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[Preprint]. 2024 Feb 26:rs.3.rs-3812442.
doi: 10.21203/rs.3.rs-3812442/v1.

The Primary Cilium and its Hedgehog Signaling in Nociceptors Contribute to Inflammatory and Neuropathic Pain

Lindsey A Fitzsimons  1   2 , Larissa Staurengo-Ferrari  3 , Oliver Bogen  3 , Dioneia Araldi  3 , Ivan J M Bonet  3 , Ethan E Jordan  1   2 , Jon D Levine  3 , Kerry L Tucker  1   2
Affiliations

The Primary Cilium and its Hedgehog Signaling in Nociceptors Contribute to Inflammatory and Neuropathic Pain

Lindsey A Fitzsimons et al. Res Sq. .

Abstract

The primary cilium, a 1-3 μm long hair-like structure protruding from the surface of almost all cells in the vertebrate body, is critical for neuronal development and also functions in the adult. As the migratory neural crest settles into dorsal root ganglia (DRG) sensory neurons elaborate a single primary cilium at their soma that is maintained into adult stages. While it is not known if primary cilia are expressed in nociceptors, or their potential function in the mature DRG neuron, recent studies have shown a role for Hedgehog, whose signaling demonstrates a dependence on primary cilia, in nociceptor sensitization. Here we report the expression of primary cilia in rat and mouse nociceptors, where they modulate mechanical nociceptive threshold, and contribute to inflammatory and neuropathic pain. When siRNA targeting Ift88, a primary cilium-specific intraflagellar transport (IFT) protein required for ciliary integrity, was administered by intrathecal injection, in the rat, it resulted in loss of Ift88 mRNA in DRG, and primary cilia in neuronal cell bodies, which was associated with an increase in mechanical nociceptive threshold, and abrogation of hyperalgesia induced by the pronociceptive inflammatory mediator, prostaglandin E2, and painful peripheral neuropathy induced by a neurotoxic chemotherapy drug, paclitaxel. To provide further support for the role of the primary cilium in nociceptor function we also administered siRNA for another IFT protein, Ift52. Ift52 siRNA results in loss of Ift52 in DRG and abrogates paclitaxel-induced painful peripheral neuropathy. Attenuation of Hedgehog-induced hyperalgesia by Ift88 knockdown supports a role for the primary cilium in the hyperalgesia induced by Hedgehog, and attenuation of paclitaxel chemotherapy-induced neuropathy (CIPN) by cyclopamine, which attenuates Hedgehog signaling, suggests a role of Hedgehog in CIPN. Our findings support a role of nociceptor primary cilia in the control of mechanical nociceptive threshold and in inflammatory and neuropathic pain, the latter, at least in part, Hedgehog dependent.

Keywords: Chemotherapy-induced Neuropathy; Cyclopamine; Hedgehog; Hyperalgesia; Inflammation; Nociceptor; PGE2; Paclitaxel; Pain; Primary Cilium; Sonic Hedgehog; siRNA.

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Conflict of interest statement

Conflict of interest statement The authors declare no competing interests. Additional Declarations: There is NO Competing Interest.

