Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.
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.
- SourceAvailable from: Dhiman Ghosh[Show abstract] [Hide abstract]
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.
Exogenous ?-synuclein fibrils seed the formation
of Lewy body-like intracellular inclusions
in cultured cells
Kelvin C. Luk, Cheng Song, Patrick O’Brien, Anna Stieber, Jonathan R. Branch, Kurt R. Brunden, John Q. Trojanowski,
and Virginia M.-Y. Lee1
Center for Neurodegenerative Disease Research, Institute on Aging, Department of Pathology and Laboratory Medicine, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104-4283
Edited by Thomas C. Su ¨dhof, Stanford University School of Medicine, Palo Alto, CA, and approved September 29, 2009 (received for review July 17, 2009)
Cytoplasmic inclusions containing ?-synuclein (?-Syn) fibrils, referred
to as Lewy bodies (LBs), are the signature neuropathological hall-
marks of Parkinson’s disease (PD). Although ?-Syn fibrils can be
fibrillar ?-Syn inclusions similar to authentic LBs in cultured cells has
not been achieved. We show here that intracellular ?-Syn aggrega-
tion can be triggered by the introduction of exogenously produced
recombinant ?-Syn fibrils into cultured cells engineered to overex-
press ?-Syn. Unlike unassembled ?-Syn, these ?-Syn fibrils ‘‘seeded’’
recruitment of endogenous soluble ?-Syn protein and their conver-
sion into insoluble, hyperphosphorylated, and ubiquitinated patho-
logical species. Thus, this cell model recapitulates key features of LBs
in human PD brains. Also, these findings support the concept that
intracellular ?-Syn aggregation is normally limited by the number of
active nucleation sites present in the cytoplasm and that small
quantities of ?-Syn fibrils can alter this balance by acting as seeds for
Parkinson’s disease ? pathology ? protein misfolding
association of ?-Syn with lipid membranes and enrichment at
synaptic terminals suggest a role in synaptic maintenance and
neurotransmitter release (1, 2), the precise physiological functions
of ?-Syn remain uncertain. Also, animals lacking the ?-Syn gene
(SNCA) show no obvious defects (3). In contrast, intracellular
accumulations comprised of highly organized ?-Syn amyloid fibrils
define a family of neurological disorders (the synucleinopathies)
that includes Parkinson’s disease (PD), dementia with Lewy bodies
iron accumulation type 1 (4).
Purified ?-Syn readily assembles into amyloid-like fibrils
similar to those in LBs under defined conditions in vitro and has
been studied extensively (5, 6). Fibrillization occurs through a
two-step polymerization process, whereby soluble monomer is
converted into conformationally distinct oligomeric intermedi-
ates, which then serve as nuclei for subsequent elongation (7).
Curiously, although ?-Syn aggregation and pathology are prom-
inent in humans and in animal models of synucleinopathies
(8–10), overexpression of ?-Syn in neuronal and nonneuronal
cells, as well as primary neurons derived from ?-Syn transgenic
mice, does not lead to significant ?-Syn inclusion formation (11).
Indeed, this absence of cell models that recapitulate the mor-
phological and biochemical features of LBs is a serious imped-
iment to elucidating the pathological events or disease pathways
leading to ?-Syn aggregation in vivo.
Given its naturally unfolded state, we hypothesized that even
highly elevated levels of ?-Syn overexpression in cultured cells
may not generate sufficient amounts of oligomeric or protofi-
brillar nuclei required to seed fibril elongation. We tested this
hypothesis by asking whether or not cellular ?-Syn could be
recruited and converted into insoluble forms with features
lpha-synuclein (?-Syn) is a highly soluble natively unfolded
protein expressed throughout the CNS. Although the close
typical of intracellular LB-like inclusions after the introduction
of ?-Syn nucleating structures into the cytoplasm. Our findings
here demonstrate that introduction of exogenously assembled
?-Syn fibrils catalyzes intracellular ?-Syn aggregation in various
cells engineered to overexpress ?-Syn. Although monomeric and
oligomeric ?-Syn showed little effect, ?-Syn fibrils rapidly re-
cruited endogenous soluble ?-Syn protein, converting this into
detergent-insoluble inclusions. Also, pathological hyperphos-
phorylated and ubiquitinated ?-Syn species were featured abun-
dantly in fibril-seeded inclusions; thus, recapitulating key fea-
tures of human LBs in a cell culture model.
