Translational Stroke Research

, Volume 9, Issue 3, pp 201–213 | Cite as

Inactivation of NSF ATPase Leads to Cathepsin B Release After Transient Cerebral Ischemia

  • Dong Yuan
  • Chunli Liu
  • Jiang Wu
  • Bingren HuEmail author
Original Article


Neurons have extraordinary large cell membrane surface area, thus requiring extremely high levels of intracellular membrane-trafficking activities. Consequently, defects in the membrane-trafficking activities preferentially affect neurons. A critical molecule for controlling the membrane-trafficking activities is the N-ethylmaleimide-sensitive factor (NSF) ATPase. This study is to investigate the cascade of events of NSF ATPase inactivation, resulting in a massive buildup of late endosomes (LEs) and fatal release of cathepsin B (CTSB) after transient cerebral ischemia using the 2-vessel occlusion with hypotension (2VO+Hypotension) global brain ischemia model. Rats were subjected to 20 min of transient cerebral ischemia followed by 0.5, 4, 24, and 72 h of reperfusion. Neuronal histopathology and ultrastructure were examined by the light and electron microscopy, respectively. Western blotting and confocal microscopy were utilized for analyzing the levels, redistribution, and co-localization of Golgi apparatus and endosome or lysosome markers. Transient cerebral ischemia leads to delayed neuronal death that occurs at 48–72 h of reperfusion mainly in hippocampal CA1 and neocortical (Cx) layers 3 and 5 pyramidal neurons. During the delayed period, NSF ATPase is irreversibly trapped into inactive protein aggregates selectively in post-ischemic neurons destined to die. NSF inactivation leads to a massive buildup of Golgi fragments, transport vesicles (TVs) and late endosomes (LEs), and release of the 33 kDa LE type of CTSB, which is followed by delayed neuronal death after transient cerebral ischemia. The results support a novel hypothesis that transient cerebral ischemia leads to NSF inactivation, resulting in a cascade of events of fatal release of CTSB and delayed neuronal death after transient cerebral ischemia.


N-ethylmaleimide sensitive factor ATPase (NSF) SNAREs Brain ischemia-reperfusion injury Membrane trafficking  Cathepsin B (CTSB) Golgi fragments Transport vesicle Late endosome Lysosome. 



N-ethylmaleimide sensitive factor ATPase


Soluble NSF attachment protein receptors


Soluble NSF attachment protein


Cathepsin B


Transport vesicles


Late endosome






Mitochondrial outer membrane permeabilization


Ischemia-reperfusion injury


Dentate gyrus


Electron microscopy


Vesicle transport through interaction with t-SNAREs homolog 1B


Trans-Golgi network membrane protein 38 kDa



This work was supported by National Institutes of Health (NIH) grants: NS36810, NS40407, and NS097875; by Veteran Affair Merit grant: I01BX001696; and by the American Heart Association 0940042N-5 to B.R.H.

Compliance with Ethical Standards

Conflict of Interest

Dong Yuan, Chunli Liu, and Bingren Hu declare no conflict of interest.

Ethical Approval

This article does not contain any studies with human subjects. All the experimental procedures involving using animals were approved by the Animal Use and Care Committee in the University of Maryland School of Medicine.


