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Metabolic regulator LKB1 is crucial for Schwann cell–mediated axon maintenance

Abstract

Schwann cells (SCs) promote axonal integrity independently of myelination by poorly understood mechanisms. Current models suggest that SC metabolism is critical for this support function and that SC metabolic deficits may lead to axonal demise. The LKB1–AMP-activated protein kinase (AMPK) kinase pathway targets several downstream effectors, including mammalian target of rapamycin (mTOR), and is a key metabolic regulator implicated in metabolic diseases. We found through molecular, structural and behavioral characterization of SC-specific mutant mice that LKB1 activity is central to axon stability, whereas AMPK and mTOR in SCs are largely dispensable. The degeneration of axons in LKB1 mutants was most dramatic in unmyelinated small sensory fibers, whereas motor axons were comparatively spared. LKB1 deletion in SCs led to abnormalities in nerve energy and lipid homeostasis and to increased lactate release. The latter acts in a compensatory manner to support distressed axons. LKB1 signaling is essential for SC-mediated axon support, a function that may be dysregulated in diabetic neuropathy.

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Figure 1: Conditional deletion of LKB1 in SCs.
Figure 2: Metabolic alterations and progressive axonopathy in LKB1-SCKO nerves.
Figure 3: Axon degeneration in LKB1-iSCKO mutants.
Figure 4: Preservation of motor and loss of sensory axons in LKB1-SCKO mutants.
Figure 5: Absence of demyelination in LKB1-SCKO mutants.
Figure 6: Direct perturbation of AMPK and mTORC1 in SCs does not cause axon losses.
Figure 7: Characterization of downstream pathways and mitochondria in LKB1-SCKO nerves.
Figure 8: Augmented lactate release in LKB1-SCKO nerves supports axonal maintenance.

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References

  1. Nave, K.A. Myelination and the trophic support of long axons. Nat. Rev. Neurosci. 11, 275–283 (2010).

    CAS  PubMed  Article  Google Scholar 

  2. Beirowski, B. Concepts for regulation of axon integrity by enwrapping glia. Front. Cell. Neurosci. 7, 256 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  3. Nave, K.A. Myelination and support of axonal integrity by glia. Nature 468, 244–252 (2010).

    CAS  Article  PubMed  Google Scholar 

  4. Fünfschilling, U. et al. Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 485, 517–521 (2012).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  5. Lee, Y. et al. Oligodendroglia metabolically support axons and contribute to neurodegeneration. Nature 487, 443–448 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Viader, A. et al. Aberrant Schwann cell lipid metabolism linked to mitochondrial deficits leads to axon degeneration and neuropathy. Neuron 77, 886–898 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Said, G. Diabetic neuropathy–a review. Nat. Clin. Pract. Neurol. 3, 331–340 (2007).

    PubMed  Article  Google Scholar 

  8. Eckersley, L. Role of the Schwann cell in diabetic neuropathy. Int. Rev. Neurobiol. 50, 293–321 (2002).

    CAS  PubMed  Article  Google Scholar 

  9. Chowdhury, S.K., Smith, D.R. & Fernyhough, P. The role of aberrant mitochondrial bioenergetics in diabetic neuropathy. Neurobiol. Dis. 51, 56–65 (2013).

    CAS  PubMed  Article  Google Scholar 

  10. Shackelford, D.B. & Shaw, R.J. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat. Rev. Cancer 9, 563–575 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Hardie, D.G., Ross, F.A. & Hawley, S.A. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 13, 251–262 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Lizcano, J.M. et al. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23, 833–843 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Bright, N.J., Thornton, C. & Carling, D. The regulation and function of mammalian AMPK-related kinases. Acta Physiol. (Oxf.) 196, 15–26 (2009).

    CAS  Article  Google Scholar 

  14. Steinberg, G.R. & Kemp, B.E. AMPK in health and disease. Physiol. Rev. 89, 1025–1078 (2009).

    CAS  PubMed  Article  Google Scholar 

  15. Chowdhury, S.K., Dobrowsky, R.T. & Fernyhough, P. Nutrient excess and altered mitochondrial proteome and function contribute to neurodegeneration in diabetes. Mitochondrion 11, 845–854 (2011).

    CAS  PubMed  Article  Google Scholar 

  16. Inoki, K., Kim, J. & Guan, K.L. AMPK and mTOR in cellular energy homeostasis and drug targets. Annu. Rev. Pharmacol. Toxicol. 52, 381–400 (2012).

    CAS  PubMed  Article  Google Scholar 

  17. Shaw, R.J. LKB1 and AMP-activated protein kinase control of mTOR signalling and growth. Acta Physiol. (Oxf.) 196, 65–80 (2009).

