Adipo-glial signaling mediates metabolic adaptation in peripheral nerve regeneration

Abstract

The peripheral nervous system harbors a remarkable potential to regenerate after acute nerve trauma. Full functional recovery, however, is rare and critically depends on peripheral nerve Schwann cells that orchestrate breakdown and resynthesis of myelin and, at the same time, support axonal regrowth. How Schwann cells meet the high metabolic demand required for nerve repair remains poorly understood. We here report that nerve injury induces adipocyte to glial signaling and identify the adipokine leptin as an upstream regulator of glial metabolic adaptation in regeneration. Signal integration by leptin receptors in Schwann cells ensures efficient peripheral nerve repair by adjusting injury-specific catabolic processes in regenerating nerves, including myelin autophagy and mitochondrial respiration. Our findings propose a model according to which acute nerve injury triggers a therapeutically targetable intercellular crosstalk that modulates glial metabolism to provide sufficient energy for successful nerve repair.

Keywords: Schwann cell; adipocytes; energy metabolism; leptin; leptin receptor; metabolic adaptation; mitochondrial respiration; myelin autophagy; myelinophagy; nerve repair; oxidative phosphorylation; peripheral nerve injury; regeneration; remyelination.

Graphical abstract

Figure 1

Schwann cells upregulate mitochondrial respiration after peripheral nerve injury (A) Experimental strategy for nerve crush surgeries. After unilateral crush surgery at the proximal sciatic nerve, the distal sciatic nerve is collected at indicated time points. At 1 week post nerve crush (1 wpc), the distal nerve is in a peak degenerative phase, whereas axonal regrowth is about to start. At 2 wpc, degeneration is still present, but axonal regrowth has progressed and remyelination is initiated. At 4 wpc, axonal regeneration and remyelination are largely completed. (B) Schematic representation of the genetic strategy to generate Schwann cell-specific mitochondria reporter mice (left). Schwann cell mitochondria are labeled by Dendra2 (green), Schwann cell cytoplasm by S100 (orange), myelin by MBP (magenta), and nuclei by 4',6-diamidino-2-phenylindole (DAPI) (blue). (C) Sciatic nerve cross sections of mice from (B), healthy, 1, 2, and 4 wpc (left, Schwann cell mitochondrial Dendra2 in green, Schwann cell nuclei SOX10 in magenta, and all nuclei DAPI in blue; scale bars, 10 μm). The Dendra2 area (in percent) is normalized to the total area of the sections (left quantification) and to SOX10-postive Schwann cell numbers (right quantification). (D) Western blot analyses and Dendra2 quantification of distal sciatic nerve endoneurial protein lysates from reporter mice from (B) revealed an increase at 1, 2, and 4 wpc. Hexokinase 1 and 2 (HK1 and HK2) show a reduction at the indicated time points. Quantification was performed relative to whole protein stain (WPS), which was identified as the most stable loading control (Figure S1A, one-way ANOVA with Dunnett’s post test). (E) Electron microscopic assessment of Schwann cell mitochondria in distal sciatic nerves at 2 and 4 wpc for mitochondrial size, number (normalized to Schwann cell cytoplasmic area), and space occupancy in Schwann cells. Representative electron micrographs of nerve cross sections are shown on top and the quantifications below. Arrowheads point to mitochondria. Data are derived from Lepr-ctrl mice (see Figure 3A and re-used in Figure 4D [nested one-way ANOVA with post test]). (F) Representative respirometric traces of the oxygen consumption rate (OCR) in sciatic nerve endoneuria from contra- (healthy) and ipsilateral (injured) sites of adult wild-type mice at 2 wpc. Traces represent the oxygen flux per sciatic nerve mass in mg (top panel). Quantification shown in bottom panel for complex I substrates, complex I and II, and maximal electron transfer system (ETS_max) (n = 4 per group, unpaired t tests; M, malate; P, pyruvate; G, glutamate; S, succinate; Cytc, cytochrome c; U, chemical uncoupler/CCCP, carbonyl cyanide m-chlorophenyl hydrazone; Rot, rotenone; Ama, antimycin A).

