Advertisement

Measuring Mitochondrial Dysfunction Caused by Soluble α-Synuclein Oligomers

  • Eric S. LuthEmail author
  • Irina G. Stavrovskaya
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1948)

Abstract

Accumulation of misfolded αSyn and mitochondrial dysfunction are central features of Parkinson’s disease. Growing evidence points to a relationship between these two phenomena as oligomeric α-synuclein (αSyn) can interact with mitochondria and impair their function. Standardization of methods to prepare αSyn oligomers and isolate functional mitochondria will facilitate efforts to expand upon early findings. Here we present detailed protocols for preparing soluble αSyn oligomers; for isolating functional mitochondria from mouse tissue; and for simultaneously measuring several aspects of mitochondrial physiology. These protocols will benefit future studies aimed at characterizing the mitotoxicity of αSyn species isolated from the brains of synucleinopathy patients as well as efforts to identify small molecules and genetic or environmental alterations that prevent αSyn-induced mitochondrial dysfunction.

Key words

α-Synuclein Oligomer Mitochondria Mitochondrial dysfunction Calcium retention Parkinson’s disease 

References

  1. 1.
    Bartels T, Choi JG, Selkoe DJ (2011) α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature 477:107–110CrossRefGoogle Scholar
  2. 2.
    Westphal CH, Chandra SS (2013) Monomeric synucleins generate membrane curvature. J Biol Chem 288:1829–1840CrossRefGoogle Scholar
  3. 3.
    Luth ES, Bartels T, Dettmer U et al (2015) Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity. Biochemistry 54:279–292CrossRefGoogle Scholar
  4. 4.
    Gould N, Mor DE, Lightfoot R et al (2014) Evidence of native α-synuclein conformers in the human brain. J Biol Chem 289:7929–7934CrossRefGoogle Scholar
  5. 5.
    Wang L, Das U, Scott DA et al (2014) α-Synuclein multimers cluster synaptic vesicles and attenuate recycling. Curr Biol 24:2319–2326CrossRefGoogle Scholar
  6. 6.
    Iljina M, Tosatto L, Choi ML et al (2016) Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein. Sci Rep 6:33928CrossRefGoogle Scholar
  7. 7.
    Chartier-Harlin M-C, Kachergus J, Roumier C et al (2004) Alpha-synuclein locus duplication as a cause of familial Parkinson’s disease. Lancet (London, England) 364:1167–1169CrossRefGoogle Scholar
  8. 8.
    Singleton AB, Farrer M, Johnson J et al (2003) Alpha-Synuclein locus triplication causes Parkinson’s disease. Science 302:841–841CrossRefGoogle Scholar
  9. 9.
    Kara E, Lewis PA, Ling H et al (2013) α-Synuclein mutations cluster around a putative protein loop. Neurosci Lett 546:67–70CrossRefGoogle Scholar
  10. 10.
    Spillantini MG, Schmidt ML, Lee VM et al (1997) Alpha-synuclein in Lewy bodies. Nature 388:839–840CrossRefGoogle Scholar
  11. 11.
    Dettmer U, Newman AJ, Soldner F et al (2015) Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation. Nat Commun 6:7314CrossRefGoogle Scholar
  12. 12.
    Danzer KM, Haasen D, Karow AR et al (2007) Different species of alpha-synuclein oligomers induce calcium influx and seeding. J Neurosci 27:9220–9232CrossRefGoogle Scholar
  13. 13.
    Winner B, Jappelli R, Maji SK et al (2011) In vivo demonstration that alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A 108:4194–4199CrossRefGoogle Scholar
  14. 14.
    Karpinar DP, Balija MBG, Kügler S et al (2009) Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson’s disease models. EMBO J 28:3256–3268CrossRefGoogle Scholar
  15. 15.
    Volles MJ, Lansbury PT (2003) Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson’s disease. Biochemistry 42:7871–7878CrossRefGoogle Scholar
  16. 16.
    Fusco G, Chen SW, Williamson PTF et al (2017) Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers. Science 358:1440–1443CrossRefGoogle Scholar
  17. 17.
    Schapira AH, Cooper JM, Dexter D et al (1989) Mitochondrial complex I deficiency in Parkinson’s disease. Lancet (London, England) 1:1269CrossRefGoogle Scholar
  18. 18.
    Parker WD, Boyson SJ, Parks JK (1989) Abnormalities of the electron transport chain in idiopathic Parkinson’s disease. Ann Neurol 26:719–723CrossRefGoogle Scholar
  19. 19.
    Keeney PM, Xie J, Capaldi RA et al (2006) Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 26:5256–5264CrossRefGoogle Scholar
  20. 20.
    Devi L, Raghavendran V, Prabhu BM et al (2008) Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem 283:9089–9100CrossRefGoogle Scholar
  21. 21.
    Martin LJ, Pan Y, Price AC et al (2006) Parkinson’s disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death. J Neurosci 26:41–50CrossRefGoogle Scholar
  22. 22.
    Stichel CC, Zhu X-R, Bader V et al (2007) Mono- and double-mutant mouse models of Parkinson’s disease display severe mitochondrial damage. Hum Mol Genet 16:2377–2393CrossRefGoogle Scholar
  23. 23.
    Hsu LJ, Sagara Y, Arroyo A et al (2000) Alpha-synuclein promotes mitochondrial deficit and oxidative stress. Am J Pathol 157:401–410CrossRefGoogle Scholar
  24. 24.
    Büttner S, Habernig L, Broeskamp F et al (2013) Endonuclease G mediates α-synuclein cytotoxicity during Parkinson’s disease. EMBO J 32:3041–3054CrossRefGoogle Scholar
  25. 25.
    Parihar MS, Parihar A, Fujita M et al (2009) Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells. Int J Biochem Cell Biol 41:2015–2024CrossRefGoogle Scholar
  26. 26.
    Shavali S, Brown-Borg HM, Ebadi M et al (2008) Mitochondrial localization of alpha-synuclein protein in alpha-synuclein overexpressing cells. Neurosci Lett 439:125–128CrossRefGoogle Scholar
  27. 27.
    Luth ES, Stavrovskaya IG, Bartels T et al (2014) Soluble, prefibrillar α-synuclein oligomers promote complex I-dependent, Ca2+-induced mitochondrial dysfunction. J Biol Chem 289:21490–21507CrossRefGoogle Scholar
  28. 28.
    Luk KC, Song C, O’Brien P et al (2009) Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A 106:20051–20056CrossRefGoogle Scholar
  29. 29.
    Volpicelli-Daley LA, Luk KC, Patel TP et al (2011) Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72:57–71CrossRefGoogle Scholar
  30. 30.
    Luk KC, Kehm V, Carroll J et al (2012) Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338:949–953CrossRefGoogle Scholar
  31. 31.
    Polinski NK, Volpicelli-Daley LA, Sortwell CE et al (2018) Best practices for generating and using alpha-Synuclein pre-formed fibrils to model Parkinson’s disease in rodents. J Parkinson’s Dis 68:1–20Google Scholar
  32. 32.
    Stavrovskaya IG, Baranov SV, Guo X et al (2010) Reactive gamma-ketoaldehydes formed via the isoprostane pathway disrupt mitochondrial respiration and calcium homeostasis. Free Radic Biol Med 49:567–579CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiologySimmons UniversityBostonUSA
  2. 2.Department of NeurologyColumbia University Medical CenterNew YorkUSA

Personalised recommendations