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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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Activation of TrkA and p75NTR signaling by hNGF mutants.Western blot and densitometric analysis of (A) TrkA (Y490), (B)                            Akt/S473), and (C) PLC-γ1 (Y783) phosphorylation, in extracts from                            BALB/C 3T3 TrkA cells, stimulated by 100 ng/ml of hNGF and hNGFR100                            mutants. D, Western blot of phospho-TrkA (Y490), Akt (S473), PLC-γ1                            (Y783) and Erks (T204/Y202) in PC12 cells, stimulated by 5 ng/ml of hNGF                            and hNGFR100E mutant. E, Densitometric analysis of phospho-TrkA. F,                            Densitometric analysis of phospho-Akt. G, Densitometric analysis of                            phospho-PLC-γ1. H, Densitometric analysis of phospho-Erks. I,                            Densitometric analysis of phospho c-jun in hippocampal neurons. The                            experiments were performed in triplicate. Bars represent the mean                            ± s.e.m.
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pone-0017321-g001: Activation of TrkA and p75NTR signaling by hNGF mutants.Western blot and densitometric analysis of (A) TrkA (Y490), (B) Akt/S473), and (C) PLC-γ1 (Y783) phosphorylation, in extracts from BALB/C 3T3 TrkA cells, stimulated by 100 ng/ml of hNGF and hNGFR100 mutants. D, Western blot of phospho-TrkA (Y490), Akt (S473), PLC-γ1 (Y783) and Erks (T204/Y202) in PC12 cells, stimulated by 5 ng/ml of hNGF and hNGFR100E mutant. E, Densitometric analysis of phospho-TrkA. F, Densitometric analysis of phospho-Akt. G, Densitometric analysis of phospho-PLC-γ1. H, Densitometric analysis of phospho-Erks. I, Densitometric analysis of phospho c-jun in hippocampal neurons. The experiments were performed in triplicate. Bars represent the mean ± s.e.m.

Mentions: The phosphorylation of residue Tyr490 of TrkA, recruits Shc and activates different downstream cascades, including the Ras/MAP kinase cascade (reviewed in [25], [38], [39], [40], [41] and Fig. S2). The phosphorylation of this residue by hNGFR100W and by hNGFR100E mutants, determined with a site-specific anti- phosphoTrkA antibody, was reduced by 70% with respect to that induced by hNGF, or by other hNGFR100 mutants, in 3T3-TrkA cells (Fig. 1A), but only slightly affected in PC12 cells (Fig. 1D,E). TrkA-dependent signaling linked to neuronal survival is channeled, via the phosphatidylinositol 3-kinase (PI3-K) through the downstream Akt pathway [38], [40]. The activation of Akt in 3T3-TrkA and PC12 cells, by different hNGF proteins, was analyzed with antibodies against active Akt. All hNGFR100 mutants, including R100W and R100E mutants, were equally effective as wild type hNGF in activating this pathway in both cell lines (Fig. 1B,D,F). The phosphorylation of residue Tyr 785 of TrkA, recruits PLC-γ1, inducing its phosphorylation at residue Y783 [38], [40]. In 3T3-TrkA cells, hNGFR100W and hNGFR100E were completely unable to induce the phosphorylation of PLC- γ1 with respect to hNGF (Fig. 1C), unlike other hNGFR100 mutants, confirming that hNGFR100W and hNGFR100E share similar properties. A significant reduction of PLC-γ1 activation by hNGFR100E proteins was also found in PC12 cells (Fig. 1E,G). TrkA activation by NGF leads to the activation of ERK1 and 2 kinases [39], [40]. In PC12 cells the major contribution to overall ERK activation is through RAP-1 and B-Raf, rather than through Ras [42], [43]. The activation of Erks in PC12 cells by hNGFR100E was analyzed with antibodies against active Erks (residues Thr202/Tyr204). The hNGFR100E mutant induced a significantly lower activation of Erks, with respect to hNGF (Fig. 1E,H), which is not due to a different time course (data not shown).


