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Ptchd1 mediates opioid tolerance via cholesterol-dependent effects on μ-opioid receptor trafficking

Nature Neuroscience volume 25, pages 1179–1190 (2022)Cite this article

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Abstract

Repeated exposure to opioids causes tolerance, which limits their analgesic utility and contributes to overdose and abuse liability. However, the molecular mechanisms underpinning tolerance are not well understood. Here, we used a forward genetic screen in Caenorhabditis elegans for unbiased identification of genes regulating opioid tolerance which revealed a role for PTR-25/Ptchd1. We found that PTR-25/Ptchd1 controls μ-opioid receptor trafficking and that these effects were mediated by the ability of PTR-25/Ptchd1 to control membrane cholesterol content. Electrophysiological studies showed that loss of Ptchd1 in mice reduced opioid-induced desensitization of neurons in several brain regions and the peripheral nervous system. Mice and C. elegans lacking Ptchd1/PTR-25 display similarly augmented responses to opioids. Ptchd1 knockout mice fail to develop analgesic tolerance and have greatly diminished somatic withdrawal. Thus, we propose that Ptchd1 plays an evolutionarily conserved role in protecting the μ-opioid receptor against overstimulation.

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Fig. 1: Forward genetic screen in C. elegans identifies PTR-25/Ptchd1 as a regulator of opioid tolerance.
Fig. 2: Knockout of Ptchd1 in mice enhances opioid efficacy and eliminates tolerance.
Fig. 3: Ptchd1 regulates opioid efficacy and response desensitization.
Fig. 4: Molecular mechanism of Ptchd1 action in MOR regulation.
Fig. 5: Role of cholesterol in mediating the effects of Ptchd1 on MOR desensitization in an endogenous neuronal setting.
Fig. 6: Proposed mechanism for Ptchd1 regulation of MOR signaling and opioid tolerance.

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Data availability

The data that support the findings of this study are published online as Source data files for all display items and are provided with this paper. Additional information is available from the corresponding authors upon reasonable request.

Code availability

Multi-Worm Tracker is an automated behavioral tracking instrument that uses custom software. Additional information about custom software is available from the corresponding authors (B.G.) upon reasonable request.

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Acknowledgements

We thank N. Martemyanova for producing and maintaining mice examined in this study and members of the Grill and Martemyanov laboratories for valuable input. This work was supported by NIH grants no. DA040406 and no. DA048036 (to B.G. and K.A.M.), no. DA036596 and no. EY028033 (to K.A.M.), and no. DA047771 (to H.M.S.). B.G.’s work is made possible in part by a generous gift in honor of Timothy Jackson. We thank the University of Kansas Genome Sequencing Core, which is supported by an NIH Center of Biomedical Research Excellence Grant (no. GM103638).

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Author notes

  1. These authors contributed equally: Nycole Maza, Dandan Wang, Cody Kowalski.

Authors and Affiliations

  1. Department of Neuroscience, UF Scripps Biomedical Research, Jupiter, FL, USA

    Nycole Maza, Cody Kowalski, Hannah M. Stoveken, Maria Dao, Omar K. Sial, Andrew C. Giles & Kirill A. Martemyanov

  2. Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, USA

    Dandan Wang & Brock Grill

  3. Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA

    Brock Grill

  4. Department of Pharmacology, University of Washington School of Medicine, Seattle, WA, USA

    Brock Grill

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Contributions

N.M., D.W., C.K., H.M.S., M.D., O.K.S. and A.C.G. performed experiments. B.G. and K.A.M. conceived the project, oversaw implementation and wrote the manuscript. All authors participated in editing the manuscript.

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Correspondence to Brock Grill or Kirill A. Martemyanov.

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Competing interests

B.G. and K.A.M. have filed a patent on the utility of Ptchd1 and modulation of neuronal cholesterol for therapeutic purposes. They are also stockholders in Evodenovo Inc., which has financial interests in intellectual property associated with this study.