Figure 2

Leptin signaling coincides with the oxidative shift in injured peripheral nerves (A) Strategy to identify upstream regulators of metabolic adaptation in Schwann cells of injured nerves employing phospho-protein explorer arrays. (B) Identification of regulated pathways in injured nerves as retrieved from (A). Shown is the ranking of the top 20 inversely regulated pathways between ipsi- and contralateral sciatic nerve endoneuria sorted by activation Z score (ingenuity pathway analysis [IPA] comparison analysis). (C) Identified putative upstream regulators of regulated pathways after nerve injury. Ranking of the top six upstream regulators sorted by activation Z score revealed leptin signaling as the strongest candidate. Candidates were determined by IPA upstream analysis based on the comparison analysis from (B) (p value cutoff: p < 10⁻¹⁰). (D) Analyses of mRNA expression of leptin (Lep) and leptin receptor (Lepr) genes in different compartments of the sciatic nerve (epineuria, yellow; perineuria, pink; endoneuria, blue in schematic representation of a sciatic nerve cross section) from adult wild-type mice across different days post crush (n = 3–5 per group, expression normalized to ctrl levels, one-way ANOVA with Dunnett’s post test). (E) Immunohistochemical stainings and quantification of LEPR in tibial nerve cross sections from contra- (healthy) and ipsilateral (injured 1 wpc) sites of adult wild-type mice. Plasma membranes (wheat germ agglutinin [WGA], green), LEPR (magenta), and cell nuclei (DAPI, blue) are shown in the representative images (scale bars, 10 μm; n = 3 per group, Student’s t test). (F) BaseScope in situ hybridization for Lepr mRNA encoding the long isoform of the leptin receptor, Ob-Rb (magenta), on a tibial nerve cross section at 1 wpc. Schwann cell cytoplasm (S100, green) and cell nuclei (DAPI, blue; scale bars, 5 μm). (G) Schematic representation of the genetic strategy and experimental plan for Schwann cell-specific cJun conditional knockout mice (cJun-cKO). (H) Lepr mRNA expression at 1 wpc in healthy and injured tibial nerves of ctrl and cJun-cKO mice (standardized to Ankrd27 and Canx, one-way ANOVA with Tukey’s post hoc test). (I) Immunohistochemistry in healthy and injured (1 wpc) nerves of control and cJun-cKO mice against SOX10 (green) and cJUN (magenta, top row) and against SOX10 (green) and LEPR (magenta, bottom row, one-way ANOVA with Tukey’s post hoc test; scale bars, 10 μm).

Figure 3

Ablation of Lepr from Schwann cells impairs regeneration after acute nerve injury (A) Schematic representation of the genetic strategy and experimental plan for conditional Lepr knockout mice. (B) Immunohistochemical stainings of tibial nerve cross sections (upper left panels) of contra- (healthy) and ipsilateral (4 wpc) sites from adult control (Lepr-ctrl) and Schwann cell Lepr knockout mice (Lepr-cKO). Plasma membranes (WGA, green), leptin receptor protein (LEPR, magenta), and cell nuclei (DAPI, blue) are depicted (scale bars, 2 μm). Cumulative fluorescence intensities of WGA and LEPR stainings (lower left panels) were assessed across the cell membrane (bottom). Quantification (right, group mean with SD; mean per animal [large circles] and individual data points [small circles] are shown) was performed by determining peak-to-peak distances between WGA and LEPR fluorescent signals (n = 3 per group, nested one-way nested ANOVA with Tukey’s post hoc test). (C) Impaired functional recovery of Lepr-cKO mice after injury as revealed by DigiGait walking analysis and calculation of the sciatic nerve functional index (SFI) of adult control (Lepr-ctrl, magenta) and Schwann cell Lepr knockout mice (Lepr-cKO, green, n = 7 per group, two-way ANOVA). (D) Electrophysiological recordings of healthy and injured (4 wpc) Lepr-cKO (green) mice compared with controls (magenta) after nerve injury. Representative electroneurographic traces (left) and calculation of the nerve conduction velocity (NCV) and compound muscle action potential (CMAP, as area under the curve, right panels) are shown (n = 6–7 per group, one-way ANOVA with Tukey’s post hoc test). (E) Representative electron micrographs (left; scale bars, 10 μm) of ultra-thin tibial nerve cross sections from adult control (Lepr-ctrl) and Schwann cell Lepr knockout (Lepr-cKO) mice before (healthy) and 2 and 4 weeks after crush (2 and 4 wpc). Quantifications (right) of the number of amyelinated and myelinated axons, macrophages, and Schwann cells that contain myelin degeneration profiles was performed and expressed per cross sectional area (21.536 μm², n = 4–7 per group, one-way ANOVA with Tukey’s post hoc test). (F) Analysis of myelin sheath thickness by calculation of the g-ratio from data in (E) (n = 4–7 per group, one-way ANOVA with Sidak’s post hoc test). (G) Western blot analysis (left) and quantification (right) in sciatic nerve endoneurium lysates of contra- (healthy) and ipsilateral sites from control (Lepr-ctrl, magenta) and Schwann cell Lepr knockout (Lepr-cKO, green) mice at 2 wpc. The ratio of LC3b-II over LC3b-I was calculated, for the p62 protein abundance was normalized to WPS as loading control, n = 3–4 per group, one-way ANOVA with Tukey’s post hoc test.

