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A glycolytic shift in Schwann cells supports injured axons

Nature Neuroscience volume 23, pages 1215–1228 (2020)Cite this article

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A Publisher Correction to this article was published on 16 October 2020

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Abstract

Axon degeneration is a hallmark of many neurodegenerative disorders. The current assumption is that the decision of injured axons to degenerate is cell-autonomously regulated. Here we show that Schwann cells (SCs), the glia of the peripheral nervous system, protect injured axons by virtue of a dramatic glycolytic upregulation that arises in SCs as an inherent adaptation to axon injury. This glycolytic response, paired with enhanced axon–glia metabolic coupling, supports the survival of axons. The glycolytic shift in SCs is largely driven by the metabolic signaling hub, mammalian target of rapamycin complex 1, and the downstream transcription factors hypoxia-inducible factor 1-alpha and c-Myc, which together promote glycolytic gene expression. The manipulation of glial glycolytic activity through this pathway enabled us to accelerate or delay the degeneration of perturbed axons in acute and subacute rodent axon degeneration models. Thus, we demonstrate a non-cell-autonomous metabolic mechanism that controls the fate of injured axons.

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Fig. 1: SCs stabilize injured axons.
Fig. 2: Glycolytic upregulation in SCs on axonal injury.
Fig. 3: Enhanced glycolytic flux and lactate extrusion in injury-activated SCs.
Fig. 4: Glycolytic SCs are metabolically coupled to injured axons and antagonize axonal degeneration.
Fig. 5: mTOR inactivation in SCs results in accelerated axonal degeneration.
Fig. 6: mTOR in SCs promotes the SC glycolytic shift on injury.
Fig. 7: The mTORC1–Hif1α/c-Myc axis in SCs protects injured axons.
Fig. 8: Axonal protection through mTORC1 hyperactivity in SCs.

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Source data are provided with this paper. Additional source data underlying Figs. 1–8 and the results presented in the extended data and supplementary figures are available from the corresponding author upon reasonable request.

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PhosphoSitePlus v.6.5.9.2 powered by Cell Signaling Technology is available at https://www.phosphosite.org.

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Acknowledgements

We thank R. Loren, T. Bierschenk, the animal husbandry staff of the Laboratory Animal Shared Resources at the Roswell Park Comprehensive Cancer Center for technical assistance with mouse colony maintenance, C. Deppmann for help with microfluidic devices, L. Feltri for help with rat SC cultures, S. James for assistance with digital droplet PCR analysis, E.D. Abel for the conditional GLUT1 mice, F. Alt for the conditional c-Myc mice, M. Gambello for the conditional Tsc2 mice, V. Pachnis for the iSox10Cre mice and the Michigan Regional Comprehensive Metabolomics Resource Core for help with the metabolomics analysis. This study was supported by Muscular Dystrophy Association grant nos. 577844 (to B.B.) and 292306 (to E.B.), GBS/CIDP Foundation International grant no 81463 (to E.B.), and start-up funding for B.B. provided through Empire State Development Corporation for Hunter James Kelly Research Institute grant nos. W753 and U446 and the Hunter’s Hope Foundation. This work was also supported by a National Institutes of Health/National Cancer Institute grant no. P30CA016056.

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Author notes

  1. Keit Men Wong

    Present address: Department of Pediatrics and Adolescent Medicine, Division of Pediatric Neurology, University Medical Center Göttingen, Göttingen, Germany

Authors and Affiliations

  1. Hunter James Kelly Research Institute, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA

    Elisabetta Babetto, Keit Men Wong & Bogdan Beirowski

  2. Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA

    Elisabetta Babetto

  3. Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA

    Bogdan Beirowski

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Contributions

B.B. conceived and supervised the study. B.B., E.B. and K.M.W. designed and planned the experiments. E.B. performed the experiments and analyzed and interpreted the data for Figs. 1, 4b,f,g, 5a and 8c, and Extended Data Fig. 5 and Supplementary Fig. 1. B.B. and K.M.W. performed the experiments and analyzed and interpreted the data for Figs. 2, 3, 4a,c–e, 5b–j, 6, 7 and 8a,b,d–i, and Extended Data Figs. 1–4, 6–10 and Supplementary Figs. 2–5. B.B. and E.B. supervised K.M.W. B.B. and E.B. prepared the figures. B.B. and E.B. wrote the manuscript.

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Correspondence to Bogdan Beirowski.

