title: Schwann cell autophagy counteracts the onset and chronification of neuropathic pain.
abstract: Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long-lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3-methyladenine administration or as occurs in activating molecule in Beclin-1-regulated autophagy transgenic mice (Ambra1(+/gt)), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox-sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.
authors.affiliation: CNR - National Research Council, Cell Biology and Neurobiology Institute, Rome, Italy Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Fondazione Santa Lucia, Rome, Italy Department of Technology and Health, Istituto Superiore di Sanita', Rome, Italy Department of Therapeutic Research and Medicine Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanita', Rome, Italy Medical School, University Campus-Biomedico, Rome, Italy Department of Experimental Medicine and Surgery, Division of Biochemistry, University of "Tor Vergata", Rome, Italy Dulbecco Telethon Institute, Department of Biology, University of "Tor Vergata", Rome, Italy San Raffaele Institute, Sulmona, L'Aquila, Italy Center of Integrated Metabolomics, Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Marinelli
authors.forename: Francesca
authors.initials: F
authors.lastname: Nazio
authors.forename: Antonella
authors.initials: A
authors.lastname: Tinari
authors.forename: Laura
authors.initials: L
authors.lastname: Ciarlo
authors.forename: Marcello
authors.initials: M
authors.lastname: D'Amelio
authors.forename: Luisa
authors.initials: L
authors.lastname: Pieroni
authors.forename: Valentina
authors.initials: V
authors.lastname: Vacca
authors.forename: Andrea
authors.initials: A
authors.lastname: Urbani
authors.forename: Francesco
authors.initials: F
authors.lastname: Cecconi
authors.forename: Walter
authors.initials: W
authors.lastname: Malorni
authors.forename: Flaminia
authors.initials: F
authors.lastname: Pavone
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D039481:CD11b Antigen / Q000378:metabolism; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins / Q000235:genetics; D010147:Pain Measurement; D012583:Schwann Cells / Q000473:pathology* / Q000648:ultrastructure; D012584:Sciatic Nerve / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D012585:Sciatica / Q000235:genetics / Q000473:pathology* / Q000503:physiopathology*; D020123:Sirolimus / Q000494:pharmacology; D013997:Time Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C521754:Ambra1 protein, mouse; D039481:CD11b Antigen; D005904:Glial Fibrillary Acidic Protein; D007166:Immunosuppressive Agents; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins; C025946:3-methyladenine; D000225:Adenine; D020123:Sirolimus
keywords: Autophagic flux; Behaviour; Chronic pain; Glia; Mice; Wallerian degeneration
references.citation: Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. PAIN® 1988;33:87-107.
references.citation: Blair IA. Lipid hydroperoxide-mediated DNA damage. Exp Gerontol 2001;36:1473-1481.
references.citation: Bridges D, Thompson SWN, Rice ASC. Mechanisms of neuropathic pain. Br J Anaesth 2001;87:12-26.
references.citation: Bulleid NJ, Ellgaard L. Multiple ways to make disulfides. Trends Biochem Sci 2011;36:485-492.
references.citation: Calle E, Berciano MT, Fernández R, Lafarga M. Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy. Acta Neuropathol 1999;97:143-155.
references.citation: Calvo M, Dawes JM, Bennett DL. The role of the immune system in the generation of neuropathic pain. Lancet Neurol 2012;11:629-642.
references.citation: Cecconi F, Levine B. The role of autophagy in mammalian development: cell makeover rather than cell death. Dev Cell 2008;15:344-357.
references.citation: Chen G, Ke Z, Xu M, Liao M, Wang X, Qi Y, Zhang T, Frank JA, Bower KA, Shi X, Luo J. Autophagy is a protective response to ethanol neurotoxicity. Autophagy 2012;8:1577-1589.
references.citation: Cheng C, Zochodne DW. In vivo proliferation, migration and phenotypic changes of Schwann cells in the presence of myelinated fibers. Neuroscience 2002;115:321-329.
references.citation: Chiarugi P. Src redox regulation: there is more than meets the eye. Mol Cells 2008;26:329-337.
references.citation: Di Bartolomeo S, Corazzari M, Nazio F, Oliverio S, Lisi G, Antonioli M, Pagliarini V, Matteoni S, Fuoco C, Giunta L, D'Amelio M, Nardacci R, Romagnoli A, Piacentini M, Cecconi F, Fimia GM. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J Cell Biol 2010;191:155-168.
references.citation: Dubový P. Wallerian degeneration and peripheral nerve conditions for both axonal regeneration and neuropathic pain induction. Ann Anat 2011;193:267-275.
references.citation: Farioli-Vecchioli S, Saraulli D, Costanzi M, Leonardi L, Cinà I, Micheli L, Nutini M, Longone P, Oh SP, Cestari V, Tirone F. Impaired terminal differentiation of hippocampal granule neurons and defective contextual memory in PC3/Tis21 knockout mice. PLoS One 2009;4:e8339.
references.citation: Figueroa-Romero C, Sadidi M, Feldman EL. Mechanisms of disease: the oxidative stress theory of diabetic neuropathy. Rev Endocr Metab Disord 2008;9:301-314.