Figure 4

Leptin receptor signaling induces mitochondrial respiration in remyelinating Schwann cells (A) Bulk RNA sequencing of healthy and injured nerves from control (ctrl) and Lepr-cKO (cKO) mice with heat map of differentially expressed genes (left). Three patterns were derived from differential gene expression analysis at 2 and 4 wpc. First, genes up in ctrl but downregulated in cKO at 2 and 4 wpc (pattern 1); second, genes downregulated in cKO at 2 wpc and upregulated at 4 wpc (pattern 2); and third, genes upregulated in cKO at 2 but downregulated at 4 wpc (pattern 3). Top 30 dysregulated processes from pattern 1 (right) were ranked according to the normalized enrichment score (NES) using WebGestaltR (version 0.4.4) for Reactome pathways (n = 4 per group). (B) Temporal expression patterns between Lepr-ctrl (magenta) and Lepr-cKO (green) are shown. Solid lines connect the average expression of individual genes associated with the mitochondrial gene set for oxidative phosphorylation (OxPhos, as extracted from MitoCarta). (C) Western blot analyses (left) and quantifications (right) of oxidative phosphorylation protein complexes I–V (OxPhos I–V) protein abundances in sciatic nerve endoneurium lysates of contra- (healthy) and ipsilateral (4 wpc) sites from adult control (ctrl) and Schwann cell Lepr knockout (cKO) mice (n = 3–4 per group, one-way ANOVA with Tukey’s post hoc test; WPS used as loading control). (D) Representative electron micrographs of tibial nerve cross sections from contra- (healthy) and ipsilateral (2 and 4 wpc) sites of adult control (Lepr-ctrl, CT) and Schwann cell Lepr knockout (Lepr-cKO, KO) mice at 2 and 4 wpc (top; scale bars, 1 μm). Mitochondria per area, mitochondrial size, and the relative occupancy of mitochondria per Schwann cell cytoplasmic area were quantified (bottom). Data of control samples (Lepr-ctrl) were re-used in Figure 1E (n = 3–5 per group; mean ± SD per group; individual means per animal [large circles] and mitochondrial means per Schwann cell [small circles]; nested one-way ANOVA with Tukey’s post hoc test). (E) Representative respirometric traces of the oxygen consumption rate (OCR) in sciatic nerve endoneuria from healthy and injured (4 wpc) sites of control and Lepr-cKO mice at 4 wpc. Traces represent the oxygen flux per sciatic nerve mass in mg (top). Quantification shown in bottom panel for complex I substrates, complex I and II, and maximal electron transfer system (ETS_max) (n = 5 per group, one-way ANOVA and Holm-Šidák multiple comparisons; M, malate; P, pyruvate; G, glutamate; S, succinate; Cytc, cytochrome c; U, chemical uncoupler/CCCP, carbonyl cyanide m-chlorophenyl hydrazone; Rot, rotenone; Ama, antimycin A).

Figure 5

Leptin promotes autophagy and mitochondrial respiration ex vivo (A) Schematic presentation of the experimental setup to investigate the impact of leptin on nerve metabolism in ex vivo sciatic nerve cultures. (B) Histological assessment of nerve explant semi-thin cross sections after 6 days ex vivo (6 div) and quantification of the ratio of degenerative myelin profiles (ovoids) over intact myelin structures in leptin-treated (green) compared with nontreated (gray) nerves. Nerve explants treated with 3-MA (red) displayed almost no myelin degeneration. Representative semi-thin images are shown on the left (scale bars, 10 μm), quantification on the right (n = 5–6 nerves per group, one-way ANOVA with Tukey’s post test). (C) Western blot analysis of LC3B autophagic flux in nerve explants upon leptin treatment in WT (left) and Lepr-cKO nerves (middle). Nerve explants were either left nontreated or treated with recombinant leptin or lysosomal blocker NH₄Cl or both recombinant leptin and NH₄Cl for 6 days. Quantifications are shown on the right (data expressed as fold change, n = 3 per group, Student’s t test). (D–F) Seahorse respirometry and measurement of the oxygen consumption rate (OCR) in WT (D and F) or Lepr-cKO (E) nerve explants after 6 days ex vivo (6 div). Explants were left untreated (black, D and E), treated with leptin (green, D–F), treated with 3-MA (red, D), treated with both leptin and 3-MA (orange, D), treated with the fatty acid oxidation inhibitor etomoxir (ETO, red, F), or treated with both leptin and etomoxir (orange, F). OCR traces are depicted on top, and quantification of the basal and maximal OCR is shown at the bottom (n = 4–5 per group, one-way ANOVA with Tukey’s post test).

