Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite–like aggregates

This article has been updated

Abstract

This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of the Lewy bodies and Lewy neurites found in Parkinson's disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addition of preformed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds the recruitment of endogenous α-syn into aggregates characterized by detergent insolubility and hyperphosphorylation. Aggregate formation follows a lag phase of 2–3 d, followed by formation in axons by days 4–7, spread to somatodendritic compartments by days 7–10 and neuron death 14 d after PFF addition. Here we provide methods and highlight the crucial steps for PFF formation, PFF addition to cultured hippocampal neurons and confirmation of aggregate formation. Neurons derived from various brain regions from nontransgenic and genetically engineered mice and rats can be used, allowing interrogation of the effect of specific genes on aggregate formation.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Electron micrographs of PFFs before and after sonication.
Figure 3: Typical results seen at Step 12 after the sedimentation assay of PFFs.
Figure 4: Visualization of PFF-induced formation of α-syn aggregates using an antibody to p-α-syn.
Figure 5: Immunofluorescence of α-syn aggregates.
Figure 6: Visualization of exogenously added PFFs and p-α-syn aggregates formed from endogenous α-syn.
Figure 7: Typical immunoblotting results seen after sequential extraction of neurons in 1% (vol/vol) TX-100 followed by SDS.

Similar content being viewed by others

Change history

  • 01 September 2016

    Since the publication of this protocol, the safe handling procedures for α-synuclein fibrils have been updated (ref. 1). 1% SDS (wt/vol) should be used to inactivate α-synuclein fibrils in place of NaOH, which does not appear to work. We also recommend performing the sonication step in a biosafety level 2 hood. Please see ref. 1 for further information on the safe handling of fibrils. 1. Bousset, L. et al. An efficient procedure for removal and inactivation of alpha-synuclein assemblies from laboratory materials. J. Parkinsons Dis. 6, 143–151 (2016).

References

  1. McNaught, K.S., Shashidharan, P., Perl, D.P., Jenner, P. & Olanow, C.W. Aggresome-related biogenesis of Lewy bodies. Eur. J. Neurosci. 16, 2136–2148 (2002).

    Article  PubMed  Google Scholar 

  2. Kopito, R.R. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10, 524–530 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Kramer, M.L. & Schulz-Schaeffer, W.J. Presynaptic α-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies. J. Neurosci. 27, 1405–1410 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Luk, K.C. & Lee, V.M. Modeling Lewy pathology propagation in Parkinson's disease. Parkinsonism Relat. Disord. 20 (suppl. 1), S85–S87 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Bendor, J.T., Logan, T.P. & Edwards, R.H. The function of α-synuclein. Neuron 79, 1044–1066 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Kaufman, S.K. & Diamond, M.I. Prion-like propagation of protein aggregation and related therapeutic strategies. Neurotherapeutics 10, 371–382 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. George, S., Rey, N.L., Reichenbach, N., Steiner, J.A. & Brundin, P. α-Synuclein: the long distance runner. Brain Pathol. 23, 350–357 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Luk, K.C. et al. Exogenous α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc. Natl. Acad. Sci. USA 106, 20051–20056 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Masliah, E. et al. Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders. Science 287, 1265–1269 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Giasson, B.I. et al. Neuronal α-synucleinopathy with severe movement disorder in mice expressing A53T human α-synuclein. Neuron 34, 521–533 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Lee, M.K. et al. Human α-synuclein–harboring familial Parkinson's disease-linked Ala-53→Thr mutation causes neurodegenerative disease with α-synuclein aggregation in transgenic mice. Proc. Natl. Acad. Sci. USA 99, 8968–8973 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Spillantini, M.G. et al. Filamentous α-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies. Neurosci. Lett. 251, 205–208 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Baba, M. et al. Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies. Am. J. Pathol. 152, 879–884 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Fujiwara, H. et al. α-Synuclein is phosphorylated in synucleinopathy lesions. Nat. Cell Biol. 4, 160–164 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Volpicelli-Daley, L.A. et al. Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72, 57–71 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Desplats, P. et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of α-synuclein. Proc. Natl. Acad. Sci. USA 106, 13010–13015 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Frost, B., Jacks, R.L. & Diamond, M.I. Propagation of tau misfolding from the outside to the inside of a cell. J. Biol. Chem. 284, 12845–12852 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ren, P.H. et al. Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. Nat. Cell Biol. 11, 219–225 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tanik, S.A., Schultheiss, C.E., Volpicelli-Daley, L.A., Brunden, K.R. & Lee, V.M. Lewy body-like α-synuclein aggregates resist degradation and impair macroautophagy. J. Biol. Chem. 288, 15194–15210 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dryanovski, D.I. et al. Calcium entry and α-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons. J. Neurosci. 33, 10154–10164 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Taguchi, K. et al. Differential expression of α-synuclein in hippocampal neurons. PLoS ONE 9, e89327 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Buerli, T. et al. Efficient transfection of DNA or shRNA vectors into neurons using magnetofection. Nat. Protoc. 2, 3090–3101 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Zeitelhofer, M. et al. High-efficiency transfection of mammalian neurons via nucleofection. Nat. Protoc. 2, 1692–1704 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Campenot, R.B., Lund, K. & Mok, S.A. Production of compartmented cultures of rat sympathetic neurons. Nat. Protoc. 4, 1869–1887 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Luk, K.C. et al. Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338, 949–953 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Beaudoin, G.M. III . et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741–1754 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Seibenhener, M.L. & Wooten, M.W. Isolation and culture of hippocampal neurons from prenatal mice. J. Vis. Exp. 10.3791/3634 (26 July 2012).