  1. 1.
    Smith ML, Auer RN, Siesjo BK. The density and distribution of ischemic brain injury in the rat following 2–10 min of forebrain ischemia. Acta Neuropathol (Berl). 1984;64:319–32.CrossRefGoogle Scholar
  2. 2.
    Wang W, Redecker C, Bidmon HJ, Witte OW. Delayed neuronal death and damage of GDNF family receptors in CA1 following focal cerebral ischemia. Brain Res. 2004;1023:92–101.CrossRefPubMedGoogle Scholar
  3. 3.
    Horn M, Schlote W. Delayed neuronal death and delayed neuronal recovery in the human brain following global ischemia. Acta Neuropathol. 1992;85:79–87.CrossRefPubMedGoogle Scholar
  4. 4.
    Kirino T, Sano K. Fine structural nature of delayed neuronal death following ischemia in the gerbil hippocampus. Acta Neuropathol. 1984;62:209–18.CrossRefPubMedGoogle Scholar
  5. 5.
    Hu BR, Martone ME, Jones YZ, Liu CL. Protein aggregation after transient cerebral ischemia. The Journal of Neuroscience. 2000;20:3191–1999.CrossRefPubMedGoogle Scholar
  6. 6.
    Hu BR, Janelidze S, Ginsberg MD, Busto R, Perez-Pinzon M, Sick TJ, et al. Protein aggregation after focal brain ischemia and reperfusion. J Cereb Blood Flow Metab. 2001;21:865–75.CrossRefPubMedGoogle Scholar
  7. 7.
    Liu CL, Ge P, Zhang F, Hu BR. Co-translational protein aggregation after transient cerebral ischemia. Neurosci. 2005;134:1273–84.Google Scholar
  8. 8.
    Zhang F, Liu CL, Hu BR. Irreversible aggregation of protein synthesis machinery after focal brain ischemia. J Neurochem. 2005;98:102–12.CrossRefGoogle Scholar
  9. 9.
    Wang D, Chan CC, Cherry S, Hiesinger PR. Membrane trafficking in neuronal maintenance and degeneration. Cell Mol Life Sci. 2013;70(16):2919–34.CrossRefPubMedGoogle Scholar
  10. 10.
    Yuan D, Liu C, Hu B. Dysfunction of membrane trafficking leads to CTSB release and brain ischemia-reperfusion injury. Transl Stroke Res. 2017; in press.Google Scholar
  11. 11.
    Morgan A, Burgoyne RD. Is NSF a fusion protein? Trends Cell Biol. 1995;5:335–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Mohtashami M, Stewart BA, Boulianne GL, Trimble WS. Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behaviouralparalysis. J Neurochem. 2001;77:1407–17.CrossRefPubMedGoogle Scholar
  13. 13.
    Robinson LJ, Aniento F, Gruenberg J. NSF is required for transport from early to late endosomes. J Cell Sci. 1997;110:2079–87.PubMedGoogle Scholar
  14. 14.
    Dalal S, Rosser MFN, Cyr DM, Hanson PI. Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway. Glick B, ed. Mol Biol Cell. 2004;15(2):637–48.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Offenhauser C, Lei N, Roy S, Collins BM, Stow JL, Murray RZ. Syntaxin 11 binds Vti1b and regulates late endosome to lysosome fusion in macrophages. Traffic. 2011;12:762–73.CrossRefPubMedGoogle Scholar
  16. 16.
    Luzio JP, Gray SR, Bright NA. Endosome-lysosome fusion. Biochem Soc Trans. 2010;38:1413–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Kunwar AJ, Rickmann M, Backofen B, Browski SM, Rosenbusch J, Schöning S, et al. Lack of the endosomal SNAREs vti1a and vti1b led to significant impairments in neuronal development. PNAS U S A. 2011;108:2575–80.CrossRefGoogle Scholar
  18. 18.
    Ponten U, Ratcheson RA, Salford L, Siesjö BK. Optimal freezing conditions for cerebral metabolites in rats. J. Neurochem. 1973;21:1127–38.CrossRefPubMedGoogle Scholar
  19. 19.
    Luo T, Roman P, Liu C, Sun X, Park Y, Hu B. Upregulation of the GEF-H1 Pathway after Transient Cerebral Ischemia. Experimental neurology. 2015;263:306–13.CrossRefPubMedGoogle Scholar
  20. 20.
    Brunger AT. Structure of proteins involved in synaptic vesicle fusion in neurons. Annu Rev. Biophys Biomol Struct. 2001;30:157–1571.CrossRefPubMedGoogle Scholar
  21. 21.
    Malhotra V, Orci L, Glick BS, Block MR, Rothman JE. Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack. Cell. 1988;54:221–7.CrossRefPubMedGoogle Scholar
  22. 22.
    May AP, Whiteheart SW, Weis WI. Unraveling the mechanism of the vesicle transport ATPase NSF, the N-ethylmaleimide-sensitive factor. J Biol Chem. 2001;276:21,991–4.CrossRefGoogle Scholar
  23. 23.
    Whiteheart SW, Matveeva EA. Multiple binding proteins suggest diverse functions for the N-ethylmaleimide sensitive factor. J Struct Biol. 2004;146:32–43.CrossRefPubMedGoogle Scholar
  24. 24.