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Extended data

Extended Data Fig. 1 Glycolytic and fermentative enzyme expression in SCs after nerve injury.

a–i, Representative immunofluorescence for the indicated metabolic components on longitudinal frozen sections from uninjured control nerve segments and axotomized distal sciatic nerve stumps at the shown post-injury times. HK2: Hexokinase 2 (a). GPI: Glucose-6-phosphate isomerase (b). ALDA: Aldolase A (c). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase (d). PGK1: Phosphoglycerate kinase 1 (e). PGAM1: Phosphoglycerate mutase 1 (f). ENO1: Enolase 1 (g). PKM1: Pyruvate kinase M1 (h). LDHB: Lactate dehydrogenase B (i). Arrows depict colocalization in SCs. Scale bars: 50μm. The experiments for each component were reproduced three times independently with similar results.

Extended Data Fig. 2 Enzymes driving mitochondrial glucose catabolism in SCs are not upregulated upon nerve lesion.

a, b, Representative immunofluorescence using wide-field fluorescence microscopy (a) and confocal microscopy (b, merged z-projections) for the indicated mitochondrial enzymes on longitudinal frozen sections from uninjured control nerve segments and axotomized distal sciatic nerve stumps at the shown post-injury times. PDH: Pyruvate dehydrogenase complex. CS: Citrate synthase. IDH/IDH3a: Isocitrate dehydrogenase catalytic subunit α. α-KGDH/OGDH: Alpha-ketoglutarate dehydrogenase also known as 2-oxoglutarate dehydrogenase E1 component, mitochondrial. Note prominent CS, IDH, and α-KGDH axonal staining in uninjured control nerves, in addition to the SC-derived signals. The axonal staining weakens in injured preparations due to axonal degeneration. Red: respective mitochondrial enzyme. Green: PLP-EGFP. Blue: DAPI. Scale bars: 50μm (a), 100μm (b). The experiments for each mitochondrial enzyme were reproduced three times independently with similar results. c, d, Left: Representative images of longitudinal sections from uninjured control nerve segments and axotomized distal sciatic nerve stumps stained for the activity of isocitrate dehydrogenase (IDH, c) and succinate dehydrogenase (SDH, d) (formazan formation) with superimposition of DAPI signals (cyan). Scale bars: 50μm. Right: Densitometric quantification of formazan intensity representing IDH or SDH enzyme activity, respectively, on nerve sections. Note markedly reduced IDH and SDH enzyme activities following nerve injury, in contrast to increased glycolytic enzyme activities (Error bars represent s.e.m. n = 3 mice for each graph). Statistical evaluation in c and d was performed using Student’s t-test, unpaired, two-tailed.

Source data

Extended Data Fig. 3 Nerve lactate metabolism following nerve injury.

a, Model for axonal consumption of lactate released by SCs in the nerve stump distal to site of axotomy. b, Lactate concentrations in lysates of uninjured control nerve segments and distal sciatic nerve stumps following axotomy (Error bars represent s.e.m. n = 3 mice per condition for 6, 12, 24, 36, and 72 h after axotomy, n = 4 mice per condition for 48 h after axotomy, *P = 0.0097, **P = 0.0446, ***P = 0.0057). c, Left: Scheme for measuring extracellular monocarboxylate release of uninjured and injured peripheral nerve segments by extracellular flux analysis in Seahorse islet capture microplates. Right: ECAR traces of control uninjured and axotomized nerve segments at the indicated post-injury times (Error bars represent s.e.m. n = 3 mice for uninjured nerve segments and n = 4 mice for injured for 24 h after axotomy, n = 7 mice for uninjured nerve segments and n = 8 mice for injured for 48 h after axotomy, n = 9 mice for uninjured and injured nerve segments for 72 h after axotomy). d, Relative glucose injection–induced maximum ECARs in uninjured control and axotomized nerve segments reflecting extracellular concentrations of glucose-derived monocarboxylates. Note extracellular accumulation of glucose-derived monocarboxylates as axonal degeneration proceeds. (Error bars represent s.e.m. n = 3 mice for uninjured nerve segments and n = 4 mice for injured for 24 h after axotomy, n = 7 mice for uninjured nerve segments and n = 8 mice for injured for 48 h after axotomy, n = 9 mice for uninjured and injured nerve segments for 72 h after axotomy). Statistical evaluation in b and d was performed using multiple Student’s t-tests, unpaired, two-tailed.

