The process of Lewy body formation, rather than simply alpha-synuclein fibrillization, is the major...
Transcript of The process of Lewy body formation, rather than simply alpha-synuclein fibrillization, is the major...
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The process of Lewy body formation, rather than simply alpha-synuclein
fibrillization, is the major driver of neurodegeneration in synucleinopathies
Anne-Laure Mahul-Mellier1, Johannes Burtscher1, Niran Maharjan1, Laura Weerens1,
Marie Croisier2, Fabien Kuttler3, Marion Leleu4, Graham Knott2, and Hilal A. Lashuel1*
1Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 2BioEM Core Facility and Technology Platform, EPFL, Lausanne 1015, Switzerland. 3BiomolecularScreening Core Facility and Technology Platform, EPFL, Lausanne 1015, Switzerland. 3 Gene expression Core Facility and Technology Platform, EPFL, Lausanne 1015, Switzerland.
*To whom correspondence should be addressed at: Laboratory of Molecular and Chemical
Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de
Lausanne, 1015 Lausanne. Tel: +41216939691, Fax: +41216939665, Email:
Running title:
The grammar of Lewy body formation.
Keywords: alpha‐synuclein (α‐synuclein), Parkinson’s disease, aggregation, post‐
translational modification (PTM), mitochondria, synapse
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Abstract
Parkinson’s disease (PD) is characterized by the accumulation of misfolded alpha-synuclein
(α-syn) into intraneuronal inclusions named Lewy bodies (LB). Although it is widely believed
that α-syn plays a central role in the pathogenesis of PD and synucleinopathies, the processes
that govern α-syn fibrillization and LB formation in the brain remain poorly understood. In this
work, we sought to reverse engineer LBs and dissect the spatiotemporal events involved in
their biogenesis at the genetic, molecular, biochemical, structural, and cellular levels. Toward
this goal, we took advantage of a seeding-based model of α-syn fibril formation in primary
neurons and further developed this model to generate the first neuronal model that reproduces
the key events leading to LB formation; including seeding, fibrillization, and the formation of
LB-like inclusions that recapitulate many of the biochemical, structural, and organizational
features of LBs found in post-mortem human PD brain tissues. Next, we applied an integrative
approach combining confocal and correlative light-electron microscopy (CLEM) imaging
methods with biochemical profiling of α-syn species and temporal proteomic and
transcriptomic analyses to dissect the molecular events associated with LB formation and
maturation and to elucidate their contributions to neuronal dysfunctions and
neurodegeneration in PD and synucleinopathies. The results from these studies demonstrate
that LB formation involves a complex interplay between α-syn fibrillization, post-translational
modifications, and interactions between α-syn aggregates and membranous organelles,
including mitochondria and the autophagosome and endolysosome. Furthermore, we
demonstrate that the process of LB formation and maturation, rather than simply fibril
formation, is the major driver of neurodegeneration through disruption of cellular functions and
inducing mitochondria damage and deficits, as well as synaptic dysfunctions. Having a
neuronal model that allows for unlinking of the key processes involved in LB formation is
crucial for elucidating the molecular and cellular determinants of each process and their
contributions to neuronal dysfunction and degeneration in PD and synucleinopathies. Such a
model is essential to efforts to identify and investigate the mode of action and toxicity of drug
candidates targeting α-syn aggregation and LB formation.
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Introduction
The intracellular accumulation of aggregated forms of alpha-synuclein (α-syn) in neurons and
glial cells represents one of the main pathological hallmarks of Parkinson’s disease (PD) and
related synucleinopathies, including dementia with Lewy bodies (DLB) and multiple system
atrophy (MSA)1. PD and DLB are characterized by the presence of Lewy bodies (LB) and
Lewy neurites (LN) in subcortical and cortical neurons, while in MSA, α-syn inclusions are
mainly detected in glial cells and are referred to as glial cytoplasmic inclusions (GCIs)2,3.
Although it is widely believed that α-syn plays a central role in the pathogenesis of
synucleinopathies, the molecular and cellular processes that trigger and govern the
misfolding, fibrillization, LB formation, and spread of α-syn in the brain remain poorly
understood. Furthermore, whether the processes of α-syn fibrillization and inclusion formation
are protective or neurotoxic remains a subject of active investigation and debate. Addressing
this knowledge gap is crucial to 1) advancing our understanding of the molecular mechanisms
underpinning the pathogenesis of PD and other synucleinopathies; 2) guiding the
development of preclinical models that reproduce the full-spectrum or well-defined features of
the human pathology; and 3) developing more effective diagnostic and therapeutic strategies
for the management and treatment of these devastating diseases.
In-depth post-mortem neuropathological examinations of human brains from patients
with LB disorders and other synucleinopathies have revealed the existence of different sub-
types of pathological inclusions that are enriched in aggregated forms of α-syn, including
fibrils3-8. This has led to the hypothesis that aggregation of α-syn has a primary role in the
formation of LBs and other α-syn pathological aggregates. However, the absence of
experimental models that reproduce all the stages of LB formation and maturation has limited
our ability to decipher the different processes involved in LB formation and the contribution of
these processes to the pathogenesis of PD and synucleinopathies. To address this knowledge
gap, it is crucial to develop cellular and/or animal models that not only produce α-syn
aggregates but also recapitulate the process of LB formation at the biochemical, structural,
and organizational levels. One advantage of cellular models is that they offer unique
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opportunities to apply advanced imaging and omics approaches to elucidate in great detail the
key events associated with α-syn aggregation and LB formation and maturation and to
correlate these events with alteration in cellular pathways and functions/dysfunctions.
Until recently, the great majority of cellular models of synucleinopathies were based on
overexpression of WT or mutant α-syn alone, with other proteins, or coupled to treatment with
other stress inducers (e.g., toxins, proteasome inhibitors)9-19. In many cases, these conditions
were sufficient to induce α-syn accumulation and aggregation. However, very few studies
investigated the extent to which these aggregates reproduce the cardinal biochemical,
structural, and organizational features of LBs and other pathological states of α-syn in PD
brains. The formation of LB-like structures is usually assessed using a limited number of
methods to indirectly assess the aggregation properties of α-syn (e.g., proteinase K
resistant9,16 and binding to the amyloid-specific dye, thioflavin S13) and immunoreactivity for
selected LB markers10,12-15,17,19, such as p62, ubiquitin (Ub), and phosphorylated α-syn
(pS129), to define their LB-like properties. When electron microscopy (EM) was used, it clearly
demonstrated that the α-syn inclusions do not share the compositional complexity and
morphological features of the LBs, despite their immunoreactivity to the standard LB
markers13,20,21. These findings suggest that the great majority of α-syn cellular models
reproduce some aspects of α-syn aggregation or fibril formation, but do not recapitulate all of
the key events leading to LB formation and maturation.
Recently, it was shown that exogenously adding pre-formed fibrils of α-syn at
nanomolar concentration can act as a seed to initiate the misfolding and aggregation of
endogenous α-synuclein in both cellular22,23 and animal models24. This neuronal seeding
model is the first model that shows the formation and accumulation over time of long
filamentous aggregates. Although this seeding model enables the induction of α-syn
fibrillization in neurons, in the absence of α-syn overexpression, the transition from fibrils to
LB-like structures has not been reported, even though these aggregates are immunoreactive
for the standard LB makers (e.g., pS129 α-synuclein, p62, and ubiquitin). These observations
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suggest that the neuronal seeding model, as initially developed, is a suitable model for
investigating the processes involved in fibrils formations but not LB formation.
Given that LBs have been consistently shown to have complex composition and
organization and usually contain other non-proteinaceous material (lipids)25,26 and
membranous organelles8,27-32, we hypothesized that the transition from fibrils to LB might
require time and could be driven by post-translational modifications and structural
rearrangements of the fibrils. By extending the characterization of this neuronal seeding model
from 11–14 days to 21 days, we were eventually able to observe the transition from fibrils to
α-syn rich inclusions that recapitulate the biochemical, morphological, and structural features
of the bona fide human LBs, including the recruitment of membranous organelles and
accumulation of phosphorylated and C-terminally truncated α-syn aggregates33-38.
By employing an integrated approach using confocal and CLEM imaging, as well as
quantitative proteomic and transcriptomic analyses, we were able to investigate with
spatiotemporal resolution the biochemical and structural changes associated with each step
involved in LB biogenesis, from seeding to fibrillization to LB formation and maturation. Our
ability to uncouple the different stages of LB formation in this model provided unique
opportunities to investigate the cellular and functional changes that occur at each stage, thus
paving the way for elucidating how the different events associated with LB formation and
maturation contribute to α-syn-induced toxicity and neurodegeneration. Our work shows that
the process of LB formation and maturation, rather than simply α-syn fibril formation, is a major
contributor to α-syn-induced toxicity and neurodegeneration in PD and synucleinopathies. We
believe that this new model of LB formation can be further developed and used as a powerful
platform to further investigate the molecular determinants and cellular pathways that regulate
the stages of LB formation and maturation and to screen for therapeutic agents based on the
modulation of these pathways.
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Results
At early stage, α-syn seeded aggregates resemble bona fide human LBs
histochemically but not at the structural and organizational levels
To investigate the mechanisms of LB formation and maturation, we initially took advantage of
a cellular seeding-based model of synucleinopathies developed by Virginia Lee and
coworkers22,23. As a first step, we sought to determine to what extent this model reproduces
LB formation. Extracellular sonicated α-syn pre-formed fibrils (PFFs, length ~50–100 nm)
(Figure S1A-E) were added at a nanomolar concentration (70 nM) to primary neuronal cultures
(Figures 1A). Once internalized into the neurons, the PFFs serve as seeds and induced
endogenous α-syn to misfold and form intracellular aggregates in a time-dependent manner
(Figures 1B and S1F). To establish the spatio-temporal features of α-syn seeded aggregates
formed in neurons, we used ICC (Figures 1C-J and S3A-E) combined with high content
imaging analysis (HCA), which allows quantitative assessment of the level of α-syn-pS129 in
MAP2 positive neurons (Figure S3F-I). The epitopes of the antibodies used in our study are
summarized in Supplemental Figure 2.
Consistent with previous studies, we observed that PFF-treated neurons only started
to exhibit α-syn-pS129 positive inclusions 4 days after the addition of α-syn seeds (Figure
S3A). These rare aggregates were exclusively detected in neurites (Figure S3A). After 7 days
of treatment (D7), we observed an accumulation of filamentous-like α-syn aggregates in
neurites with a limited number of cell-body inclusions (Figures 1C and S3B, G-I). At 14 days
(D14), the number of filamentous aggregates continued to significantly increase in neurites
but also in the neuronal cell bodies where they were distributed in the vicinity of the nucleus
(Figures 1D and S3C, G-I).
