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Atg7 Knockout Alleviated the Axonal Injury of Neuro-2a Cells Induced by Tri-Ortho-Cresyl Phosphate

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Abstract

Autophagy is believed to be essential for the maintenance of axonal homeostasis in neurons. However, whether autophagy is causally related to the axon degeneration in organophosphorus-induced delayed neuropathy (OPIDN) still remains unclear. This research was designed to investigate the role of autophagy in axon degeneration following tri-ortho-cresyl phosphate (TOCP) in an in vitro model. Differentiated wild-type and Atg7−/− neuro-2a (N2a) cells were treated with TOCP for 24 h. Axonal degeneration in N2a cells was quantitatively analyzed; the key molecules responsible for axon degeneration and its upstream signaling pathway were determined by Western blotting and real-time PCR. The results found that Atg7−/− cells exhibited a higher resistance to TOCP insult than wild-type cells. Further study revealed that TOCP caused a significant decrease in pro-survival factors NMNATs and SCG10 and a significant increase in pro-degenerative factor SARM1 in both cells. Notably, Atg7−/− cells presented a higher level of pro-survival factors and a lower level of pro-degenerative factors than wild-type cells in the same setting of TOCP administration. Moreover, DLK-MAPK pathway was activated following TOCP. Altogether, our results suggest that autophagy is able to affect TOCP-induced axonal injury via regulating the balance between pro-survival and pro-degenerative factors, providing a promising avenue for the potential therapy for OPIDN patients.

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References

  • Abou-Donia MB (1981) Organophosphorus ester-induced delayed neurotoxicity. Annu Rev Pharmacol Toxicol 21:511–548

    Article  CAS  PubMed  Google Scholar 

  • Abou-Donia MB (1995) Involvement of Cytoskeletal Proteins in the Mechanisms of Organophosphorus Ester-Induced Delayed Neurotoxicity. Clin Exp Pharmacol 22:358–359

  • Araki T, Sasaki Y, Milbrandt J (2004) Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 305:1010–1013

    Article  CAS  PubMed  Google Scholar 

  • Beirowski B, Adalbert R, Wagner D, Grumme DS, Addicks K, Ribchester RR, Coleman MP (2005) The progressive nature of Wallerian degeneration in wild-type and slow Wallerian degeneration (WldS) nerves. BMC Neurosci 6:6

    Article  PubMed  PubMed Central  Google Scholar 

  • Bertolote JM, Fleischmann A, Eddleston M, Gunnell D (2006) Deaths from pesticide poisoning: a global response. The British journal of psychiatry : the journal of mental science 189:201–203

    Article  CAS  Google Scholar 

  • Bischoff A (1970) Ultrastructure of tri-ortho-cresyl phosphate poisoning in the chicken. II. Studies on spinal cord alterations. Acta Neuropathol 15:142–155

    Article  CAS  PubMed  Google Scholar 

  • Bouldin TW, Cavanagh JB (1979a) Organophosphorus neuropathy. I. A teased-fiber study of the spatio-temporal spread of axonal degeneration. Am J Pathol 94:241–252

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bouldin TW, Cavanagh JB (1979b) Organophosphorus neuropathy. II. A fine-structural study of the early stages of axonal degeneration. Am J Pathol 94:253–270

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chen JX, Sun YJ, Wang P, Long DX, Li W, Li L, Wu YJ (2013) Induction of autophagy by TOCP in differentiated human neuroblastoma cells lead to degradation of cytoskeletal components and inhibition of neurite outgrowth. Toxicology 310:92–97

    Article  CAS  PubMed  Google Scholar 

  • Cheng HC, Kim SR, Oo TF, Kareva T, Yarygina O, Rzhetskaya M, Wang C, During M, Talloczy Z, Tanaka K, Komatsu M, Kobayashi K, Okano H, Kholodilov N, Burke RE (2011) Akt suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy. J Neurosci 31:2125–2135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Classen W, Gretener P, Rauch M, Weber E, Krinke GJ (1996) Susceptibility of various areas of the nervous system of hens to TOCP-induced delayed neuropathy. Neurotoxicology 17:597–604

    CAS  PubMed  Google Scholar 

  • Conforti L, Gilley J, Coleman MP (2014) Wallerian degeneration: an emerging axon death pathway linking injury and disease. Nat Rev Neurosci 15:394–409

