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Combinatorial proteomics and transcriptomics identify AMPK in the control of the axonal regeneration programme of DRG sensory neurons after spinal injury

Guiping Kong, Luming Zhou, Elisabeth Serger, Ilaria Palmisano, Francesco De Virgiliis, Thomas H Hutson, Eilidh Mclachlan, Anja Freiwald, Paolo La Montanara, Kirill Shkura, Radhika Puttagunta, Simone Di Giovanni
doi: https://doi.org/10.1101/661488
Guiping Kong
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UKHertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, GermanyGraduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Germany
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Luming Zhou
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UKHertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, GermanyGraduate School for Cellular and Molecular Neuroscience, University of Tuebingen, Germany
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Elisabeth Serger
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Ilaria Palmisano
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Francesco De Virgiliis
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Thomas H Hutson
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Eilidh Mclachlan
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Anja Freiwald
Proteomics Core Facility, IMB, Mainz, Germany
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Paolo La Montanara
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Kirill Shkura
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK
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Radhika Puttagunta
Hertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, GermanyUniversity of Heidelberg, Heidelberg, Germany
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Simone Di Giovanni
Centre for Restorative Neuroscience, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UKHertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, Germany
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  • For correspondence: s.di-giovanni@imperial.ac.uk
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SUMMARY

Regeneration after injury occurs in axons that lie in the peripheral nervous system but it fails in the central nervous system limiting functional recovery. Despite recent progress, the signalling response to injury of peripheral versus central projecting axons that might underpin this differential regenerative ability is currently largely uncharacterized. To fill this knowledge gap, here we combined axoplasmic proteomics from peripheral sciatic or central projecting dorsal root axons from sciatic DRG neurons. Proteomics was combined with cell body RNAseq to compare axonal and soma responses between a spinal regeneration-incompetent versus sciatic regeneration-competent nerve injury. This allowed the identification of injury-dependent signalling pathways uniquely represented in peripheral versus central projecting sciatic DRG axons. RNAseq and proteomics analysis suggested AMPK as a putative regulator of axonal regenerative signalling pathways. AMPK immunoprecipitation followed by mass spectrometry from DRG suggested that the 26S proteasome and its regulatory subunit PSMC5 preferentially interact with AMPKα for proteasomal degradation following sciatic axotomy. Mechanistically, we found that the proteasome and CaMKIIα-dependent proteasomal subunit PSMC5 regulates AMPKα1 protein expression. Finally, conditional deletion of AMPKα1 promoted multiple regenerative signalling pathways and robust axonal growth across the injured spinal cord, suggesting inhibition of AMPK as a novel regenerative target following spinal injury.

HIGHLIGHTS

  • Axoplasmic proteomics from sciatic or centrally projecting branches of sciatic DRG identifies unique protein enrichment and signalling pathways, including prior and subsequent to a spinal regeneration-incompetent versus sciatic regeneration-competent axonal injury

  • Combined axoplasmic DRG proteomics and cell body RNAseq analysis suggest AMPK as a central regulator controlling axonal regeneration

  • The 26S proteasome and the 19S regulatory subunit PSMC5 interact with AMPKα following sciatic axotomy. PSMC5 regulates AMPKα1 protein levels

  • AMPKα1 conditional deletion enhances robust axonal growth following SCI

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted June 06, 2019.
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Combinatorial proteomics and transcriptomics identify AMPK in the control of the axonal regeneration programme of DRG sensory neurons after spinal injury
Guiping Kong, Luming Zhou, Elisabeth Serger, Ilaria Palmisano, Francesco De Virgiliis, Thomas H Hutson, Eilidh Mclachlan, Anja Freiwald, Paolo La Montanara, Kirill Shkura, Radhika Puttagunta, Simone Di Giovanni
bioRxiv 661488; doi: https://doi.org/10.1101/661488
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Combinatorial proteomics and transcriptomics identify AMPK in the control of the axonal regeneration programme of DRG sensory neurons after spinal injury
Guiping Kong, Luming Zhou, Elisabeth Serger, Ilaria Palmisano, Francesco De Virgiliis, Thomas H Hutson, Eilidh Mclachlan, Anja Freiwald, Paolo La Montanara, Kirill Shkura, Radhika Puttagunta, Simone Di Giovanni
bioRxiv 661488; doi: https://doi.org/10.1101/661488

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