Figures

Figure 1.
Figure 1.. Rat DRG neurons elaborate primary cilia, in vivo.
(A-D) Immunohistofluorescence analysis of adult rat DRGs in vivo, using confocal microscopy. Histological sections were labeled with antibodies recognizing ARL13B (A-D, green), ɣ-tubulin (A-D, red), and Fox-3 (A, NeuN, greyscale), TrkA (B, greyscale), or CGRP (C, greyscale). (D) IB4 staining is indicated in greyscale. (A-D) Cell nuclei are marked by DAPI (blue). Red and yellow arrowheads indicate neuronal and non-neuronal primary cilia, respectively. (A-D) Scale bar 3 μm.
Figure 2.
Figure 2.. Targeted knockdown of Ift88 in rat DRG leads to a loss of primary cilia and a decrease in the average length of retained cilia.
(A) siRNA-mediated knockdown of Ift88. The histogram depicts the level of Ift88 expression in rats treated with either a control siRNA (control) or the siRNA targeting Ift88 (Ift88 knockdown). Gene expression was determined with SYBR green RT-PCR and values (mean ± 95% CI) represent expression relative to GAPDH (the housekeeping gene). Rats treated with siRNA targeting Ift88 demonstrate a significant reduction in Ift88 mRNA in lumbar DRG, compared to those treated with the control siRNA (unpaired two-tailed Student’s t-test, P <0.0001, t(10)=7.88, n=6). One way ANOVA with Tukey comparison was used to analyze for differences between groups. (B) Confocal immunohistofluorescence images of L4 rat DRGs stained for the ciliary axoneme (αArl13b, green), the basal body (anti-ɣ-tubulin, red), and the nucleus (DAPI, blue). Left panels, control siRNA. Right panels, Ift88-targeted siRNA. Lower panels represent magnifications of the green boxes (green star) indicated in the upper panels. Magnification/zoom factor indicated. Scale bar 100 and 10 μm (top and bottom panels, respectively). (C, D) Quantification of percentage ciliation (C) and ciliary length (D) in micrometers (μm) in L4 rat DRGs (rDRG) injected with control (CTL) vs Ift88-targeted (KD) siRNA. **: P < 0.01.
Figure 3.
Figure 3.. Effect of siRNA targeting Ift88 on mechanical nociceptive threshold and paclitaxel-induced hyperalgesia (CIPN).
Male rats were treated i.t. with one of 3 doses of siRNA (2, 5 or 10 μg/20 μl) targeting Ift88 or negative control siRNA, 10 mg/20ml for 3 consecutive days. Mechanical nociceptive threshold was evaluated before siRNA injection was started and again at 24, 48, and 72 h after treatment was finished. 72 h after the last siRNA injection (day 0), paclitaxel (1 mg/kg, i.p.) was administered (days 0, 2, 4 and 6) and the mechanical nociceptive threshold evaluated on days 0, 7, 14, 21, and 28. (A,B) Administration of siRNA targeting Ift88 increases mechanical nociceptive threshold when compared with the vehicle control group. (A) Mechanical nociceptive threshold is presented as absolute values in grams. Data is reported as mean ± SEM, treatment F(3,80) = 34.54, ****P <0.0001: neg siRNA vs siIft88 2 μg, neg siRNA vs siIft88 5 μg or neg siRNA vs siIft88 10 μg. (B) Mechanical nociceptive threshold is represented as percentage change from baseline threshold. Data shown as mean ± SEM, treatment F(3,60) = 15.80, ****P <0.0001: neg siRNA vs siIft88 2 μg, neg siRNA vs siIft88 5 μg or neg siRNA vs siIft88 10 μg. (C,D) The magnitude of paclitaxel-induced hyperalgesia was markedly attenuated in rats treated with siRNA targeting mIft88. (C) Mechanical nociceptive threshold is presented as absolute values in grams. Data shown as mean ± SEM, treatment F(3,100) = 192.0, ****P <0.0001: neg siRNA vs siIft88 2 μg, neg siRNA vs siIft88 5 μg or neg siRNA vs siIft88 10 μg. (D) Data shown as mean ± SEM, treatment F(3,100) = 77.50, ****P <0.0001: neg siRNA vs siIft88 2 μg, neg siRNA vs siIft88 5 μg or neg siRNA vs siIft88 10 μg. Two-way repeated-measures ANOVAs were used to compare negative control siRNA and siRNA targeting Ift88 mRNA groups, n = 6 paw in each group.
Figure 4.
Figure 4.. Role of IFT52 in paclitaxel-induced mechanical hyperalgesia.