    CAS  Article  Google Scholar 

  18. Barnes, A.P. et al. LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons. Cell 129, 549–563 (2007).

    CAS  PubMed  Article  Google Scholar 

  19. Granot, Z. et al. LKB1 regulates pancreatic beta cell size, polarity, and function. Cell Metab. 10, 296–308 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Boehlke, C. et al. Primary cilia regulate mTORC1 activity and cell size through Lkb1. Nat. Cell Biol. 12, 1115–1122 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. van der Velden, Y.U. et al. The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish. Proc. Natl. Acad. Sci. USA 108, 4358–4363 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. Contreras, C.M. et al. Loss of Lkb1 provokes highly invasive endometrial adenocarcinomas. Cancer Res. 68, 759–766 (2008).

    CAS  PubMed  Article  Google Scholar 

  23. Gan, B. et al. Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells. Nature 468, 701–704 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Jessen, N. et al. Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function. Biochim. Biophys. Acta 1802, 593–600 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Nakada, D., Saunders, T.L. & Morrison, S.J. Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells. Nature 468, 653–658 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Patti, G.J., Tautenhahn, R. & Siuzdak, G. Meta-analysis of untargeted metabolomic data from multiple profiling experiments. Nat. Protoc. 7, 508–516 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Chrast, R., Saher, G., Nave, K.A. & Verheijen, M.H. Lipid metabolism in myelinating glial cells: lessons from human inherited disorders and mouse models. J. Lipid Res. 52, 419–434 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Viader, A. et al. Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function. J. Neurosci. 31, 10128–10140 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Shorning, B.Y. & Clarke, A.R. LKB1 loss of function studied in vivo. FEBS Lett. 585, 958–966 (2011).

    CAS  PubMed  Article  Google Scholar 

  30. Napoli, I. et al. A central role for the ERK-signaling pathway in controlling Schwann cell plasticity and peripheral nerve regeneration in vivo. Neuron 73, 729–742 (2012).

    CAS  PubMed  Article  Google Scholar 

  31. Arthur-Farraj, P.J. et al. c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration. Neuron 75, 633–647 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Gwinn, D.M. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Corradetti, M.N., Inoki, K., Bardeesy, N., DePinho, R.A. & Guan, K.L. Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome. Genes Dev. 18, 1533–1538 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Lai, L.P., Lilley, B.N., Sanes, J.R. & McMahon, A.P. Lkb1/Stk11 regulation of mTOR signaling controls the transition of chondrocyte fates and suppresses skeletal tumor formation. Proc. Natl. Acad. Sci. USA 110, 19450–19455 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. Sherman, D.L. et al. Arrest of myelination and reduced axon growth when Schwann cells lack mTOR. J. Neurosci. 32, 1817–1825 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Goebbels, S. et al. Genetic disruption of Pten in a novel mouse model of tomaculous neuropathy. EMBO Mol. Med. 4, 486–499 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Carling, D., Sanders, M.J. & Woods, A. The regulation of AMP-activated protein kinase by upstream kinases. Int. J. Obes. (Lond.) 32 (suppl. 4): S55–S59 (2008).

    CAS  Article  Google Scholar 

  38. Woods, A. et al. Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2, 21–33 (2005).

    CAS  PubMed  Article  Google Scholar 

  39. Herrero-Martín, G. et al. TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells. EMBO J. 28, 677–685 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  40. Momcilovic, M., Hong, S.P. & Carlson, M. Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro. J. Biol. Chem. 281, 25336–25343 (2006).

    CAS  PubMed  Article  Google Scholar 

  41. Shackelford, D.B. et al. LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin. Cancer Cell 23, 143–158 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Jaleel, M. et al. Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate. FEBS Lett. 579, 1417–1423 (2005).

    CAS  PubMed  Article  Google Scholar 

  43. Sun, X., Gao, L., Chien, H.Y., Li, W.C. & Zhao, J. The regulation and function of the NUAK family. J. Mol. Endocrinol. 51, R15–R22 (2013).

    CAS  PubMed  Article  Google Scholar 

  44. Obrosova, I.G., Fathallah, L., Lang, H.J. & Greene, D.A. Evaluation of a sorbitol dehydrogenase inhibitor on diabetic peripheral nerve metabolism: a prevention study. Diabetologia 42, 1187–1194 (1999).

    CAS  PubMed  Article  Google Scholar 

  45. Stevens, M.J., Obrosova, I., Cao, X., Van Huysen, C. & Greene, D.A. Effects of DL-alpha-lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy. Diabetes 49, 1006–1015 (2000).

    CAS  PubMed  Article  Google Scholar 

  46. Laden, B.P. & Porter, T.D. Inhibition of human squalene monooxygenase by tellurium compounds: evidence of interaction with vicinal sulfhydryls. J. Lipid Res. 42, 235–240 (2001).

    CAS  PubMed  Article  Google Scholar 

  47. Hofmeijer, J., Franssen, H., van Schelven, L.J. & van Putten, M.J. Why are sensory axons more vulnerable for ischemia than motor axons? PLoS ONE 8, e67113 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Ramji, N., Toth, C., Kennedy, J. & Zochodne, D.W. Does diabetes mellitus target motor neurons? Neurobiol. Dis. 26, 301–311 (2007).

