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: 9.81). 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.

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    ABSTRACT: Human α-Synuclein (α-Syn) is a natively unstructured protein, whose aggregation into amyloid fibrils is associated with Parkinson's disease (PD) pathogenesis. The mutations of α-Syn, E46K, A53T and A30P have been linked to familial form of PD. The in vitro aggregation studies suggest that increased propensity to form non-fibrillar oligomers is the shared property of these familial PD associated mutants. However the structural basis of the altered aggregation propensities of these PD associated mutants is not yet clear. To understand this, we studied the site-specific structural dynamics of wild type (wt) α-Syn and its three PD mutants (A53T, E46K and A30P). Tryptophan (Trp) was substituted at the N-terminus, central hydrophobic region and C-terminus of all α-Syns. Using various biophysical techniques including time-resolved fluorescence studies, we show that irrespective of similar secondary structure and early oligomerization propensities, familial PD associated mutations alter the site-specific microenvironment, solvent exposure and conformational flexibility of the protein. Our results further show that the common structural feature of the three PD associated mutants is more compact and rigid sites at their N- and C-terminus compared to wt α-Syn, which may facilitate the formation of partially folded intermediate that eventually leads to their increased oligomerization propensities. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    The Journal of biological chemistry. 01/2015;
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    ABSTRACT: Formation and accumulation of misfolded protein aggregates are a central hallmark of several neurodegenerative diseases. In Parkinson's disease (PD), the aggregation-prone protein alpha-synuclein (α-syn) is the culprit. In the past few years, another piece of the puzzle has been added with data suggesting that α-syn may self-propagate, thereby contributing to the progression and extension of PD. Of particular importance, it was the seminal observation of Lewy bodies (LB), a histopathological signature of PD, in grafted fetal dopaminergic neurons in the striatum of PD patients. Consequently, these findings were a conceptual breakthrough, generating the "host to graft transmission" hypothesis, also called the "prion-like hypothesis." Several in vitro and in vivo studies suggest that α-syn can undergo a toxic templated conformational change, spread from cell to cell and from region to region, and initiate the formation of "LB-like aggregates," contributing to the PD pathogenesis. Here, we will review and discuss the current knowledge for such a putative mechanism on the prion-like nature of α-syn, and discuss about the proper use of the term prion-like.
    Frontiers in Neuroanatomy 12/2014; 8:159. · 4.18 Impact Factor
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    ABSTRACT: Converging lines of evidence suggest that cell-to-cell transmission and the self-propagation of pathogenic amyloidogenic proteins play a central role in the initiation and the progression of several neurodegenerative disorders. This "prion-like" hypothesis has been recently reported for α-synuclein, a presynaptic protein implicated in the pathogenesis of Parkinson's disease (PD) and related disorders. This review summarizes recent findings on α-synuclein prion-like propagation, focusing on its transmission, seeding and degradation and discusses some key questions that remain to be explored. Understanding how α-synuclein exits cells and propagates from one brain region to another will lead to the development of new therapeutic strategies for the treatment of PD, aiming at slowing or stopping the disease progression.
    Experimental neurobiology. 12/2014; 23(4):324-36.

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