The newly formed aggregates observed at D7 and D14 were all positive for the two
well-established LB markers, p62 (Figure 1E-F) and ubiquitin (Ub) (Figure 1G-H)39 and were
also stained by the Amytracker tracer, a fluorescent dye that binds specifically the β-sheet
structure of amyloid-like protein aggregates (Figure 1I-J). Although previous studies have
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suggested the formation of specific morphologies (strains) of α-syn aggregates in seeding
models, our ICC data revealed the formation of aggregates that are heterogeneous in size,
shape, and subcellular distribution in the same population of PFF-treated neurons (Figures
1C-J and S3B-C). Biochemical analysis of the insoluble fraction of the PFF-treated neurons
confirmed the accumulation of both truncated and high molecular weight (HMWs) α-syn
species that were positively stained with a pan-synuclein antibody (SYN-1) and with an
antibody against pS129 (Figures 1K and S7). Interestingly, these insoluble fractions displayed
a potent seeding activity in neuronal primary culture (Figure S4). Compared to recombinant
PFFs, we did not observe differences in the morphology and the subcellular distribution of the
seeded-aggregates formed after addition of “native aggregates” (Figures S3C and S4B).
These findings demonstrate that the newly formed pS129 positive α-syn filamentous
aggregates contain α-syn seeding competent species, most likely in a fibrillar species.
To determine if the newly formed aggregates share the structural and morphological
properties of the bona fide LBs observed in PD brains, we performed Electron Microscopy
(EM) and immunogold labeling studies against α-syn pS129 at D14. As shown in Figure 1L,
the newly formed aggregates in neurons appear as loosely organized uniform filaments
(Figure 1L, bottom panel). Interestingly, at this point, we did not observe significant lateral
association or clustering of the fibrils in the form of inclusions. Although previous studies have
referred to the α-syn aggregates formed at 11–14 days post-treatment with PFFs as LB-like
inclusions, our ICC and EM studies, which are consistent with previous studies using the same
model23,40,41 and tools (e.g., antibodies and LB markers), clearly demonstrate the formation of
predominantly α-syn fibrils, rather than LB-like inclusions. LBs are highly organized round
structures that are composed of not only α-syn fibrils but also other proteins42, lipids25,26, and
membranous organelles, including lysosomal structures and mitochondria8,28,29,31,43. The
absence of such structures at D14, even though the fibrillar aggregates are immunoreactive
for the classical markers used to define LBs, pS129, ubiquitin, and p62, suggests that these
markers cannot be used to accurately distinguish between α-syn fibrillar aggregates and
mature LBs.
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At late stage, α-syn seeded fibrillar aggregates rearrange into inclusions that
morphologically resemble to the bona fide human LBs
We hypothesized that the rearrangement and transition of the newly formed α-syn
fibrils into LB-like inclusions might require more time. Therefore, we sought to investigate the
structural and morphological properties of the newly formed aggregates over an extended
period, up to 21 days (D21) post-treatment with PFFs (DIV 28). Interestingly, at D21, ICC
combined with a HCA approach showed a significant increase in the number of aggregates
detected in the neuronal cell bodies between D14 and D21 (Figure S3F-H). Furthermore, we
observed major changes in the morphology of the newly formed aggregates. The seeded
aggregates, up to 14 days of formation, were exclusively detected as long filamentous-like
structures located either in the neurites and/or the neuronal cell bodies (Figure S3B-C).
However, at D21, we identified three different morphologies; filamentous (~30%), ribbon-like
(~45%), and round LB-like inclusions (~ 22%) (Figure S3D-E). The formation of these types
of aggregates in seeded neurons has not been reported, as previous studies limited their
analysis up to D14. The α-syn rounded inclusions observed at D21 were all positive for p62,
Ub, and the amyloid-specific dye (Amytracker) (Figure 1M-P). Furthermore, we also showed
the presence of several classes of lipids in the pS129-positive inclusions using fluorescent
probes (Figure S3 J-O). This finding is in line with several studies which have previously
documented the presence of lipids within LBs and α-syn pathological inclusions observed in
post-mortem PD human brain tissues25,26,44-47.
Finally, the LB-like inclusions were exclusively detected in the vicinity of the nucleus
(Figures 1M-P and S3D-E). After D14, no further significant accumulation of α-syn aggregates
was observed in the neurites (Figure S3G). Altogether, our data suggest that α-syn fibrillar
aggregates initially formed in the neuronal extensions (D4) are transported over time (D7–
D21) to the perinuclear region, where they eventually accumulate as LB-like inclusions (D21).
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The process of inclusion formation is accompanied by the sequestration of lipids,
organelles and endomembranes structures
The transition from filamentous aggregates into ribbon and round-like aggregates suggests
that the newly formed fibrils undergo major structural changes over time. To determine if these
changes are associated with the evolution and maturation of α-syn fibrils into LBs, we next
turned to correlative light and electron microscopy (CLEM) to characterize the ultrastructural
properties of the seeded α-syn aggregates formed in neurons after 7, 14, or 21 days post-
treatment with WT PFFs (Figures 1Q, 2 and S5A). As shown in Figure 2, the CLEM data
revealed a marked reorganization of the newly formed α-syn fibrils over time. Indeed, at D7,
EM images of neurites positive for pS129-positive α-syn seeded aggregates showed
predominantly single fibril filaments distant from each other (Figures 2Aa). Aligned and
randomly ordered fibrils were observed in the same neurite (Figures 2Aa). In comparison, in
neurite negative for α-syn seeded aggregates, we observed the classical organization of the
microtubules that appear as aligned bundle of long filaments parallel to the axis of the neurites
(Figures 2Ab). In-depth measurements of the diameters of the filament-like structures
observed in neurites confirmed that the newly formed α-syn fibrils exhibited an average
diameter of 11.94 ± 3.97 nm (standard deviation, SD) which was significantly smaller than the
average diameter of the microtubules (18.67 ± 2.94 nm) (Figure 2Ab, inset). Moreover, the
range of fibril lengths (from 500 nm to 2 μM per slide section) (Figure 2A) confirmed that the
fibrils detected in the cytosol were not simply internalized α-syn PFFs (size ~40-120 nm,
Figure S5B) but rather newly formed fibrils resulting from the seeding and fibrillization of
endogenous α-syn. At D7, although most of the newly formed α-syn fibrils were observed in
the neuritic extensions, pS129-positive α-syn aggregates were also detected in the cell bodies
of few neurons (Figure 1C-I).
CLEM showed, in the same neuronal cell body, α-syn fibrils randomly organized (Figure 2B-
C, blue arrows) or ordered as long tracks of parallel bundles of filaments (Figure 2B-C, red
arrows). At D14, most α-syn filaments were reorganized into laterally associated and tightly
packed bundles of fibrils (Figure 2D-E, red arrows).
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In some inclusions, these laterally associated bundles were seen to start to associate with or
encircle organelles, including mitochondria, autophagosomes, and others endolysosomal-like
vesicles (Figure 2D-E). Single fibrils, loosely distributed in the neuronal cell body, were also
detected close to these organelles (Figure 2D-E).
The sequestration of organelles and membranous structures by α-syn fibrillar
aggregates seems to gradually increase over time between D14 and D21 (Figures 2D-F and
S5F). At D21, both the ribbon-like aggregates (Figures 3A-B, highlighted in red and S6A) and
the LB-like inclusions (Figures 2F, 3C-D, highlighted in red and S6B) were composed of α-syn
filaments (highlighted in black) but also of a high number of mitochondria (highlighted in green)
and several types of vesicles, such as autophagosomes, endosome and lysosome-like
vesicles (highlighted respectively in yellow, pink and purple) (Figures 2F, 3A-D and S6A-D).
Interestingly, the mitochondria were either sequestered inside the inclusions or organized at
the periphery of the inclusions. The presence of these organelles inside the LB-like inclusions
was confirmed by ICC using specific markers for the late endosomes/lysosomes (Figure 3E,
LAMP1), the mitochondria (Figure 3G, Tom20) and the autophagosome vesicles (Figure 1M,
p62, and 3F, LAMP2A). BiP protein localized to the endoplasmic reticulum was also partially
colocalizing to the LB-like inclusions (Figure 3H). Our data strongly suggest that the process
of inclusion formation is not only driven by the mechanical assembly of newly formed α-syn
fibrils but seems to be accompanied by the sequestration or active recruitment of other
proteins, membranous structures, and organelles over time. This is in line with previous
studies showing that LBs from PD brains are not only composed of α-syn filaments and
proteinaceous material42,48,49 but also contain a mixture of vesicles, mitochondria, and others
organelles 8,27-31,43 as well as lipids25,26.
Formation and maturation of LB-like inclusions require the lateral association and the
fragmentation of the newly formed α-syn fibrils over time
The lateral association of the newly formed fibrils at D14 might represent an early stage in the
process of packaging fibrillar aggregates into LB-like inclusions. Also, the length of the newly
formed fibrils significantly decreased over time during the evolution of the inclusions (Figure
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S5G-J). At D7, the neurites contained long filaments of α-syn reaching up to 2 μm in length
(Figures 2Aa, 2B-C and S5G, J). Strikingly, at D14, the size of α-syn fibrils was significantly
reduced to an average length of 400 nm (Figures 2D-E and S5H, J). This was even more
obvious in the compact inclusions observed in neurons treated for 21 days, in which only very
short filaments were detected with an average size of 300 nm (Figures 2F-G and S5F, I-J).
This suggests that the lateral association of the newly formed fibrils and their packing into
higher-organized aggregates might require fragmentation of α-syn into shorter filaments. The
presence of C-terminally truncated species of α-syn was confirmed by Western blotting (WB),
as evidenced by the detection of the 12 kDa band by the pan α-syn antibody (SYN-1, epitope
91-99) but not by the antibody against pS129 (Figure S7). This is in line with our recent findings
showing that specific post-fibrillization C-terminal cleavages serve as key regulators in the
processing of α-syn seeds and the growth of newly formed α-syn fibrils in the neuronal seeding
model33.
Altogether, our findings suggest that the formation of α-syn inclusions, in the context
of this neuronal seeding model, requires a sequence of events starting with 1) the
internalization of PFF seeds, followed by 2) the initiation of the seeding, 3) fibril elongation and
post-fibrillization modification (phosphorylation at S129 and ubiquitination), 4) the
fragmentation of the newly formed fibrils, which facilitates 5) the structural reorganization of
the fibrils, their lateral assembly, and their packing into higher-order aggregates along with 6)
the recruitment and sequestration of organelles and endomembranes (Figure 9). It is likely
that many of these events are regulated by post-fibrillization post-translational dependent
interactions between α-syn fibrils and other proteins and organelles. To the best of our
knowledge, this is the first report of the formation of inclusions that recapitulate many of the
cardinal features of LBs, including the presence of fibrillar α-syn, lipids, and membranous
organelles.
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Quantitative proteomics reveals the temporal sequestration of membranous organelle,
synaptic, and mitochondrial proteins into LB-like inclusions
Having demonstrated by CLEM imaging that the maturation of the newly formed fibrils (D14)
into LB-like inclusions (D21) is accompanied by the active and gradual recruitment and
sequestration of organelles and endomembranes over time (D14 to D21), we next sought to
gain further insight into the molecular interactions and mechanisms that govern LB formation
and maturation by mapping the proteome of α-syn inclusions over time. Toward this goal, we
performed quantitative proteomic analysis on the insoluble fractions of PBS or PFF-treated
neurons after 7, 14, or 21 days of treatment (Figure 4). The proteomic data generated from
three independent experiments are presented as a volcano plot to depict the differentially
expressed proteins based on the mean intensity differences versus t-test probability.