    CAS  PubMed  Article  Google Scholar 

  49. Zochodne, D.W., Verge, V.M., Cheng, C., Sun, H. & Johnston, J. Does diabetes target ganglion neurones? Progressive sensory neurone involvement in long-term experimental diabetes. Brain 124, 2319–2334 (2001).

    CAS  PubMed  Article  Google Scholar 

  50. Schmidt, R.E. Neuropathology and pathogenesis of diabetic autonomic neuropathy. Int. Rev. Neurobiol. 50, 257–292 (2002).

    CAS  PubMed  Article  Google Scholar 

  51. Bardeesy, N. et al. Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation. Nature 419, 162–167 (2002).

    CAS  PubMed  Article  Google Scholar 

  52. Feltri, M.L. et al. P0-Cre transgenic mice for inactivation of adhesion molecules in Schwann cells. Ann. NY Acad. Sci. 883, 116–123 (1999).

    CAS  PubMed  Article  Google Scholar 

  53. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    CAS  Article  PubMed  Google Scholar 

  54. Mallon, B.S., Shick, H.E., Kidd, G.J. & Macklin, W.B. Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development. J. Neurosci. 22, 876–885 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Doerflinger, N.H., Macklin, W.B. & Popko, B. Inducible site-specific recombination in myelinating cells. Genesis 35, 63–72 (2003).

    CAS  Article  PubMed  Google Scholar 

  56. Rodríguez, C.I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140 (2000).

    PubMed  Article  Google Scholar 

  57. Dasgupta, B. et al. The AMPK beta2 subunit is required for energy homeostasis during metabolic stress. Mol. Cell. Biol. 32, 2837–2848 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Viollet, B. et al. The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity. J. Clin. Invest. 111, 91–98 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Jørgensen, S.B. et al. Knockout of the alpha2 but not alpha1 5′-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranosidebut not contraction-induced glucose uptake in skeletal muscle. J. Biol. Chem. 279, 1070–1079 (2004).

    PubMed  Article  CAS  Google Scholar 

  60. Lesche, R. et al. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis 32, 148–149 (2002).

    CAS  PubMed  Article  Google Scholar 

  61. Viader, A., Chang, L.W., Fahrner, T., Nagarajan, R. & Milbrandt, J. MicroRNAs modulate Schwann cell response to nerve injury by reinforcing transcriptional silencing of dedifferentiation-related genes. J. Neurosci. 31, 17358–17369 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Babetto, E., Beirowski, B., Russler, E.V., Milbrandt, J. & DiAntonio, A. The Phr1 ubiquitin ligase promotes injury-induced axon self-destruction. Cell Reports 3, 1422–1429 (2013).

    CAS  PubMed  Article  Google Scholar 

  63. Beirowski, B. et al. Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling. Proc. Natl. Acad. Sci. USA 108, E952–E961 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  64. Beirowski, B. et al. Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo. J. Neurosci. 29, 653–668 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Mack, T.G.A. et al. Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat. Neurosci. 4, 1199–1206 (2001).

    CAS  PubMed  Article  Google Scholar 

  66. Hunter, D.A. et al. Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. J. Neurosci. Methods 166, 116–124 (2007).

    PubMed  PubMed Central  Article  Google Scholar 

  67. Yanes, O., Tautenhahn, R., Patti, G.J. & Siuzdak, G. Expanding coverage of the metabolome for global metabolite profiling. Anal. Chem. 83, 2152–2161 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. Yang, K., Cheng, H., Gross, R.W. & Han, X. Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics. Anal. Chem. 81, 4356–4368 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. Miller, B.R. et al. A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat. Neurosci. 12, 387–389 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. Golden, J.P. et al. RET signaling is required for survival and normal function of nonpeptidergic nociceptors. J. Neurosci. 30, 3983–3994 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We are grateful to R. DePinho (University of Texas MD Anderson Cancer Center) for the conditional Lkb1 mice, L. Wrabetz (Hunter James Kelly Research Institute) and A. Messing (University of Wisconsin-Madison) for the P0-cre transgenic mice, and the Genome Technology Access Center (GTAC) in the Department of Genetics for help with genomic analysis. We thank members of the Holtzman laboratory for help with glucose and lactate measurements, members of the Solnica-Krezel laboratory for assistance with live imaging, and Y. Sasaki and J. Gerdts for comments on the manuscript. This work was supported by an European Molecular Biology Organization (EMBO) long-term fellowship (B.B.), Muscular Dystrophy Association (MDA) Development Award (B.B.), American-Italian Cancer Foundation (AICF) postdoctoral research fellowship (E.B.); US National Institutes of Health (NIH) grants NS040745, NS087306 (J.M.) and AG13730 (J.M.), PPG 2P01 HL057278 (R.W.G), R21NS059566 (J.P.G), AG0 038036 (G.J.P.); and MDA grant 237041 (J.M.). This study was also supported by NIH Neuroscience Blueprint Center core grant P30 NS057105 and the HOPE Center for Neurological Disorders, Washington University.