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Extended data

Extended Data Fig. 1 TgMOR; bgg10 mutants show impaired tolerance to fentanyl and morphine.

a, Time course showing animal speed after application of different concentrations of fentanyl (arrow) for tgMOR animals at 1st exposure (naïve animals). b, Time course of fentanyl doses for tgMOR after repeated (5x) exposure to negative control vehicle. c, Time course of fentanyl doses for tgMOR after repeated (5x) exposure to fentanyl (10 μM). d, Time course of fentanyl doses for tgMOR; bgg10 mutants at 1st exposure (naïve animals). e, Time course of fentanyl doses for tgMOR; bgg10 after repeated (5x) exposure to vehicle. f, Time course of fentanyl doses for tgMOR; bgg10 after repeated (5x) exposure to fentanyl (10 μM). g, Dose-response curves showing tgMOR animals develop tolerance after repeated (5x) exposure to morphine (300 μM). h, Quantitation of opioid efficacy (EC50 values from g) shows decreased morphine efficacy and development of tolerance by tgMOR animals after repeated (5x) exposure to morphine. For a-h, automated MWT was used to acquire all data. Plotted is mean data for n = 12 wells (4-5 animals/well) for all conditions and genotypes. Data for 12 wells were derived from 3 independent experiments. For g and h, data points and histograms are means. n = 3 independent experiments with each experiment derived from 4 wells (4-5 animals/well) per dose. For g, error bars are ± SEM. For h, error bars are ± SD and data were analyzed using one-way ANOVA and Bonferroni’s post hoc test. ***p < 0.001.

Source data

Extended Data Fig. 2 RSBP-1/R7BP and FRPR-13 show differential effects on opioid tolerance.

a, Dose-response curves showing tgMOR; rsbp-1 mutants do not develop tolerance after repeated (5x) exposure to fentanyl (10 μM). b, Quantitation of opioid efficacy (EC50 values from a) shows similar fentanyl efficacy and absence of tolerance in tgMOR; rsbp-1 mutants after repeated (5x) exposure to fentanyl. c, Dose-response curves showing tgMOR; frpr-13 mutants develop tolerance after repeated (5x) exposure to fentanyl (10 μM). d, Quantitation of opioid efficacy (EC50 values from c) shows decreased fentanyl efficacy and development of tolerance by tgMOR; frpr-13 mutants after repeated (5x) exposure to fentanyl. For a-d, data points and histograms are means ± SD. n = 3 independent experiments with each experiment derived from 4 wells (4-5 animals/well) per dose for all genotypes. For b and d, data were analyzed using one-way ANOVA and Bonferroni’s post hoc test. ***p < 0.001.

Source data

Extended Data Fig. 3 Altered opioid responsiveness of tgMOR; bgg10.

a, Representative traces of individual animal movement acquired using MWT after application of fentanyl (10 μM) for indicated time and genotypes. b, Time course of fentanyl response (10 μM, arrow) for indicated genotypes. Arrow denotes fentanyl application. Note only tgMOR and tgMOR; bgg10 animals respond to fentanyl. TgMOR; bgg10 mutants are hypersensitive reaching maximum paralysis after 45 minutes in fentanyl. For b, plotted is mean data for n = 12 wells (4–5 animals/well) for all genotypes. Data for 12 wells were derived from three independent experiments. Data were analyzed using two-way ANOVA and Bonferroni’s post hoc test. ***p < 0.001.

Source data

Extended Data Fig. 4 Mutations in tgMOR; bgg10 occur in PTR-25/F43D9.1, a conserved member of the Patched protein family.

a, Plot showing analysis of whole genome sequence data with mapped region of chromosome III (red and grey bars) containing bgg10. b, Gene diagram showing Q1032stop nonsense mutation (red arrow) in ptr-25 (F43D9.1) contained in mapped region from tgMOR; bgg10 mutants. c, PTR-25 protein with Sterol Sensing Domain (SSD, yellow), Patched family domain (blue) and arginine rich domain (grey). bgg10 mutation, Q1032stop, highlighted (red arrow). d, Phylogenetic analysis of Patched family proteins including all major subfamilies: PTC/PTCH, NCR/NPC, DISP/PTCHD2, PTCHD1/4 (shaded in green) and the PTR group which is heavily expanded in C. elegans. Note PTR-25 has closest homology to mammalian PTCHD1/4. Protein sequences from human (black), mouse (light blue), fly (dark blue) and C. elegans (red) were aligned with Clustal W. Phylogenetic tree was constructed using maximum likelihood method with 1000 bootstrap replicates.