Intracellular ?-Syn Fibrils, but Not Soluble ?-Syn Species, Seed the
Formation of LB-Like Inclusions. To effectively serve as seeds for
aggregation, exogenously added ?-Syn fibrils must first localize
and persist for a sufficient period to allow recruitment of
endogenous ?-Syn and promote growth of inclusions. Recent
studies have revealed that fibrils comprised of polyglutamine
repeats, as well as of tau protein accumulating in various
neurodegenerative disorders, can be internalized by cells on
addition to culture medium (12, 13), although the mechanism by
which fibrils reach the cytoplasm is not clearly understood.
We investigated whether monomeric and fibrillar forms of
fluorescently labeled ?-Syn (?-Syn594) could be efficiently in-
troduced into the cytoplasm of QBI-293 cells to generate a cell
culture model system of LB formation. In contrast to prior
studies (12–14), we were unable to demonstrate meaningful
internalization of either monomeric or fibrillar ?-Syn594by mere
addition of protein preparations to the culture medium. How-
ever, intracellular ?-Syn594was readily detectable in cells by
fluorescence- and differential interference contrast (DIC)-
microscopy 24 h after transduction using cationic-liposomes
optimized for intracellular protein delivery (Fig. 1 A–F; Fig. S1
f–k) (15). Transduced ?-Syn594monomers were transiently dis-
tributed throughout the cytoplasm as small punctate structures,
whereas internalized ?-Syn594preformed-fibrils (PFFs) were
present as large irregular foci. Although nearly all monomeric
?-Syn594was degraded by 48 h, ?-Syn594PFFs persisted for days
within cells. When unlabeled WT ?-Syn PFFs (Fig. S1a) were
transduced into QBI-293 cells stably expressing WT ?-Syn
(QBI-WT-Syn cells), we observed large cytoplasmic inclusions
Author contributions: K.C.L., K.R.B., J.Q.T., and V.M.-Y.L. designed research; K.C.L., C.S.,
P.O., A.S., and J.R.B. performed research; K.C.L. contributed new reagents/analytic tools;
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
November 24, 2009 ?
vol. 106 ?
no. 47 ?
within 48 h by using multiple antibodies against ?-Syn that
differed from the smaller irregular foci observed shortly after
PFF transduction (Figs. 1 G–L and 2). Confocal microscopy
confirmed that these inclusions resided within the boundaries of
the plasma membrane (Fig. 1 J–O). Similar results were also
obtained in cells expressing a disease-associated ?-Syn mutants
Inclusions were detectable as early as 24 h after PFF treatment
and typically round in shape, ranging from 2 to 5 ?m in diameter
(Figs. 1 and 2). More than 40% of PFF-transduced QBI-WT-Syn
cells harbored these LB-like inclusions (Fig. 1P). Notably, only one
abutted or indented the nucleus in most cells. Interestingly, no
inclusions were detected in cells transduced with nonfibrillar ?-Syn
comprised of either monomers or oligomers generated by incuba-
tion with dopamine (Figs. S1e and S2 d–l) despite previous reports
that certain species of oligomeric ?-Syn may initiate intracellular
?-Syn aggregation (17). Similarly, transduction with an unrelated
soluble protein (?-galactosidase) did not result in inclusion forma-
tion. Thus, exogenously generated ?-Syn fibrils specifically induce
intracellular inclusion formation.