  29. Irwin, D.J. et al. Neuropathologic substrates of Parkinson disease dementia. Ann. Neurol. 72, 587–598 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Churchyard, A. & Lees, A.J. The relationship between dementia and direct involvement of the hippocampus and amygdala in Parkinson's disease. Neurology 49, 1570–1576 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Braak, H. et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging 24, 197–211 (2003).

    Article  PubMed  Google Scholar 

  32. Banker, G.A. Trophic interactions between astroglial cells and hippocampal neurons in culture. Science 209, 809–810 (1980).

    Article  CAS  PubMed  Google Scholar 

  33. Bousset, L. et al. Structural and functional characterization of two α-synuclein strains. Nat. Commun. 4, 2575 (2013).

    Article  PubMed  Google Scholar 

  34. Murphy, D.D., Rueter, S.M., Trojanowski, J.Q. & Lee, V.M. Synucleins are developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons. J. Neurosci. 20, 3214–3220 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Giasson, B.I., Murray, I.V., Trojanowski, J.Q. & Lee, V.M. A hydrophobic stretch of 12 amino acid residues in the middle of α-synuclein is essential for filament assembly. J. Biol. Chem. 276, 2380–2386 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Murray, I.V. et al. Role of α-synuclein carboxy-terminus on fibril formation in vitro. Biochemistry 42, 8530–8540 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Fauvet, B. et al. α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer. J. Biol. Chem. 287, 15345–15364 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Conway, K.A., Harper, J.D. & Lansbury, P.T. Jr. Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid. Biochemistry 39, 2552–2563 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Kloepper, K.D., Woods, W.S., Winter, K.A., George, J.M. & Rienstra, C.M. Preparation of α-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms. Protein Expr. Purif. 48, 112–117 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Bellon, A. et al. Decontamination of prions in a plasma product manufacturing environment. Transfusion 54, 1028–1036 (2013).

    Article  PubMed  Google Scholar 

  41. Murphy, R.G. et al. Alkaline hydrolysis of mouse-adapted scrapie for inactivation and disposal of prion-positive material. J. Anim. Sci. 87, 1787–1793 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Luk, K.C. et al. Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. J. Exp. Med. 209, 975–986 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A.B. West for allowing us to create a of video of him demonstrating the sonication of the fibrils. We also thank the reviewers whose careful attention to details and suggestions greatly improved this protocol. This study was supported by US National Institutes of Health grant no. P50 NS053488 to V.M.-Y.L.

Author information

Authors and Affiliations

Authors

Contributions

L.A.V.-D. carried out the experiments that formed the basis of the protocol; K.C.L. provided the electron microscopy images of the sonicated fibrils; V.M.-Y.L. supervised the project; L.A.V.-D., K.C.L. and V.M.-Y.L. provided intellectual input that contributed to the development of the protocol; L.A.V.-D. wrote the paper; and K.C.L. and V.M.-Y.L. provided valuable editorial input.

Corresponding author

Correspondence to Laura A Volpicelli-Daley.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

41596_2014_BFnprot2014143_MOESM272_ESM.mp4

This video demonstrates sonication PFFs using a probe tip sonicator, a critical step for the success of this protocol. (MP4 28075 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Volpicelli-Daley, L., Luk, K. & Lee, VY. Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite–like aggregates. Nat Protoc 9, 2135–2146 (2014). https://doi.org/10.1038/nprot.2014.143

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2014.143

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing