Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells - PubMed (original) (raw)
Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells
Kelvin C Luk et al. Proc Natl Acad Sci U S A. 2009.
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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Intracellular fibrils seed α-Syn aggregation. (A–F) Monomeric fluorescently labeled α-Syn (α-Syn594) or α-Syn594 PFFs were delivered into QBI-WT-Syn cells by using cationic-liposome reagent (Bioporter). Cells were passaged after 4 h and visualized under DIC and fluorescence-microscopy after fixation at 24 h. Both transduced α-Syn594 monomer (A–C, arrowheads) and fibrils (D–F, arrows) could readily be detected within cell boundaries (dashed lines) indicating efficient intracellular delivery. (G–L) QBI cells stably expressing A53T-Syn were transduced with WT-α-Syn PFFs, fixed at 48 h, and immunostained with either a pan-α-Syn antibody (SNL4; G) or a monoclonal antibody specifically recognizing misfolded α-Syn (Syn506; J). Colabeling with Alexa Fluor 488 conjugated PHΑ-L (PHA) was used to reveal the plasma membrane. Confocal microscopy shows large α-Syn-positive intracellular inclusions in fibril-seeded cells (G–I and J–L). (M–O) A 3D view of an inclusion-bearing cell reconstructed from serial confocal images. Removal of the PHA signal (green) reveals the juxtanuclear position of a single prominent α-Syn aggregate and confirmed its intracellular location. (P) Quantification of α-Syn inclusions in QBI-WT-Syn cells seeded with either WT-Syn monomer or PFFs (data from three separate transductions from two independent experiments; n > 500 cells per condition). [Scale bars, 10 μm (F); 6 μm (G); 15 μm (j).]
Fig. 2.
Seeded inclusions resemble human LBs. LBs in the cingulate cortex of a PD patient with dementia showing strong immunoreactivity for α-Syn (A), phosphorylated α-Syn (pSyn) (E), ubiquitin (Ubi) (I), and positive staining with the amyloid-specific dye ThS (M). Intracellular inclusions formed by seeding with recombinant WT-α-Syn fibrils also stained positively with a monoclonal antibody specific to misfolded α-Syn (Syn 506; B–D). Inclusions were also strongly phosphorylated (F–H) and ubiquitinated (J–L), and detectable by using additional α-Syn antibodies SNL4 (G) and Syn303 (K). Confocal images demonstrating intense ThS labeling in the core region of inclusions, in contrast to phosphorylation, which was strongest in the periphery (N–P). Cell boundary is indicated in white. [Scale bars, 10 μm (A, E, I, and M); 10 μm (D, H, and L); 5 μm (P).]
Fig. 3.
Soluble endogenous Syn is recruited into inclusions by fibrils. (A–C) QBI-WT-Syn cells were seeded with fibrils generated by using recombinant α-Syn containing a C-terminal Myc-tag. Double staining for Myc and anti-α-SynpSer129 (pSyn) revealed that fibril seeds form the core of inclusions whereas pSyn predominates in the periphery regions. (D) Immunoblot of detergent-soluble (TriX) and detergent-insoluble (SDS) fractions of cell lysates from control unseeded QBI-WT-Syn cells. (E and F) Lysates from cells transduced with Syn-Myc fibrils contained both WT (black arrowhead) and Syn-Myc (white arrowhead) in the insoluble fraction, indicating that α-Syn originating from the cell comprise the majority of α-Syn within inclusions. A smear representing high molecular weight α-Syn species (**) could also be detected in the SDS fraction. Remaining Syn-Myc seeds within the SDS-fraction of transduced lysates were immunoprecipitated with anti-Myc (9E10). (G) Antibodies against α-Syn (SNL-4) indicate efficient pull-down of Syn-Myc seeds. (H) Probing with anti-pSyn indicates that phosphorylation occurs overwhelmingly in endogenous α-Syn but not exogenous fibrils. (I and J) Inclusions detected in cells stably expressing Myc-tagged α-Syn (QBI-Syn-Myc) transduced with WT α-Syn PFFs. Positive staining for Myc (I) and Syn303 (J) indicates that inclusions contain α-Syn of cellular origin. [Scale bars, 2.5 μm (A); 5 μm (I).] *, IgG light chain; FT, flow-through fraction.