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Authors and Affiliations

Authors

Contributions

B.B. and E.B. designed and performed experiments and analyzed data. J.P.G. performed some behavioral studies, Y.-J.C. and G.J.P. metabolomics, and K.Y. and R.W.G. lipidomics analysis. J.M. supervised the project. B.B. and J.M. wrote the manuscript.

Corresponding authors

Correspondence to Bogdan Beirowski or Jeffrey Milbrandt.

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Competing interests

R.W.G. has financial relationships with LipoSpectrum and Platomics Inc.

Integrated supplementary information

Supplementary Figure 1 Inactivation of LKB1 in mutant mice.

(a) Schematic illustration of the conditional LKB1 allele (grey squares depict exons, and black triangles loxP sequences), and the recombined allele in SCs expressing Cre recombinase (not drawn to scale). (b) Fluorescence in situ hybridization (FISH) results demonstrating LKB1 transcripts (puncta) in longitudinal sciatic nerve section in close vicinity to MBP RNA in control SCs (red arrows). There is complete absence of LKB1 transcript signal in the P30 LKB1-SCKO nerve section as compared to the LKB1fl/fl control sample. Scale bar: 50 μm (overview), 10 μm (higher power). (c) Left: Graph showing reduced body weights in female LKB1-SCKO mutants at 5, 6, and 10-16 months of age (p<0.05) (N=5-19 mice per genotype and age). Right: Graph showing reduced body weights in male LKB1-SCKO mutants at 6-12, and 15-16 months of age (p<0.05) (N=5-17 mice per genotype and age)

Supplementary Figure 2 Dysmyelination in LKB1-SCKO mutant mice.

(a) Representative traces of sciatic nerve CMAPs recorded from a foot muscle after proximal and distal stimulation in P30 mice. Arrows indicate the onset of the CMAPs. Compared to controls, mutants display prolonged latencies indicating reduced nerve conduction velocity. (b) Light and electron microscopy (3rd row) of sciatic nerve transverse sections from LKB1fl/fl control and LKB1-SCKO mice at the indicated ages. Note reduced numbers of myelinated axons, bundles of unsorted axons (red arrow), and incomplete enwrapping of axons (green arrows) in mutant preparations. Scale bars: 20 μm (light microscopy); 4 μm (electron microscopy) (c) Increased cumulative g-ratio in sciatic nerves from P30 LKB1-SCKO mutants reflecting retarded compact myelin formation. N=3-4 mice per genotype (d) Intensities of individual western blot bands for Nrg1, ERBB2, phospho-ERBB2, and ERBB3 were densitometrically quantified and normalized to β-actin loading control. See also Fig. 1e. N=3-4 mice per genotype

Supplementary Figure 3 Normal SC polarity in P60 LKB1-SCKO nerves.

Light microscopy of longitudinal sciatic nerve semithin sections (1st and 3rd column) from P60 LKB1fl/fl control and LKB1-SCKO mutant mice shows normal appearance of nodes of Ranvier (arrows in 2nd row) with adjacent paranodal regions, and grossly normal structure of Schmidt-Lanterman incisures (arrows in 3rd row) despite hypomyelination in the mutants. Similarly, fluorescence immunolabeling of P60 tibial nerve teased fiber preparations (2nd and 4th column) shows normal localization of nodal (Nav1.6), paranodal (Caspr), and juxtaparanodal (K(v)1.2) markers in mutant fibers. Scale bars: 50 μm (overview semithin light microscopy), 10 μm (high power semithin light microscopy), 5 μm (fluorescence microscopy)

Supplementary Figure 4 Metabolomics in LKB1-SCKO nerves.

Cloud plots (generated with metabolomics XCMS software) summarizing dysregulated metabolic features in extracts from P60 LKB1-SCKO sciatic nerves (11 mutant mice) as compared to controls (11 mice). Cloud plots for distinct chromatography (C18, HILIC) and mass spectrometry methods (+/- ESI) are shown. Each data point represents a metabolic feature as defined by a unique coordinate pair of mass-to-charge ratios (m/z) (y-axis) and chromatographic retention-times (x-axis). Features graphed above the x-axis are up-regulated, whereas metabolic features below the x-axis are down-regulated. The size of the data points reflects the fold-change values (i.e. features with higher fold change have larger radii), and points with black border indicate putative metabolite hits identified in the METLIN database. The color of the feature depends on how high or low the feature’s p-value is within all the graphed features. Thus, features that have low statistic p-value appear dark while features with high p-value are light. Interactive cloud plots with metabolite annotations are accessible on the XCMS online website.

Supplementary Figure 5 Normal SC numbers in atrophic nerves from LKB1-SCKO mice.