Extended Data Fig. 5 Validation of PTR-25/PTCHD1 as causal gene mutated in tgMOR; bgg10 animals that results in opioid hypersensitivity.

a, Time course of fentanyl application for indicated genotypes. Arrow indicates application of fentanyl (10 μM). TgMOR; ptr-25 CRISPR animals phenocopied hypersensitivity in tgMOR; bgg10 mutants. b, Native rescue with PTR-25 restored opioid sensitivity of tgMOR; bgg10 animals. c, Pan-neuronal rescue (rab-3 promoter) with PTR-25 reversed opioid sensitivity of tgMOR; bgg10 mutants. d, Time course of fentanyl application (5 μM, arrow) for indicated genotypes. Note transgenic expression of human PTCHD1 using the native ptr-25 promoter rescues opioid hypersensitivity of tgMOR; bgg10 animals. e, Dose-response curves with fentanyl showing transgenic human PTCHD1 decreased opioid sensitivity of tgMOR; bgg10 animals. For a-d, plotted is mean data for n = 12 wells (4-5 animals/well) for all genotypes. For e, n = 3 independent experiments with each experiment derived from 4 wells (4-5 animals/well) per dose for all genotypes. Data points are means ± SD. For a-d, data were analyzed by two-way ANOVA with Bonferroni’s post hoc test. ***p < 0.001, * p < 0.05.

Source data

Extended Data Fig. 6 Behavioral characterization of opioid responsiveness of Ptchd1 KO mice.

a, Evaluation of activity levels by tracking distance traveled in an open field. Data are from 7 WT and 6 Ptchd1 KO mice. b, Evaluation of activity levels by calculating total distance traveled in an open field over 120 minutes (from panel a). Data are from 7 WT and 6 Ptchd1 KO mice. c, Evaluation of activity levels by tracking distance traveled in an open field in first 10 minutes and last 10 minutes from panel a. Data are from 7 WT and 6 Ptchd1 KO mice. d, Evaluation of activity levels by tracking distance traveled in an open field after naltrexone or saline injection. Data are from 7 WT and 6 Ptchd1 KO mice. e, Analysis of time course of morphine analgesia by hot plate assay upon systemic morphine administration. Data are from 7 WT and 6 Ptchd1 KO mice. f, Evaluation of morphine induced analgesia in hot plate assay upon intracerebroventricular (ICV) injection of morphine. Data are from 7 male mice per genotype. g, Analysis of time course of morphine analgesia in the hot plate assay following ICV injection of morphine. Data are from 7 male mice per genotype. h, Evaluation of analgesia by hot plate assay with systemic fentanyl administration. Data are from 7 male mice per genotype. i, Analysis of time course of fentanyl analgesia by hot plate assay from the experiment in panel d. j, Evaluation of chronic analgesic tolerance by hot plate assay. Data are from 7 male mice per genotype. k, Quantification of analgesic efficacy reduction as a difference in the MPE between session 1 and 10 from panel j. Data are from 7 male mice per genotype. l, Evaluation of opioid induced hyperalgesia by hot plate assay. Baseline response latencies of mice receiving repeated fentanyl injections (in panel j) prior to drug administration. m, Quantification of opioid induced hyperalgesia as a difference in the baseline latencies between session 1 and 10 from panel l. Data are from 7 male mice per genotype. n, Evaluation of acute analgesic tolerance by hot plate assay. Mice received repeated morphine injections (20 mg/kg) 120 minutes apart. Data are from 5 WT and 7 Ptchd1 KO mice. o, Quantification of analgesic efficacy reduction as a difference in the MPE between session 1 and 2 from panel n. Data are from 5 WT and 7 Ptchd1 KO mice. p, Evaluation of analgesia by tail immersion assay after intrathecal administration of morphine. Data are from 9 WT and 5 Ptchd1 KO mice. q, Evaluation of analgesic tolerance by tail immersion assay. Mice received repeated intrathecal morphine injections (3.5 μg) over 5 days. Data are from 9 WT and 5 Ptchd1 KO mice. r, Quantification of analgesic efficacy reduction as a difference in the MPE between session 1 and 5 from panel q. Data are from 9 WT and 5 Ptchd1 KO mice. For panel a, d, and q, statistical comparisons were performed by 2-way ANOVA and Šídák post-hoc test. For panel c, e, g, i, j, l, and p, statistical comparisons were performed by 2-way ANOVA and Bonferroni’s post hoc comparison. For panels f, h, and n, statistical comparisons were performed by multiple unpaired two tailed Student’s t-test with Bonferroni’s post hoc. For b, k, m, o, and r, statistical comparisons were performed by unpaired two tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mean values with S.E.M. errors are shown. MPE, maximum possible effect.