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)

Activation of TrkA and p75NTR signaling by hNGF mutants.Western blot and densitometric analysis of (A) TrkA (Y490), (B)                            Akt/S473), and (C) PLC-γ1 (Y783) phosphorylation, in extracts from                            BALB/C 3T3 TrkA cells, stimulated by 100 ng/ml of hNGF and hNGFR100                            mutants. D, Western blot of phospho-TrkA (Y490), Akt (S473), PLC-γ1                            (Y783) and Erks (T204/Y202) in PC12 cells, stimulated by 5 ng/ml of hNGF                            and hNGFR100E mutant. E, Densitometric analysis of phospho-TrkA. F,                            Densitometric analysis of phospho-Akt. G, Densitometric analysis of                            phospho-PLC-γ1. H, Densitometric analysis of phospho-Erks. I,                            Densitometric analysis of phospho c-jun in hippocampal neurons. The                            experiments were performed in triplicate. Bars represent the mean                            ± s.e.m.
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getmorefigures.php?uid=PMC3046150&req=5

pone-0017321-g001: Activation of TrkA and p75NTR signaling by hNGF mutants.Western blot and densitometric analysis of (A) TrkA (Y490), (B) Akt/S473), and (C) PLC-γ1 (Y783) phosphorylation, in extracts from BALB/C 3T3 TrkA cells, stimulated by 100 ng/ml of hNGF and hNGFR100 mutants. D, Western blot of phospho-TrkA (Y490), Akt (S473), PLC-γ1 (Y783) and Erks (T204/Y202) in PC12 cells, stimulated by 5 ng/ml of hNGF and hNGFR100E mutant. E, Densitometric analysis of phospho-TrkA. F, Densitometric analysis of phospho-Akt. G, Densitometric analysis of phospho-PLC-γ1. H, Densitometric analysis of phospho-Erks. I, Densitometric analysis of phospho c-jun in hippocampal neurons. The experiments were performed in triplicate. Bars represent the mean ± s.e.m.
Mentions: The phosphorylation of residue Tyr490 of TrkA, recruits Shc and activates different downstream cascades, including the Ras/MAP kinase cascade (reviewed in [25], [38], [39], [40], [41] and Fig. S2). The phosphorylation of this residue by hNGFR100W and by hNGFR100E mutants, determined with a site-specific anti- phosphoTrkA antibody, was reduced by 70% with respect to that induced by hNGF, or by other hNGFR100 mutants, in 3T3-TrkA cells (Fig. 1A), but only slightly affected in PC12 cells (Fig. 1D,E). TrkA-dependent signaling linked to neuronal survival is channeled, via the phosphatidylinositol 3-kinase (PI3-K) through the downstream Akt pathway [38], [40]. The activation of Akt in 3T3-TrkA and PC12 cells, by different hNGF proteins, was analyzed with antibodies against active Akt. All hNGFR100 mutants, including R100W and R100E mutants, were equally effective as wild type hNGF in activating this pathway in both cell lines (Fig. 1B,D,F). The phosphorylation of residue Tyr 785 of TrkA, recruits PLC-γ1, inducing its phosphorylation at residue Y783 [38], [40]. In 3T3-TrkA cells, hNGFR100W and hNGFR100E were completely unable to induce the phosphorylation of PLC- γ1 with respect to hNGF (Fig. 1C), unlike other hNGFR100 mutants, confirming that hNGFR100W and hNGFR100E share similar properties. A significant reduction of PLC-γ1 activation by hNGFR100E proteins was also found in PC12 cells (Fig. 1E,G). TrkA activation by NGF leads to the activation of ERK1 and 2 kinases [39], [40]. In PC12 cells the major contribution to overall ERK activation is through RAP-1 and B-Raf, rather than through Ras [42], [43]. The activation of Erks in PC12 cells by hNGFR100E was analyzed with antibodies against active Erks (residues Thr202/Tyr204). The hNGFR100E mutant induced a significantly lower activation of Erks, with respect to hNGF (Fig. 1E,H), which is not due to a different time course (data not shown).

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