Source data

Extended Data Fig. 4 Conditional mutant mice lacking key glucose metabolism regulators in SCs show overtly normal nerve structure.

a–c, Western blot analysis (cropped blot images) of sciatic nerve lysates from indicated 8-weeks-old control and mutant mice with the indicated genotypes probed with the shown antibodies (Error bars represent s.e.m. n = 3 mice per genotype for each graph. Each dot represents measurement from sciatic nerve lysate from one mouse). d–f, Representative electron micrographs of transverse sciatic nerve sections from 8-weeks-old control and mutant mice with the indicated genotypes. Scale bars: 2μm. g–i, Quantification of myelinated axons in sciatic nerves from indicated 8-weeks-old control and mutant mice (Error bars represent s.e.m. n = 4 mice per genotype for each graph). j–l, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from indicated 8-weeks-old control and mutant mice (Error bars represent s.e.m. n = 4 mice per genotype for j and n = 3 mice per genotype for k, l). m, Representative immunofluorescence for the indicated glycolytic regulators on longitudinal frozen sections from control uninjured nerves and axotomized distal sciatic nerve stumps of mice with the indicated genotypes 36 h after nerve transection injury (blue: DAPI). Scale bars: 50μm. The experiments for each glycolytic regulator were reproduced three times independently with similar results. Statistical evaluation in a–c and g–l was performed using Student’s t-test, unpaired, two-tailed.

Source data

Extended Data Fig. 5 Additional analysis of MCT inhibitors in co-cultures.

a, b, Representative immunofluorescence (confocal (a) and wide-field fluorescence (b)) microscopy of control uninjured axons under the indicated conditions. Note normal appearance of axon structure after 24 h of treatment with the indicated MCT inhibitors. Scale bars: 50μm. The experiments for each condition were reproduced three times independently with similar results. c, Box and whiskers plots (maximum, 25th percentile, median, 75th percentile, minimum) of axon survival 24 h following axotomy under the indicated conditions (BAY-8002: β-III-tub and NF-H, Neurons: n = 37 DRG neurite preparations, Neurons+SCs DMSO: n = 44 DRG neurite preparations, Neurons+SCs BAY-8002: n = 38 DRG neurite preparations, all DRG neurite preparations from four experimental sets performed on different days. UK-5099: β-III-tub, Neurons: n = 53 DRG neurite preparations, Neurons+SCs DMSO: n = 57 DRG neurite preparations, Neurons+SCs UK-5099: n = 38 DRG neurite preparations, all DRG neurite preparations from five experimental sets performed on different days (four experimental sets performed on different days for Neurons+SCs UK-5099). NF-H, Neurons: n = 53 DRG neurite preparations, Neurons+SCs DMSO: n = 49 DRG neurite preparations, Neurons+SCs UK-5099: n = 36 DRG neurite preparations, all DRG neurite preparations from five experimental sets performed on different days (four experimental sets performed on different days for Neurons+SCs UK-5099)). Statistical evaluation was performed using One-way-ANOVA and Sidak’s multiple comparisons tests.

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Extended Data Fig. 6 Upregulation of AMPK activity in injury-activated SCs, and analysis of nerves from conditional mutant mice lacking AMPK or mTOR activity in SCs.

a, Western blot analysis (cropped blot images) of lysates from uninjured control nerve segments and axotomized distal sciatic nerve stumps from C57Bl/6 J mice showing AMPK activity at different times following nerve transection. Note marked AMPK activation as reflected by increased p-AMPKα phosphorylation at Thr172 already 10 min after nerve injury. Individual lanes represent pooled data from at least three mice. b, Representative immunofluorescence for p-AMPKα (Thr172) on longitudinal frozen section from axotomized distal sciatic nerve stump 12 h after nerve injury. Arrows depict colocalization in SCs. Scale bar: 20μm. The experiment was reproduced three times independently with similar results. c, f, Quantification of myelinated axons in sciatic nerves from indicated 8-weeks-old control and mutant mice (Error bars represent s.e.m. n = 4 mice per genotype for each graph). d, g, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from indicated 8-weeks-old control and mutant mice (Error bars represent s.e.m. n = 4 mice per genotype for d and n = 3 mice per genotype for g). e, Quantitative analysis of relative axon survival in distal sciatic nerve stumps 36 h after axotomy in mice with the indicated genotypes (Error bars represent s.e.m. n = 4 mice per genotype). h, Quantification of SC nuclei in sciatic nerve cross sections in 8-weeks-old mice with the indicated genotypes (Error bars represent s.e.m. n = 4 mice per genotype). i, j, Representative semithin and electron micrographs (last panel with pseudocoloring) of transverse sciatic nerve sections of distal nerve stumps from mice with the indicated genotypes at different time points after sciatic nerve transection (i) with corresponding quantifications of relative axon survival (j). Electron micrographs show pseudocoloring of intact (turquoise) and degenerated (magenta) myelinated fibers. Note accelerated axonal degeneration in the mTORfl/fl; P0Cre mutants (Error bars represent s.e.m. n = 3 mice for mTORfl/fl for 0 and 36 h after axotomy, n = 4 mice for mTORfl/fl for 24 and 48 h after axotomy, n = 3 mice for mTORfl/fl; P0Cre for 0 h after axotomy, n = 4 mice for mTORfl/fl; P0Cre for 24 and 36 h after axotomy, n = 6 mice for mTORfl/fl; P0Cre for 48 h after axotomy, *P = 0.003, ** P < 0.0001). Scale bars: 10μm. Statistical evaluation in c–h was performed using Student’s t-test, unpaired, two-tailed, and in j using multiple Student’s t-tests, unpaired, two-tailed.

