Neuroscience

Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

Department of Anesthesia, McGill University, Montreal, QC, Canada
Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada
Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC, Canada
Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada
Centre de recherche sur le cerveau et l’apprentissage (CIRCA), Université de Montréal, Montréal, Québec, Canada
Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast, BT9 7AE, UK.
Department of Neuroscience and Center for Advanced Pain Studies, University of Texas at Dallas, Dallas, 75080
Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA
Biomedical Research Institute, Foundation for Research and Technology-Hellas, University Campus, 45110 Ioannina, Greece
Department of Psychology, Faculty of Science, McGill University, Montreal, QC, Canada

https://doi.org/10.7554/eLife.100451.1

Open access
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Figures and data

Transcriptional and translational analysis of gene expression using Ribo-seq.
(A) A schematic diagram showing the spared nerve injury (SNI) assay. S: Sural branch, T: Tibial branch and CP: Common peroneal branch. (B) An illustration of the ribosome profiling technique (Ribo-seq). Scatter plot shows ribosomal footprint (rFP) log₂ fold change (FC) as a function of mRNA log₂ fold change for dorsal root ganglia (DRG, C) and spinal cord (SC, E), at day 4 and day 63 post-SNI. Each dot is a gene. Fold change evaluated between SNI and sham conditions. Color coding indicate modality of differential gene expression control, either at the transcriptional level (mRNA, magenta) or at the translational level (rFP, blue). (D) Number of genes showing changes at mRNA and rFP levels across two independent biological replicates. The rFP/mRNA ratio for each condition is shown above the columns. (F) Pathway analyses of translationally regulated genes in the SC at day 63 post-SNI in the KEGG and Reactome databases.

Targeting spinal translation alleviates pain hypersensitivity at the late stage after peripheral nerve injury.
(A) A schematic showing the regulation of eIF4E via mTORC1/4E-BP1 and MAPKs/MNK pathways. eIF4E ASO (i.c.v) reduces eIF4E protein levels in the spinal cord (B) but not DRG (C) two weeks after administration (n = 4/group). (D) Time course of ASO (eIF4E and control) administration after SNI. The effect of ASO on von Frey (E, n = 9/group) and Mouse Grimace Scale (MGS) (F, n = 9/10 mice per group). (G) Time course of ASO administration before SNI and its effect on the von Frey (H, n = 11/12 mice per group) and MGS (I, n = 11/12 mice per group) tests. An unpaired two-tailed t-test was used in B, C, F, and I. Two-way ANOVA followed by Tukey’s post-hoc comparison was used in E and H. Each data point represents an individual animal. Data are plotted as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns – not significant.

Assessment of protein synthesis using metabolic labeling.
(A) Illustration of protein synthesis assessment using FUNCAT. Anisomycin (100 mg/kg, i.p. injection 1 h before AHA injection) treatment blocked AHA incorporation (n = 3 mice per group), demonstrating the validity of the approach. AHA signal in the superficial spinal cord (laminae I-III) was quantified in inhibitory neurons (Pax2⁺) and excitatory neurons (Pax2^-/NeuN⁺) at day 4 (C, n = 6 mice per group) and day 60 (D, n = 5 mice per group) post-SNI. An unpaired two-tailed t-test was used. Each data point represents an individual animal. Data are plotted as mean ± s.e.m. *p < 0.05, **p < 0.01, ns – not significant.

Cell-type-specific profiling of spinal gene expression after peripheral nerve injury.
(A) A schematic describing the TRAP approach to assess gene expression in specific cell types. Confirmation of specificity of IP fractions to inhibitory neurons in L10a-eGFP; Gad2^Cre mouse line (B) and to excitatory neurons in L10a-eGFP; Tac1^Cre mouse line (C). Dual flashlight plots (left) show the strictly standardized mean difference (SSMD) versus log₂ FC for genes in IP samples and panels on the right show the top 15 upregulated and downregulated genes for inhibitory neurons at day 4 (D) and 60 (E), and Tac1⁺ excitatory neurons at day 4 (F) and 60 (G) post-SNI. Positive Log₂ FC indicates increased expression in SNI compared to sham mice. Parameters for defining data as upregulated in SNI are indicated at the top. (H) The number of altered genes in each condition (GAD2 D60: SNI versus sham day 60 in GAD2⁺ neurons; GAD2 D4: SNI versus sham day 4 in GAD2⁺ neurons; Tac1 D60: SNI versus sham day 60 in Tac1⁺ neurons; and Tac1 D4: SNI versus sham day 4 in Tac1⁺ neurons).

