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Taking pain out of NGF: a "painless" NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrophic activity.

Capsoni S, Covaceuszach S, Marinelli S, Ceci M, Bernardo A, Minghetti L, Ugolini G, Pavone F, Cattaneo A - PLoS ONE (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: European Brain Research Institute, Rome, Italy.

ABSTRACT
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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hNGF mutant bioactivity on survival and differentiation of PC12                            cells, neuroblastoma SH-SY5Y cells and chick DRG neurons.In Panels A–C, PC12 cells were plated in presence of 100 ng/ml of                            hNGF (B) or hNGFR100E (C) and the number of PC12 processes evaluated                            (J). In panels D-F, PC12cells were primed with 50 ng/ml of hNGF (E) or                            hNGFR100E (F) for 1 week and replated for 2 days in presence of 10 ng/ml                            of either hNGF or hNGFR100E. Negative controls (A,D) are represented by                            cells incubated in absence of hNGF or hNGFR100E. (G) Untreated human                            neuroblastoma SH-SY5Y cells are induced to differentiate when treated                            for 7 days with 100 ng/ml of hNGF (H), or with hNGFR100E (I). The mutant                            hNGFR100E is as effective as wild type hNGF in determining the survival                            and differentiation of chick embryo dorsal root ganglia sensory neurons,                            after a 48 hrs exposure.
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pone-0017321-g002: hNGF mutant bioactivity on survival and differentiation of PC12 cells, neuroblastoma SH-SY5Y cells and chick DRG neurons.In Panels A–C, PC12 cells were plated in presence of 100 ng/ml of hNGF (B) or hNGFR100E (C) and the number of PC12 processes evaluated (J). In panels D-F, PC12cells were primed with 50 ng/ml of hNGF (E) or hNGFR100E (F) for 1 week and replated for 2 days in presence of 10 ng/ml of either hNGF or hNGFR100E. Negative controls (A,D) are represented by cells incubated in absence of hNGF or hNGFR100E. (G) Untreated human neuroblastoma SH-SY5Y cells are induced to differentiate when treated for 7 days with 100 ng/ml of hNGF (H), or with hNGFR100E (I). The mutant hNGFR100E is as effective as wild type hNGF in determining the survival and differentiation of chick embryo dorsal root ganglia sensory neurons, after a 48 hrs exposure.

Mentions: The neurotrophic activity of the R100E mutant was studied, at first, in rat PC12 pheochromocytoma and in human SH-SY5Y neuroblastoma cell lines. The time course and extent of neuronal differentiation of naïve PC12 cells incubated with hNGF or hNGFR100E for one week was by and large identical (Fig. 2A–C, J). Priming of PC12 cells with hNGF or hNGFR100E for one week equally induced NGF dependency upon neurotrophin removal (data not shown), and survival and differentiation of primed rat PC12 cells, induced by 50 ng/ml hNGF and hNGFR100E addition after replating, was identical, both in terms of number of surviving and differentiating cells and of time course and extent of neurite outgrowth (Fig. 2D–F). Also in human SH-SY5Y neuroblastoma cells [45], hNGF, and hNGFR100E were similarly effective in neurite outgrowth induction (Fig. 2G–I).


Taking pain out of NGF: a "painless" NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrophic activity.

Capsoni S, Covaceuszach S, Marinelli S, Ceci M, Bernardo A, Minghetti L, Ugolini G, Pavone F, Cattaneo A - PLoS ONE (2011)

hNGF mutant bioactivity on survival and differentiation of PC12                            cells, neuroblastoma SH-SY5Y cells and chick DRG neurons.In Panels A–C, PC12 cells were plated in presence of 100 ng/ml of                            hNGF (B) or hNGFR100E (C) and the number of PC12 processes evaluated                            (J). In panels D-F, PC12cells were primed with 50 ng/ml of hNGF (E) or                            hNGFR100E (F) for 1 week and replated for 2 days in presence of 10 ng/ml                            of either hNGF or hNGFR100E. Negative controls (A,D) are represented by                            cells incubated in absence of hNGF or hNGFR100E. (G) Untreated human                            neuroblastoma SH-SY5Y cells are induced to differentiate when treated                            for 7 days with 100 ng/ml of hNGF (H), or with hNGFR100E (I). The mutant                            hNGFR100E is as effective as wild type hNGF in determining the survival                            and differentiation of chick embryo dorsal root ganglia sensory neurons,                            after a 48 hrs exposure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046150&req=5

pone-0017321-g002: hNGF mutant bioactivity on survival and differentiation of PC12 cells, neuroblastoma SH-SY5Y cells and chick DRG neurons.In Panels A–C, PC12 cells were plated in presence of 100 ng/ml of hNGF (B) or hNGFR100E (C) and the number of PC12 processes evaluated (J). In panels D-F, PC12cells were primed with 50 ng/ml of hNGF (E) or hNGFR100E (F) for 1 week and replated for 2 days in presence of 10 ng/ml of either hNGF or hNGFR100E. Negative controls (A,D) are represented by cells incubated in absence of hNGF or hNGFR100E. (G) Untreated human neuroblastoma SH-SY5Y cells are induced to differentiate when treated for 7 days with 100 ng/ml of hNGF (H), or with hNGFR100E (I). The mutant hNGFR100E is as effective as wild type hNGF in determining the survival and differentiation of chick embryo dorsal root ganglia sensory neurons, after a 48 hrs exposure.
Mentions: The neurotrophic activity of the R100E mutant was studied, at first, in rat PC12 pheochromocytoma and in human SH-SY5Y neuroblastoma cell lines. The time course and extent of neuronal differentiation of naïve PC12 cells incubated with hNGF or hNGFR100E for one week was by and large identical (Fig. 2A–C, J). Priming of PC12 cells with hNGF or hNGFR100E for one week equally induced NGF dependency upon neurotrophin removal (data not shown), and survival and differentiation of primed rat PC12 cells, induced by 50 ng/ml hNGF and hNGFR100E addition after replating, was identical, both in terms of number of surviving and differentiating cells and of time course and extent of neurite outgrowth (Fig. 2D–F). Also in human SH-SY5Y neuroblastoma cells [45], hNGF, and hNGFR100E were similarly effective in neurite outgrowth induction (Fig. 2G–I).

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.

View Article: PubMed Central - PubMed

Affiliation: European Brain Research Institute, Rome, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus