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, 31 (6), 2113-24

Spinal Phosphinositide 3-kinase-Akt-mammalian Target of Rapamycin Signaling Cascades in Inflammation-Induced Hyperalgesia

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Spinal Phosphinositide 3-kinase-Akt-mammalian Target of Rapamycin Signaling Cascades in Inflammation-Induced Hyperalgesia

Qinghao Xu et al. J Neurosci.

Abstract

Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.

Figures

Figure 1.
Figure 1.
Effects of intrathecal wortmannin on intraplantar carrageenan-induced pain behaviors. A, C, Time course over 360 min of PWL to thermal stimulation in the injected paw before (baseline, time 0) and after carrageenan. Intrathecal (IT) vehicle (3% DMSO, 10 μl) or wortmannin (Wort; 4.5 μg in A; 1.5 and 4.5 μg in B) was administered 15 min before (A) or 130 min after (C) intraplantar carrageenan (2%, 100 μl). B, D, Histograms represent the hyperalgesia index from 0 to 360 min. E, Time course over 240 min of PWT to tactile stimulation in the injected paw before and after carrageenan. F, Histogram represents the hyperalgesia index from 0 to 240 min intrathecal vehicle or wortmannin (4.5 μg) was given 15 min before carrageenan. The data are presented as mean ± SEM of six to eight rats per group. *p < 0.05 compared with the vehicle by t test (B, F) or one-way ANOVA (D).
Figure 2.
Figure 2.
Effects of intrathecal injection of PI3K–Akt inhibitors on Formalin-induced flinching behaviors. A, C, Time course over 60 min of flinch responses after Formalin (2.5%, 50 μl) paw injection in rats with intrathecal injection of vehicle (3% DMSO, 10 μl, 15 min before Formalin) or wortmannin (Wort; 0.5–4.5 μg) (A) or vehicle (3% DMSO), PI-103 (0.5 μg), or Akt-I-1/2 (3 μg) (C). Each point represents the number of flinches in a 5 min block. B, D, Histograms represent the total number of flinches in each phase: phase I, 1–9 min; and phase II, 10–60 min. The data are presented as mean ± SEM of six to eight rats per group. *p < 0.05 compared with vehicle (3% DMSO) by one-way ANOVA, followed by Tukey's post hoc test.
Figure 3.
Figure 3.
Effects of intrathecal rapamycin on Formalin or carrageenan-induced pain behaviors. A, Time course over 60 min of flinch responses after Formalin (2.5%, 50 μl). Each point represents the number of flinches in a 5 min block. B, Histogram represents the total number of flinches in each phase: phase I, 1–9 min; and phase II, 10–60 min. C, E, Time course of PWT to tactile stimulation (C) and PWL to thermal stimulation (E) in the injected paw before (baseline, time 0) and after carrageenan (2%, 100 μl). D, F, Histograms represent the hyperalgesia index over the testing period (240 min in D and 180 min in F). Intrathecal vehicle (3% DMSO) or rapamycin (Rapa; 4.5 μg) was administered 15 min before Formalin or carrageenan. The data are presented as mean ± SEM of six to eight rats per group. *p < 0.05 compared with the vehicle by t test.
Figure 4.
Figure 4.
Distribution and cellular localization of pAkt immunoreactivity in the ipsilateral spinal cord dorsal horn after intraplantar carrageenan. A–C, Immunoreactivity of pAkt is increased in the ipsilateral dorsal horn 1 h after intraplantar carrageenan (carr; 2%, 100 μl) compared with naive. A pronounced increase is seen in laminae I, III, and IV. Ten confocal planes along the z-axis are projected (20 μm depth). D–L, Cell-type-specific immunolabeling of pAkt in the ipsilateral spinal cord dorsal horn. Single focal plane images of pAkt (green) with NeuN (D–F, red), GFAP (G–I, red), or OX42 (J–L, red) are shown. Arrows indicate colocalization of pAkt with the respective cell markers (yellow). Scale bars: A–C, 250 μm; D–L, 50 μm.
Figure 5.
Figure 5.
Distribution and cellular localization of pmTOR, pS6, p4EBP, and pAkt immunoreactivity in the ipsilateral spinal cord dorsal horn after intraplantar carrageenan. A, Immunoreactivity of pmTOR, pS6, and p4EBP in the ipsilateral dorsal horn. Ten confocal planes along the z-axis are projected (20 μm depth). B, Cell-type-specific immunolabeling of pmTOR, pS6, and p4EBP in the ipsilateral dorsal horn. Single focal plane images are shown. Arrows indicate colocalization of the phospho-proteins with the respective cell markers (yellow). C, Double labeling of pAkt, pmTOR, and pS6 in dorsal horn neurons at 1 h after intraplantar carrageenan. Sections are counterstained with TO-PRO-3 (blue) to label cell nuclei. Single focal plane images are shown. Scale bars: A, 250 μm; B, 50 μm; C, 20 μm.
Figure 6.
Figure 6.
Distribution and cellular localization of p4EBP (catalog #2855; Cell Signaling Technology) immunoreactivity in the ipsilateral spinal cord dorsal horn after intraplantar carrageenan. A, Immunoreactivity of p4EBP in the ipsilateral dorsal horn. Ten confocal planes along the z-axis are projected (20 μm depth). B, Cell-type-specific immunolabeling of p4EBP in the ipsilateral dorsal horn at 1 h after intraplantar carrageenan. Single focal plane images are shown. Arrows indicate colocalization of the phospho-proteins with the respective cell markers (yellow). Scale bars: A, 250 μm; B, 50 μm.
Figure 7.
Figure 7.
Carrageenan-induced changes in spinal levels of pAkt and pmTOR assessed by Western blot. A, C, Representative Western blots showing levels of pAkt, total Akt, and β-actin (b-actin) (A) and pmTOR, total mTOR, and β-tubulin (C) in the ipsilateral lumbar spinal dorsal quadrant at different time points after intraplantar carrageenan. B, D, Histograms represent the mean phosphor-protein levels with respect to the naive group. The data are presented as mean ± SEM of three to five rats per group. *p < 0.05 compared with naive by one-way ANOVA, followed by Tukey's post hoc test.
Figure 8.
Figure 8.
Formalin-induced changes in spinal levels of pAkt, pmTOR, and pS6 ribosomal protein. A, C, Representative Western blots showing levels of pAkt, total Akt, and β-actin (b-actin) (A) and pmTOR, total mTOR, and β-tubulin (C) in the ipsilateral lumbar spinal dorsal quadrant at different time points after Formalin. B, D, Histograms represent the mean pAkt or pmTOR level with respect to the naive group. E, Immunoreactivity of pS6 ribosomal protein in the spinal dorsal horn in naive and Formalin-injected animals. F, Histogram represents number of neurons labeled with pS6 in laminae I/II, III/IV, and V/VI. The data are presented as mean ± SEM of three to five rats per group. *p < 0.05 compared with naive by one-way ANOVA, followed by Tukey's post hoc test (B, D) or t test (F).
Figure 9.
Figure 9.
Effects of intrathecal wortmannin on intraplantar carrageenan or Formalin-induced pAkt. A, C, Representative Western blots showing pAkt, total Akt, and β-actin (b-actin) in the ipsilateral lumbar spinal dorsal quadrant at 1 h after carrageenan (A) or 30 min after Formalin (C). Rats received intrathecal vehicle (3% DMSO, 10 μl) or wortmannin (wort; 4.5 μg) 15 min before carrageenan or Formalin. B, D, Histograms represent the mean pAkt level with respect to the vehicle group. The data are presented as mean ± SEM of four to eight rats per group. *p < 0.05 compared with vehicle by t test. E, Immunoreactivity pAkt was reduced by intrathecal wortmannin compared with vehicle in dorsal horn at 1 h after intraplantar carrageenan. Scale bar, 250 μm.
Figure 10.
Figure 10.
Effects of intrathecal wortmannin or rapamycin on intraplantar carrageenan-induced phosphorylation of 4EBP and S6 ribosomal protein. A, C, Representative Western blots showing p4EBP, total 4EBP, and β-actin (b-actin) (A) and pS6, total S6, and β-actin (C) in the ipsilateral lumbar spinal dorsal quadrant at 1 h after carrageenan. Rats received intrathecal vehicle (3% DMSO, 10 μl) or wortmannin (wort; 4.5 μg) 15 min before carrageenan. E, Western blots showing pS6, total S6, and β-actin in rats received intrathecal vehicle (3% DMSO, 10 μl) or rapamycin (rapa; 3 μg) before carrageenan. B, D, F, Histograms represent the mean p4EBP and pS6 levels with respect to the vehicle group. The data are presented as mean ± SEM of four or five rats per group. *p < 0.05 compared with vehicle by t test.
Figure 11.
Figure 11.
pAkt, pmTOR, and pS6 are colocalized with NK1R in laminae I, III, and IV. A, Confocal images of pAkt or pmTOR (green) and NK1R (red) at 1 h after carrageenan. In each case, a single NK1R-labeled neuron is seen also pAkt or pmTOR positive. Sections are counterstained with TO-PRO-3 (blue) to label cell nuclei. B, Confocal images of pAkt, pmTOR, or pS6 (green) and NK1R (red) at 30 min after intrathecal SP (30 nmol). A large lamina III/IV neuron is shown. NK1R internalization can be seen on the dendrites and soma. Single focal plane images are shown. Scale bars, 20 μm.
Figure 12.
Figure 12.
Effects of intrathecal wortmannin or rapamycin on spinal SP-induced pain behaviors. A, C, Time course over 60 min of PWL to thermal stimulation in the injected paw before (baseline, time 0) and 10, 20, 30, and 60 min after intrathecal (IT) SP (30 nmol). Animals received intrathecal wortmannin (Wort) 15 min before intrathecal SP (A) or rapamycin (Rapa) 2 h before intrathecal SP (C). B, D, The histograms represent the hyperalgesia index over testing period of 60 min. *p < 0.05 compared with vehicle by one-way ANOVA, followed by Tukey's post hoc test (B) or t test (D). Data are presented as mean ± SEM; n = 7 or 8 for each group.
Figure 13.
Figure 13.
Effect of intrathecal wortmannin or rapamycin on intrathecal SP-induced pAkt and pS6 in spinal cord assessed by Western blot. A, A representative blot of pAkt, total Akt, and β-actin (b-actin) in the lumbar dorsal cord at 30 min after intrathecal (IT) SP (30 nmol). C and E show the Western blots of pAkt, total Akt, and β-actin (C) and pS6, total S6, and β-actin (E) in the lumbar spinal dorsal cord at 30 min after intrathecal SP. These animals received intrathecal vehicle (3% DMSO, 10 μl) or wortmannin (wort; 4.5 μg) 15 min before SP. G, A Western blot of pS6, total S6, and β-actin in rats that received intrathecal vehicles only, vehicle + SP, or rapamycin + SP. Intrathecal vehicle (3% DMSO, 10 μl) or rapamycin (rapa; 3 μg) was administered 15 min before SP. B, D, F, H, Histograms represent the mean pAkt or pS6 level with respect to the vehicle group. The data are presented as mean ± SEM of three to six rats per group. *p < 0.05 compared with vehicle by t test (B, D, F) or one-way ANOVA, followed by Tukey's post hoc test (H).
Figure 14.
Figure 14.
Schematic illustration of potential role of PI3K–Akt–mTORC1 cascade in regulating the excitability of dorsal horn neurons including NK1R projection neurons. Peripheral tissue injury and inflammation induces neurotransmitter release in the spinal cord dorsal horn, such as SP. SP activates NK1 receptors on the dorsal horn neurons, leading to the activation of the PI3K–Akt–mTORC1 cascade and phosphorylation of their substrates. mTORC1 may regulate translation of certain mRNAs, thereby enhancing the excitability of these neurons. The identities of these mRNAs are not known. Previous reports suggest certain protein kinases, ion channels, neurotropic factors, and receptors could be among the candidates (Eberwine et al., 2002; Skup, 2008).

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