At D7, after multi comparisons correction using the Bonferroni method (FDR threshold
<0.05, magnitude of proteins changes above 2 and a –log10 p-value >0.05), only α-syn and
the Phospholipase C Beta 1 (Plcb1) were shown to be significantly enriched in the insoluble
fraction with a nine- and two-fold increase in the insoluble fraction of the PFF-treated neurons,
respectively (Figure S8A). No proteins from the endomembrane compartments were
significantly enriched in the insoluble fraction of the PFF-treated neurons at D7. This finding
is in line with our CLEM observations showing that at an early stage, α-syn seeded aggregates
were mainly detected in the neurites as long filaments that did not appear to interact with
intracellular organelles.
Next, we examined the protein contents of the α-syn inclusions at the intermediate
stage, prior to (D14) and after (D21) the formation of LB-like inclusions (Figure 4). As shown
in Figure 4A, the volcano plots show that 633 proteins and 568 proteins were significantly
upregulated in the insoluble fraction of the PFF-treated neurons at D14 and D21, respectively.
These proteins were then classified by cellular component (Figure 4B) and biological
processes (Figure 4C) using the gene ontology (GO) term analysis and the database for
annotation, visualization, and integrated discovery (DAVID) analysis and pathways (Figure
S8B) using the Ingenuity Pathway Analysis (IPA). Classifications of the proteins by cellular
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compartment (Figure 4B) showed that the insoluble fractions of the PFFs-treated neurons at
D14 and D21 were highly enriched by proteins that belong to the endomembrane
compartments mainly from the mitochondria, the endoplasmic reticulum (ER), the Golgi and
to a lesser extent the endolysosomal apparatus or synapses. These data are in line with the
CLEM imaging showing that from D14 the newly formed fibrils start to interact with and
encircled several organelles, including mitochondria, autophagosome, endosomes,
lysosomes-like vesicles, and others endomembranous structures (Figures 2D) that are found
later to be sequestered in the LB-like inclusions at D21 (Figures 2F and 3). Finally, several
cytoskeletal proteins, including microtubule and myosin complexes, were also significantly
enriched at D14 and D21 in the insoluble fraction of the PFF-treated neurons (Figure 4B). It is
noteworthy that the close resemblance between the protein contents of the insoluble fraction
of PFF-treated neurons at D14 and D21 is expected given that at D21 only ~22% of the
positive α-syn aggregates have completely transitioned to inclusions with an LB-like
morphology (Figure S3E).
Formation and maturation of LB-like inclusions are dynamic processes which involve
proteins sequestration and alterations of key signaling pathways rather than simply α-
syn fibrillization
Several studies have shown that protein aggregation and inclusion formation could contribute
to neurodegeneration and disease progression through both gain of toxic function and loss of
normal protein function, possibly through aberrant interactions involving protein aggregation
or the sequestration of functional proteins within pathological inclusions50-54. Therefore, to gain
insight into the mechanisms and pathways that are involved and affected by α-syn fibrillization
and LB formation and maturation, the list of proteins found enriched in the PFF-treated
neurons (Figure 4A) was further classified by biological processes (Figure 4C) and signaling
pathways (Figure S8B). At the early stage of fibrils formation (D7), only α-syn and Plcb1
proteins were significantly enriched in the insoluble fraction of the PFF-treated neurons. At
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this stage, no biological processes were found to be altered (Figure S8A). Cellular
compartment analysis showed that the insoluble fractions of PFF-treated neurons at D14 and
D21 contained a large number of cytoskeletal proteins including several tubulin sub-units,
microtubule-associated proteins, and motor proteins (kinesin, dynein, and myosin) (Figure
4B). This cluster of proteins was associated with the enrichment of several biological
processes, including actin cytoskeleton organization, axonogenesis, and dendritic spine
development (Figure 4C). The top canonical signaling pathways were also related to axonal
guidance and microtubule regulation by the stathmin proteins (Figure S8B). The accumulation
of the cytoskeletal proteins in the neuronal insoluble fraction is concomitant with the
redistribution of newly formed α-syn aggregates from the neurites to the perinuclear region
(Figures 1C-P, S3B-H). This suggests that the retrograde trafficking of newly formed α-syn
fibrils is accompanied by the sequestration of the proteins involved in axonal transport.
Impaired axonal transport has been shown to have dramatic effects on the intracellular
trafficking of proteins, vesicles, and organelles55,56. In line with this hypothesis, our temporal
proteomic analysis revealed that, at D14 and D21, the most highly enriched terms for the
biological processes were related to intracellular protein transport, including ER to Golgi
mediated transport and the vesicle-mediated transport but also the nucleocytoplasmic protein
transport (Figure 4C). Overall, the sequestration of proteins related to the intracellular
transport inside α-syn inclusions seems to result in an impairment in the trafficking of
organelles and vesicles, such as mitochondria, endosomes, lysosomes, and the synaptic
vesicles55,56, that eventually accumulate inside α-syn inclusions as evidenced by CLEM
imaging (Figures 2 and 3). In line with this hypothesis, our proteomic data showed perturbation
of the biological processes and signaling pathways related to mitochondria and synaptic
compartments (Figures 4C and S8B). Interestingly, not only proteins involved in mitochondrial
transport were enriched in the insoluble fraction of the PFF-treated neurons but also proteins
related to mitochondrial dynamics, the mitochondrial apoptotic pathway, and the energy
metabolism pathways (Tricarboxylic acid cycle, ATP and NADH metabolisms). This indicates
that the process of inclusion formation that occurs throughout D14 to D21 might dramatically
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15
alter mitochondrial physiology, suggesting that mitochondrial dysfunction could be a major
contributor to neurodegeneration in PD and synucleinopathies.
The upregulation of biological processes related to synaptic homeostasis (Figure 4C)
was due to the accumulation of both pre and post-synaptic proteins (Figure 4E) mostly at D21
(Figure 4D). The processes involved in synaptic transmission, synapse assembly and synapse
maturation (Figure 4C) were associated with an upregulation of the long-term synaptic
depression and potentiation pathways but also of the endocannabinoid and CXCR4 signaling
that regulate synapse function57 (Figure S8B). Several signaling pathways related to
neurotoxicity were detected as upregulated in our analyses at D14, such as the Huntington’s
disease signaling, mitochondrial dysfunction, Apoptosis signaling, the ER stress pathway,
autophagy, and the neuroinflammation signaling pathway (Figure S8B).
Our proteomic and CLEM data provide strong evidence that formation of LB does not
occur through simply the continued formation, growth, and assembly of α-syn fibrils but instead
arises as a result of complex α-syn aggregation-dependent events that involve the active
recruitment and sequestration of proteins and organelles over time. This process, which
occurs mainly between 14-21 days, rather than simply fibril formation, which occurs as early
as 7 days, seems to trigger a cascade of cellular processes, including changes in the
physiological properties of the mitochondria and the synapses, that could ultimately lead to
neuronal dysfunctions and toxicity.
Transcriptomic analysis reveals multiple changes in gene expression during the
formation of the newly formed fibrils and their maturation into LB-like inclusions
Next, to better understand the mechanisms of dysfunctions that ultimately lead to
neurodegeneration, we investigated the transcriptomic changes in response to PFFs
treatment over time. RNA sequencing (RNA-Seq) technology was used to explore the
genome-wide transcript profiling in PBS and PFF-treated neurons after 7, 14, or 21 days of
treatment. Data were generated from three independent experiments. RNA-Seq analysis
(FDR threshold <0.01) identified a large number of genes that were differentially expressed
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between the PBS- and the PFF-treated neurons (Figure S9A). Interestingly, global
transcriptome changes increased over time in the PFF-treated neurons. At D7, only 75
differentially expressed genes were identified, while at D14 the total number of genes
increased to 435. At D21, 1017 genes were differentially expressed (Figure S9A), of which
455 were upregulated and 562 were downregulated (Figure S9A).
We first investigated the potential enrichment of cellular component (GO term analysis)
for the differentially expressed genes in PFF-treated neurons in cellular compartment. At D7,
the differentially expressed genes encoded for proteins located at the synapses, in the axons,
or the secretory and exocytic vesicles (Figure 5A). These genes are thought to play regulatory
roles in the neurogenesis processes, including the organization, the growth, and the extension
of the axons and dendrites (Figure 5B). Thus, the presence of the newly formed fibrils that are
mainly detected in the neuritic extensions at this stage could perturb the development and the
differentiation of the neurites. At D14, among the differentially expressed genes, 329 genes
(106 upregulated, 223 downregulated) were linked to the synaptic, neuritic, and vesicular
cellular compartments (Figure 5A). Further analyses showed that these genes were
associated with multiple biological processes, including neurogenesis, calcium homeostasis,
synaptic homeostasis (organization, plasticity, and neurotransmission), cytoskeleton
organization, response to stress, and neuronal cell death process (Figure 5B).
At D21, in addition to the axon and vesicles, our transcriptomic data showed an
enrichment of genes that encode for proteins present in several cellular compartments,
including the ion channels complex, plasma membrane protein complex, and the cell-cell
junctions. More importantly, our data show that the expression level of 217 synaptic related
genes was dramatically changed over time with a marked difference between D14 and D21
(Figures 5B, D, and S10B).
Most of the differentially expressed synaptic genes were significantly downregulated
between D14 and D21 (Figures 5D and S9B). Strikingly, at D21, ~20% of the genes
differentially expressed in the PFF-treated neurons were related to synaptic functions,
including neurotransmission processes and synapse organization. Also, at D21, biological
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processes related to the response to oxidative stress and mitochondria, including
mitochondrion disassembly, mitophagy, and mitochondria depolarization, were significantly
enriched (Figure 5B-C).
Finally, both transcriptomic and proteomic analyses revealed that shared biological
processes (Figures 4C and 5B) are mostly involved in the cytoskeletal, synaptic,
mitochondrial, and neuronal cell death functions. Altogether, our results show that alterations
of the mitochondrial and synaptic functions, both at the proteomic and transcriptomic levels,
occur primarily throughout D14 to D21, thus coinciding with inclusion formation, LB maturation,
and cell death.
Dynamics of Lewy body formation and maturation induce mitochondrial alterations
To validate our findings that mitochondrial dysfunctions are associated with the formation and
the maturation of the LB-like inclusions, we assessed the mitochondrial activity over time in
PFF-treated neurons. ICC for mitochondrial markers (VDAC1, TOM20, and TIM23) revealed
strong co-localization of mitochondria with α-syn pS129-positive aggregates starting from D14
after PFF exposure. To assess whether this recruitment of mitochondrial components
decreased mitochondrial function, we applied a combined protocol of high-resolution
respirometry with Amplex Red based fluorometry to measure the production of mitochondrial
reactive oxygen species (ROS).
Routine respiration of intact cells was significantly reduced at D21 (Figure 6B), while it was
similar to PBS-treated control cells at the other assessed time points. Plasma membranes
were subsequently permeabilized using digitonin, and substrates feeding into NADH-linked
respiration were supplied. In the absence (NL) and presence (NP) of ADP, these respirational
states did not significantly differ across all tested time points of PFF- and PBS-treatment.