    Article  CAS  PubMed  Google Scholar 

  • Essuman K, Summers DW, Sasaki Y, Mao X, DiAntonio A, Milbrandt J (2017) The SARM1 toll/interleukin-1 receptor domain possesses intrinsic NAD+ cleavage activity that promotes pathological axonal degeneration. Neuron 93(1334–1343):e1335

    Google Scholar 

  • Gerdts J, Brace EJ, Sasaki Y, DiAntonio A, Milbrandt J (2015) SARM1 activation triggers axon degeneration locally via NAD(+) destruction. Science 348:453–457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gerdts J, Summers DW, Milbrandt J, DiAntonio A (2016) Axon self-destruction: new links among SARM1, MAPKs, and NAD+ metabolism. Neuron 89:449–460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gerdts J, Summers DW, Sasaki Y, DiAntonio A, Milbrandt J (2013) Sarm1-mediated axon degeneration requires both SAM and TIR interactions. J Neurosci 33:13569–13580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gilley J, Coleman MP (2010) Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons. PLoS Biol 8:e1000300

    Article  PubMed  PubMed Central  Google Scholar 

  • Grenningloh G, Soehrman S, Bondallaz P, Ruchti E, Cadas H (2004) Role of the microtubule destabilizing proteins SCG10 and stathmin in neuronal growth. J Neurobiol 58:60–69

    Article  CAS  PubMed  Google Scholar 

  • Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441:885–889

    Article  CAS  PubMed  Google Scholar 

  • Harvey MJ, Sharma RP (1980) Organophosphate cytotoxicity: the effects on protein metabolism in cultured neuroblastoma cells. J Environ Pathol Toxicol 3:423–436

    CAS  PubMed  Google Scholar 

  • Hausherr V, van Thriel C, Krug A, Leist M, Schobel N (2014) Impairment of glutamate signaling in mouse central nervous system neurons in vitro by tri-ortho-cresyl phosphate at noncytotoxic concentrations. Toxicol Sci 142:274–284

    Article  CAS  PubMed  Google Scholar 

  • Johnson GL, Lapadat R (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298:1911–1912

    Article  CAS  PubMed  Google Scholar 

  • Jortner BS, Ehrich M (1987) Neuropathological effects of phenyl saligenin phosphate in chickens. Neurotoxicology 8:303–314

    CAS  PubMed  Google Scholar 

  • Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441:880–884

    Article  CAS  PubMed  Google Scholar 

  • Levine B (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lotti M (1991) The pathogenesis of organophosphate polyneuropathy. Crit Rev Toxicol 21:465–487

    Article  CAS  PubMed  Google Scholar 

  • Lunn ER, Perry VH, Brown MC, Rosen H, Gordon S (1989) Absence of Wallerian degeneration does not hinder regeneration in peripheral nerve. Eur J Neurosci 1:27–33

    Article  CAS  PubMed  Google Scholar 

  • Mack TG, Reiner M, Beirowski B, Mi W, Emanuelli M, Wagner D, Thomson D, Gillingwater T, Court F, Conforti L, Fernando FS, Tarlton A, Andressen C, Addicks K, Magni G, Ribchester RR, Perry VH, Coleman MP (2001) Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci 4:1199–1206

    Article  CAS  PubMed  Google Scholar 

  • Miller BR, Press C, Daniels RW, Sasaki Y, Milbrandt J, DiAntonio A (2009) A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat Neurosci 12:387–389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741

    Article  CAS  PubMed  Google Scholar 

  • Osterloh JM, Yang J, Rooney TM, Fox AN, Adalbert R, Powell EH, Sheehan AE, Avery MA, Hackett R, Logan MA, MacDonald JM, Ziegenfuss JS, Milde S, Hou YJ, Nathan C, Ding A, Brown RH Jr, Conforti L, Coleman M, Tessier-Lavigne M, Zuchner S, Freeman MR (2012) dSarm/Sarm1 is required for activation of an injury-induced axon death pathway. Science 337:481–484

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Raff MC, Whitmore AV, Finn JT (2002) Axonal self-destruction and neurodegeneration. Science 296:868–871

    Article  CAS  PubMed  Google Scholar 

  • Ravikumar B, Duden R, Rubinsztein DC (2002) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet 11:1107–1117

    Article  CAS  PubMed  Google Scholar 

  • Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, Massey DC, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqi FH, Underwood BR, Winslow AR, Rubinsztein DC (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90:1383–1435