Fibril-Seeded Intracellular ?-Syn Inclusions Display Properties of LBs
in Human Disease. Immunostaining of PFF-transduced QBI-WT-
intensely stained inclusions surrounded by diffuse cytoplasmic
?-Syn, indicating that ?-Syn was a major constituent of these
with antibodies recognizing misfolded forms of ?-Syn (Syn506 and
Syn303; Fig. 2 C and K), suggesting that ?-Syn present within
inclusions assumes pathological conformations similar to ?-Syn in
LBs of PD, but distinct from normal cellular ?-Syn.
To further confirm that transduced ?-Syn PFFs are intracellular
and to determine whether the resulting seeded-inclusions recapit-
ulate posttranslational modifications found in human LBs, we next
asked whether ?-Syn within inclusions inside transduced cells
immunofluorescence with anti-?-SynpSer129revealed that nearly all
inclusions showed strong immunoreactivity for ?-Syn phosphory-
lated at Ser-129, thereby resembling authentic LBs in PD (Fig. 2
after transduction with ?-Syn PFFs in other cell lines overexpress-
ing ?-Syn, including HeLa and SH-SY5Y neuroblastoma cells
% cells w/ inclusions
cently labeled ?-Syn (?-Syn594) or ?-Syn594PFFs were delivered into QBI-WT-Syn
transduced ?-Syn594monomer (A–C, arrowheads) and fibrils (D–F, arrows) could
readily be detected within cell boundaries (dashed lines) indicating efficient
with WT-?-Syn PFFs, fixed at 48 h, and immunostained with either a pan-?-Syn
antibody (SNL4; G) or a monoclonal antibody specifically recognizing misfolded
?-Syn (Syn506; J). Colabeling with Alexa Fluor 488 conjugated PH?-L (PHA) was
used to reveal the plasma membrane. Confocal microscopy shows large ?-Syn-
of an inclusion-bearing cell reconstructed from serial confocal images. Removal
?-Syn aggregate and confirmed its intracellular location. (P) Quantification of
(data from three separate transductions from two independent experiments;
n ? 500 cells per condition). [Scale bars, 10 ?m (F); 6 ?m (G); 15 ?m (j).]
Intracellular fibrils seed ?-Syn aggregation. (A–F) Monomeric fluores-
Lewy BodiesSeeded Inclusions
phorylated ?-Syn (pSyn) (E), ubiquitin (Ubi) (I), and positive staining with the
amyloid-specific dye ThS (M). Intracellular inclusions formed by seeding with
specific to misfolded ?-Syn (Syn 506; B–D). Inclusions were also strongly phos-
phorylated (F–H) and ubiquitinated (J–L), and detectable by using additional
?-Syn antibodies SNL4 (G) and Syn303 (K). Confocal images demonstrating in-
which was strongest in the periphery (N–P). Cell boundary is indicated in white.
[Scale bars, 10 ?m (A, E, I, and M); 10 ?m (D, H, and L); 5 ?m (P).]
Seeded inclusions resemble human LBs. LBs in the cingulate cortex of a
www.pnas.org?cgi?doi?10.1073?pnas.0908005106Luk et al.
(Fig. S4), indicating that this phenomenon extends to multiple cell
types. The majority of the ?-Syn inclusions were also ubiquitinated,
as evidenced by ubiquitin-immunostaining (Fig. 2 I–L). Because
recombinant ?-Syn PFFs are neither phosphorylated or ubiquiti-
nated before transduction, these modifications occur de novo after
the transduction process and further confirm the intracellular
location of ?-Syn inclusions, as well as their verisimilitude to ?-Syn
fibrils found in PD brains. Staining with the amyloid-specific dye
thioflavin S (ThS) revealed that most inclusions contained signif-
icant ?-pleated sheet content like that found in authentic LBs
(Fig. 2 M–P). Together, these observations indicate that the intra-
cellular ?-Syn inclusions in our cell culture system closely resemble
LBs in PD with respect to their key defining features.
