CoChat
Sign Up
Cited by 731Parkinson's Disease Mechanisms and TreatmentsAlzheimer's disease research and treatmentsNuclear Receptors and Signaling

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

Open access
Volpicelli-Daley, Laura A, Luk, Kelvin C, Lee, Virginia M-Y·2014·Nature Protocols
DOI 10.1038/nprot.2014.143PMC PMC4372899
Open this paper in CoChat — free account

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.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/nprot.2014.143

Cites

4 of 4 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Cellular and animal studies have provided a model for prion-like spread of α-synuclein from the gut to the brain via the enteric nervous system, chiefly through the vagus nerve.

    “Third, cellular and animal studies have provided a model for prion-like spread of α-synuclein from the gut to the brain via the enteric nervous system, chiefly through the vagus nerve [15–17].”

    From The gut–brain axis in early Parkinson’s disease: from prodrome to prevention (2025) · cites this paper as [15]

    The Introduction section on seeded aggregate formation (page not provided) describes α-synuclein fibrils seeding aggregates that spread within cultured neurons, supporting the cellular-model portion of the claim. The paper does not describe spread from the gut to the brain through the enteric nervous system or vagus nerve. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 38

  1. [1]McNaught KS Shashidharan P Perl DP Jenner P Olanow CW Aggresome-related biogenesis of Lewy bodies Eur J Neurosci 2002 16 2136 2148 12473081
  2. [2]Kopito RR Aggresomes, inclusion bodies and protein aggregation Trends Cell Biol 2000 10 524 530 11121744
  3. [3]Kramer ML Schulz-Schaeffer WJ Presynaptic alpha-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies J Neurosci 2007 27 1405 1410 17287515 PMC6673583
  4. [4]Luk KC Lee VM Modeling Lewy pathology propagation in Parkinson's disease Parkinsonism Relat Disord 2014 20 Suppl 1 S85 S87 24262196 PMC4034060
  5. [5]Bendor JT Logan TP Edwards RH The function of alpha-synuclein Neuron 2013 79 1044 1066 24050397 PMC3866954
  6. [6]Kaufman SK Diamond MI Prion-like propagation of protein aggregation and related therapeutic strategies Neurotherapeutics 2013 10 371 382 23801258 PMC3701767
  7. [7]George S Rey NL Reichenbach N Steiner JA Brundin P alpha-Synuclein: the long distance runner Brain Pathol 2013 23 350 357 23587141 PMC3674536
  8. [8]Masliah E Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders Science 2000 287 1265 1269 10678833
  9. [9]Giasson BI Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein Neuron 2002 34 521 533 12062037
  10. [10]Lee MK Human alpha-synuclein-harboring familial Parkinson's disease-linked Ala-53 -->Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice Proc Natl Acad Sci U S A 2002 99 8968 8973 12084935 PMC124407
  11. [11]Spillantini MG Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies Neurosci Lett 1998 251 205 208 9726379
  12. [12]Baba M Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies Am J Pathol 1998 152 879 884 9546347 PMC1858234
  13. [13]Fujiwara H alpha-Synuclein is phosphorylated in synucleinopathy lesions Nat Cell Biol 2002 4 160 164 11813001
  14. [14]Volpicelli-Daley LA Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death Neuron 2011 72 57 71 21982369 PMC3204802
  15. [15]Desplats P Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein Proc Natl Acad Sci U S A 2009 106 13010 13015 19651612 PMC2722313
  16. [17]Luk KC Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells Proc Natl Acad Sci U S A 2009 106 20051 20056 19892735 PMC2785290
  17. [18]Ren PH Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates Nat Cell Biol 2009 11 219 225 19151706 PMC2757079
  18. [20]Dryanovski DI Calcium entry and alpha-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons J Neurosci 2013 33 10154 10164 23761910 PMC3682382
  19. [21]Taguchi K Differential expression of alpha-synuclein in hippocampal neurons PLoS One 2014 9 e89327 24586691 PMC3934906
  20. [22]Buerli T Efficient transfection of DNA or shRNA vectors into neurons using magnetofection Nat Protoc 2007 2 3090 3101 18079708
  21. [23]Zeitelhofer M High-efficiency transfection of mammalian neurons via nucleofection Nat Protoc 2007 2 1692 1704 17641634
  22. [24]Jiang M Chen G High Ca2+-phosphate transfection efficiency in low-density neuronal cultures Nat Protoc 2006 1 695 700 17406298
  23. [25]Campenot RB Lund K Mok SA Production of compartmented cultures of rat sympathetic neurons Nat Protoc 2009 4 1869 1887 20010935
  24. [26]Luk KC Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice Science 2012 338 949 953 23161999 PMC3552321
  25. [27]Beaudoin GM 3rd Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex Nat Protoc 2012 7 1741 1754 22936216
  26. [28]Seibenhener ML Wooten MW Isolation and culture of hippocampal neurons from prenatal mice J Vis Exp 2012 PMC3476399 22871921
  27. [29]Irwin DJ Neuropathologic substrates of Parkinson disease dementia Ann Neurol 2012 72 587 598 23037886 PMC3484250
  28. [30]Churchyard A Lees AJ The relationship between dementia and direct involvement of the hippocampus and amygdala in Parkinson's disease Neurology 1997 49 1570 1576 9409348
  29. [31]Braak H Staging of brain pathology related to sporadic Parkinson's disease Neurobiol Aging 2003 24 197 211 12498954
  30. [33]Bousset L Structural and functional characterization of two alpha-synuclein strains Nat Commun 2013 4 2575 24108358 PMC3826637
  31. [34]Murphy DD Rueter SM Trojanowski JQ Lee VM Synucleins are developmentally expressed, and alpha-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons J Neurosci 2000 20 3214 3220 10777786 PMC6773130
  32. [36]Murray IV Role of alpha-synuclein carboxy-terminus on fibril formation in vitro Biochemistry 2003 42 8530 8540 12859200
  33. [37]Fauvet B alpha-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer J Biol Chem 2012 287 15345 15364 22315227 PMC3346117
  34. [38]Conway KA Harper JD Lansbury PT Jr Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid Biochemistry 2000 39 2552 2563 10704204
  35. [39]Kloepper KD Woods WS Winter KA George JM Rienstra CM Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms Protein Expr Purif 2006 48 112 117 16564705
  36. [40]Bellon A Decontamination of prions in a plasma product manufacturing environment Transfusion 2013 54 1028 1036 24032663
  37. [41]Murphy RG Alkaline hydrolysis of mouse-adapted scrapie for inactivation and disposal of prion-positive material J Anim Sci 2009 87 1787 1793 19098230
  38. [42]Luk KC Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice J Exp Med 2012 209 975 986 22508839 PMC3348112