(a, b) Quantification of SC numbers (a) (DAPI-positive SC nuclei) and numbers of TUNEL-positive cells (b) on entire transverse sciatic nerve section from control and mutant mice at the indicated ages (N=4-6 mice per genotype and age for counts with both assays). No significant differences in SC numbers and numbers of TUNEL-positive cells were observed. (c, d) Representative fluorescence micrographs of TUNEL-labeled transverse sciatic nerve sections from control and mutant mice at the indicated ages. Note absence of TUNEL signal on micrographs of sciatic nerves from both genotypes. In contrast, TUNEL-positive cells are abundant in control DNAse I treated samples and in nerves from PTEN-SCKO neuropathy mutants (positive control) (d). Scale bars: 40 μm (e) Tail-suspended LKB1fl/fl control and LKB1-SCKO mouse (6 and 12 months of age). Note hindlimb clasping in 12-month-old LKB1-SCKO mouse (red arrows), but not in younger 6-month-old mutant. (f) Confocal microscopy (longitudinal z-series projections of YFP-labelled axoplasm) of sciatic nerves (whole-mount preparations) from 16-month-old control (LKB1fl/fl(Thy1.2-YFP-16)) and mutant (LKB1fl/fl:P0Cre+(Thy1.2-YFP-16)) mouse. Note axonal continuity interruption and swelling of axons in the mutant. Scale bar: 100 μm (g) Example semithin light microscopy of sciatic nerve transverse sections from 5-month-old control and mutant mice. Note substantially smaller size of the LKB1-SCKO mutant nerve. Scale bar: 100 μm (h) Fluorescence microscopy of sciatic nerves (whole-mount preparations with DAPI counterstaining) from 5-month-old control (LKB1fl/fl:PLP-EGFP) and mutant (LKB1fl/fl:P0Cre+:PLP-EGFP) mouse. Scale bar: 100 μm

Supplementary Figure 6 No myelin alterations in LKB1-iSCKO mutants prior to axon degeneration.

(a) PCR analysis of genomic DNA isolated from sciatic nerves from vehicle (1) or tamoxifen (2) treated control mice, and tamoxifen treated LKB1fl/fl: PLP-CreERT mutant (LKB1-iSCKO) (3). Arrow indicates recombination of the floxed LKB1 allele in LKB1-iSCKO nerve 11 months after first tamoxifen administration. (b) Western blot of sciatic nerve lysates from vehicle (1) or tamoxifen (2) treated control mice, and tamoxifen treated LKB1fl/fl: PLP-CreERT mutant (LKB1-iSCKO) (3). Note reduction of LKB1 protein expression in the LKB1-iSCKO sample, 11 months after first tamoxifen administration. The expression of structural myelin proteins is not affected. Data are representative for results obtained from 3 mice per group. (c) Reduced rotarod performance of 12-month-old LKB1-iSCKO mutants in comparison to age-matched carrier-treated control mice. N = 6-8 mice per group (d) Light microscopy of transverse sciatic nerve sections from vehicle treated control mouse and tamoxifen treated LKB1-iSCKO mutant 5 months after first injection shows normal nerve morphology with no axon degeneration and no changes in myelination. Scale bars: 20 μm (e) Sciatic nerve g-ratio scatter plots (left) and cumulative g-ratios (right) from tamoxifen-treated LKB1-iSCKO mutants are similar to those in vehicle treated control animals (11 months after first injection) indicating no overt changes in myelination despite the presence of axon degeneration at this time point. N=3 mice per group (f) Representative electron micrograph of sciatic nerve transverse section from tamoxifen treated LKB1-iSCKO mouse 11 months after first injection shows normal appearance of compact myelin and associated SCs (blue arrows) of intact axons in the presence of degenerating unmyelinated fibers (red arrow) in Remak bundles. Scale bar: 2 μm (g) Quantification of nerve conduction velocities in vehicle treated control mice and tamoxifen treated LKB1-iSCKO 3 and 10 months after the first injection shows no significant differences. N=5-7 mice per group

Supplementary Figure 7 Analysis of motor performance and muscle structure in LKB1-SCKO mice.

(a, b) Graphs showing quantification of forelimb grip strength (a) and results from hanging wire tests (b) in LKB1fl/fl control and LKB1-SCKO mutants at the indicated ages. Each data point represents the experimental results from an individual mouse, and continuous lines indicate the means for each genotype. There are no significant differences between control and mutant mice in both tests. Grip strength: N=4-8 mice per genotype and age; hanging wire: N=5-9 mice per genotype and age (c) Graph showing cumulative rotarod performance of LKB1fl/fl control and LKB1-SCKO mice at the indicated ages. Each data point represents the sum of 5 trials (in sec) of individual mouse in accelerated rotarod test, and continuous lines indicate the means for each genotype. Note reduced performance of LKB1-SCKO mutants at 13-16 months of age (p<0.05). N=4-9 mice per genotype and age (d) No significant differences in pole climb and turn performance between 10-month-old LKB1fl/fl control and LKB1-SCKO mice demonstrating absence of overt coordination deficits in aged mutants. N=5 mice per genotype for each test (e) Transverse gastrocnemius muscle sections (hematoxylin and eosin stain) are indistinguishable between 16-month-old control and mutant mice. Inset: Note normal appearance of individual skeletal muscle fibers in mutant muscle with peripheral localization of nuclei and uniform size. Scale bar: 100 μm (overviews), 20 μm (inset) (f) Photographs of dissected posterior limbs (coat removed) from control and mutant mice at the indicated ages. Note that there is no overt muscle wasting and atrophy in mutant extremities in contrast to many other neuropathy models.

Supplementary Figure 8 Further morphological and electrophysiological analysis of motor axons in LKB1-SCKO mutants.

(a) Epifluorescence (left) and confocal microscopy (right) of whole-mount preparations of flexor digitorum brevis muscles from 16-month-old control LKB1fl/fl(Thy1.2-YFP-16) and mutant LKB1fl/fl:P0Cre+(Thy1.2-YFP-16) mice. Green: YFP; red: TRITC-α-bungarotoxin. Note full preservation of intramuscular axon projections in the overview micrograph of the mutant muscle (green axon arbors). Additionally, note normal apposition between green axon terminals and red postsynaptic signals indicating preserved neuromuscular junctions (arrows) in these mice. Scale bars: 200 μm (epifluorescence); 20 μm (confocal) (b) Left: Epifluorescence microscopy of extensor hallucis longus (EHL) muscles from 16-month-old LKB1fl/fl(Thy1.2-YFP-16) control and LKB1-SCKO(Thy1.2-YFP-16) mutant mice. Green: YFP; red: TRITC-α-bungarotoxin. Note full preservation of intramuscular axon projections in the mutant (green axon arbors). Scale bar: 200 μm. Right: Quantification of total numbers of intact neuromuscular junctions in EHL muscles from 16-month-old LKB1fl/fl(Thy1.2-YFP-16) control and LKB1-SCKO(Thy1.2-YFP-16) mutant mice. N=3 mice per genotype (c) No changes in CMAP amplitudes in 12-month-old LKB1-SCKO mutants as compared to age-matched controls. (d) Light microscopy and quantification of motor fibers in L3 ventral spinal cord roots from 12-month-old control and LKB1-SCKO mice. Scale bar: 50 μm (e) Light microscopy and quantification of motor fibers in quadriceps nerves from 18-month-old control and LKB1-SCKO mice. Scale bar: 50 μm

Supplementary Figure 9 Further assessment of sensory deficits in LKB1-SCKO mutants.

(a) Significantly increased withdrawal latencies to heat stimuli in Hargreaves test in 12-month-old LKB1-SCKO mutants, consistent with degeneration of unmyelinated C-fiber axons in the mutants. N=9-11 female mice per genotype (b) Quantification of abnormal behavior to hind paw acetone stimulus in 12-month-old LKB1-SCKO mutants. N=8-11 female mice per genotype (c) Results of von Frey mechanical sensitivity analysis in 12-month-old control and LKB1-SCKO mutants. N=9-10 female mice per genotype (d) Light microscopy and quantification of sensory myelinated fibers in L3 dorsal spinal cord roots from 12-month-old control and LKB1-SCKO mice. Scale bar: 100 μm; N=6-7 mice per genotype (e) Light microscopy and quantification of sensory myelinated fibers in saphenous nerves from 18-month-old control and LKB1-SCKO mice. Note marked atrophy in mutant nerve secondary to axon loss. Scale bar: 50 μm; N=5 mice per genotype (f) Left: Fluorescence microscopy of L1-L5 dorsal root ganglia (whole-mount preparations) from 12-month-old control LKB1fl/fl(Thy1.2-YFP-16) and mutant LKB1fl/fl:P0Cre+(Thy1.2-YFP-16) mice. Note normal appearance and regular numbers of YFP-labelled DRG neuron bodies in mutant preparations. Scale bar: 200 μm. Right: Quantification of YFP-labelled sensory neuron bodies in L3 dorsal root ganglia from 12-month-old control LKB1fl/fl(Thy1.2-YFP-16) and mutant LKB1fl/fl:P0Cre+(Thy1.2-YFP-16) mice shows no deficit in the mutant. N=5-6 mice per genotype (g) Axon width distribution profile in sciatic nerves from control and mutant mice at P90. N=6 mice per genotype.

Supplementary Figure 10 Improvement of g-ratio deficits and absence of neuroinflammation in LKB1-SCKO nerves.

(a) Sciatic nerve g-ratios as a function of axon diameter. Note improvement of g-ratio deficits at 12 months of age as compared to 6 months of age in LKB1-SCKO mutants. N=3-5 mice per genotype and age (b-d) Quantification of Iba1 or CD68-positive macrophages and CD3 labelled T-lymphocytes in transverse frozen sections of 12-month-old control and LKB1-SCKO mutant mice (additionally expressing Thy1.2-YFP-16). There are no significant differences in immune cell densities between control and mutant preparations. Fluorescence micrographs show representative examples from individual immunolabeling experiments. Insets depict typical examples of Iba1, CD68, and CD3-positive immune cells (arrows). N=6 mice per genotype. Scale bars: 50 μm

Supplementary Figure 11 Elevated c-Jun expression in LKB1-SCKO nerves.