Exogenous Fibrils Seed Intracellular Inclusion Formation via Recruit-
ment and Conversion of Soluble Cytoplasmic ?-Syn. Given the sub-
stantial size of the intracellular inclusions within fibril-transduced
inclusion periphery, we surmised that endogenously expressed
hypothesis, and to determine the relative contributions of exoge-
nous and cellular ?-Syn to the formation of intracellular inclusions,
recombinant Myc-tagged ?-Syn (Fig. S1d). Double-immunofluo-
rescence with anti-Myc (labeling the internalized ?-Syn PFFs) and
anti-?-SynpSer129revealed that Myc-positive PFF seeds are located
at the center of inclusions (Fig. 3 A–C). Also, Myc-labeled cores
were surrounded by a region labeled with ?-SynpSer129that was
devoid of Myc staining, indicating that this phosphorylated ?-Syn,
which represents a significant proportion of inclusions, was com-
prised largely, if not exclusively, of endogenous ?-Syn. Indeed, the
lack of colocalization between Myc and ?-SynpSer129suggested that
the ?-Syn PFF seeds were not significantly phosphorylated after
The ?-Syn within LBs isolated from human PD brains has been
demonstrated to be insoluble (10, 19). Endogenous ?-Syn from
untransduced QBI-WT-Syn cells was highly soluble and entirely
recovered after 1% Triton X-100 extraction (Fig. 3D). However,
transduction with ?-Syn-Myc PFFs led to the appearance of a
significant amount of Triton-insoluble WT ?-Syn as well as higher
Mr species, which required SDS for solubilization (Fig. 3E). A
weaker band consistent with ?-Syn-Myc was also recovered in the
SDS fractions of ?-Syn-Myc PFF-transduced cells, the identity of
which was confirmed on reprobing blots with anti-Myc antibody
(Fig. 3 E and F). The relative intensities of the endogenous ?-Syn
band, which are several fold-higher than ?-Syn-Myc in the SDS-
soluble fraction from transduced cells (Fig. 3E), further support
that the intracellular aggregates are comprised largely of recruited
endogenous ?-Syn. The insolubility of endogenous ?-Syn recruited
by ?-Syn-Myc PFFs was also apparent by immunofluorescence
after Triton X-100 extraction of transduced QBI-WT-Syn cells,
which effectively removed all diffuse cytoplasmic ?-Syn while
leaving inclusions intact as shown by immunostaining with anti-?-
SynpSer129or Syn506 (Fig. S5 a–f).
Inclusion Formation Is Mediated by the Core Amyloid-Forming Region
of ?-Syn. Concordant with our colocalization data, we were unable
to detect significant phosphorylation of Myc-tagged ?-Syn PFFs
after immunoprecipitation with anti-Myc and probing with
?-SynpSer129antibody, further confirming that exogenously intro-
duced ?-Syn fibrils are not phosphorylated (Fig. 3 G and H).
Consistent with the majority of ?-Syn found within detergent-
origin, cells stably expressing ?-Syn-Myc also formed Myc-positive
inclusions after transduction with WT ?-Syn fibrils (Fig. 3 I–K).
To better understand the molecular interaction between re-
cruited endogenous ?-Syn and the PFFs, we transduced QBI-
A53T-Syn cells with fibrils lacking either the N-terminal (?-
Syn21–140) or C-terminal (?-Syn1–120) region of ?-Syn. These
PFFs are indistinguishable from WT ?-Syn PFFs on EM eval-
uation ((Figs. S1 a–c and S5g). Transduction with N or C
terminus truncated ?-Syn PFFs resulted in robust formation of
inclusions that were detected by antibodies that recognizing only
endogenous full-length ?-Syn. For example, Syn303 recognizes
an N-terminal epitope not present in ?-Syn21–140PFFs (Fig. 4 A
and B), whereas ?-Syn1–120PFFs lack the epitope detected by
?-Syn in the Triton-insoluble fractions from these cells confirms
that recruitment has a central role in ?-Syn inclusion formation
insoluble fraction, which parallels studies of pathological ?-Syn in
s l l e
+ Syn-Myc PFF
containing a C-terminal Myc-tag. Double staining for Myc and anti-?-SynpSer129
(pSyn) revealed that fibril seeds form the core of inclusions whereas pSyn pre-
dominates 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 ?-Syn originating from the cell comprise the
seeds within the SDS-fraction of transduced lysates were immunoprecipitated
with anti-Myc (9E10). (G) Antibodies against ?-Syn (SNL-4) indicate efficient
and J) Inclusions detected in cells stably expressing Myc-tagged ?-Syn (QBI-Syn-
Myc) transduced with WT ?-Syn PFFs. Positive staining for Myc (I) and Syn303 (J)
?m (I).]*, IgG light chain; FT, flow-through fraction.