Addition of succinate, drove overall respiration of all other groups to significantly higher
levels than neurons at D21. This was the case for both maximum oxidative phosphorylation
capacity (NSP) and maximum electron transport system capacity (NSE) in the uncoupled state
(by CCCP). Blocking NADH-linked respiration by rotenone, yielding maximal succinate-driven
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respiration in the CCCP uncoupled state, maintained the significantly lower respiration of
neurons at D21. Strikingly, the generation of mitochondrial ROS was significantly decreased
at D7 but returned to baseline levels after longer periods of PFF treatment (Figure 6B, lower
panel). In line with respiration results, analysis of proteomic data revealed significant
recruitment of oxidative phosphorylation system components into aggregates starting from
D14 (Figure 6C). Complexes I, II, and V were strongly affected up to D21, with complex II
subunit SDHC being robustly detected in aggregates at D14 and D21 (Figure 6D). The strong
sequestration of complex II components into aggregates might explain the stronger effects of
α-syn pathology on succinate- than on NADH-linked substrate driven respiration.
The reduced mitochondrial ROS production at D7 (Figure 6B) correlated with the
apparent lack of mitochondrial proteins involved in oxidative stress responses in aggregates
(Figure 6D). Coinciding with the appearance of proteins and components of the JNK pathway
in the aggregates, mitochondrial ROS production increased to baseline levels. Mitochondrial
membrane potential was measured from attached cells by TMRE fluorometry (Figure 6E), and
in line with respiration data, deterioration of mitochondrial membrane potential was apparent
only at D21. At this time point, we also observed a slight reduction of (membrane-potential
independent) binding of Mitotracker green to mitochondrial proteins (Figure 6F), indicating
moderate loss of mitochondrial density.
To investigate the effects of the aggregation process on selected mitochondrial
proteins in more detail, WB analyses were performed and the expression levels were
assessed at distinct time points of this process (Figure 6G): outer mitochondrial membrane
proteins (mitofusin 2 and VDAC1), matrix protein (Citrate synthase), inner mitochondrial
membrane proteins, including OPA1 and subunits of oxidative phosphorylation complexes
(OXPHOS), known to be labile when the respective complexes are not assembled. At D7, no
altered mitochondrial protein levels were observed despite reduced mitochondrial ROS
production and increases in α-syn pS129 and HMW species (Figure 6G). At D14, the levels
of the investigated mitochondrial proteins were still essentially unaltered, while significant
reductions were observed at D21 for pro-fusion protein OPA1 (long and short isoforms) and
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mitofusin 2 (Figure 6G). Similar effects were observed for subunits of oxidative
phosphorylation complexes, in particular, complex I subunit NDUFB8 (Figure 6G). Importantly,
levels of VDAC1 and citrate synthase, common markers of mitochondrial density, remained
relatively unaffected, even in this period of massive respirational deficits and dropping
mitochondrial membrane potential.
Synaptic dysfunction is primarily linked to the formation and the maturation of the LB-
like inclusions
Our transcriptomic and proteomic data strongly suggest that the reorganization of the newly
formed fibrils into LB-like inclusions are concomitant not only with mitochondria defects but
also with synaptic dysfunctions including neurotransmission, synaptic organization, and
plasticity (Figures 4C and 5B). Therefore, we assessed the protein levels of pre- and post-
synaptic markers [(respectively Synapsin I and Post-synaptic Density 95 (PSD95)]. We
measured a dramatic reduction of these synaptic markers at D21 (Figure 7A).
ICC also revealed that the density of the synapses was reduced in neurons having an
aggregates burden (Figure 7B-C). The loss of synapses correlates with the marked increase
in neuronal cell death observed at D21.
Finally, both proteomic and transcriptomic data suggested dysregulation of the mitogen-
activated protein kinase (MAPK) signaling pathways and the recruitment of the MAP kinases
ERK1 (MAPK3) and ERK2 (MAPK1) within α-syn inclusions over time. These findings are
especially relevant as the ERK signaling pathway is involved in regulating mitochondria fission
and integrity58-60 and synaptic plasticity61-64, and is also an important player in PD
pathogenesis65-67. WB analysis showed a significant reduction of the total protein level of the
extracellular signal-regulated (ERK 1/2) proteins at D21. Interestingly most of the ERK proteins
that were still expressed in neurons at D21 were highly phosphorylated, suggesting that the
ERK pathway is still activated at the late stage of LB formation. Finally, ICC revealed that p-
ERK 1/2 decorated the newly formed fibrils at D14 before being sequestered in the LB-like
inclusions at D21 (Figure 7D). Also, our data showed that p-ERK 1/2 was only associated with
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the newly formed aggregates localized in the neuronal cell bodies, as evidenced by the
absence of p-ERK 1/2 signal near the neuritic aggregates at D7 and D14. At D21, the granular
p-ERK 1/2 immunoreactivity was mostly detected at the periphery of the inclusions as
observed in the bona fide LBs found in human PD brain tissue68,69.
The formation and maturation of the LB-like inclusions are associated with neuronal
cell death
We next assessed how the processes involved in the formation, remodeling, and interactome
of α-syn aggregates could impact on the health of the cells at the distinct phases of α-syn
fibrillization from the early stage of fibrils formation (D7), through the packing of the newly
formed fibrils into aggregates (D7 to D14) and their maturation into LB-like inclusions (D14 to
D21).
First, it is important to note that treatment of α-syn KO neurons with the PFFs (70 nM)
did not induce neuronal death, even at D21, confirming that α-syn PFFs treatment and uptake
is not toxic to the neurons (Figure S10). Cell death was only detectable in WT neurons treated
with PFFs (Figure 8A-G). The first signs of toxicity in this neuronal population were only
observed from D14, as indicated by the significant activation of caspase 3, a key marker of
apoptosis.
From D14 to D21, the level of activated caspase 3 was further increased (Figure 8A-
B). The loss of plasma membrane integrity was observed starting at D19, as reflected by the
release of Lactate dehydrogenase (LDH) that significantly increased at D21 (Figure 8B-C).
ICC combined with HCA confirmed the neuronal loss at D21, as evident by the significant
decrease in the number of neuronal cell bodies and neurites (Figure 8D-E). In all the cell death
assays, the presence of α-syn positive aggregates was confirmed by the measurement of
pS129 level in MAP2-positive neurons (Figure 8F).
Strikingly, our transcriptomic analysis revealed that 156 genes involved in signaling
pathways related to the regulation of neuronal cell death were also significantly changed over
time in PFF-treated neurons (Figure 8G). These genes started to be significantly dysregulated
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at D14. At D21, 70 genes involved in cell death signaling pathways were upregulated in PFF-
treated neurons compared to PBS-treated neurons (Figure 8G). Among these genes, 24 were
directly related to apoptotic pathways, which is in line with our cell death assays showing
significant activation of caspase 3 at D21. Between D14 and D21, 26 genes were differentially
expressed in PFF-treated neurons (Figure 8H).
Our data also revealed a significant reduction of α-syn mRNA levels at D21 (Figure 8I),
concomitant with the downregulation of the transcript level of the Polo-like kinase 2 (Plk2)
(Figure 8J), the major kinase involved in the phosphorylation of α-syn at S129 residue. This
suggests that several cellular responses are deployed by the neurons to prevent further
aggregation and inclusion formation, either by downregulation of the protein level of α-syn or
its phosphorylation state.
Overall, our data suggest a progressive neurodegeneration that coincides with the first signs
of organelle accumulation and assembly of the LB-like inclusions.
Altogether our data demonstrate that the formation of fibrillar α-syn positive aggregates
and their maturation into highly organized LB-like inclusions are accompanied by
mitochondrial defects, synaptic dysfunctions, and the dysregulation of associated signaling
pathways that ultimately lead to toxicity and neurodegeneration.
Discussion
The majority of existing cellular models are models of α-syn fibril formation rather than
LBs
Several in vitro assays70-73 and cellular and animal models12,17,22,24,71,74 of α-syn aggregation
and pathology formation have been developed and are commonly used to study these
processes. While many of these models reproduce specific aspects or stages of LB pathology
formation, none has been shown to form pathological aggregates exhibiting the biochemical
and organizational complexity of α-syn pathologies, including LBs, found in post-mortem
brains of patients with synucleinopathies8,25,26,29-31,39,43,74,75. For the most part, classification of
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22
α-syn aggregates as having LB-like features has been limited to very few biomarkers12,13,17,
namely pS129, ubiquitin, and p62 immunoreactivity and the detection of high molecular weight
aggregates in WB13,22,40,41. In very few cases, detailed studies were performed to characterize
the nature of the aggregates and their morphological properties. Failure of these models to
reproduce the transition of α-syn from the aggregated/fibrillar stage to LB-like inclusions has
hampered efforts to elucidate the molecular determinants and cellular pathways that regulate
the different stages of LB pathology formation and to determine the role of this process in the
pathogenesis of PD.
The use of PFFs to seed the aggregation of endogenous α-syn has enabled the reliable
induction of α-syn fibril formation in neurons and other cell types74,76. Based on the use of the
LB markers discussed above, these seeding models are often presented as models of LB
pathology. However, EM studies22,40,41 of the seeded aggregates, including our own (Figure
2A-F), clearly show the presence of mainly fibrillar aggregates at the time points usually used
to assess seeding in this model (10–14 days) (Figures 1-2). These observations demonstrate
that the α-syn modifications and interacting proteins commonly used to define LB pathology
represent markers of α-syn fibril formation and, not necessarily, LB pathology, although these
markers persist with α-syn fibrils during their transition to LBs and other types of α-syn
pathologies.
Extending the seeding process enabled the reconstitution of LB-like inclusions in
neurons.
While investigating the mechanism of seeding and pathology spreading in mice inoculated
with PFFs, we observed a time-dependent shift in the morphology and localization of α-syn
pathology from a filamentous neuritic fibrillar pathology to predominantly compact α-syn in cell
body inclusions. Similarly, we observed that by simply extending the seeding process in
primary neurons from 14 to 21 days, we began to see major changes in the localization and
biochemical properties of the fibrils that eventually led to the formation rounded LB-like
inclusions (Figures 2F, S5F and 3) that exhibit similar biochemical, structural, and architectural
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23
features as patient-derived LBs, as previously described by EM7,8,30,31,46,75,77,78, IHC- or
ICC39,42,79-based imaging, WB34-38, and proteomic48,49 analyses. This includes the cleavage33
and accumulation of truncated fibrillar α-syn species34-38, the presence of many of the proteins
found in LBs42,48,49 and the active recruitment of membranous organelles (e.g., mitochondria,
the endolysosomal apparatus, and autophagolysosome-like vesicles)8,25,26,29-31,39,43,74,75,78,80,81.