Cited by

Sorted by citations

Authors
Volpicelli-Daley, L. A., Luk, K. C., & Lee, V. M.-Y.
Affiliations
University of Pennsylvania Health System (US)
ORCID
Venue
Nature Protocols
Publisher
Springer Science and Business Media LLC
Published
2014-08-14
Volume
9
Issue
9
Pages
2135-2146
ISSN
  • 1750-2799 (ISSN-L)
  • 1754-2189
DOI
10.1038/nprot.2014.143
PMC
PMC4372899
OpenAlex
W1989913002
Cited by
731
References
4
Citations by year
2026: 61 · 2025: 85 · 2024: 107 · 2023: 63 · 2022: 103 · 2021: 71 · 2020: 79 · 2019: 57 · 2018: 42 · 2017: 32 · 2016: 23 · 2015: 5 · 2014: 3
FWCI
13.70
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Protocols (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institute of Neurological Disorders and Stroke (P50NS053488, R01NS064934)
Topics
  • Health Sciences › Medicine › Neurology › Parkinson's Disease Mechanisms and Treatments
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Nuclear Receptors and Signaling
Keywords
alpha-synuclein, preformed fibrils, Lewy bodies, Lewy neurites, Parkinson's disease, synucleinopathies, primary neuronal cultures, hippocampal neurons, neuronal cell death
MeSH terms
Animals, Fluorescent Antibody Technique, Models, Biological, Immunoblotting, Neurites, Lewy Bodies, Cell Culture Techniques, Multiprotein Complexes, Mice, Rats, alpha-Synuclein

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review