Abstract
Regeneration after injury occurs in axons that lie in the peripheral nervous system but fails in the central nervous system, thereby limiting functional recovery. Differences in axonal signalling in response to injury that might underpin this differential regenerative ability are poorly characterized. Combining axoplasmic proteomics from peripheral sciatic or central projecting dorsal root ganglion (DRG) axons with cell body RNA-seq, we uncover injury-dependent signalling pathways that are uniquely represented in peripheral versus central projecting sciatic DRG axons. We identify AMPK as a crucial regulator of axonal regenerative signalling that is specifically downregulated in injured peripheral, but not central, axons. We find that AMPK in DRG interacts with the 26S proteasome and its CaMKIIα-dependent regulatory subunit PSMC5 to promote AMPKα proteasomal degradation following sciatic axotomy. Conditional deletion of AMPKα1 promotes multiple regenerative signalling pathways after central axonal injury and stimulates robust axonal growth across the spinal cord injury site, suggesting inhibition of AMPK as a therapeutic strategy to enhance regeneration following spinal cord injury.
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Data availability
The axoplasmic proteomics and AMPK IP–MS data have been deposited in the ProteomeXchange Consortium under accession codes PXD013297 and PXD013318. The pipeline used for the proteomics and AMPK IP analysis is available at: https://github.com/intgenomics/191015.proteomics_analysis. Source data are provided with this paper.
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Acknowledgements
We acknowledge start-up funds from the Department of Brain Sciences, Imperial College London (S.D.G.), and the Hertie Foundation for financial support (S.D.G.); the International Spinal Research Trust (Nathalie Rose Barr PhD awards to E.S and E.M.); Wings for Life (S.D.G.); the Deutsche Forschungsgemeinschaft (S.D.G.); the Medical Research Council (S.D.G.); and Rosetrees Trust (S.D.G.). The research was supported by the National Institute for Health Research Imperial Biomedical Research Centre (S.D.G.). The views expressed are those of the author(s) and not necessarily those of the National Health Service, the National Institute for Health Research or the Department of Health. The Q Exactive Plus mass spectrometer was funded by Deutsche Forschungsgemeinschaft INST 247/766-1 FUGG. We would also like to thank Mike Fainzilber for critical discussion of the data and the manuscript.
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G.K. designed and performed experiments, performed data analysis and wrote the paper; L.Z. designed and performed experiments and performed data analysis; E.S. performed experiments and data analysis; I.P. performed data analysis; F.D.V. performed experiments; T.H.H. performed experiments; E.M. performed data analysis; A.F. performed the mass spectrometry experiments and data analysis; P.L.M. performed experiments; K.S. performed data analysis; R.P. provided experimental advice and edited the paper and S.D.G. designed experiments, provided funding and wrote the paper.
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Extended data
Extended Data Fig. 1 Venn diagrams show high neuronal enrichment of axoplasm proteins.
a, Area-proportional Venn diagram showing the overlap between the proteins identified in our axoplasm dataset and previously published DRG neuron or Schwann cell specific transcriptomic datasets. Red: peripheral axoplasm; pink: central axoplasm. b, Area-proportional Venn diagram showing the overlap between proteins represented in our axoplasm dataset and previously published DRG neuron or Oligodendrocyte specific transcriptomic datasets.
Extended Data Fig. 2 Injury to the DRG peripheral and central axons elicit differential protein expression profiles.
a, Heat map of the log2 ratio of differentially expressed proteins (FDR < 0.05) identified by mass spectrometry in the axoplasmic extract from peripheral and central DRG axons. Comparisons include peripheral nerve after sciatic nerve axotomy (SNA) vs sham (control injury); central branches after dorsal column axotomy (DCA) vs Lam (control injury). Red and Blue indicates up- and down-regulated proteins, respectively. b, Venn diagram shows the number of differentially expressed proteins following SNA and DCA (FDR < 0.05, absolute log2 ratio > 0.58) and how many proteins are overlapped between these two compartments after injury. c–f, Heatmap graphs show Gene ontology (GO) analysis of differentially expressed proteins following SNA and DCA. Differentially expressed proteins were selected with cut off FDR < 0.05, log2 ratio > 0.58 (Red) or log2 ratio < -0.58 (Blue). Gene ontology was performed by DAVID. Only enriched GO items with Fisher’s exact P value < 0.05 were selected and categories that share the same protein groups were combined in one category. Categories in orange of (c, e, f) are known to be regulated by or to regulate AMPK.
Extended Data Fig. 3 Regeneration associated genes and immunoblotting validation of axoplasmic protein expression identified by mass spectrometry.
a, Bar graphs show axoplasmic proteins belonging to RAGs with FDR < 0.05, log2 ratio > 0.58 (SNA vs Sham) that are plotted in a log2 ratio scale. b, d, Immunoblots show validation of axoplasmic proteins identified by mass spectrometry after SNA vs Sham or DCA vs Lam (SN: sciatic nerve; SC: spinal cord). c, e, Bar graphs show quantification of immunoblots in (b) and (d) respectively. n = 3 independent experiments. The expression level of each protein was quantified after normalization to GAPDH. Values represent means ± SEM. Two-tailed unpaired Welch’s t-test.