Although previous reports have established the presence of lipids in LBs from post-mortem
PD tissues using different techniques, including Coherent Anti-Stokes Raman Scattering
(CARS) microscopy45,46 or synchrotron Fourier transform infrared micro-
spectroscopy (FTIRM)47, very few studies have explored the different types of lipids and their
distribution in LBs. Using different probes that target different types of lipids, we identified
several classes of lipids that colocalize within the LB-like structures. Consistent with our
confocal and CLEM imaging data, we observed enrichment of lipids (methyl ester staining)
that are specific for mitochondria, ER and Golgi apparatus membranes within the LB-like
inclusions. In addition, the phospholipids (cell membranes)26, sphingolipids (ER and Golgi
complex)26, neutral lipids25,26 (lipid droplets) and the cholesteryl esters26 (cholesterol transport)
which have been previously reported as components of the bona fide LB inclusions were also
detected reinforcing the similarities between the patient-derived LB pathologies and the LB-
like inclusions formed in the seeding model. These findings underscore the critical importance
of reassessing the role of lipids in the different stages of α-syn aggregation and LB formation
and maturation.
Altogether our findings are in agreement with ultrastructure-based studies showing that LBs
imaged in the substantia nigra27,29,31,46,75,78, the hippocampal CA2 region82, or the Stellate
ganglion8 from PD patients or non-PD patients30 exhibit organized structures that are enriched
in deformed and damaged mitochondria, cytoskeletal components, and lipidic structures,
including vesicles, fragmented membranes of organelles, and presynaptic structures.
Recently, in substantia nigra, CLEM approach showed some LBs rich in lipids and
membranous structures but devoid of α-syn fibrils46. However, a STED microscopy study of
similar LBs conducted by the same group revealed the presence of a highly dense and
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organized shell of phosphorylated α-syn at the periphery of these LBs45. These observations
raise the possibility that the CLEM procedure could have led to the disruption and removal of
this peripheral aggregated pS129-α-syn. One alternative explanation that is consistent with
our findings in the neuronal model is that the fibrils become increasingly fragmented over time
(Figure 2, ~50–350 nm in length at D21 in PFF-treated neurons). Such small fibrils, which are
also C-terminally truncated33, might not be recognized and/or could be missed, especially
when C-terminal antibodies were used to identify LBs (pS129 antibody or total α-syn antibody
against amino acids 115–122)46. We cannot rule out the possibility that LB structures devoid
of fibrillar α-syn aggregates exist and could represent a rare type of α-syn pathologies in PD
and synucleinopathies. Indeed, several reports have confirmed the existence of a large
morphological spectrum of LBs3,30,39,75,77,83-85.
Altogether our results suggest that the neuronal seeding model recapitulates many of
the key events and processes that govern α-syn seeding, aggregation, and LB formation
(Figures S13 and 9). This model also allows disentangling of the two processes of fibril
formation and Lewy body formation, thus paving the way for systematic investigation of the
molecular and cellular determinants of each process and their contributions to neuronal
dysfunction and degeneration.
The proteome of the neuronal LB-like inclusions overlaps with that of PD and points to
failure of the protein degradation machinery to clear α-syn inclusions
To further characterize the pathological relevance of the inclusions formed in the neuronal
seeding model, we compared our proteomic data with results from previous studies on the
proteome of brainstem or cortical LBs from PD human brain tissues. Approximately one-fourth
of the proteins identified in our inclusion proteomic data at D14 and D21 were previously
described as components of the LBs based on immunohistochemical-based studies 1,42,86
(Figure S11). When comparing our proteomic data of the LB-like inclusions with the proteome
of human LBs, we found that ~15–20% of the proteome overlaps with that of LBs from human
PD brain tissues48,49 (Figure S6). This is not surprising given the differences in the time of LB
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25
inclusion formation and maturation between primary neurons and in the human brain. It is also
evident from our CLEM studies that the neuronal LB-like inclusions have not reached the same
mature stage as the PD LBs. Interestingly, even previous studies that investigated the protein
content of human cortical LBs showed ~20% of similarities in the proteome of the inclusions.
Overall, most of the proteins found in common between LBs from PD human brain tissues and
the LB-like inclusions from the neuronal seeding model could be classified into three main
categories: 1) proteins from the mitochondria and synaptic compartments or belonging to the
endomembrane system; 2) cytoskeleton constituents and proteins associated with the
intracellular trafficking of proteins and vesicles and the nucleocytoplasmic transport; and 3)
proteins involved in the protein quality control (PQC) and the degradation/clearance
machineries (Figures S11–S12). These results suggest that the sequestration of proteins
related to the intracellular transport inside α-syn inclusions might result in the trafficking
impairment of organelles and vesicles, such as mitochondria, endosomes, lysosomes, and
the synaptic vesicles. Interestingly, dysfunction of the intracellular trafficking system and, in
particular, defects in the ER-Golgi caused by α-syn have been shown to underlie the
pathogenesis of PD at the early stage56. Previously published proteomic analyses using the
same neuronal seeding model also indicated a potential role of the microtubule-dependent
transport of vesicles and organelles87 during the process of aggregate formation (Figure S8C).
Finally, the accumulation of large components of the proteins involved in the PQC and
degradation/clearance machinery inside LB-like inclusions and the human bona fide LBs point
toward a potential failure of the protein degradation machinery to clear off α-syn inclusions,
leading to the intracellular accumulation of the aggregates as failed aggresomes-like
inclusions. In line with this hypothesis, it has been shown that a conditional KO of 26S
proteasome in mice led to the formation of LB-like inclusions consisting of filamentous α-syn,
mitochondria and membrane-bound vesicles in neurons88. This is also consistent with our
proteomic study showing a marked enrichment of the proteasomal system between D14 and
D21 in the insoluble fractions PFFs-treated neurons (Figures 4 and S12). Altogether these
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findings highlight the potential role of the proteasomal system in driving the formation of the
LB.
The processes associated with LB formation and maturation, rather than simply α-syn
fibril formation, are the major drivers of α-syn toxicity in neurons
Several studies have suggested that the formation of LBs represents a protective mechanism
whereby aggregated and potentially toxic α-syn species are actively recruited into aggresome-
like structures to prevent their aberrant interactions with other cytosolic proteins and their
deleterious effects on cellular organelles89. A protective role for LBs is plausible if one
assumes that this process is efficient. However, if this process stalls for any reason, then this
will likely expose neurons to deleterious effects mediated by the presence of toxic proteins
and damaged organelles and vesicles. To test this hypothesis, it is crucial to develop a
neuronal and animal model system that enables uncoupling of the different stages of α-syn
aggregation, fibrillization, and LB formation. One key advantage of neuronal models, as shown
in this study, is that they permit detailed investigation of the molecular and cellular changes
that occur during LB formation with high temporal resolution.
Our results show that formation of α-syn fibrils occurs as early as days 4–7, primarily
in neurites, where they accumulate as long filamentous fibrils (Figures 1C–J and 2Aa). At the
early stage of the seeding and fibrillization process (D7), the presence of these newly formed
fibrils did not result in significant alteration of the proteome (Figures 4 and S8A), and induced
only limited genetic and molecular perturbations (Figures 4 and 5) that did not impact neuronal
viability up to D14 (Figures 8A-F and S13). These findings are in line with previous studies
where no cell death was reported before D1422,40,41,90,91 and suggest that α-syn fibrillization is
not sufficient to trigger neuron death, despite the large accumulation of α-syn fibrils throughout
the cytosol of neurons.
However, the progressive accumulation of the filamentous fibrils in the neuronal cell
bodies from D7 to D14 was accompanied by the shortening of the fibrils, their lateral
association, and their interaction with surrounding organelles (Figure 2). These structural
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changes were concomitant with significant perturbations at the proteomic (Figure 4) and
transcriptomic levels (Figure 5). Despite the interaction of the newly formed fibrils with a large
number of proteins associated with cytoskeleton organization, mitochondrial functions,
intracellular trafficking of proteins, vesicles, and nucleocytoplasmic transport, only the early
stages of cell death were activated at D14 as evidenced by the significant activation of
caspase 3 without yet the loss of plasma membrane integrity (Figures 5A-F and S13). The
enrichment of proteins related to the chaperones machineries, the autophagy-lysosomal
pathway, and the ubiquitin-proteasome system in the insoluble fraction of the PFF-treated
neurons at D14 (Figures S12-S13) could suggest that the engagement of the protein quality
control machinery and other related processes represent early cellular responses to prevent
the formation and/or the accumulation of the fibrils in the cytosol. Similarly, transcriptomic
analyses revealed that the formation of α-syn seeded aggregates induced (between D7 and
D14) major dysregulations in the expression of genes involved in neurogenesis, calcium and
synaptic homeostasis, cytoskeleton organization, response to stress, and neuronal cell death
process (Figure 5). Therefore, the lack of noticeable toxic effects during the fibrillization
process (D7–D14) might reflect the ability of neurons to activate multiple pathways to counter
any negative effects induced by the fibrils.
In our extended neuronal seeding model, we observed that the major changes in the
structural properties of the newly formed fibrils occur between days 14 and 21. This suggests
that alternative cellular mechanisms take over to allow the structural reorganization of the
fibrils and their sequestration within LB-like inclusions. This could represent an ideal
detoxification mechanism by reducing aberrant interactions of the fibrils with cytosolic proteins
or organelles. However, our CLEM (Figure 3) and proteomic analyses (Figure 4) showed that
these structural changes were accompanied by significant recruitment and aberrant
sequestration of intracellular proteins related to the intracellular transport inside α-syn
inclusions. This seems to result in the trafficking impairment of organelles and vesicles such
as mitochondria, endosomes, lysosomes, and the synaptic vesicles56 that eventually
accumulate within LBs-like inclusions (Figures 2-4). This process was coupled with altered
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expression of genes associated with the cytoskeleton, mitochondria, and synaptic pathways
together with an increase of the neuronal cell death pathways and neurodegeneration (Figures
5-8 and S13). Our data suggest that post-fibrillization post-translational modifications could
play important roles in these processes by regulating the interactome of the fibrils and their
interactions with cellular organelles. The sequestration of proteins and organelles eventually
leads to a widespread loss of the biological functions, ultimately resulting in
neurodegeneration. Finally, the reorganization of the newly formed fibrils into LB-like
inclusions led to a significant decrease of α-syn both at the protein (Figure S7) and mRNA
(Figure 8I) levels. While depletion of endogenous α-syn could prevent further aggregation and
inclusion formation, it could also lead to loss of important physiological function(s) of α-syn
with cellular dysfunctions as consequences.
The dynamics of LB formation and maturation induce mitochondrial alterations
The prominent accumulation of mitochondria and mitochondrial components within the LB-like
inclusions prompted us to further investigate how the various stages of α-syn fibrillization and
LB formation influence mitochondrial functions (Figure S14). Although mitochondrial
dysfunctions have been strongly implicated in PD, and other neurodegenerative diseases92,93,
the mechanisms by which mitochondrial dysfunction is induced and how it promotes pathology
formation and/or neurodegeneration are still unclear.
Based on our CLEM results and literature evidence, we propose that interactions between the
newly formed α-syn aggregates and mitochondria (D7 to D14) result in the increasing
sequestration of mitochondrial organelles and proteins during the formation and the
maturation of the LB-like inclusions leading to severe mitochondria defects observed at D21
(Figures 3-4, 6 and S14). This is in line with proteomic48,49, IHC42 and EM29,31,46,75,77
characterizations of patient LBs, for which the presence of mitochondrial proteins and entire
mitochondrial organelles have also been demonstrated.
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29
Based on our results we suggest a working model on how subtle mitochondria-related events
across the formation and maturation of LB-like inclusions might induce neurodegeneration
(Figure S14).
A reduction in mitochondrial complex I, and of the pro-fusion proteins mitofusin 2 and OPA1
protein levels by WB (Figure 7) was only observed on D21, indicating an eventual failure of
compensatory mechanisms during the formation of LB-like inclusions. These observations
correlate with reduced mitochondrial membrane potential and a breakdown of mitochondrial
respiration at this time point. Decreased respiration has already been shown in previous
studies in the neuronal seeding model. However, respiration was assessed only between 8/9
and 14 days after PFFs treatment using mouse cortical neurons94 or rat midbrain neurons95,
respectively. Importantly, in these previous studies high PFF concentrations (140nM or
350nM) were used, which in our hands induce cell death events faster. The concentration
applied here (70nM) allowed investigation at a state, where LB-like inclusions occur. While
mitochondrial respiration in these reports was assessed only in intact cells, here we measured
maximum respiratory capacities at states requiring plasma membrane permeabilization. This
allowed the additional determination of mitochondrial complexes I and complexes II to
respiration capacity. Our data are in line with post-mortem studies96-98 reporting the deficiency
of the respiratory chain in SN neurons from PD patients.
Given that we were able to detect different types of changes in mitochondrial functions and
biochemical properties during the different stages associated with α-syn aggregation,
fibrillization and formation of LB-like inclusions, we believe that the seeding model provides a
powerful platform for dissecting the role of mitochondrial dysfunctions in synucleinopathies.
Synaptic functions are impaired during the formation and the maturation of the LB
Several studies on post-mortem PD brain tissues and animal models have recently suggested
that neuritic and synaptic degeneration correlate with early disease progression99. High
presynaptic accumulation of insoluble α-syn has been proposed as a greater contributor to the
development of clinical symptoms than LBs. Our proteomic (Figure 4) and transcriptomic
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30
analyses (Figure 6) suggest that mitochondrial defects are accompanied by an alteration of
synapse-related RNA and protein levels. However, it remains unclear which pathways in the
mitochondria-synapse signaling loop is dysregulated first by the formation and the maturation
of α-syn pathological inclusions100. Consistent with postmortem studies of PD patients101,102,
during the transition from fibrils to LB-like inclusion, we showed a reduction of synaptic density,
alterations of the protein level of both pre- and post-synaptic markers (Figure 8), and
dysregulation of the synaptic transcriptome (Figure 6). Unlike mitochondrial dysfunction, which
are manifested mainly once newly formed α-syn fibrils are reorganized into LB-like inclusions
(D14-D21), early synaptic changes at the transcriptomic level were observed concomitant with
the beginning of the α-syn fibrilization process and the appearance of α-syn-seeded
aggregates inside the neuritic extensions at the early stage of the seeding model (D4–D7)
(Figures 5E and S14). This suggests that synaptic dysfunction or different pathways linked to
synaptic dysfunction are affected at the different stages of α-syn aggregation and LB formation
and occur before the early onset of neurodegeneration, supporting the hypothesis that
synaptopathy is an initial event in the pathogenesis of PD and related synucleinopathies102,103.
Synaptotoxicity reflected by the downregulation of the synaptic activity and the loss of
synapses was also recently reported in seeding neuronal models22,91.
Finally, the ERK 1/2 signaling was also shown to be upregulated during both the
formation and the maturation of the LB-like inclusions (Figure 4). The ERK 1/2 pathway
regulates neuronal cell death, oxidative stress, mitochondria fission, and integrity, as well as
synaptic plasticity65, which are the major biological processes dysregulated in PD. Similar to
what has been previously reported in bona fide LB from PD patients68,69, we found that p-ERK
1/2 was recruited inside the LB-like inclusions (Figure 7D). Therefore, dysregulation of this
central signaling pathway during the formation and the maturation of the LB-like inclusions
could affect mitochondrial and synaptic functions, potentially explaining our observation in the
neuronal seeding model and PD brains.
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31
In conclusion, our work provides a comprehensive characterization of a highly
reproducible neuronal model that recapitulates many of the key biochemical, structural, and
organizational features of LB pathologies in PD brains. Using integrative imaging approaches,
we were able to dissect the key processes involved the formation of LB-like pathologies and
provide novel insight into their contribution to neuronal dysfunction and neurodegeneration.
To our knowledge, this is the first report that shows the formation of such LB-like inclusions
and presents a comprehensive characterization of the different stages of LB formation, from
seeding to fibrillization to the formation of LB-like inclusions at the molecular, biochemical,
proteomic, transcriptomic, and structural levels. These advances, combined with the
possibility to uncouple the three key processes (seeding, fibrillization, and LB formation) and
investigate them separately, provide unique opportunities to 1) investigate the molecular
mechanisms underpinning these processes; 2) elucidate their role in α-syn-induced toxicity
and potential contributions to the pathogenesis of PD; and 3) to screen for therapeutic agents
based on the modulation of these pathways. The use of a model system that allows the
investigation of these processes over a longer period of time (e.g., iPSC derived neurons)
could enable further insights into the molecular mechanisms that regulate LB pathology
formation, maturation, and pathological diversity in PD and synucleinopathies3, especially if
the development of such model systems is driven by insight from human pathology.
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32
Material and Methods
Antibodies and compounds
Information and RRID of the primary antibodies used in this study are listed in Figure S2.
Tables include their clone name, catalog numbers, vendors, and respective epitopes.
Expression and Purification of Mouse α-syn
pT7-7 plasmids were used for the expression of recombinant mouse α-syn in E. coli. Mouse
wild type (WT) α-syn was expressed and purified using anion exchange chromatography
(AEC) followed by reverse-phase High-Performance Liquid Chromatography (HPLC) and fully
characterized as previously described104,105.
Primary culture of hippocampal neurons and treatment with mouse α-syn fibrils
Primary hippocampal neurons were prepared from P0 pups of WT mice (C57BL/6JRccHsd,
Harlan) or α-syn KO mice (C57BL/6J OlaHsd, Harlan) and cultured as previously described106.
The neurons were seeded in black, clear-bottomed, 96-well plates (Falcon, Switzerland), 6-
wells plates, or onto coverslips (CS) (VWR, Switzerland) previously coated with poly-L-lysine
0.1% w/v in water (Brunschwig, Switzerland) at a density of 300,000 cells/mL. After 5 days in
culture, the WT or α-syn KO neurons were treated with extracellular mouse α-syn fibrils to a
final concentration of 70 nM and cultured for up to 21 days as previously described22,23. All
procedures were approved by the Swiss Federal Veterinary Office (authorization number VD
3392).
Immunocytochemistry (ICC)
After α-syn PFFs treatment, HeLa cells or primary hippocampal neurons were washed twice
with PBS, fixed in 4% PFA for 20 min at RT, and then immunostained as previously
described105. The antibodies used are indicated in the corresponding legend section of each
figure. The source and dilution of each antibody can be found in Figure S2. The cells plated
on CS were then examined with a confocal laser-scanning microscope (LSM 700, Carl Zeiss
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Microscopy, Germany) with a 40× objective and analyzed using Zen software
(RRID:SCR_013672). The cells plated in black, clear-bottom, 96-well plates were imaged
using the IN Cell Analyzer 2200 (with a ×10 objective). For each independent experiment, two
wells were acquired per tested condition, and in each well, nine fields of view were imaged.
Each experiment was reproduced at least three times independently.
Quantitative high-content wide-field cell imaging analyses (HCA)
After α-syn PFFs treatment, primary hippocampal neurons plated in black, clear-bottom, 96-
well plates (BD, Switzerland) were washed twice with PBS, fixed in 4% PFA for 20 min at RT
and then immunostained as described above. Images were acquired using the Nikon 10×/
0.45, Plan Apo, CFI/60 of the IN Cell Analyzer 2200 (GE Healthcare, Switzerland), a high-
throughput imaging system equipped with a high-resolution 16-bits sCMOS camera
(2048×2048 pixels), using a binning of 2×2. For each independent experiment, duplicated
wells were acquired per condition, and nine fields of view were imaged for each well. Each
experiment was reproduced at least three times independently. Images were then analyzed
using Cell profiler 3.0.0 software (RRID:SCR_007358) for identifying and quantifying the level
of LB-like inclusions (stained with pS129 antibody) formed in neurons MAP2-positive cells, the
number of neuronal cell bodies (co-stained with MAP2 staining and DAPI), or the number of
neurites (stained with MAP2 staining).
Correlative Light and Electron Microscopy (CLEM)
Primary hippocampal neurons were grown on 35 mm dishes with alphanumeric searching
grids etched to the bottom glass (MatTek Corporation, Ashland, MA, USA) and treated with
WT PFFs. At the indicated time point, cells were fixed for 2 h with 1% glutaraldehyde and 2.0%
PFA in 0.1 M phosphate buffer (PB) at pH 7.4. After washing with PBS, ICC was performed
(for more details, see the corresponding section in the Materials and Methods). Neurons with
LB-like inclusions (positively stained for pS129) were selected by fluorescence confocal
microscopy (LSM700, Carl Zeiss Microscopy) for ultrastructural analysis. The precise position
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of the selected neuron was recorded using the alpha-numeric grid etched on the dish bottom.
The cells were then fixed further with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1
M PB at pH 7.4 for another 2 h. After washing (5 times for 5 min) with 0.1 M cacodylate buffer
at pH 7.4, cells were post-fixed with 1% osmium tetroxide in the same buffer for 1 h and
washed with double-distilled water before contrasting with 1% uranyl acetate water for 1 h.
The cells were then dehydrated in increasing concentrations of alcohol (twice at 50%, once at
70%, once at 90%, once at 95%, and twice at 100%) for 3 min each wash. Dehydrated cells
were infiltrated with Durcupan resin diluted with absolute ethanol at 1:2 for 30 min, at 1:1 for
30 min, and 2:1 for 30 min, and twice with pure Durcupan (Electron Microscopy Sciences,
Hatfield, PA, USA) for 30 min each. After 2 h of incubation in fresh Durcupan resin, the dishes
were transferred into a 65°C oven for the resin to polymerize overnight. Once the resin had
hardened, the glass CS on the bottom of the dish was removed by repeated immersion in hot
(60°C) water, followed by liquid nitrogen. The cell of interest was then located using the
alphanumeric coordinates previously recorded and a razor blade used to cut this region away
from the rest of the resin. This piece was then glued to a resin block with acrylic glue, trimmed
with a glass knife using an ultramicrotome (Leica Ultracut UCT, Leica Microsystems), and then
ultrathin sections (50–60 nm) were cut serially from the face with a diamond knife (Diatome,
Biel, Switzerland) and collected onto 2 mm single-slot copper grids coated with formvar plastic
support film. Sections were contrasted with uranyl acetate and lead citrate and imaged with a
transmission electron microscope (Tecnai Spirit EM, FEI, The Netherlands) operating at 80
kV acceleration voltage and equipped with a digital camera (FEI Eagle, FEI).