Source data

Extended Data Fig. 7 Characterization of Ptchd1 – MOR co-expression across the brain.

a, Schematic of Ptchd1:YFP and MOR-mCherry mouse genetic manipulations. In the Ptchd1-YFP mouse, exon 1 of the Ptchd1 gene has been replaced with YFP with a YFP-bovine growth hormone poly-A tail (BGH) cassette. In the MOR-mCherry mouse, mCherry was inserted just before the stop codon on exon 4. For electrophysiology and expression studies, the Ptchd1-YFP mouse was crossed with the MOR-mCherry mouse. All mice imaged were heterozygous for the MOR-mCherry allele and homozygous for the Ptchd1-YFP allele. For electrophysiology experiments, control mice were heterozygous the Ptchd1-YFP allele and the MOR-mCherry allele. Ptchd1 KO mice were homozygous for the Ptchd1-YFP allele and heterozygous for the MOR-mCherry allele. ‘−’ denotes genetically modified allele, ‘+’ is the wild-type allele. b-g, Co-expression of MOR and PTCHD1 in locus coeruleus (b-c), nucleus accumbens (d-e), and thalamus (f-g). Representative immunohistochemistry images of brain sections from Ptchd1−/− /MOR-mCherry−/+ mice showing PTCHD1-YFP and MOR-mCherry co-expression. Scale bar is 250 µm in panel b and 100 µm in d and f. Quantification was performed by counting neurons with identified presence of indicated markers in each region from n = 3 (panel c and e) and n = 4 (panel g) mice per genotype. Mean values with S.E.M. errors are shown in all bar graphs.

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Extended Data Fig. 8 Analysis of MOR co-expression with Ptchd1 and morphological effects of Ptchd1 loss.

a, In situ hybridization in brain sections from WT animals. Slices were processed for the detection of the indicated probes in dorsal root ganglia (DRG), striatum (STR) and ventral tegmental area (VTA) using RNAScope. Neuronal marker NeuN was used to identify neurons. b, Quantifications of regional size and cell density in VTA and NAc between Ptchd1 KO and control genotypes. VTA included 8,071 cells from n = 3 in control and 7,812 cells from n = 3 in Ptchd1 KO mice. NAc included 46,979 cells from n = 3 in control and 41,111 cells from n = 3 in Ptchd1 KO mice. c, Quantification of membrane capacitance from recorded DRG nociceptors (control n = 26 cells, 8 mice; Ptchd1 KO n = 34 cells, 7 mice), VTA GABAergic interneurons (control n = 22 cells, 8 mice; Ptchd1 KO n = 25 cells, 12 mice), VTA dopamine projection neurons (control n = 6 cells, 3 mice; Ptchd1 KO n = 7 cells, 4 mice), and NAc medium spiny neurons (control n = 12 cells, 4 mice; Ptchd1 KO n = 12 cells, 5 mice) between Ptchd1 KO and control genotypes. For panel b and c, the statistical comparisons were performed by unpaired two-tailed student’s t-test. *p < 0.05, n.s. is p > 0.05. Mean values with S.E.M. errors are shown.

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Extended Data Fig. 9 Electrophysiological characterization of opioid responses in Ptchd1 KO neurons.

a, Representative whole-cell current recordings of Ptchd1 KO and control DA neurons illustrating inhibition of sIPSC frequency by application of 10 μM DAMGO. b, Quantification of both baseline sIPSC frequencies and their inhibition in DA neurons upon sequential applications of increasing concentrations of DAMGO. Control n = 6 cells, 3 mice; Ptchd1 KO n = 7 cells, 4 mice. c, Schematic of recordings from medium spiny neurons (MSN) in the NAc. Activation of MOR on MSNs depresses their excitability. d, Quantification of rheobase from baseline, through two applications of 10 μM morphine and recovery. Control n = 8 cells, 4 mice and 3 cells, 3 mice; Ptchd1 KO n = 6 cells, 4 mice and 3 cells, 3 mice for the first and second application, respectively. e, Quantification of 10 μM morphine responses as the difference in rheobase. f, Quantification of desensitization between first and second morphine responses. n = 3 cells from 3 mice. For panel b, the statistical comparison was performed by 2-way ANOVA with Holm-Šídák post-hoc test. For panel e, the statistical comparison was performed by a mixed-model ANOVA with Holm-Šídák post-hoc test. For panel f, the statistical comparison was performed by an unpaired two-tailed student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mean values with S.E.M. errors are shown.