Soluble endogenous Syn is recruited into inclusions by fibrils. (A–C)
Luk et al. PNAS ?
November 24, 2009 ?
vol. 106 ?
no. 47 ?
PD brains (Fig. 4F). The ability of truncated ?-Syn PFFs to seed
further aggregate formation implies that the central portion of
?-Syn is sufficient for the recruitment and subsequent incorpora-
tion of ?-Syn into inclusions. Residing within this region is a
hydrophobic sequence (residues 71–82) that forms the core of
?-Syn fibrils (20). To test whether this segment is required for the
association of endogenous ?-Syn, we transduced WT ?-Syn PFFs
into cells stably expressing ?-Syn lacking this region. As predicted
based on our previous ?-Syn fibrillization studies (20), we were
unable to detect inclusions in transduced QBI-?-Syn?71–82cells
(Fig. 4G), thereby confirming that binding of endogenous ?-Syn to
PFFs requires the region of ?-Syn critical for fibrillization in vitro.
The observation that phosphorylated ?-Syn was present only
in the Triton-insoluble fraction suggested that this modification
occurs after recruitment to the growing inclusion. To confirm
that phosphorylation of ?-Syn at Ser-129 is not necessary for
recruitment, we transduced either ?-Syn1–120or ?-SynS129APFFs
into cells stably expressing phosphorylation-incompetent
?-SynS129A. Inclusions resembling those formed with WT ?-Syn
were detected, further revealing that phosphorylation is not
required for inclusion seeding nor the subsequent recruitment of
endogenous ?-Syn (Fig. 4 H and I).
Ultrastructure of Fibril-Seeded ?-Syn Inclusions. Cells containing
?-Syn inclusions were next examined by EM. In monomer-transduced
QBI-Syn-A53T cells, major organelles appeared intact and the cyto-
plasm was clear of any large accumulations (Fig. 5A). In contrast,
electron-dense cytoplasmic inclusions were prominent after PFF-
transduction (Fig. 5B). Inclusions were perinuclear, although no dis-
magnifications revealed the presence of distinct fibrillar structures
?10–15 nm in diameter located at the center of inclusions (Fig. 5C).
Inclusions also contained many vesicles, of which some were multila-
mellar and in contact with the fibrillar core. We used immuno-EM to
further monitor the localization of transduced ?-Syn-Myc PFFs in
of cellular ?-Syn to fibrils. At 6 h after transduction, anti-Myc staining
transduced fibrils at this stage were not associated with vesicles typical
of mature inclusions. By 16 h, PFF-transduced cells contained Myc-
positive structures that were surrounded by various membrane bound
verify that exogenously introduced PFFs reach the cytoplasmic space
where they serve as a nidus for the formation of complex intracellular
inclusions. Also, cytoplasmic vesicles appear to associate with fibrils
3 A–C), these vesicles may contain hyperphosphorylated ?-Syn and,
thus, form an integral part of inclusions.
