFR100E mutant are totally superimposable (Fig. 2K), demonstrating that the neurotrophic potency of hNGFR100E mutants is identical to that of wild type hNGF. In SCG neuronal cultures, we found that, in the context of mature hNGF, the R100E mutation did not affect hNGF ability to induce neuronal survival or dependency (blue versus green bars, Fig. 4C). On the other hand, hproNGFR100E was less effective than hproNGF in inducing SCG survival (blue versus red bars, Fig. 4C) and dependency (red versus pink bars, Fig. 4C).