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Extended Data Fig. 10 Cell biological characterization of Ptchd1 actions.

a, Representative immunofluorescence images examining co-localization of MOR with Na+/K+ ATPase. n = 8 cells from each genotype. b, Quantification of the Ptchd1 effect on co-localization based on Pearson’s correlation coefficient in confocal images of fixed cells. 0x and 2x refer to the amount of DNA transfected, where x = 0.21 µg. n = 12 cells from each genotype. Significance determined using unpaired two tailed Student’s t-test. ***p < 0.001. c, Western blot of HEK293T cells expressing HiBiT-MOR alone or with PTCHD1-flag. d, Schematic of the assay design to study the effect of C. elegans protein PTR-25 on internalization of MOR. Addition of LgBit to cells expressing HiBit-MOR forms a nanoluciferase (nLuc) enzyme. DAMGO treatment causes internalization of the complex and quenching of the luminescence. e, Quantification of DAMGO-induced internalization of HiBiT-MOR in HEK293T cells with or without PTR-25 co-expression. 0, 1x, and 2x refer to the amount of DNA transfected, where x = 0.21 µg. n = 5 biologically independent experiments. Significance determined using one-way ANOVA and Dunnett’s post-hoc. *p < 0.05. f, Schematic of assay design to study the effect of Ptchd1 on internalization of β2-adrenergic receptor (ADRB2). Addition of LgBit to cells expressing HiBit-ADR2B forms a nanoluciferase (nLuc) enzyme. Isoproterenol treatment causes internalization of the complex and quenching of the luminescence. g, Quantification of isoproterenol induced internalization of HiBit-ADRB2 in HEK293T cells with or without PTCHD1 co-expression. 0x, 1x, and 2x refer to the amount of DNA transfected, where x = 0.21 µg. n = 4 biologically independent experiments. Significance determined using one-way ANOVA and Dunnett’s post-hoc. n.s. is p > 0.05. h, Experimental design of β-arrestin recruitment assay to study the effect of Ptchd1 on type 2 vasopressin receptor (VPR2). Addition of arginine vasopressin (AVP) induces recruitment of LgBiT-tagged β-arr2 to VPR2-SmBiT to form a functional nLuc enzyme. i, Time course of Ptchd1 effect on recruitment of β-arr2 to VPR2 determined in n = 5 biologically independent experiments. j, Quantification of the effect of Ptchd1 on the maximum fold-change of β-arr2 recruitment from data in panel i. Significance determined using one-way ANOVA. n.s. is p > 0.05. k, Quantification of the initial rate of β-arr2 recruitment to MOR from data in b. 0x, 1x, and 2x refer to the amount of DNA transfected, where x = 0.21 µg. n = 5 biologically independent experiments. Significance determined using one-way ANOVA and Dunnett’s post-hoc. n.s., p > 0.05. l, Schematic of the assay design of β-arrestin recruitment assay. Addition of DAMGO induces recruitment of LgBiT-tagged β-arr2 to MOR-SmBiT to form a functional nLuc enzyme. m, Modulation of DAMGO mediated- βarr2-LgBiT recruitment to MOR-SmBiT following incubation with 4 mM MBCD or 200 µg / mL cholesterol. n = 7 biologically independent experiments for control and MBCD and n = 6 biologically independent experiments for cholesterol. n, Quantification of the maximum fold change and o, the initial recruitment rate from panel n. Significance was determined using a one-way ANOVA and Dunnett’s post-hoc. ns, p > 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001. In all graphs mean values with S.E.M. errors are shown.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

Evaluation of baseline nociception of WT mice in the hotplate test.

Supplementary Video 2

Evaluation of baseline nociception of Ptchd1 KO mice in the hotplate test.

Supplementary Video 3

Evaluation of morphine (10 mg kg−1) analgesia of WT mice in the hotplate test.

Supplementary Video 4

Evaluation of morphine (10 mg kg−1) analgesia of Ptchd1 KO mice in the hotplate test.

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Maza, N., Wang, D., Kowalski, C. et al. Ptchd1 mediates opioid tolerance via cholesterol-dependent effects on μ-opioid receptor trafficking. Nat Neurosci 25, 1179–1190 (2022). https://doi.org/10.1038/s41593-022-01135-0

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