references.citation: Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R, Corazzari M, Fuoco C, Ucar A, Schwartz P, Gruss P, Piacentini M, Chowdhury K, Cecconi F. Ambra1 regulates autophagy and development of the nervous system. Nature 2007;447:1121-1125.
references.citation: Flower RJ, Rothwell NJ. Lipocortin-1: cellular mechanisms and clinical relevance. Trends Pharmacol Sci 1994;15:71-76.
references.citation: Fortun J, Dunn WA Jr, Joy S, Li J, Notterpek L. Emerging role for autophagy in the removal of aggresomes in Schwann cells. J Neurosci 2003;23:10672-10680.
references.citation: Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol 1997;14:67-116.
references.citation: Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999;340:448-454.
references.citation: Garbay B, Heape AM, Sargueil F, Cassagne C. Myelin synthesis in the peripheral nervous system. Prog Neurobiol 2000;61:267-304.
references.citation: Gaudet AD, Popovich PG, Ramer MS. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J Neuroinflammation 2011;8:110.
references.citation: Geber C, Baumgärtner U, Schwab R, Müller H, Stoeter P, Dieterich M, Sommer C, Birklein F, Treede RD. Revised definition of neuropathic pain and its grading system: an open case series illustrating its use in clinical practice. Am J Med 2009;122:S3-S12.
references.citation: Gold R, Oelschlager M, Pepinsky RB, Sommer C, Hartung HP, Toyka KV. Increased lipocortin-1 (annexin-1) expression in the sciatic nerve of Lewis rats with experimental autoimmune neuritis. Acta Neuropathol 1999;98:583-589.
references.citation: Hayashi A, Nakashima K, Yamagishi K, Hoshi T, Suzuki A, Baba H. Localization of annexin II in the paranodal regions and Schmidt-Lanterman incisures in the peripheral nervous system. Glia 2007;55:1044-1052.
references.citation: Jiang J, Jiang J, Zuo Y, Gu Z. Rapamycin protects the mitochondria against oxidative stress and apoptosis in a rat model of Parkinson's disease. Int J Mol Med 2013;31:825-832.
references.citation: Johnson JS, Roux KJ, Fletcher BS, Fortun J, Notterpek L. Molecular alterations resulting from frameshift mutations in peripheral myelin protein 22: implications for neuropathy severity. J Neurosci Res 2005;82:743-752.
references.citation: Kamal AM, Flower RJ, Perretti M. An overview of the effects of annexin 1 on cells involved in the inflammatory process. Mem Inst Oswaldo Cruz 2005;100:39-47.
references.citation: Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717-1721.
references.citation: Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012;8:445-544.
references.citation: Lee SM, Olzmann JA, Chin LS, Li L. Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome-autophagy pathways. J Cell Sci 2011;124:3319-3331.
references.citation: Li X, Gonias SL, Campana WM. Schwann cells express erythropoietin receptor and represent a major target for Epo in peripheral nerve injury. Glia 2005;51:254-265.
references.citation: Luvisetto S, Marinelli S, Cobianchi S, Pavone F. Anti-allodynic efficacy of botulinum neurotoxin A in a model of neuropathic pain. Neuroscience 2007;145:1-4.
references.citation: Marzano V, Santini S, Rossi C, Zucchelli M, D'Alessandro A, Marchetti C, Mingardi M, Stagni V, Barilà D, Urbani A. Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity. J Proteomics 2012;75:4632-4646.
references.citation: Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci 2011;36:320-328.
references.citation: Mirsky R, Woodhoo A, Parkinson DB, Arthur-Farraj P, Bhaskaran A, Jessen KR. Novel signals controlling embryonic Schwann cell development, myelination and dedifferentiation. J Peripher Nerv Syst 2008;13:122-135.
references.citation: Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004;36:2491-2550.
references.citation: Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011;147:728-741.
references.citation: Mizushima N, Kuma A. Autophagosomes in GFP-LC3 transgenic mice. Methods Mol Biol 2008;445:119-124.
references.citation: Obara I, Tochiki KK, Géranton SM, Carr FB, Lumb BM, Liu Q, Hunt SP. Systemic inhibition of the mammalian target of rapamycin (mTOR) pathway reduces neuropathic pain in mice. PAIN® 2011;152:2582-2595.
references.citation: Opalach K, Rangaraju S, Madorsky I, Leeuwenburgh C, Notterpek L. Lifelong calorie restriction alleviates age-related oxidative damage in peripheral nerves. Rejuvenation Res 2010;13:65-74.
references.citation: Orhan CE, Onal A, Ulker S. Antihyperalgesic and antiallodynic effect of sirolimus in neuropathic pain and the role of cytokines in this effect. Neurosci Lett 2010;481:17-20.
references.citation: Perry CN, Kyoi S, Hariharan N, Takagi H, Sadoshima J, Gottlieb RA. Novel methods for measuring cardiac autophagy in vivo. Methods Enzymol 2009;453:325-342.