Immunogold Staining
Primary hippocampal neurons were grown on 35 mm dishes with alphanumeric searching
grids etched to the bottom glass (MatTek Corporation, Ashland, MA, USA) and treated with
WT PFFs. At the indicated time point, cultured cells were fixed for 2 h with a buffered mix of
paraformaldehyde (2%) and glutaraldehyde (0.2%) in PB (0.1M), then washed in PBS (0.01M).
After washing with PBS, ICC was performed as described105. Briefly, cells were incubated with
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pS129 antibody (81a, Figure S4B) for 2 h at RT diluted in the blocking solution [(3% BSA and
0.1% Triton X-100 in PBS), (PBS-BSA-T)]. After five washes with PBS-BSA-T, neurons were
incubated with the Alexa Fluor647 Fluoronanogold secondary antibody (Nanoprobes, USA) for
1 h at RT. Neurons were washed five times with PBS-BSA-T and mounted in polyvinyl alcohol
mounting medium supplemented with anti-fading DABCO reagent. Neurons with LB-like
inclusions (positively stained for pS129) were selected by fluorescence confocal microscopy
(LSM700, Carl Zeiss Microscopy) for ultrastructural analysis. The precise position of the
selected neuron was recorded using the alpha-numeric grid etched on the dish bottom. The
cells were next washed in phosphate buffer (0.1M) and fixed again in 2.5% glutaraldehyde
before being rinsed in distilled water. A few drops of a silver enhancement solution (Aurion,
Netherlands) were then added to each CS to cover all of the cells and left in the dark for 1 h
at RT. The solution was then removed, and the CS washed in distilled water followed by 0.5%
osmium tetroxide for 30 min and 1% uranyl acetate for 40 min. Following this, the CS were
dehydrated through increasing concentrations of ethanol and then transferred to 100% epoxy
resin (Durcupan, Sigma Aldrich) for 4 h and then placed upside down on a glass CS in a 60°C
oven overnight. Once the resin had cured, the CS was removed, and regions of interest were
cut away with a razor blade and mounted onto a blank resin block for thin sectioning. Serial
thin sections were cut at 50 nm thickness with a diamond knife (Diatome, Switzerland) in an
ultramicrotome (UC7, Leica Microsystems) and collected onto a pioloform support firm on
copper slot grids. These were further stained with lead citrate and uranyl acetate. Sections
were imaged with a digital camera (Eagle, FEI Company) inside a transmission electron
microscope (Tecnai Spirit, FEI Company) operating at 80 kV.
Cell lysis and WB analyses of primary hippocampal neurons
Preparation of soluble and insoluble fractions
After α-syn PFFs treatment, primary hippocampal neurons were lysed as described in
Volpicelli-Daley et al.22,23. Briefly, treated neurons were lysed in 1% Triton X-100/ Tris-buffered
saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.5) supplemented with protease inhibitor
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cocktail, 1 mM phenylmethane sulfonyl fluoride (PMSF), and phosphatase inhibitor cocktail 2
and 3 (Sigma-Aldrich, Switzerland). After sonication using a fine probe [(0.5-s pulse at an
amplitude of 20%, ten times (Sonic Vibra Cell, Blanc Labo, Switzerland], cell lysates were
incubated on ice for 30 min and centrifuged at 100,000 g for 30 min at 4°C. The supernatant
(soluble fraction) was collected while the pellet was washed in 1% Triton X-100/TBS,
sonicated as described above, and centrifuged for another 30 min at 100,000 g. The
supernatant was discarded whereas the pellet (insoluble fraction) was resuspended in 2%
sodium dodecyl sulfate (SDS)/TBS supplemented with protease inhibitor cocktail, 1 mM
PMSF, and phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, Switzerland), and sonicated
using a fine probe (0.5-s pulse at amplitude of 20%, 15 times).
Total cell lysates
After α-syn PFFs treatment, primary hippocampal neurons were lysed in 2% sodium dodecyl
sulfate (SDS)/TBS supplemented with protease inhibitor cocktail, 1 mM PMSF, and
phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, Switzerland) and boiled for 10 min. The
bicinchoninic acid (BCA) protein assay was performed to quantify the protein concentration in
the total, soluble, and insoluble fractions before addition of Laemmli buffer 4× (4% SDS, 40%
glycerol, 0.05% bromophenol blue, 0.252 M Tris-HCl pH 6.8, and 5% β-mercaptoethanol).
Proteins from the total, soluble, and the insoluble fractions were then separated on a 16%
tricine gel, transferred onto a nitrocellulose membrane (Fisher Scientific, Switzerland) with a
semi-dry system (Bio-Rad, Switzerland), and immunostained as previously described105.
Proteomic identification of the proteins enriched in the insoluble fraction of the PFF-
treated neurons
Primary hippocampal neurons were treated with PBS or PFFs for 7, 14, or 21 days. Cells were
lysed at the indicated time-points into the soluble and insoluble fractions as described above.
Proteins from the insoluble fraction were then separated by SDS-PAGE on a 16%
polyacrylamide gel, which was then stained with Coomassie Safestain (Life Technologies,
Switzerland). Each gel lane was entirely sliced, and proteins were in-gel digested as previously
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described107. Peptides were desalted on stageTips108 and dried under a vacuum concentrator.
For LC-MS/MS analysis, resuspended peptides were separated by reversed phase
chromatography on a Dionex Ultimate 3000 RSLC nano UPLC system connected in-line with
an Orbitrap Lumos (Thermo Fisher Scientific, Waltham, USA). Label-free quantification (LFQ)
was performed using MaxQuant 1.6.0.1109 against the UniProt mouse database (UniProt
release 2017_05). Perseus was used to highlight differentially quantified proteins110. Reverse
proteins, contaminants, and proteins only identified by sites were filtered out. Biological
replicates were grouped together. Protein groups containing a minimum of two LFQ values in
at least one group were conserved. Empty values were imputed with random numbers from a
normal distribution (Width: 0.5 and Downshift: 1.7). Significant hits were determined by a
volcano plot-based strategy, combining t-test p-values with ratio information111. Significance
curves in the volcano plot corresponding to a SO value of 0.4 (D7 and D21) or 0.5 (D14) and
0.05 FDR were determined by a permutation-based method. Further graphical displays were
generated using a homemade program written in R (https://www.R-project.org/). Table 1
contains the final results.
Quantification of cell death in primary hippocampal neurons
Primary hippocampal neurons were plated in a 96-well plate and treated with α-syn PFFs (70
nM or up to 500 nM). After 7, 14, and 21 days of treatment, cell death was quantified using
complementary cell death assays.
Quantification of active caspase 3
The CaspaTag fluorescein caspase 3 activity kit (ImmunoChemistry Technologies, MN, USA)
allows the detection of active effector caspase (caspase 3) in living cells. These kits use the
specific fluorochrome peptide inhibitor (FAM-DEVD-fmk) of the caspase 3 (FLICA). This probe
passively enters the cells and binds irreversibly to the active caspases. Neurons were washed
twice with PBS and incubated for 30 min at 37°C with FAM-DEVD-FMK in accordance with
the supplier's instructions. Fluorescein emission was quantified using a Tecan infinite M200
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Pro plate reader (Tecan, Maennedorf, Switzerland) with excitation and emission wavelengths
of 487 nm and 519 nm respectively.
Quantification of LDH release
Using a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Switzerland), the lactic
acid dehydrogenase (LDH) released into culture supernatants was measured following the
manufacturer’s instructions. After a 30 min coupled enzymatic reaction, which results in the
conversion of a tetrazolium salt (INT) into a red formazan product, the amount of color formed,
that is proportional to the number of damaged cells, was measured using a Tecan infinite
M200 Pro plate reader (Tecan, Maennedorf, Switzerland) at a wavelength of 490 nm.
Quantitative real-time RT-PCR and transcriptomic analysis
Primary hippocampal neurons were treated with WT PFFs or PBS buffer for 7, 14, and 21
days. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Switzerland) according to
the manufacturer's protocol. The concentration of each sample was measured using a
NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), and the purity was confirmed
using the ratios at (260/280) nm and (260/230) nm.
Quantitative real-time RT-PCR
RNA (2 µg) was used to synthesize cDNA using the High-Capacity RNA-to-cDNA Kit (Life
Technologies) following the manufacturer's instructions. To quantify α-syn mRNAs levels, we
used the SYBR Green PCR master mix (Pack Power SYBR Green PCR mix, Life
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39
Technologies). Q-PCR was performed using the following primers synthesized by Microsynth
(Balgach, Switzerland):
Gene Forward sequence (5’-3’) Reverse sequence (5’-3’)
SNCA AATGTTGGAGGAGCAGTGGT GGCATGTCTTCCAGGATTCC
β-actin TTGTGATGGACTCCGGAGAC TGATGTCACGCACGATTTCC
GAPDH AACGACCCCTTCATTGACCT TGGAAGATGGTGATGGGCTT
Forty cycles of amplification were then performed in an ABI Prism 7900 (Applied Biosystem,
Foster City, USA) using a 384-well plate, which allowed the simultaneous analysis of the
genes of interest and the housekeeping genes (β-actin and GAPDH) that serve as references
for the normalization step. For each independent experiment, triplicated wells were acquired
per condition, and each experiment was reproduced at least three times independently. To
quantify the expression level of the genes of interest in the different conditions tested, the
comparative 2-ΔΔCT method was used where ΔΔCT = ΔCT(target gene)−ΔCT(reference gene)
and ΔCT = CT(target gene)−CT(reference gene). Results were expressed as the fold change
relative to control neurons (2-ΔΔCT). The geNorm method (RRID: SCR_006763,
https://genorm.cmgg.be/) was performed to assess the most stable reference gene that should
be used for the normalization of the gene expression112.
Temporal transcriptomic analysis
Libraries for mRNA-seq were prepared according to manufacturer’s instructions with the
TruSeq stranded mRNA kit (Illumina) starting from 300 ng of good-quality total RNAs (RNA
quality scores >8.9 on the TapeStation 4200). Libraries were subsequently loaded at 1.44 pM
on two High Output flow cells (Illumina) and sequenced in a NextSeq 500 instrument (Illumina)
according to manufacturer instructions, yielding 75 nt paired-end reads.
RNA-seq reads were aligned to the mm10 mouse reference genome
(GRCm38.p5) with STAR (version 2.5)113. An average of 52 million of uniquely mapped reads
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40
per sample was then assigned to genes (gencode primary assembly v16) using HTSeq-
count (version 0.9)114. The analysis was focused on protein-coding and antisense RNA genes
only, representing a total of 23,057 studied genes. Lowly expressed genes (CPM < 5) and
genes present in less than three samples were excluded prior to further processing.
Normalization and differential expression analysis of the remaining 11,767 genes were done
with the R package DEseq2 (version 1.2)115, using the internal DEseq and the
results methods. Genes with an absolute log2 fold-change greater than 1 and an FDR less
than 0.01 were considered as significantly differentially expressed. Table 2
[combined_DEseq2_FC1_FDR_0_01_PFatLeastOnce.xls] contains the final results. Tables
3-4 contains respectively analyses of mitochondrial and synaptic genes significantly enriched.
