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A role for Piezo2 in EPAC1-dependent mechanical allodynia.

Eijkelkamp N, Linley JE, Torres JM, Bee L, Dickenson AH, Gringhuis M, Minett MS, Hong GS, Lee E, Oh U, Ishikawa Y, Zwartkuis FJ, Cox JJ, Wood JN - Nat Commun (2013)

Bottom Line: Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton.Piezo2 knockdown also enhanced thresholds for light touch.Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. N.Eijkelkamp@umcutrecht.nl

ABSTRACT
Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

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Epac signalling-mediated allodynia is Piezo2 dependent and does not require Nav1.8 expressing nociceptors.WT and nociceptor-depleted mice (Nav1.8-DTA) received an intraplantar injection of 8-pCPT (12.5 nmol per paw) and time course of (a) allodynia (n=8) was determined. WT and Nav1.8-DTA mice received different doses of 8-pCPT (b, n=4–8) or 6-Bnz-cAMP (d, 6-Bnz-cAMP, n=4–8) and mechanical sensitivity was determined 5 h after intraplantar injection. Duration of 8-pCPT (c, n=4-8) or 6-Bnz-cAMP-induced (e, n=4–8) mechanical allodynia determined as the earliest time point in which the 50% threshold is equal or higher than the average baseline 50% threshold—2x standard deviation. (f) Time course of 6-Bnz-cAMP-induced (12.5 nmol per paw) allodynia (n=8). Repeated measures of allodynia: genotype: P<0.001, time: P<0.001, interaction: P<0.05. (g–h) Piezo2 or mismatch antisense ODNs were administered intrathecally 5, 3, 2, and 1 day before behavioural experiments. (g) Two days after the last injection L2-L5 DRG Piezo2 mRNA levels were determined by quantitative RT-PCR. (h) Time course of 8-pCPT-induced allodynia was determined using von Frey. (i) To verify role of Piezo2 in touch perception, one day after the last antisense ODN injection L2–L6 DRG Piezo1/2 mRNA expression levels were determined by quantitative RT-PCR. (j) Sensitivity to touch was determined using Von Frey filaments. Data are expressed as mean±s.e.m. (a–f, h)Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (g,i) Data are analysed by t-test. (j) Data were analysed using one-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001.
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f7: Epac signalling-mediated allodynia is Piezo2 dependent and does not require Nav1.8 expressing nociceptors.WT and nociceptor-depleted mice (Nav1.8-DTA) received an intraplantar injection of 8-pCPT (12.5 nmol per paw) and time course of (a) allodynia (n=8) was determined. WT and Nav1.8-DTA mice received different doses of 8-pCPT (b, n=4–8) or 6-Bnz-cAMP (d, 6-Bnz-cAMP, n=4–8) and mechanical sensitivity was determined 5 h after intraplantar injection. Duration of 8-pCPT (c, n=4-8) or 6-Bnz-cAMP-induced (e, n=4–8) mechanical allodynia determined as the earliest time point in which the 50% threshold is equal or higher than the average baseline 50% threshold—2x standard deviation. (f) Time course of 6-Bnz-cAMP-induced (12.5 nmol per paw) allodynia (n=8). Repeated measures of allodynia: genotype: P<0.001, time: P<0.001, interaction: P<0.05. (g–h) Piezo2 or mismatch antisense ODNs were administered intrathecally 5, 3, 2, and 1 day before behavioural experiments. (g) Two days after the last injection L2-L5 DRG Piezo2 mRNA levels were determined by quantitative RT-PCR. (h) Time course of 8-pCPT-induced allodynia was determined using von Frey. (i) To verify role of Piezo2 in touch perception, one day after the last antisense ODN injection L2–L6 DRG Piezo1/2 mRNA expression levels were determined by quantitative RT-PCR. (j) Sensitivity to touch was determined using Von Frey filaments. Data are expressed as mean±s.e.m. (a–f, h)Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (g,i) Data are analysed by t-test. (j) Data were analysed using one-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001.

Mentions: Nav1.8+ sensory neurons comprise 85% of nociceptors and are essential for detecting noxious mechanical stimuli as well as for development of inflammatory hyperalgesia, but not neuropathic pain24. To test whether Nav1.8+ sensory neurons are required for 8-pCPT-induced mechanical allodynia, we used diphtheria toxin to kill these sensory neurons (Nav1.8-DTA24). As reported24, baseline sensitivity to touch was similar in WT and Nav1.8-DTA mice (Fig. 7a). Importantly, the course of 8-pCPT-induced mechanical allodynia in mice in which Nav1.8 nociceptors were ablated was indistinguishable from control littermates (Fig. 7a). The absence of an effect of Nav1.8 nociceptor depletion on 8-pCPT-induced mechanical allodynia was independent of the dose of 8-pCPT; at any dose of 8-pCPT tested (12.5 pmol per paw–12.5 nmol per paw) mice showed no notable difference in magnitude (Fig. 7b) or duration (Fig. 7c) of mechanical allodynia in Nav1.8-DTA mice compared with control littermates.