(a) Western blots of P60 sciatic nerve lysates probed with the indicated antibodies show increased c-Jun levels in LKB1-SCKO nerves. Data are representative for results obtained from at least 3 mice per genotype. (b) Immunofluorescence on transverse frozen sciatic nerve (P60) sections shows higher numbers of SCs expressing c-Jun (arrows) in the mutant sample as compared to control. Insets show individual c-Jun-positive SCs from boxed area. Scale bars: 30 μm

Supplementary Figure 12 Inactivation of mTOR in SCs does not cause axon loss.

(a) Schematic illustration of the conditional mTOR allele (grey squares depict exons, and black triangles depict loxP sequences) and the recombined allele in SCs expressing Cre recombinase (not drawn to scale). (b) Electron microscopy of sciatic nerve transverse sections from 12-month-old control and mTOR-SCKO mutants. Note marked thinning of compact myelin in the mutant, indicating hypomyelination. Arrow depicts abnormal Remak bundle with lacking segregation of individual unmyelinated axons in the mutant. No axon degeneration is observed despite these pathological changes. Scale bars: 5 μm (left); 2 μm (right) (c) Sciatic nerve g-ratios are shown as a function of axon diameter (scatter plot) and cumulative g-ratios (bar graph) in 12-month-old control mice and mTOR-SCKO mutants. Note the increased g-ratio in these mutants, indicative of hypomyelination. N=3 mice per genotype (d) Quantification of myelinated and unmyelinated axons in sciatic nerves from 12-month-old control and mTOR-SCKO mice shows no difference in total axon numbers. N=6 mice per genotype

Supplementary Figure 13 Generation and initial characterization of SC-specific AMPK knockout mice.

(a) Left: Schematic illustration of the breeding scheme for generation of AMPKβ1/2-SCKO mutants. The conditional AMPKβ1 allele (grey squares depict exons, and black triangles depict loxP sequences) and the recombined allele is shown (not drawn to scale). Right: Western blot analysis of sciatic nerves from control and AMPKβ1/2-SCKO mice using the indicated antibodies showing reduced expression of both AMPKα and AMPKβ isoforms in the mutant. Data are representative for results obtained from at least 3 mice per genotype. (b) Left: Schematic illustration of the breeding scheme for generation of AMPKα1/2-SCKO mutants. The conditional AMPKα2 allele and the recombined allele is shown (not drawn to scale). Right: Western blot analysis of sciatic nerves from control and AMPKα1/2-SCKO mice using the indicated antibodies showing reduced expression of both AMPKα and AMPKβ isoforms in the mutant. Data are representative for results obtained from at least 3 mice per genotype. (c) Normal accelerated rotarod performance in 18-month-old AMPKβ1/2-SCKO mutant mice. N=15-17 mice per genotype (d) Normal reaction latencies to thermal stimulus in hot-plate tests in 18-month-old AMPKβ1/2-SCKO mice indicating preserved integrity of small unmyelinated sensory axons. N=7-12 mice per genotype

Supplementary Figure 14 Further characterization of SC-specific AMPK knockout mice.

(a) Upper: sciatic nerve g-ratios are shown as a function of axon diameter (scatter plot) at P60 and in 18-month-old AMPKβ1/2-SCKO mutants. Lower: increased cumulative g-ratios in 12- and 18-month-old mutants, but not at P60. N=3-5 mice per genotype (b) Semithin light microscopy of sciatic nerve transverse sections from 12-month-old control and AMPKα1/2-SCKO mutant mice. Note hypomyelination, but normal numbers of axons in the mutant. Scale bar: 20 μm (c) Quantification of myelinated and unmyelinated axons in sciatic nerves from 12-month-old control and AMPKα1/2-SCKO mice shows no difference in total axon numbers. N=6 mice per genotype (d) Sciatic nerve g-ratios are shown as a function of axon diameter (scatter plot) and cumulative g-ratios (bar graph) in 12-month-old AMPKα1/2-SCKO mutants. Note the increased g-ratio in these animals, indicative of hypomyelination. N=2-3 mice per genotype

Supplementary Figure 15 Induction of Tak1 signaling in LKB1-SCKO nerves.

(a) Western blot analysis of P30 sciatic nerve lysates probed with the indicated antibodies shows strong induction of Tak1 signaling (as witnessed by decreased total Tak1 and elevated p-Tak1(T184/187) levels) in the mutant nerve, while no difference in CamKKα/β signaling is detectable. Data are representative for results obtained from at least 4 mice per genotype. (b) Densitometric quantification of p-Tak1(T184/187)/ total Tak1 ratios in P30 nerves. Integrated band intensities from individual western blot bands were measured. N=4 mice for each genotype tested (c) p-Tak1(T184/187) western blot analysis of sciatic nerve lysates at the indicated ages shows sustained induction of Tak1 signaling in mutant nerves from aged LKB1-SCKO mice.

Supplementary Figure 16 Mitochondrial abnormalities in LKB1-deficient SCs.

