Lee Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Center for Neurodegenerative Disease Research, Institute on Aging, Philadelphia, PA 19104-4283, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2009; 106(47):20051-6. DOI: 10.1073/pnas.0908005106
Source: PubMed

ABSTRACT Cytoplasmic inclusions containing alpha-synuclein (alpha-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although alpha-Syn fibrils can be generated from recombinant alpha-Syn protein in vitro, the production of fibrillar alpha-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular alpha-Syn aggregation can be triggered by the introduction of exogenously produced recombinant alpha-Syn fibrils into cultured cells engineered to overexpress alpha-Syn. Unlike unassembled alpha-Syn, these alpha-Syn fibrils "seeded" recruitment of endogenous soluble alpha-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular alpha-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of alpha-Syn fibrils can alter this balance by acting as seeds for aggregation.

Download full-text


Available from: Kelvin Luk, Feb 04, 2015
22 Reads
  • Source
    • "Several others were also able to verify those observations with synthetic fibrils (Danzer et al., 2009; Luk et al., 2009; Volpicelli- Daley et al., 2011) or by co-culture experiments using different CNS cell types (Hansen et al., 2011). Transmission to second order neurons through anterograde axonal transport by live-cell imaging and immunofluorescence was also observed (Freundt et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of fatal neurodegenerative disorders affecting several mammalian species being Creutzfeldt-Jacob Disease (CJD) the most representative in human beings, scrapie in ovine, Bovine Spongiform Encephalopathy (BSE) in bovine and Chronic Wasting Disease (CWD) in cervids. As stated by the "protein-only hypothesis", the causal agent of TSEs is a self-propagating aberrant form of the prion protein (PrP) that through a misfolding event acquires a β-sheet rich conformation known as PrP(Sc) (from scrapie). This isoform is neurotoxic, aggregation prone and induces misfolding of native cellular PrP. Compelling evidence indicates that disease-specific protein misfolding in amyloid deposits could be shared by other disorders showing aberrant protein aggregates such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic lateral sclerosis (ALS) and systemic Amyloid A amyloidosis (AA amyloidosis). Evidences of shared mechanisms of the proteins related to each disease with prions will be reviewed through the available in vivo models. Taking prion research as reference, typical prion-like features such as seeding and propagation ability, neurotoxic species causing disease, infectivity, transmission barrier and strain evidences will be analyzed for other protein-related diseases. Thus, prion-like features of amyloid β peptide and tau present in AD, α-synuclein in PD, SOD-1, TDP-43 and others in ALS and serum α-amyloid (SAA) in systemic AA amyloidosis will be reviewed through models available for each disease. Copyright © 2015. Published by Elsevier B.V.
    Virus Research 04/2015; 207. DOI:10.1016/j.virusres.2015.04.014 · 2.32 Impact Factor
  • Source
    • "␣-synuclein intracellular inclusions underwent several modifications characteristic of LBs, such as hyperphosphorylation, ubiquitination and accumulation of cytoplasmic vesicles around the periphery of the inclusions. Moreover, exogenous fibrils were shown to rapidly recruit and convert endogenous soluble protein into detergentinsoluble inclusions closely resembling LBs, thus suggesting that fibrillar seeds may have a central role in the initial formation of LBs and other disease-associated inclusions (Luk et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transmissible spongiform encephalopathies (TSE) are a heterogeneous group of neurodegenerative disorders. The common feature of these diseases is the pathological conversion of the normal cellular prion protein (PrP(C)) into a β-structure-rich conformer-termed PrP(Sc). The latter can induce a self-perpetuating process leading to amplification and spreading of pathological protein assemblies. Much evidence suggests that PrP(Sc) itself is able to recruit and misfold PrP(C) into the pathological conformation. Recent data have shown that recombinant PrP(C) can be misfolded in vitro and the resulting synthetic conformers are able to induce the conversion of PrP(C) into PrP(Sc)in vivo. In this review we describe the state-of-the-art of the body of literature in this field. In addition, we describe a cell-based assay to test synthetic prions in cells, providing further evidence that synthetic amyloids are able to template conversion of PrP into prion inclusions. Studying prions might help to understand the pathological mechanisms governing other neurodegenerative diseases. Aggregation and deposition of misfolded proteins is a common feature of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and other disorders. Although the proteins implicated in each of these diseases differ, they share a common prion mechanism. Recombinant proteins are able to aggregate in vitro into β-rich amyloid fibrils, sharing some features of the aggregates found in the brain. Several studies have reported that intracerebral inoculation of synthetic aggregates lead to unique pathology, which spread progressively to distal brain regions and reduced survival time in animals. Here, we review the prion-like features of different proteins involved in neurodegenerative disorders, such as α-synuclein, superoxide dismutase-1, amyloid-β and tau. Copyright © 2014. Published by Elsevier B.V.
    Virus Research 10/2014; DOI:10.1016/j.virusres.2014.10.020 · 2.32 Impact Factor
  • Source
    • "In cell assays, AD- and PD-associated protein aggregates show similarities to prions in terms of (i) binding to or uptake by cells of Aβ [96], tau [28, 63] or α-synuclein aggregates [63, 91], (ii) seeding in or on cells of Aβ [96], tau [63, 80] or α-synuclein aggregates [54, 63, 91], and (iii) intercellular spread of tau [28] and α-synuclein deposition [34]. In contrast with prions, aggregated Aβ and α-synuclein species have not yet been reported to show sustained self-propagation in multiple serial cell culture passages. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The misfolding and aggregation of endogenous proteins in the central nervous system is a neuropathological hallmark of Alzheimer's disease (AD), Parkinson's disease (PD), as well as prion diseases. A molecular mechanism referred to as "nucleation-dependent aggregation" is thought to underlie this neuropathological phenomenon. According to this concept, disease-associated protein particles act as nuclei, or seeds, that recruit cellular proteins and incorporate them, in a misfolded form, into their growing aggregate structure. Experimental studies have shown that the aggregation of the AD-associated proteins amyloid-β (Aβ) and tau, and of the PD-associated protein α-synuclein, can be stimulated in laboratory animal models by intracerebral (i.c.) injection of inocula containing aggregated species of the respective proteins. This has raised the question of whether AD or PD can be transmitted, like certain human prion diseases, between individuals by self-propagating protein particles potentially present on medical instruments or in blood or blood products. While the i.c. injection of inocula containing AD- or PD-associated protein aggregates was found to cause neuronal damage and clinical abnormalities (e.g., motor impairments) in some animal models, none of the studies published so far provided evidence for a transmission of severe or even fatal disease. In addition, available epidemiological data do not indicate a transmissibility of AD or PD between humans. The findings published so far on the effects of experimentally transmitted AD- or PD-associated protein seeds do not suggest specific precautionary measures in the context of hemotherapy, but call for vigilance in transfusion medicine and other medical areas.
    Acta Neuropathologica 07/2014; 128(4). DOI:10.1007/s00401-014-1324-9 · 10.76 Impact Factor
Show more