A role for Piezo2 in EPAC1-dependent mechanical allodynia.

Eijkelkamp N, Linley JE, Torres JM, Bee L, Dickenson AH, Gringhuis M, Minett MS, Hong GS, Lee E, Oh U, Ishikawa Y, Zwartkuis FJ, Cox JJ, Wood JN - Nat Commun (2013)

Epac signalling-mediated allodynia is Piezo2 dependent and does not require Nav1.8 expressing nociceptors.WT and nociceptor-depleted mice (Nav1.8-DTA) received an intraplantar injection of 8-pCPT (12.5 nmol per paw) and time course of (a) allodynia (n=8) was determined. WT and Nav1.8-DTA mice received different doses of 8-pCPT (b, n=4–8) or 6-Bnz-cAMP (d, 6-Bnz-cAMP, n=4–8) and mechanical sensitivity was determined 5 h after intraplantar injection. Duration of 8-pCPT (c, n=4-8) or 6-Bnz-cAMP-induced (e, n=4–8) mechanical allodynia determined as the earliest time point in which the 50% threshold is equal or higher than the average baseline 50% threshold—2x standard deviation. (f) Time course of 6-Bnz-cAMP-induced (12.5 nmol per paw) allodynia (n=8). Repeated measures of allodynia: genotype: P<0.001, time: P<0.001, interaction: P<0.05. (g–h) Piezo2 or mismatch antisense ODNs were administered intrathecally 5, 3, 2, and 1 day before behavioural experiments. (g) Two days after the last injection L2-L5 DRG Piezo2 mRNA levels were determined by quantitative RT-PCR. (h) Time course of 8-pCPT-induced allodynia was determined using von Frey. (i) To verify role of Piezo2 in touch perception, one day after the last antisense ODN injection L2–L6 DRG Piezo1/2 mRNA expression levels were determined by quantitative RT-PCR. (j) Sensitivity to touch was determined using Von Frey filaments. Data are expressed as mean±s.e.m. (a–f, h)Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (g,i) Data are analysed by t-test. (j) Data were analysed using one-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001.
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f7: Epac signalling-mediated allodynia is Piezo2 dependent and does not require Nav1.8 expressing nociceptors.WT and nociceptor-depleted mice (Nav1.8-DTA) received an intraplantar injection of 8-pCPT (12.5 nmol per paw) and time course of (a) allodynia (n=8) was determined. WT and Nav1.8-DTA mice received different doses of 8-pCPT (b, n=4–8) or 6-Bnz-cAMP (d, 6-Bnz-cAMP, n=4–8) and mechanical sensitivity was determined 5 h after intraplantar injection. Duration of 8-pCPT (c, n=4-8) or 6-Bnz-cAMP-induced (e, n=4–8) mechanical allodynia determined as the earliest time point in which the 50% threshold is equal or higher than the average baseline 50% threshold—2x standard deviation. (f) Time course of 6-Bnz-cAMP-induced (12.5 nmol per paw) allodynia (n=8). Repeated measures of allodynia: genotype: P<0.001, time: P<0.001, interaction: P<0.05. (g–h) Piezo2 or mismatch antisense ODNs were administered intrathecally 5, 3, 2, and 1 day before behavioural experiments. (g) Two days after the last injection L2-L5 DRG Piezo2 mRNA levels were determined by quantitative RT-PCR. (h) Time course of 8-pCPT-induced allodynia was determined using von Frey. (i) To verify role of Piezo2 in touch perception, one day after the last antisense ODN injection L2–L6 DRG Piezo1/2 mRNA expression levels were determined by quantitative RT-PCR. (j) Sensitivity to touch was determined using Von Frey filaments. Data are expressed as mean±s.e.m. (a–f, h)Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (g,i) Data are analysed by t-test. (j) Data were analysed using one-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001.
Mentions: Nav1.8+ sensory neurons comprise 85% of nociceptors and are essential for detecting noxious mechanical stimuli as well as for development of inflammatory hyperalgesia, but not neuropathic pain24. To test whether Nav1.8+ sensory neurons are required for 8-pCPT-induced mechanical allodynia, we used diphtheria toxin to kill these sensory neurons (Nav1.8-DTA24). As reported24, baseline sensitivity to touch was similar in WT and Nav1.8-DTA mice (Fig. 7a). Importantly, the course of 8-pCPT-induced mechanical allodynia in mice in which Nav1.8 nociceptors were ablated was indistinguishable from control littermates (Fig. 7a). The absence of an effect of Nav1.8 nociceptor depletion on 8-pCPT-induced mechanical allodynia was independent of the dose of 8-pCPT; at any dose of 8-pCPT tested (12.5 pmol per paw–12.5 nmol per paw) mice showed no notable difference in magnitude (Fig. 7b) or duration (Fig. 7c) of mechanical allodynia in Nav1.8-DTA mice compared with control littermates.

Bottom Line: Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton.Piezo2 knockdown also enhanced thresholds for light touch.Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. N.Eijkelkamp@umcutrecht.nl

ABSTRACT
Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

Show MeSH
Related in: MedlinePlus