We examined the consequences of LB-like ?-Syn inclusions
on cellular architecture and organization. Although we were
unable to detect colocalization of ?-SynpSer129-positive inclu-
sions with various cytoskeletal markers, inclusions were pos-
itive for Hsp70 and Hsp90 (Fig. 5 G–I; Fig. S6), members of
the heat-shock protein family widely reported to accumulate in
LBs and misfolded protein inclusions in multiple neurodegen-
erative disorders. In contrast to phosphorylated ?-Syn, which
was distributed primarily at the periphery of inclusions but
excluded from the core, Hsp70 was present throughout inclu-
sions, suggesting that chaperones recognize both ?-Syn PFF
seeds and subsequently recruited endogenous ?-Syn. Last, we
examined whether the presence of LB-like inclusions in QBI-
Syn-A53T cells disrupts trafficking pathways, including ER-
Golgi transport and secretory function as recently shown in
yeast models of ?-Syn overexpression (21, 22). Immunostain-
ing for ?-SynpSer129and the Golgi matrix protein GM130 in
?-Syn PFF-transduced cells revealed that the majority of ?-Syn
inclusions were located near the cis-Golgi (Fig. 5 J–L). Sig-
nificantly, GM130 staining in inclusion-bearing cells exhibited
a dispersed pattern compared with the dense stacked mor-
phology in cells without inclusions. Quantitative analyses of
the mean Golgi area and intensity also revealed significant
1-120 PFF 21-140 PFF
N- or C-terminal regions in seeds. (A–D) QBI-A53T-Syn
cells were transduced with ?-Syn PFFs lacking either the
N-terminal (?-Syn21–140) or C-terminal domain (?-Syn1–
120). Transduced cells were immunostained using anti-
bodies recognizing either the extreme N-terminal
(Syn303; A) or C-terminal (Syn211; C); thus, detecting
only endogenous ?-Syn. Both truncated forms of fibrils
recruited cellular ?-Syn as indicated by inclusion forma-
tion. Inclusions were phosphorylated, indicating that
their formation does not depend on seed phosphoryla-
tion or interaction with membranes. (E and F) Lysates
from cells transduced with either WT-?-Syn monomer
(Mono), full-length (Syn PFF), or truncated ?-Syn fibrils
were probed with antibodies against ?-Syn (SNL4) and
?-SynpSer129. Immunoblot with SNL-4 (E) shows full-
length endogenous ?-Syn within Triton-insoluble frac-
tions of cells transduced with WT, ?-Syn21–140, and
by fibril seeds. Transduction also resulted in the appear-
ance of high molecular weight ?-Syn species (*) consis-
tent with ubiquitination. Fibril transduction also led to a
dramatic increase in the amount of phosphorylated
?-Syn found almost exclusively within SDS-soluble frac-
tions (F Upper) consistent with its location within insol-
uble inclusions. GAPDH loading controls are also shown
(F Lower). (G) QBI-?71-82-Syn cells transduced with WT
fibrils did not form inclusions, indicating that the core
with PFFs prepared from ?-Syn-S129A (H) or ?-Syn1–120(I), which lack this phosphorylation site. Double immunostaining with Syn506 and SNL4 indicate the
formation of misfolded ?-Syn inclusions. [Scale bars, 5 ?m (A–D); 5 ?m (G–I).]
Inclusion formation does not require the ?-Syn
www.pnas.org?cgi?doi?10.1073?pnas.0908005106Luk et al.
differences between cells with and without ?-Syn inclusions
(Fig. 5 M and N). Thus, as in yeast models of synucleinopathies,
seeded ?-Syn inclusions alter normal cellular processes.
formation in ?-Syn overexpressing cells can be initiated by the
presence of fibrillar ?-Syn seeds. Once inside cells, fibrillar seeds
actively recruit and convert soluble endogenous ?-Syn into a
misfolded state, leading to the formation and growth of deter-
gent-insoluble structures closely resembling LBs in the brains of
patients with PD and other synucleinopathies. Importantly, the
?-Syn inclusions in our cell culture model also undergo several
modifications characteristic of human LBs, including hyperphos-
phorylation, ubiquitination, and the accumulation of cytoplas-
mic vesicles around the periphery of the inclusions. The striking
morphological and biochemical similarities between LBs and the
intracellular accumulations in this model suggest that fibrillar
and other disease-associated filamentous inclusions. Also, the
accumulation of assembly-competent ?-Syn nucleation seeds
may be an important rate-limiting factor for LB formation.