(A) siRNA-mediated knockdown of Ift52 mRNA. The histogram depicts the level of Ift52 mRNA in rats treated with either control siRNA or siRNA targeting Ift52. Gene expression was determined with SYBR green RT-PCR and values (mean ± 95% CI) represent expression relative to GAPDH (the housekeeping gene). Rats treated with siRNA targeting Ift52 demonstrate a significant reduction in Ift52 mRNA levels in their lumbar DRG compared to those treated with the control siRNA (unpaired two-tailed Student’s t-test, P <0.0001, t(10)=11.21, n=6). (B,C) Male rats were treated i.t. with siRNA targeting Ift52 (10 μg/day) for 3 consecutive days. 72 h after the last siRNA injection, paclitaxel (1 mg/kg, i.p.) was administered on days 0, 2, 4 and 6. Mechanical nociceptive threshold was evaluated before siRNA treatment was started (baseline), and again on days −3, −2, −1, 3, 5, 7, 14, 21 and 28 days after paclitaxel injection. (B) Magnitude of hyperalgesia is expressed as absolute values of mechanical nociceptive threshold, in grams. Ift52 siRNA attenuates paclitaxel induced CIPN. Data are expressed as means ± SEM, n = 6 paws in each group. F(9,90) = 29.38, ****P <0.0001, ***P <0.0002, **P <0.0028; when Ift52 siRNA group was compared with control siRNA group; two-way repeated-measures ANOVA followed by Bonferroni multiple comparison test. (C) The magnitude of hyperalgesia expressed as percentage reduction from the baseline mechanical nociceptive thresholds. Ift52 siRNA attenuates paclitaxel induced CIPN. Data are expressed as means ± SEM, n=6 paws in each group. F(8,80)=12.87, ***P = 0.0005 **P <0.0089 *P = 0.0143; when the Ift52 siRNA group was compared with the control siRNA group; two-way repeated-measures ANOVA followed by Bonferroni multiple comparison test.
Figure 5.
Figure 5.. Pronociceptive effect of Shh is primarily cilium-dependent.
(A,B) Male rats were treated i.t. with siRNA targeting Ift88 (10 μg/day) for 3 consecutive days. 72 h after the last siRNA injection, rats received i.d. or i.gl. injections of recombinant Shh (200 ng) and mechanical nociceptive threshold was assessed at 0.5, 1, 3, and 5 h after injection. (A) The hyperalgesic effect of Shh injected i.d. was significantly reduced when the expression of Ift88 was downregulated through siRNA-mediated knockdown. Data shown as mean ± SEM, treatment F(1,40) = 73.26, time F(4,50) = 6.861, ****P <0.01: siIFT88, Shh i.d. vs siRNA neg, Shh i.d.. (B) The hyperalgesic effect of i.gl. Shh was also significantly reduced when the expression of Ift88 was downregulated with siRNA. Data shown as mean ± SEM, treatment F(1,40) = 48.13, time F(3,40) = 2.286, ****P <0.01: IFT88 siRNA, Shh i.gl. vs siRNA neg, Shh i.gl. Two-way repeated-measures ANOVA followed by Bonferoni’s test, n=6 paws in each group. (C, D) Male rats received paclitaxel every other day for a total of 4 injections, on days 0, 2, 4, and 6 (1 mg/kg × 4, i.p.). Seven days after the first administration of paclitaxel, rats were treated with i.d. (10 μg/5 μl) or i.gl. (10 μg/5 μl) cyclopamine. As a control, the contralateral paw or contralateral lumbar DRG received vehicle (saline plus DMSO 2%). Mechanical nociceptive threshold was evaluated before and 7 days after paclitaxel injection (before cyclopamine injection, ~0h), and again 0, 0.5, 1, 3, and 5 h after i.d. or i.gl. cyclopamine injection. (C) Paclitaxel-induced hyperalgesia was markedly attenuated in the male rats treated i.d. with cyclopamine. Data shown as mean ± SEM, treatment F(1,50) = 28.28, time F(4,50) = 4.154, **P <0.01: cyclopamine i.d. vs vehicle i.d. groups. (D) The magnitude of paclitaxel-induced hyperalgesia was markedly attenuated in male rats treated with i.gl. cyclopamine. Data shown as mean ± SEM, treatment F(1,50) = 57.38, time F(4,50) = 4.748, **P <0.01: cyclopamine i.d. vs vehicle i.d..
Figure 6.
Figure 6.. Ift88 siRNA attenuates hyperalgesia induced by i.d. PGE2.
Male rats were treated i.t. with siRNA for IFT88 mRNA for 3 consecutive days in a dose of 10 μg/day. 72 h after the last siRNA injection, PGE2 was administered i.d. (100 ng/5 μl/paw). Mechanical nociceptive threshold was evaluated before siRNA treatment was started (baseline), and again 72 h after the last siRNA treatment, and then 30 min after PGE2. (A) As shown in Figure 3, siRNA targeting Ift88 led to a significant increase in baseline nociceptive threshold, expressed in grams. Data shown as means ± SEM, Time F(1,20) = 21.73 ***P < 0.0001: siIFT88 vs baseline or vs siRNA neg control. Two way-repeated measures ANOVA followed by Bonferroni’s test, n = 6 paws of each group. (B, C) Treatment with Ift88 siRNA also resulted in a substantial reduction in hyperalgesia induced by i.d. PGE2. (B) The magnitude of hyperalgesia is expressed as percentage reduction from the baseline mechanical nociceptive threshold before siRNA treatment. Data shown as mean ± SEM, unpaired t-test: t = 5.846, df = 10; ***P < 0.001: siIFT88 vs siRNA neg control, n = 6 paws of each group. (C) The magnitude of hyperalgesia is expressed as % reduction from the baseline mechanical nociceptive thresholds read 72 h after the last treatment with siRNA. Data are expressed as mean ± SEM, unpaired t-test: t = 3.260, df=10; **P < 0.01: siIFT88 vs siRNA neg, n = 6 paws of each group.
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