Activation of 4E-BP1-dependent translation in inhibitory neurons promotes plasticity and causes pain hypersensitivity.
(A) A schematic of mTORC1 pathway. Ablation of 4E-BP1 in GAD2 (B, 4E-BP1 cKO: Eif4ebp1^fl/fl;Gad2^Cre, Control: Gad2^Cre, n = 9/10) and PV (C, 4E-BP1 cKO: Eif4ebp1^fl/fl;Pv^Cre, Control: Pv^Cre, n = 8/11) neurons induces mechanical but not heat hypersensitivity. (C) Recording from PV neurons in spinal cord slices (identified by the expression of L10a-eGFP) shows that the ablation of 4E-BP1 in PV neurons (4E-BP1 cKO: Eif4ebp1^fl/fl: L10a-eGFP: Pv^Cre, Control: L10a-eGFP: Pv^Cre, n = 8/8 mice) induces a decrease in firing frequency (D) and an increase in rheobase (E). No change in membrane capacitance (F), resting membrane potential (RMP, G), and input resistance (Rin, H) were found. AAVs (AAV-CAG-DIO-eGFP-eIF4E-shRNAmir or AAV-CAG-DIO-EGFP-scrambled-shRNAmir) were injected into the parenchyma of the dorsal horn of Pv^Cre mice (illustration and time course are shown in I, n = 8/group), preventing the SNI-induced decrease in PV neuron firing frequency (J) and elevation of rheobase (K). No changes were found in capacitance (L), RMP (M), and Rin (N). An unpaired two-tailed t-test was used in B, C, E-H. Two-way ANOVA followed by Tukey’s post-hoc comparison was used in J-N. Each data point represents an individual animal. Data are plotted as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ns – not significant.

Activation of 4E-BP1-dependent translation in excitatory neurons does not induce pain hypersensitivity.
No changes were observed in mechanical (von Frey) and heat (radiant heat paw-withdrawal) thresholds in mice lacking 4E-BP1 in Vglut2 (A, 4E-BP1 cKO: Eif4ebp1^fl/fl;Vglut2^Cre, Control: Vglut2^Cre) and Tac1 neurons (B, 4E-BP1 cKO: Eif4ebp1^fl/fl;Tac1^Cre, Control: Tac1^Cre, n = 7/8 mice). An unpaired two-tailed t-test. Data are plotted as mean ± s.e.m. ns – not significant.

Volcano plots showing changes in mRNA (top), ribosome footprint (rFP, middle), and translational efficiency (TE, bottom) levels in the DRG and SC tissues at day 4 post-SNI (A) and day 63 post-SNI (B). π-values⁵⁹ calculated as log₂(FC) · -log₁₀(P), given an expression fold-change (FC; X axis) and its associated P-value (P; Y axis). Statistical significance at the alpha=0.2 level; decreased (magenta) or increased fold-change (green) expression in SNI versus sham.

Confirmation of 4E-BP1 and eIF4E downregulation.
(A) Lumbar spinal cord tissue from 4E-BP1 cKO GAD2 (Eif4ebp1^fl/fl: L10a-eGFP: Gad2^Cre) and Control (L10a-eGFP: Gad2^Cre) mice was immunostained for 4E-BP1. eGFP expression indicates GAD2⁺ neurons (marked by white arrows). (B) Lumbar spinal cord tissue from 4E-BP1 cKO PV (Eif4ebp1^fl/fl: L10a- eGFP: Pv^Cre) and Control (L10a-eGFP: Pv^Cre) mice were immunostained for 4E-BP1. eGFP expression indicates PV⁺ neurons (marked by white arrows). (C) Lumbar spinal cord tissue from 4E-BP1 cKO Vglut2 (Eif4ebp1^fl/fl: Vglut2^Cre) and Control (Pv^Cre) mice was immunostained for 4E-BP1. Excitatory neurons were identified as NeuN⁺/Pax2^- (white arrows mark inhibitory neurons). (D) Lumbar spinal cord tissue from 4E-BP1 cKO Tac1 (Eif4ebp1^fl/fl: L10a-eGFP: Tac1^Cre) and Control (L10a-eGFP: Tac1^Cre) mice was immunostained for 4E-BP1. eGFP expression indicates Tac1⁺ neurons (marked by white arrows). (E) AAVs (AAV9-CAG-DIO-eGFP-eIF4E-shRNAmir and AAV9-CAG-DIO-eGFP-scrambled) were injected into the lumbar spinal cord of Pv^Cre mice and immunohistochemistry against eIF4E was performed 14 days later. Scale bar is 20 µm in all images. An unpaired two-tailed t-test was used. Each data point represents an individual animal. Data are plotted as mean ± s.e.m. *p < 0.05, **p < 0.01.

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