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Extended Data Fig. 7 Analysis of nerves from mutant mice lacking key mTOR components in SCs.

a, Western blot mTORC1 activity (reflected by S6 phosphorylation at Ser240/244) analysis of sciatic nerve lysates (cropped blot images) from control and mTORfl/fl; iSox10Cre mice (30 days after last tamoxifen administration) probed with the indicated antibodies (Error bars represent s.e.m. n = 3 mice per genotype. Each dot represents measurement from sciatic nerve lysate from one mouse). b, Representative semithin micrographs of transverse sciatic nerve sections from 12-weeks-old control and mTORfl/fl; iSox10Cre mice 30 days following last tamoxifen administration. Note indistinguishable nerve structure between control and mutant mice. Scale bar: 50μm. c, Quantification of SC nuclei in sciatic nerve cross sections from mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 5 mice for mTORfl/fl and n = 7 mice for mTORfl/fl; iSox10Cre). d, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 3 mice per genotype). e, f, Western blot analysis (cropped blot images) of sciatic nerve lysates from indicated control and mutant mice (age 5 days in e and 8 weeks in f) probed with the shown antibodies (Error bars represent s.e.m. n = 3 mice per genotype in e and n = 4 mice per genotype in f. Each dot represents measurement from sciatic nerve lysate from one mouse). g, Quantification of myelinated axons in sciatic nerves from indicated 8-weeks-old control and mutant mice (Error bars represent s.e.m. n = 4 mice per genotype for each graph). h, Representative immunofluorescence using the indicated markers on longitudinal frozen sections of distal sciatic nerve stumps from control and Rictorfl/fl; P0Cre mutant mice 36 h after nerve transection injury. Note normal induction of mTORC1 activity in SCs of Rictor-deficient mice as reflected by indistinguishable p-S6 (Ser240/244) immunoreactivity. Scale bar: 50μm. The experiment was reproduced three times independently with similar results. Statistical evaluation in a, c–g was performed using Student’s t-test, unpaired, two-tailed.

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Extended Data Fig. 8 Nerves from mutant mice with depletion of c-Myc and/or Hif1α in SCs show no abnormalities of myelinated axons.

a, Representative immunofluorescence using the indicated antibodies on longitudinal sections of axotomized sciatic nerve stumps from control and c-Mycfl/fl; iSox10Cre mutant mice (30 days after last tamoxifen administration) 36 h after nerve transection injury. Note largely abolished induction of c-Myc expression in SCs (S100 + ) of c-Mycfl/fl; iSox10Cre mice. Scale bar: 50μm. The experiment was reproduced three times independently with similar results. b, Quantification of myelinated axons in sciatic nerves from 12-weeks-old mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 4 mice per genotype). c, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from 12-weeks-old mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 3 mice per genotype). d, Western blot analysis (cropped blot images) of sciatic nerve lysates from 8-weeks-old mice with the indicated genotypes probed with the shown antibodies (Error bars represent s.e.m. n = 3 mice per genotype for each graph. Each dot represents measurement from sciatic nerve lysate from one mouse). e, Representative immunofluorescence using the indicated markers on longitudinal frozen sections of axotomized sciatic nerve stumps from control and Hif1αfl/fl; P0Cre mutant mice 36 h after nerve transection injury. Note largely abolished induction of Hif1α expression in Hif1αfl/fl; P0Cre mice. Scale bar: 50μm. The experiment was reproduced three times independently with similar results. f, Representative electron micrographs of transverse sciatic nerve sections from 8-weeks-old control and Hif1αfl/fl; P0Cre mice. Note indistinguishable nerve ultrastructure between control and mutant mice. Scale bar: 2μm. g, Quantification of myelinated axons in sciatic nerves from 8-weeks-old mice with the indicated genotypes (Error bars represent s.e.m. n = 4 mice per genotype for each graph). h, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from 8-weeks-old mice with the indicated genotypes (Error bars represent s.e.m. n = 3 mice per genotype). i, Representative semithin (left) and electron micrographs (right) of transverse sciatic nerve sections from distal nerve stumps of mice with the indicated genotypes 36 h after sciatic nerve transection with pseudocoloring of intact (turquoise) and degenerated (magenta) myelinated fibers. Scale bars: 10μm. The experiment was reproduced three times independently with similar results. j, Representative electron micrographs of transverse sciatic nerve sections from 12-weeks-old control and Hif1αfl/fl; c-Myc fl/fl; iSox10Cre mice 30 days following the last tamoxifen administration. Note indistinguishable nerve ultrastructure between control and mutant mice. Scale bar: 2μm. k, Quantification of myelinated axons in sciatic nerves from 12-weeks-old mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 4 mice per genotype). l, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from 12-weeks-old mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 3 mice per genotype). Statistical evaluation in b–d, g, h, k, l was performed using Student’s t-test, unpaired, two-tailed.