Extended Data Fig. 4 AMPKα1 expression in NF200, PARV, CGRP and IB4 positive DRG neurons.
a, Representative fluorescence images of immunostaining for AMPKα1 and parvalbumin (PARV) or CGRP, or IB4+ in DRG neurons. n = 3. Scale bar, 100 μm. b, Representative fluorescence images of immunostaining for AMPKα1 and parvalbumin (PARV) in DRG neurons following Sham and SNA. n = 3. Scale bar, 100 μm. c, Percentage of NF200, PARV, CGRP or IB4 positive neurons expressing AMPKα1. d, Quantification of immunostaining for AMPKα1 expression level of (b). n = 3 mice each group. Each mouse represents an independent replicate. The relative AMPKα1 expression level was quantified after normalization to the background (secondary antibody only). Values represent means ± SEM. Two-tailed unpaired Welch’s t-test.
Extended Data Fig. 5 AMPKα1 expression in DRG neurons following AMPKα1 conditional deletion or overexpression.
a, Representative fluorescence images of immunostaining for GFP; AMPKα1 and DAPI in cultured DRG cells dissected from AMPKα1 floxed mice 48 h after transfection with AAV-GFP or AAV-Cre-GFP. n = 3. Scale bar, 100 μm. b, Representative fluorescence images of immunostaining for GFP; AMPKα1 and DAPI in cultured DRG cells after electroporation with GFP plasmid or AMPKα1 plasmid at 48 h. Arrows show non-pycnotic DAPI positive nuclei. Scale bar, 50 μm. c, Quantification of the percentage of cells with non-pycnotic nuclei (b). n = 3 independent experiments. Values represent means ± SEM. Two-tailed unpaired Welch’s t-test.
Extended Data Fig. 6 AMPKα1 and AMPKα2 interaction with PSMC5.
a, b, Immunoblots for PSMC5, AMPKα1 and AMPKα2 after AMPKα1 and AMPKα2 IP from DRG. Repeated twice with similar results.
Extended Data Fig. 7 In vivo AMPKα1 conditional deletion in DRG neurons.
a, Representative images of AMPKα1 and GFP immunostaining in DRG sections 5 weeks following AAV-GFP or AAV-Cre-GFP sciatic nerve injection. Scale bar, 50 μm. b, Representative images of AMPKα2 and GFP immunostaining in DRG sections 5 weeks following AAV-GFP or AAV-Cre-GFP sciatic nerve injection. Arrowheads mark GFP positive neurons showing the presence or loss of AMPKα2 staining. Scale bar, 50 μm. c, Quantification of AMPKα1 level of (a). n = 3 mice. Values represent means ± SEM. Two-tailed unpaired Welch’s t-test. d, Quantification of AMPKα2 level of (b). n = 3 mice. Values represent means ± SEM. Two-tailed unpaired Welch’s t-test.
Extended Data Fig. 8 GFP and dextran co-localization in DRG neurons.
a, Representative images of DRG sections from AAV-Cre-GFP sciatic nerve injected mice co-immunostained with antibodies against GFP and Dextran. Scale bar, 50 μm. b, c, Quantification of the percentage of GFP+ and Dextran+ / GFP+ cells following AAV-GFP or AAV-Cre-GFP. AAV-GFP, n = 13 mice; AAV-Cre-GFP, n = 10 mice. Percentage of GFP positive cells was calculated as the ratio of GFP+ versus TUJ1+ cells; percentage of Dextran+/GFP+ was calculated as the ratio of Dextran+/GFP+ versus TUJ1+ cells. Values represent means ± SEM. Two-tailed unpaired Welch’s t-test. d, Longitudinal spinal cord section 5 weeks after SCI showing axonal labelling across the injured dorsal columns following deletion of AMPKα1. Dorsal column axons are labelled by sciatic nerve injected Dextran. Asterisk indicates the lesion epicentre. D; dorsal; V; ventral, C; caudal; R; rostral. Scale bar; 250 μm. Similar results were found in eight AMPKα1 conditionally deleted mice. The quantification source data is provided in statistical source data, Fig. 8b.
Supplementary information
Supplementary Information
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AMPK IP–MS
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Kong, G., Zhou, L., Serger, E. et al. AMPK controls the axonal regenerative ability of dorsal root ganglia sensory neurons after spinal cord injury. Nat Metab 2, 918–933 (2020). https://doi.org/10.1038/s42255-020-0252-3
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DOI: https://doi.org/10.1038/s42255-020-0252-3
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