Although the precise series of events leading to inclusion for-
mation remain unclear, our data indicate ?-Syn recruitment
depends on the presence of an amyloidogenic sequence. To-
gether with the observation that the majority of ?-Syn within
inclusions is endogenous, these findings suggest that endogenous
?-Syn recruitment to fibrillar ?-Syn seeds underlies the forma-
tion of these inclusions in our cell culture system, and we
of LBs in PD and related synucleinopathies.
The absolute number of nucleation sites introduced into indi-
vidual cells in our model has not been determined, although our
biochemical data suggest that the amount of protein transduced
represents a minor fraction of the endogenous ?-Syn pool. Thus,
small quantities of misfolded and fibrillar ?-Syn may be sufficient
to seed aggregation in the context of long-lived postmitotic cells
such as neurons. However, little is known regarding how fibrillar
nuclei initially arise in neurons and glia in vivo. Misfolded ?-Syn
could arise in a cell-autonomous manner via increased synthesis as
seen in individuals with ?-Syn gene amplification (23) or by
mutations that accelerate ?-Syn misfolding itself (e.g., the familial
A53T mutation) (16). Generation of rapidly aggregating C-
terminally truncated ?-Syn species, as reported in PD brains (24),
may also contribute to this process. Likewise, impairment of ?-Syn
degradation pathways or insults that alter the degradation or
of seeds. Indeed, our results indicate that, even in rapidly dividing
and convert endogenous ?-Syn.
Another possibility is that ?-Syn seeds enter from neighboring
cells or the extracellular space as suggested by recent studies
in the release and uptake of soluble ?-Syn species (14). Supporting
in a progressive temporospatial pattern between closely connected
?-Syn inclusions, suggesting that pathology is conferred by prox-
imity to pathological tissue (26). However, it remains unclear
whether released ?-Syn or some other agent is responsible for the
development of ?-Syn deposits in the transplanted cells. Signifi-
cantly, we were unable to detect inclusion formation in ?-Syn
overexpressing cells, even when cocultured in direct contact with
cells already containing prominent inclusions (Fig. S7). Our data,
together with the observation that ?-Syn pathology within grafts is
seen after extended periods, suggest that transmission of misfolded
seeds is a rare event.
The capacity for exogenously introduced amyloids to seed intra-
cellular aggregation has also been recently reported for two neu-
+ inclusion- inclusion
Mean intensity (% Max)
+ inclusion - inclusion
Mean area (µm2)
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
recruiting endogeous ?-Syn. Multilamellar bodies are also present in the sur-
are localized to the perinuclear region 6 h after treatment (D). At 16 h, vesicular
organelles, likely containing ?-Syn, are recruited to PFF seeds (D and E). [Scale
bars, 2 ?m (A, B, and E); 400 nm (C); 0.5 ?m (D and F).] Double-immunostaining
against pSyn and Hsp70 (G–I) reveal molecular chaperones colocalized to inclu-
sions 48 h after transduction with Syn-Myc PFFs. Whereas pSyn is found at the
label inclusions and the Golgi matrix, respectively. Cells lacking aggregates dis-
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 ?-Syn inclusions.
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).
Luk et al.PNAS ?
November 24, 2009 ?
vol. 106 ?
no. 47 ?
rodegenerative disease-related proteins. In contrast to our results
with ?-Syn, fibrils comprised of either tau (13) or polyglutamine-
expanded proteins (12) appear to be actively taken into cells,
including neurons, without reagent-mediated transduction. Signif-
icantly, when injected into the brains of transgenic mice expressing
WT tau, which do not otherwise develop tau lesions, tissue ho-
mogenates containing misfolded mutant tau induce conforma-
tional changes and tau neuropathology, even in areas beyond the
injection site (27). It is not clear why ?-Syn fibrils were not
efficiently introduced into cells in the absence of transduction
reagent, although it is likely that not all fibrils are internalized in a
similar manner. For example, it has been suggested that tau
are internalized via an endosome-independent process (12). It is
also possible that different cell types employ different mechanisms
of fibril internalization.