    Brandon E, Szul T, Alvarez C, Grabski R, Benjamin R, Kawai R, et al. On and off membrane dynamics of the endoplasmic reticulum-golgi tethering factor p115 in vivo. Mol Biol Cell. 2006;17:2996–3008.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Liu CL, Hu BR. Alterations of N-Ethylmaleimide-Sensitive ATPase Following Transient Cerebral Ischemia. Neuroscience. 2004;128:767–74.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Antón-Fernández A, Aparicio-Torres G, Tapia S, DeFelipe J, Muñoz A. Morphometric alterations of Golgi apparatus in Alzheimer’s disease are related to tau hyperphosphorylation. Neurobiol Dis. 2017;97(Pt A):11–23.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Turk V, Stoka V, Vasiljeva O, Renko M, Sun T, Turk B, et al. Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta. 1824;2012:68–88.Google Scholar
  28. 28.
    Huotari J, Helenius A. Endosome maturation. EMBO J. 2011;30:3481–500.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Pungercar JR, Caglic D, Sajid M, Dolinar M, Vasiljeva O, Pozgan U, et al. Autocatalytic processing of procathepsin B is triggered by proenzyme activity. The FEBS journal. 2009;276:660–8.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Block MR, Glick BS, Wilcox CA, Wieland FT, Rothman JE. Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. Proc Natl Acad Sci U S A. 1988;85:7852–6.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Hong HK, Chakravarti A, Takahashi JS. The gene for soluble N-ethylmaleimide sensitive factor attachment protein {alpha} is mutated in hydrocephaly with hop gait (hyh) mice. Proc Natl Acad Sci U S A. 2004;101:1748–53.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Https:// Scholar
  33. 33.
    Diaz R, Mayorga LS, Weidman PJ, Rothman JE, Stahl PD. Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport. Nature. 1989;339:398–400.CrossRefPubMedGoogle Scholar
  34. 34.
    Wattenberg BW, Raub TJ, Hiebsch RR, Weidman PJ. The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation. J Cell Biol. 1992;118:1321–32.CrossRefPubMedGoogle Scholar
  35. 35.
    Acharya U, Jacobs R, Peters JM, Watson N, Farquhar MG, Malhotra V. The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion events. Cell. 1995;82:895–904.CrossRefPubMedGoogle Scholar
  36. 36.
    Naslavsky N, McKenzie J, Altan-Bonnet N, Sheff D, Caplan S. EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology. J Cell Sci. 2009;122:389–400.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Mullock BM, Bright NA, Fearon CW, Gray SR, Luzio J. Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent. J Cell Biol. 1998;140(3):591–601.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Petanceska S, Burke S, Watson SJ, Devi L. Differential distribution of messenger RNAs for cathepsins B, L and S in adult rat brain: an in situ hybridization study. Neuroscience. 1994;59:729–38.CrossRefPubMedGoogle Scholar
  39. 39.
    Gómez-Sintes R, Ledesma MD, Boya P. Lysosomal cell death mechanisms in aging. Ageing Res Rev. 2016;32:150–168.Google Scholar
  40. 40.
    Repnik U, Stoka V, Turk V, Turk B. Lysosomes and lysosomal cathepsins in cell death. Biochim Biophys Acta. 1824;2012:22–33.Google Scholar
  41. 41.
    Jakobson M, Jakobson M, Llano O, Palgi J, Arumäe U. Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons. Cell Death Dis. 2013;4:e777.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Akhtar RS, Ness JM, Roth KA. Bcl-2 family regulation of neuronal development and neurodegeneration. Biochim Biophys Acta. 1644;2004:189–203.Google Scholar
  43. 43.
    Serrano-Puebla A, Boya P. Lysosomal membrane permeabilization in cell death: new evidence and implications for health and disease. Ann N Y Acad Sci. 2016;1371:30–44.CrossRefPubMedGoogle Scholar
  44. 44.
    Prunell GF, Mathiesen T, Svendgaard NA. Experimental subarachnoid hemorrhage: cerebral blood flow and brain metabolism during the acute phase in three different models in the rat. Neurosurgery. 2004;54:426–36.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC (Outside the USA) 2017
corrected publication May/2018

Authors and Affiliations

  1. 1.Department of Neurology, The First Teaching HospitalJilin UniversityChangchunChina
  2. 2.Department of Anesthesiology and Neurology, Shock Trauma and Anesthesiology Research CenterUniversity of Maryland School of MedicineBaltimoreUSA
  3. 3.Veterans Affairs Maryland Health Center SystemBaltimoreUSA

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