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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
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
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).
Kopito, R.R. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10, 524–530 (2000).
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).
Luk, K.C. & Lee, V.M. Modeling Lewy pathology propagation in Parkinson's disease. Parkinsonism Relat. Disord. 20 (suppl. 1), S85–S87 (2014).
Bendor, J.T., Logan, T.P. & Edwards, R.H. The function of α-synuclein. Neuron 79, 1044–1066 (2013).
Kaufman, S.K. & Diamond, M.I. Prion-like propagation of protein aggregation and related therapeutic strategies. Neurotherapeutics 10, 371–382 (2013).
George, S., Rey, N.L., Reichenbach, N., Steiner, J.A. & Brundin, P. α-Synuclein: the long distance runner. Brain Pathol. 23, 350–357 (2013).
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).
Masliah, E. et al. Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders. Science 287, 1265–1269 (2000).
Giasson, B.I. et al. Neuronal α-synucleinopathy with severe movement disorder in mice expressing A53T human α-synuclein. Neuron 34, 521–533 (2002).
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).
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).
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).
Fujiwara, H. et al. α-Synuclein is phosphorylated in synucleinopathy lesions. Nat. Cell Biol. 4, 160–164 (2002).
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).
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).
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).
Ren, P.H. et al. Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. Nat. Cell Biol. 11, 219–225 (2009).
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).
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).
Taguchi, K. et al. Differential expression of α-synuclein in hippocampal neurons. PLoS ONE 9, e89327 (2014).
Buerli, T. et al. Efficient transfection of DNA or shRNA vectors into neurons using magnetofection. Nat. Protoc. 2, 3090–3101 (2007).
Zeitelhofer, M. et al. High-efficiency transfection of mammalian neurons via nucleofection. Nat. Protoc. 2, 1692–1704 (2007).
Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).
Campenot, R.B., Lund, K. & Mok, S.A. Production of compartmented cultures of rat sympathetic neurons. Nat. Protoc. 4, 1869–1887 (2009).
Luk, K.C. et al. Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338, 949–953 (2012).
Beaudoin, G.M. III . et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741–1754 (2012).
Seibenhener, M.L. & Wooten, M.W. Isolation and culture of hippocampal neurons from prenatal mice. J. Vis. Exp. 10.3791/3634 (26 July 2012).
Irwin, D.J. et al. Neuropathologic substrates of Parkinson disease dementia. Ann. Neurol. 72, 587–598 (2012).
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).
Braak, H. et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging 24, 197–211 (2003).
Banker, G.A. Trophic interactions between astroglial cells and hippocampal neurons in culture. Science 209, 809–810 (1980).
Bousset, L. et al. Structural and functional characterization of two α-synuclein strains. Nat. Commun. 4, 2575 (2013).
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).
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).
Murray, I.V. et al. Role of α-synuclein carboxy-terminus on fibril formation in vitro. Biochemistry 42, 8530–8540 (2003).
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).
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).
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).
Bellon, A. et al. Decontamination of prions in a plasma product manufacturing environment. Transfusion 54, 1028–1036 (2013).
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).
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).
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
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
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
About this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2014.143
This article is cited by
-
In vitro modulation of mTOR and mGlur5 influence α-synuclein accumulation
Molecular Brain (2024)
-
Neuropathogenesis-on-chips for neurodegenerative diseases
Nature Communications (2024)
-
HTRA1 disaggregates α-synuclein amyloid fibrils and converts them into non-toxic and seeding incompetent species
Nature Communications (2024)
-
Transcriptomic profiling of early synucleinopathy in rats induced with preformed fibrils
npj Parkinson's Disease (2024)
-
Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils
Acta Neuropathologica Communications (2023)
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.