We have expanded significantly on these recent studies, in
particular focusing on molecular processes that occur once ?-Syn
fibrils gain entry into cells. Our data support the view that the
majority of inclusions occupied a juxtanuclear position, although
they failed to colocalize with any classically defined compartment.
However, inclusions consistently colocalized with ubiquitin and
multiple chaperones, which strongly suggests that they elicit the
misfolded protein response and protein degradation pathways.
Also, the ?-Syn inclusions disrupt Golgi integrity, indicating that
insoluble ?-Syn inclusions are not benign. Further characterization
of these intracellular changes should uncover how ?-Syn inclusions
influence key cellular processes. Intriguingly, although some pre-
and cytotoxicity, our data show that inclusion formation does not
require this modification. Nonetheless, the extent of ?-Syn hyper-
phosphorylation seen in our model and human LBs suggests that it
may be an important postaggregation event.
Our findings, coupled with other recent results (12–14, 27),
strongly suggest that several different amyloid fibrils can act as
potent catalysts for the conversion of soluble proteins into amyloid
fibrils, and highlight the importance of developing agents that
prevent the formation of nucleating cores (or block further aggre-
gate growth from existing seed structures) as a therapeutic strategy
for the treatment of patients with neurodegenerative protein mis-
into the events that regulate the formation of intracellular ?-Syn
inclusions and represents an invaluable tool for further elucidating
the pathological mechanisms underlying this major family of dis-
Materials and Methods
Recombinant ?-Syn Proteins and Protein Labeling. Recombinant WT, Myc-
S1 and purified as described in ref. 20 and SI Materials and Methods.
Fibril Assembly. Fibrils were prepared in reactions (200 ?L per tube) containing
in WT ?-Syn assembly reactions. Reactions were stopped after 5 days, aliquoted,
and stored at ?80 °C until use. The presence of amyloid fibrils was confirmed by
using thioflavin fluorimetry and EM.
Cell Culture and Fibril Transduction in Mammalian Cells. Cells stably expressing
?-Syn used in this study are listed in Table S2. Generation of stable QBI-HEK-293
(QBiogene) and SH-SY5Y cell lines in SI Materials and Methods. QBI cells were
plated in 35-mm tissue culture plates and allowed to reach 80–90% confluence
for transduction experiments. Cells were transferred to serum-free media 1 h
before transduction. For each plate, 10 ?L of cationic-liposomal protein trans-
duction reagent (Bioporter; Sigma) was added to a 1.5-mL Microfuge tube and
evaporated as per the manufacturer’s guidelines. The resulting reagent dry-film
was then directly resuspended with 80 ?L of PBS containing ?-Syn fibrils (100
mixture was further diluted in OptiMEM (Invitrogen) and added to cells. Cells
were then further incubated for 4 h, washed twice with Versene and 0.5%
trypsin/EDTA to remove extracellular ?-Syn fibrils, and transferred onto 6-well
tissue culture plates or poly(D-lysine)-coated glass coverslips. Transduced cells
were maintained in media containing 0.5% FBS unless otherwise indicated.
Immunocytochemistry and ThS Staining. Fluorescence immunocytochemistry
was performed by using primary antibodies listed in Table S3. Procedures for
immuno and ThS staining are described in SI Materials and Methods.
Immunoblot Analysis and Immunoprecipitation. Procedures for protein extrac-
tions and immunoblot analyses are detailed in SI Materials and Methods.
EM. Procedures for sample preparation and acquisition of EM images are de-
scribed in SI Materials and Methods.
this manuscript, and I. P. Mills for technical assistance. This work was sup-
ported by National Institutes of Health Grants AG09215 (to V.M.-Y.L) and
NS053488 (to J.Q.T.), the Picower Foundation, and the Benaroya Foundation.
V.M.-Y.L. is the John H. Ware, III, Professor of Alzheimer’s Disease Research.
J.Q.T. is the William Maul Measey-Truman G. Schnabel, Jr., Professor of
Geriatric Medicine and Gerontology.
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www.pnas.org?cgi?doi?10.1073?pnas.0908005106Luk et al.