Fig. 4.
Inclusion formation does not require the α-Syn N- or C-terminal regions in seeds. (A–D) QBI-A53T-Syn cells were transduced with α-Syn PFFs lacking either the N-terminal (α-Syn21–140) or C-terminal domain (α-Syn1–120). Transduced cells were immunostained using antibodies recognizing either the extreme N-terminal (Syn303; A) or C-terminal (Syn211; C); thus, detecting only endogenous α-Syn. Both truncated forms of fibrils recruited cellular α-Syn as indicated by inclusion formation. Inclusions were phosphorylated, indicating that their formation does not depend on seed phosphorylation or interaction with membranes. (E and F) Lysates from cells transduced with either WT-α-Syn monomer (Mono), full-length (Syn PFF), or truncated α-Syn fibrils were probed with antibodies against α-Syn (SNL4) and α-SynpSer129. Immunoblot with SNL-4 (E) shows full-length endogenous α-Syn within Triton-insoluble fractions of cells transduced with WT, α-Syn21–140, and α-Syn1–120 PFFs, indicating that cellular α-Syn is converted by fibril seeds. Transduction also resulted in the appearance of high molecular weight α-Syn species (*) consistent with ubiquitination. Fibril transduction also led to a dramatic increase in the amount of phosphorylated α-Syn found almost exclusively within SDS-soluble fractions (F Upper) consistent with its location within insoluble inclusions. GAPDH loading controls are also shown (F Lower). (G) QBI-Δ71-82-Syn cells transduced with WT fibrils did not form inclusions, indicating that the core fibril assembly region of α-Syn is critical to recruitment and incorporation. (H and I) QBI-cells stably expressing α-Syn mutated at Ser-129 (S129A) were transduced with PFFs prepared from α-Syn-S129A (H) or α-Syn1–120 (I), which lack this phosphorylation site. Double immunostaining with Syn506 and SNL4 indicate the formation of misfolded α-Syn inclusions. [Scale bars, 5 μm (A–D); 5 μm (G–I).]
Fig. 5.
Fibril-seeded α-Syn inclusions contain vesicular bodies. (A–C) EM images of cells stably expressing A53T-α-Syn 24 h after transduction with either WT-α-Syn monomer (A) or PFFs (B and C). Electron dense inclusions (in) were found only in the cytoplasm of PFF-seeded cells. Nucleus (Nu) and cytoplasm (Cyt) are also indicated. (C) High-power magnification of the region delineated in B revealing a fibrillar core (black arrows and Inset) consistent with fibrils serving as a nidus for recruiting endogeous α-Syn. Multilamellar bodies are also present in the surrounding cytoplasm (arrowhead). (D and E) Immuno-EM using anti-Myc (9E10) in QBI-Syn-A53T cells transduced with Syn-Myc PFFs. Exogenous (Myc-tagged) fibrils are localized to the perinuclear region 6 h after treatment (D). At 16 h, vesicular organelles, likely containing α-Syn, are recruited to PFF seeds (D and E). [Scale bars, 2 μm (A, B, and E); 400 nm (C); 0.5 μm (D and F).] Double-immunostaining against pSyn and Hsp70 (G–I) reveal molecular chaperones colocalized to inclusions 48 h after transduction with Syn-Myc PFFs. Whereas pSyn is found at the periphery, Hsp70 is detected throughout inclusions, suggesting it is also recruited to PFF seeds. (J–L) Antibodies against α-SynpSer129 (J) and GM130 (K) were used to label inclusions and the Golgi matrix, respectively. Cells lacking aggregates display compact Golgi morphology (asterisks), whereas inclusion-bearing cells show fragmented GM130 staining (arrows). (M and N) Golgi dispersal was assessed by measuring the area stained by GM130 (M) and average pixel intensity (N) in Myc-Syn PFF-transduced cells with or without phosphorylated α-Syn inclusions. The majority of inclusion-bearing cells displayed fragmentation as reflected by an increase in area positive for GM130 with a concomitant decrease in staining intensity. *, P < 0.001; #, P < 0.005 t test (results obtained from three separate transduction experiments, n = 200).
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