(a) Confocal live-cell imaging analysis of primary SC cultures loaded with the mitochondrial membrane potential indicator dye TMRM (Tetramethylrhodamine, methyl ester) established from P30 and 12-month-old mice (LKB1fl/fl:PLP-EGFP control and LKB1fl/fl:P0Cre+:PLP-EGFP mutant). Graphs depict fold-changes of calculated average cumulative fluorescence intensities of TMRM signal in SCs. Note that TMRM intensities in mutant SCs from 12-month-old mice are similarly increased as compared to cells from P30 mice despite the progressive elevations in mitochondrial load. For validation the mitochondrial uncoupler CCCP was used to de-energize mitochondria and to demonstrate quenching of TMRM signal. Scale bars: 100 μm (b) Progressive increase of mitochondrial DNA copy numbers in mutant sciatic nerve preparations. *P=0.003, **P=0.010. N=4 mice per genotype and age

Supplementary Figure 17 Expression of glycolytic enzymes and glucose/lactate transporters in LKB1-SCKO nerves.

(a) Western blot analysis of control and LKB1-SCKO mutant sciatic nerve lysates using the indicated antibodies demonstrates no obvious differences in the expression of key glycolytic enzymes. Data are representative for results obtained from at least 5 mice per genotype. (b) qRT-PCR analysis shows normal levels of glucose transporters Glut1, 3, 4 and monocarboxylate transporters MCT1, 2, 4 (normalized to GAPDH expression) in sciatic nerves from P60 LKB1-SCKO mutants as compared to LKB1fl/fl control mice. N=3-4 mice per genotype (c) Western blot analysis of control and LKB1-SCKO mutant sciatic nerve lysates at the depicted ages using the indicated antibodies demonstrates no differences in the expression of MCT2 (axonal) and MCT4 (glial) proteins. Data are representative for results obtained from 3 mice per genotype. (d) Representative confocal microscopy of MCT4 (blue) immunofluorescence on Triton-X permeabilized control and LKB1-deficient SCs expressing EGFP (green) shows no difference in the expression level of the MCT4 monocarboxylate transporter. Scale bars: 5 μm (e) Normal glucose levels in LKB1-SCKO sciatic nerve preparations. N=3-6 mice per genotype and age

Supplementary Figure 18 Manipulation of lactate levels in vitro and in vivo.

(a) Phase-contrast and immunofluorescence images of control-treated (no lactate) and lactate-treated DRG neurites at the indicated concentrations and time points. There is no impairment of neuritic continuity (i.e. fragmentation) following these treatments as assessed by distinct neuritic integrity markers (neurofilament, tubulin). Scale bars: 50 μm (b) No difference in rotarod performance between vehicle-control treated and 2DG treated LKB1-SCKO mutants, 60 days after the first intraperitoneal injection. N=8 mice per group (c) No difference in body weights between vehicle-control treated LKB1-SCKO and 2DG treated LKB1-SCKO mutants on the 4th and 8th treatment week. N=10-13 mice per group and time-point

Supplementary Figure 19 Uncropped pictures of the western blots presented in the main figures

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–19 (PDF 8562 kb)

Supplementary Methods Checklist (PDF 358 kb)

Supplementary Table 1: Metabolomics analysis in LKB1-SCKO nerves.

Significantly dysregulated metabolic features (fold change >1.5; p=0.01) in P60 sciatic nerve preparations from LKB1-SCKO mice in comparison to controls as assessed by multiple chromatography (C18 and HILIC) and mass-spectrometry methods (positive and negative ion mode). Putative metabolites were identified using XCMS bioinformatics processing and the METLIN database. (XLSX 41 kb)

Supplementary Table 2: Microarray analysis in LKB1-SCKO nerves.

Genes differentially expressed in LKB1-SCKO sciatic nerves compared with controls at P60. (XLSX 22 kb)

Supplementary Table 3: Lipidomics analysis in LKB1-SCKO nerves.

Results of lipidomics measurements in P60 control and LKB1-SCKO lipid nerve preparations. (XLSX 135 kb)

Supplementary Table 4: Antibodies.

Primary and secondary antibodies used in this study. (XLSX 13 kb)

MPG movie of 18-month-old LKB1-SCKO mouse showing gait deficits and non-ambulatory behavior. (MPG 4184 kb)

41593_2014_BFnn3809_MOESM53_ESM.mpg

MPG movie of 12-month-old control mouse (Lkb1fl/fl:Plp-creERT, vehicle treated) for comparison to mutant shown in Supplementary Video 3. (MPG 3191 kb)

MPG movies of 12-month-old LKB1-iSCKO mouse (Lkb1fl/fl:Plp-creERT, tamoxifen treated).

Note non-ambulatory behavior, gait abnormalities and ataxia in the LKB1-iSCKO mutant in comparison to the control from Supplementary Video 2. (MPG 6011 kb)

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Beirowski, B., Babetto, E., Golden, J. et al. Metabolic regulator LKB1 is crucial for Schwann cell–mediated axon maintenance. Nat Neurosci 17, 1351–1361 (2014). https://doi.org/10.1038/nn.3809

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