    Article  CAS  PubMed  Google Scholar 

  • Shin JE, Miller BR, Babetto E, Cho Y, Sasaki Y, Qayum S, Russler EV, Cavalli V, Milbrandt J, DiAntonio A (2012) SCG10 is a JNK target in the axonal degeneration pathway. Proc Natl Acad Sci U S A 109:E3696-3705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song F, Han X, Zeng T, Zhang C, Zou C, Xie K (2012) Changes in beclin-1 and micro-calpain expression in tri-ortho-cresyl phosphate-induced delayed neuropathy. Toxicol Lett 210:276–284

    Article  CAS  PubMed  Google Scholar 

  • Song FY, Kou RR, Zou CS, Gao Y, Zeng T, Xie KQ (2014) Involvement of autophagy in tri-ortho-cresyl phosphate- induced delayed neuropathy in hens. Neurochem Int 64:1–8

    Article  CAS  PubMed  Google Scholar 

  • Summers DW, Milbrandt J, DiAntonio A (2018) Palmitoylation enables MAPK-dependent proteostasis of axon survival factors. Proc Natl Acad Sci U S A 115:E8746–E8754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tanida I, Ueno T, Kominami E (2004) LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 36:2503–2518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vahsen BF, Ribas VT, Sundermeyer J, Boecker A, Dambeck V, Lenz C, Shomroni O, Caldi Gomes L, Tatenhorst L, Barski E, Roser AE, Michel U, Urlaub H, Salinas G, Bahr M, Koch JC, Lingor P (2020) Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing. Cell Death Differ

  • Wakatsuki S, Tokunaga S, Shibata M, Araki T (2017) GSK3B-mediated phosphorylation of MCL1 regulates axonal autophagy to promote Wallerian degeneration. J Cell Biol 216:477–493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walker LJ, Summers DW, Sasaki Y, Brace EJ, Milbrandt J, DiAntonio A (2017) MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2. eLife 6

  • Waller A (1851) Experiments on the section of the glosso-pharyngeal and hypoglossal nerves of the frog, and observations of the alterations produced thereby in the structure of their primitive fibres. Edinb Med Surg J 76:369–376

    PubMed  PubMed Central  Google Scholar 

  • Wang Y, Song M, Song F (2018) Neuronal autophagy and axon degeneration. Cell Mol Life Sci 75:2389–2406

    Article  CAS  PubMed  Google Scholar 

  • Xie Y, Zhou B, Lin MY, Sheng ZH (2015) Progressive endolysosomal deficits impair autophagic clearance beginning at early asymptomatic stages in fALS mice. Autophagy 11:1934–1936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yan T, Feng Y, Zhai Q (2010) Axon degeneration: Mechanisms and implications of a distinct program from cell death. Neurochem Int 56:529–534

    Article  CAS  PubMed  Google Scholar 

  • Yang SH, Sharrocks AD, Whitmarsh AJ (2003) Transcriptional regulation by the MAP kinase signaling cascades. Gene 320:3–21

    Article  CAS  PubMed  Google Scholar 

  • Yang Y, Coleman M, Zhang L, Zheng X, Yue Z (2013) Autophagy in axonal and dendritic degeneration. Trends Neurosci 36:418–428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yorimitsu T, Klionsky DJ (2005) Autophagy: Molecular machinery for self-eating. Cell Death Differ 12:1542–1552

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Professor Masaaki Komatsu (Niigata University, Japan) for Atg7 knockout N2a cells.

Funding

This work was supported by National Natural Science Foundation of China (No. 81673209).

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  1. Cuiqin Zhang and Kang Kang contributed equally to this work and should be considered co-first authors.

Authors and Affiliations

  1. Institute of Toxicology, Cheeloo College of Medicine, Shandong University, 44 West Wenhua Road, Jinan, Shandong, 250012, People’s Republic of China

    Cuiqin Zhang, Kang Kang, Yisi Chen, Shulin Shan, Keqin Xie & Fuyong Song

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  1. Cuiqin Zhang
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Correspondence to Fuyong Song.

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Zhang, C., Kang, K., Chen, Y. et al. Atg7 Knockout Alleviated the Axonal Injury of Neuro-2a Cells Induced by Tri-Ortho-Cresyl Phosphate. Neurotox Res 39, 1076–1086 (2021). https://doi.org/10.1007/s12640-021-00344-y

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