references.citation: Pieroni L, Finamore F, Ronci M, Mattoscio D, Marzano V, Mortera SL, Quattrucci S, Federici G, Romano M, Urbani A. Proteomics investigation of human platelets in healthy donors and cystic fibrosis patients by shotgun nUPLC-MSE and 2DE: a comparative study. Mol Biosyst 2011;7:630-639.
references.citation: Raivich G, Makwana M. The making of successful axonal regeneration: genes, molecules and signal transduction pathways. Brain Res Rev 2007;53:287-311.
references.citation: Ramírez-Rangel I, Bracho-Valdés I, Vázquez-Macías A, Carretero-Ortega J, Reyes-Cruz G, Vázquez-Prado J. Regulation of mTORC1 complex assembly and signaling by GRp58/ERp57. Mol Cell Biol 2011;31:1657-1671.
references.citation: Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov 2012;11:709-730.
references.citation: Ryan MC, Shooter EM, Notterpek L. Aggresome formation in neuropathy models based on peripheral myelin protein 22 mutations. Neurobiol Dis 2002;10:109-118.
references.citation: Ryu SY, Hong GU, Kim DY, Ro JY. Enolase 1 and calreticulin regulate the differentiation and function of mouse mast cells. Cell Signal 2012;24:60-70.
references.citation: Sarbassov DD, Sabatini DM. Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex. J Biol Chem 2005;280:39505-39509.
references.citation: Shan SW, Tang MK, Cai DQ, Chui YL, Chow PH, Grotewold L, Lee KK. Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death. Dev Dyn 2005;233:266-281.
references.citation: Smith KJ, Kapoor R, Felts PA. Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathol 1999;9:69-92.
references.citation: Stoll G, Griffin JW, Li CY, Trapp BD. Wallerian degeneration in the peripheral nervous-system – participation of both Schwann cells and macrophages in myelin degradation. J Neurocytol 1989;18:671-683.
references.citation: Stoll G, Hartung HP. The role of macrophages in degeneration and immune-mediated demyelination of the peripheral nervous-system. Adv Neuroimmunol 1992;2:163-179.
references.citation: Strappazzon F, Vietri-Rudan M, Campello S, Nazio F, Florenzano F, Fimia GM, Piacentini M, Levine B, Cecconi F. Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy. EMBO J 2011;30:1195-1208.
references.citation: Stroikin Y, Dalen H, Lööf S, Terman A. Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material. Eur J Cell Biol 2004;83:583-590.
references.citation: Sun X, Wang X, Chen T, Li T, Cao K, Lu A, Chen Y, Sun D, Luo J, Fan J, Young W, Ren Y. Myelin activates FAK/Akt/NF-kappaB pathways and provokes CR3-dependent inflammatory response in murine system. PLoS One 2010;5:e9380.
references.citation: Tan EC, Bahrami S, Kozlov AV, Kurvers HA, Ter Laak HJ, Nohl H, Redl H, Goris RJ. The oxidative response in the chronic constriction injury model of neuropathic pain. J Surg Res 2009;152:84-88.
references.citation: Thacker MA, Clark AK, Marchand F, McMahon SB. Pathophysiology of peripheral neuropathic pain: immune cells and molecules. Anesth Analg 2007;105:838-847.
references.citation: Tuñón MJ, Sánchez-Campos S, Gutiérrez B, Culebras JM, González-Gallego J. Effects of FK506 and rapamycin on generation of reactive oxygen species, nitric oxide production and nuclear factor kappa B activation in rat hepatocytes. Biochem Pharmacol 2003;66:439-445.
references.citation: Uceyler N, Sommer C. Wallerian degeneration and neuropathic pain. Drug Discov Today 2006;3:351-356.
references.citation: Viader A, Sasaki Y, Kim S, Strickland A, Workman CS, Yang K, Gross RW, Milbrandt J. Aberrant Schwann cell lipid metabolism linked to mitochondrial deficits leads to axon degeneration and neuropathy. Neuron 2013;77:886-898.
references.citation: Wang Y, Kawamura N, Schmelzer JD, Schmeichel AM, Low PA. Decreased peripheral nerve damage after ischemia reperfusion injury in mice lacking TNF-alpha. J Neurol Sci 2008;267:107-111.
references.citation: Webber C, Zochodne D. The nerve regenerative microenvironment: early behavior and partnership of axons and Schwann cells. Exp Neurol 2010;223:51-59.
references.citation: Woo HA, Yim SH, Shin DH, Kang D, Yu DY, Rhee SG. Inactivation of peroxiredoxin I by phosphorylation allows localized H(2)O(2) accumulation for cell signaling. Cell 2010;140:517-528.
references.citation: Wrabetz L, D'Antonio M, Pennuto M, Dati G, Tinelli E, Fratta P, Previtali S, Imperiale D, Zielasek J, Toyka K, Avila RL, Kirschner DA, Messing A, Feltri ML, Quattrini A. Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice. J Neurosci 2006;26:2358-2368.
references.citation: Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. PAIN® 1983;16:109-110.
references.citation: Zito E, Melo EP, Yang Y, Wahlander Å, Neubert TA, Ron D. Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin. Mol Cell 2010;40:787-797.