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Extended Data Fig. 9 Nerves from mutant mice with depletion of TSC2 in SCs show no abnormalities of myelinated axons.

a, Representative semithin (left) and electron micrographs (right) of transverse sciatic nerve sections from 12-weeks-old control and TSC2fl/fl; iSox10Cre mice 30 days following tamoxifen administration. Scale bars: 10μm. b, Quantification of myelinated axons in sciatic nerves from mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 4 mice per genotype). c, Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in sciatic nerves from mice with the indicated genotypes 30 days following tamoxifen administration (Error bars represent s.e.m. n = 3 mice per genotype). Statistical evaluation in b and c was performed using Student’s t-test, unpaired, two-tailed.

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Extended Data Fig. 10 Additional behavioral, electrophysiological, and structural analysis of ACR-treated mice.

a, Behavioral analysis of C57Bl/6 J mice over the course of ACR intoxication (or control treatment) for 14 days. Note accentuated deterioration of motor performance (rotarod, hanging wire, grip strength) relative to sensory performance (tail flick, hot and cold plate) (Error bars represent s.e.m. n = 8 mice per group and time point, except n = 7 mice for control treatment 0 days tail flick, *P = 0.0024, **P < 0.0001 for rotarod, *P < 0.0001 for hanging wire, *P = 0.0049, **P = 0.0130, ***P < 0.0001 for grip strength). b, Weight analysis of C57Bl/6 J mice over the course of ACR intoxication (or control treatment) for 14 days (Error bars represent s.e.m. n = 5 female and n = 3 male control mice, n = 4 female and male ACR-treated mice, *P = 0.0299, **P = 0.0346). c, Analysis of CMAP amplitudes recorded in gastrocnemius muscles evoked after sciatic nerve stimulation of C57Bl/6 J mice following 14d of ACR treatment, or control treatment (Error bars represent s.e.m. n = 8 mice per group). d, Representative electron micrographs of transverse tibial nerve sections from C57Bl/6 J mice following 14d of ACR admininistration (or control treatment), with pseudocoloring of intact (turquoise) and degenerated (magenta) myelinated fiber profiles. Degenerated profiles appeared as collapsed myelinated axons with little axoplasm or axons with segregation of the axoplasm by segments of the myelin sheath (d1), deranged fibers with axoplasm constriction due to myelin infoldings and convolution (d2), and myelinated fibers with accumulation of membrane like material, multivesicular structures, and dense bodies in the axoplasm (d3). Scale bars: 2μm. e, Densities of degenerated axon profiles in tibial nerves from C57Bl/6 J mice following control treatment or 14d of ACR admininistration (Error bars represent s.e.m. n = 8 mice per group). f, Representative electron micrographs of transverse tibial nerve sections from C57Bl/6 J mice following control treatment or 14d of ACR admininistration show normal ultrastructure of unmyelinated axons in Remak bundles (‘N’ depicts nuclei of SCs forming Remak bundles). Scale bar: 2μm. The experiment was reproduced three times independently with similar results. g, Weight analysis of ACR-treated control TSC2fl/fl and mutant TSC2fl/fl; iSox10Cre mice (Error bars represent s.e.m. n = 4 female and n = 7 male mice (except n = 6 male mice for 14 days treatment time point) with genotype TSC2fl/fl, n = 8 female and n = 6 male mice with genotype TSC2fl/fl; iSox10Cre). Statistical evaluation in a, b, and g was performed using multiple Student’s t-tests, unpaired, two-tailed, and in c and e using Student’s t-test, unpaired, two-tailed.

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Supplementary Table

Supplementary Table 1. List of antibodies with validation details

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Babetto, E., Wong, K.M. & Beirowski, B. A glycolytic shift in Schwann cells supports injured axons. Nat Neurosci 23, 1215–1228 (2020). https://doi.org/10.1038/s41593-020-0689-4

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