Figure 6

Adipocyte-derived leptin is required for efficient nerve repair (A) BaseScope in situ hybridization is shown with a Lep probe (magenta) on a tibial nerve cross section from an adult wild-type mouse 1 wpc. Schwann cells (S100) and cell nuclei (DAPI) are also depicted. Blow ups show leptin mRNA in a single Schwann cell (top) and an epineurial adipocyte (bottom, identified by morphology by autofluorescence in the S100 channel; scale bars, 5 μm). (B) ELISA quantification of total leptin protein in adult healthy and crushed wild-type mice in paraneural white adipose tissue (WAT) at 1, 2, and 4 wpc (n = 4–5, one-way ANOVA with Sidak’s post test). (C) Quantification of the paraneural fat mass after nerve injury at 1, 2, and 4 wpc. Quantification is shown as ratio of ipsi- versus contralateral fat mass per animal and individual data points display ratios per animal (n = 9–13 per time point, Wilcoxon matched-pairs signed rank test for each time point). (D) Schematic representation of the genetic strategy used for the generation and use of conditional adipocyte reporter mice (left). Representative immunohistochemical staining of a longitudinal sciatic nerve section from a recombined adult adipocyte reporter mouse 4 wpc depicts reporter-positive epineurial adipocytes (TdTomato, magenta) and reporter-free endoneurial myelin (MBP, green) and cell nuclei (DAPI, blue, right). (E) Schematic representation of the genetic strategy used for the generation and use of inducible conditional adipocyte Lep knockout mice. (F) Confirmation of recombination efficiency in paraneural WAT of Lep-AcKO mice at 4 weeks post nerve crush (4 wpc) by qPCR and measurement of relative Lep mRNA expression (n = 7–10, Student’s t test). (G) ELISA for leptin in endoneuria in Lep-ctrl (gray) and Lep-AcKO (rose) at 4 wpc (n = 7–8 per group, Student’s t test). (H) DigiGait walking analysis and calculation of the sciatic nerve functional index (SFI) of the injured side at different time points post sciatic nerve crush in Lep-ctrl (gray) and Lep-AcKO (rose) mice (n = 7 per group, two-way ANOVA). (I) Representative electrophysiological traces (left) and quantification of the compound muscle action potentials (CMAPs) and nerve conduction velocities (NCVs) of contra- (healthy) and ipsilateral (4 wpc) sites from Lep-ctrl and Lep-AcKO mice at 4 wpc (n = 12–16 per group, one-way ANOVA with Tukey’s post hoc test). (J) Representative electron micrographs (left panels; scale bars, 10 μm) of tibial nerve cross sections from Lep-ctrl and Lep-AcKO mice, healthy and 2 and 4 wpc. Quantifications (right) of the number of amyelinated and myelinated axons, macrophages, and Schwann cells that contain degeneration profiles was performed and normalized to cross sectional area (21.536 μm², n = 4–5 per group, one-way ANOVA with Tukey’s post hoc test). (K) Analysis of myelin sheath thickness by calculation of the g-ratio in data from (J). Scatterplots (right) depict g-ratio data points of individual fibers plotted against the respective axon diameter (n = 3 per group, Student’s t test). (L) Representative respirometric traces of the oxygen consumption rate (OCR) in sciatic nerve endoneuria from healthy and injured (4 wpc) sites of control and Lep-AcKO mice at 4 wpc. Traces represent the oxygen flux per sciatic nerve mass in mg (left). Quantification shown in the right panel for maximal electron transfer system (ETS_max) (n = 5 per group, one-way ANOVA and Holm-Šidák multiple-comparisons; M, malate; P, pyruvate; G, glutamate; S, succinate; Cytc, cytochrome c; U, chemical uncoupler/CCCP, carbonyl cyanide m-chlorophenyl hydrazone; Rot, rotenone; Ama, antimycin A).

Figure 7

Leptin therapy supports regeneration after peripheral nerve injury (A) Therapeutic leptin treatment regime scheme after acute nerve injury. (B) ELISA for leptin in blood serum (left) and endoneuria (right) of mice receiving continuous leptin treatment (lep/lep) compared with control treatment (veh/veh) at 4 wpc (n = 6–12, Student’s t test). (C) DigiGait walking analysis and calculation of the sciatic nerve functional index (SFI) at different time points post sciatic nerve crush. Baseline was recorded 2 days before sciatic nerve crush (n = 9–10 per group, two-way ANOVA). (D) Electrophysiological measurement of the nerve conduction velocities (NCVs, left) and compound muscle action potentials (CMAPs, right) from ipsilateral sites at 4 wpc (n = 9–10 per group, one-way ANOVA with Tukey’s post hoc test). (E) Histological quantification of the number of remyelinated fibers per tibial nerve cross sections on the semi-thin level at 4 wpc (left; n = 9–10 per group, one-way ANOVA with Tukey’s post hoc test). (F) Schematic model of adipo-glial-mediated catabolism after nerve injury. Schwann cells respond to nerve injury with cJUN-dependent leptin receptor expression (1) and membrane presentation (2). Integration of circulating adipocyte-derived leptin (3) mediates oxidative phosphorylation (4) and myelinophagy (5) to support nerve repair (6).