Respirometry on detached primary neurons
Primary hippocampal neurons were plated 4 weeks before respiration experiments. At 21, 14,
or 7 days before respiration, neurons were exposed to 70 nM PFFs. Immediately before
respiration experiments, cells were carefully washed with PBS, and exposed to 0.05% trypsin
for 35–40 min, until detachment of cells was clearly visible, after which DMEM F12 medium
was used to neutralize the trypsin. Cells were gently spun down (300 g for 3 min), and the
cells of 1.5 wells of 6 well plates were resuspended in DMEM F12 and used for high-resolution
respirometry. The cell suspension was transferred to MiR05 (0.5 mM EGTA, 3 mM MgCl2, 60
mM potassium lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose
and 0.1% (w/v) BSA, pH 7.1) at a concentration of 2.5% cell suspension in MiR05 directly in
a 2 ml oxygraphy chamber. Respiration was measured in parallel to mitochondrial ROS
production (O2− and H2O2) at 37°C in the Oroboros O2k (calibrated for each experiment)
equipped with the O2K Fluo-LED2 Module (Oroboros Instruments, Austria). Mitochondrial
ROS measurements were performed as described previously116 using LEDs for green
excitation in the presence of 10 μM Amplex Red, 1 U/ml horseradish peroxidase, and 5 U/ml
superoxide dismutase in 2 ml MiR05 per O2K chamber. Calibration was performed by titrations
of 5 μL of 40 μM H2O2.
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Routine respiration and associated mitochondrial ROS production were assessed from
unpermeabilized cells, after which plasma membranes were permeabilized by application of
an optimized (optimal accessibility of substrates to mitochondria while maintaining
mitochondrial outer membranes intact) concentration of digitonin (5 μg/mL).
A substrate-uncoupler-inhibitor-titration (SUIT) protocol was applied to measure
oxygen flux at different respirational states on permeabilized cells, as described
previously117,118. Briefly, NADH-pathway (N) respiration in the LEAK and oxidative
phosphorylation (OXPHOS) state was analyzed in the presence of malate (2 mM), pyruvate
(10 mM) and glutamate (20 mM) before and after the addition of ADP (5 mM), respectively
(NL, NP). Addition of succinate (10 mM) allowed assessment of NADH- and Succinate-linked
respiration in OXPHOS (NSP) and the uncoupled state (NSE) after incremental (Δ0.5 μM)
addition of carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Inhibition of Complex I by
rotenone (0.5 μM) yielded succinate-linked respiration in the uncoupled state (SE). Tissue-
mass specific oxygen fluxes were corrected for residual oxygen consumption, Rox, measured
after additional inhibition of the mitochondrial electron transfer system, ETS, Complex III with
antimycin A. For further normalization, fluxes of all respiratory states were divided by ETS-
capacity to obtain flux control ratios, FCR.
Terminology was applied according to
http://www.mitoglobal.org/index.php/Gnaiger_2019_MitoFit_Preprint_Arch
Mitochondrial ROS values were corrected for background fluorescence, and respirational
states before the addition of uncoupler were used for analysis.
Measurement of the mitochondrial membrane potential and the mitochondrial mass in
PFF-treated neurons
Primary hippocampal neurons were plated in a 96-well plate and treated with α-syn PFFs (70
nM). After 7, 14, and 21 days of treatment, mitochondrial membrane potential and
mitochondrial mass were quantified using the TMRE (tetramethylrhodamine, ethyl ester)
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
42
assay kit (Abcam, UK) and Mitotracker Green tracer (Life Technologies, Switzerland) assays
respectively.
TMRE assay
TMRE is a permeant dye that accumulates in the active mitochondria. TMRE was added to
cells at a final concentration of 200 nM and incubated for 20 min 37°C. After a wash with PBS,
TMRE staining was quantified using a Tecan infinite M200 Pro plate reader (Tecan,
Maennedorf, Switzerland) with excitation and emission wavelengths of 549 nm and 575 nm
respectively.
Mitotracker Green assay
Mitotracker Green was added to cells at a final concentration of 20 nM and incubated for 30
min at 37°C. After a wash with PBS, TMRE staining was quantified using a Tecan infinite M200
Pro plate reader (Tecan, Maennedorf, Switzerland) with excitation and emission wavelengths
of 487 nm and 519 nm respectively.
Measurement of the synaptic area
Images were acquired on an LSM 700 microscope (Carl Zeiss Microscopy, Germany), with a
40× objective (N.A. = 1.30). The pixel size was set to 78 nm and a z-step of 400 nm; the
pinhole was set at 1 µm (equivalent 1A.U, channel 2).
To automatically analyze the z-stack images, the image analysis was performed in
Fiji119, using a custom script (in ImageJ macro language). Briefly, the script binarizes the
different channels of the image using thresholds and measures the area and perimeter.
The script binaries user selected channels of the image using a threshold, either a fixed value
or a value defined by an automatic threshold method (https://imagej.net/Auto_Threshold)
(user defined the appropriate threshold by visual inspection, c1 = DAPI, c2 = Synapsin I, c3 =
MAP2, c4 = pS129). From these resulting images, the script applies median filtering to remove
noisy pixels and outputs the final masks (user defined the radius of the median filter for each
channel, c1 = DAPI, c2 = Synapsin I, c3 = MAP2, c4 = pS129). Finally, the script measures
the area, the perimeter, and multiplies the perimeter value by the z-step (to get an estimate of
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43
the surface). The generated masks are merged and saved as an output for visual validation
of the thresholding.
Relative Quantification of WBs and Statistical Analysis
The level of total α-syn (15 kDa, 12 kDa, or HMW) or pS129-α-syn were estimated by
measuring the WB band intensity using Image J software (U.S. National Institutes of Health,
Maryland, USA; RRID:SCR_001935) and normalized to the relative protein levels of actin. All
the experiments were independently repeated three times. The statistical analyses were
performed using Student’s t-test or ANOVA test followed by a Tukey-Kramer post-hoc test
using KaleidaGraph (RRID:SCR_014980). The data were regarded as statistically significant
at p<0.05.
Acknowledgments
This work was supported by funding from EPFL and UCB (H.A.L, A.L.M, J.B, N.M, and L.W).
We thank F.A for the production and the characterization of α-syn monomers and fibrils
depicted in Figure S1. We are grateful to Dr. Arne Seitz and his staff at the Bio-imaging Core
Facility (BioP, EPFL) for their technical support and helpful discussions. We thank Dr Romain
Guiet (BioP, EPFL) for the development of a custom script (in ImageJ macro language) used
for post-processing imaging analysis. We thank the Proteomics Core Facility (EPFL): Dr Marc
Moniatte for the discussions, and Romain Hamelin and Dr. Florence Armand for handling the
samples and for their valuable discussions. We thank Dr Gerardo Turcatti and his staff at the
Biomolecular Screening Core Facility (EPFL) for the discussions and their technical support.
We thank Dr. Bastien Mangeat and his staff at Gene expression Core Facility (EPFL) for their
technical support in handling the samples. We also thank Stéphanie Rosset and Anaëlle
Dubois at the Bio-EM Core facility (EPFL) for their technical support. Figures 9 and S14 have
been prepared partially created using with BioRender.
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44
Author contributions
H.A.L conceived and supervised the study. H.A.L and A.L.M.M designed all the experiments
and wrote the paper. A.L.M.M performed and analyzed the experiments shown in Figures 1-
5, 6A, 6E-G, 7-9, and Figures S1B-S13. J.B designed, performed, and analyzed the
experiments shown in Figures 6B-D and S14. N.M analyzed the experiments shown in Figures
4 and S8, S12-S13. L.W performed and analyzed the experiments shown in Figures 6G and
7A. M.L. analyzed the transcriptomic data and participated to the Figures 4D, 5, 8G-H, I and
S9. F.K developed the pipeline for High Content Image Analysis (HCA) using Cell Profiler
software shown in Figures S3 G-I and Figure 8D-F. M.C prepared the samples for CLEM
analysis and acquired EM images in Figures 2, 3, and S5. G.K supervised the experiments
shown in Figures 1L, 2-3, and S5 and contributed to the interpretation of the data. All authors
reviewed and contributed to the writing.
Competing interests statement
Authors declare no competing financial interests in association with this manuscript.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
45
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Figures titles and Legends
Figure 1. At early stage, α-syn-seeded fibrillar aggregates share the
immunohistochemical but not the structural and morphological features of the bona
fide human LBs.
A. Seeding model in primary hippocampal neurons. 70 nM of mouse sonicated PFFs were
added to neurons at DIV 5 (days in vitro). Control neurons were treated with PBS used to
prepare PFFs. Following a defined period of 4 days (D4) during which no seeded aggregates
are detected, α-syn aggregates first appear in the neuronal extension (between D4 to D7)
before being detected in the neuronal cell bodies (D7–D14) where they eventually accumulate
(D14 to D21).
B. The formation of α-syn aggregates in the context of the neuronal seeding requires a
sequence of events starting with 1) the internalization and cleavage of PFF seeds (D0–D1)
followed by 2) the initiation of the seeding by the recruitment of endogenous α-syn (D0–D4),
3) fibril elongation along with the incorporation of PTMs, such as phosphorylation at residue
S129 (D4–D14), and 4) formation of large filamentous aggregates (D7–D14).
C-J, M-P. Temporal analysis of α-syn aggregates formed at D7 (C, E, G, I), at D14 (D, F, H,
J), and at D21(M-P) in WT neurons after addition of α-syn PFFs. Aggregates were detected
by ICC using pS129 (MJFR13 or 81a) in combination with total α-syn (epitope: 1-20) (C-D, M),
p62 (E-F, N) or ubiquitin (G-H, O) antibodies. Aggregates were also stained with the
Amytracker dye (I-J, P). Neurons were counterstained with microtubule-associated protein
(MAP2) antibody, and the nucleus was counterstained with DAPI staining. Scale bars = 10
μm.
K. WB analysis of α-syn seeded-aggregates formed 14 days after adding mouse PFFs to WT
neurons. Control neurons were treated with PBS. After sequential extractions, the insoluble
fractions of neuronal cell lysates were analyzed by immunoblotting. Total α-syn, pS129, and
actin were detected by SYN-1, pS129 (MJFR13), and actin antibodies, respectively.
Monomeric α-syn (15 kDa) is indicated by a double asterisk; C-terminal truncated α-syn (12
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53
kDa) is indicated by a single asterisk; the higher molecular weights (HMWs) corresponding to
the newly formed fibrils are detected from 25 kDa to the top of the gel.
L. At D14, neurons were fixed, and ICC with a pS129 (81a) antibody was performed and
imaged by confocal microscopy (upper images). Scale bar = 5 μm. The selected neuron was
embedded and cut by an ultramicrotome. Serial sections were examined by EM (bottom
image). Immunogold labeling allowed the detection of long filaments in the neuronal cell body
that appeared as tightly packed bundles of fibrils that were positive for pS129-α-syn. Scale
bar = 500 nm.
Q. In this study, we employed an integrated approach using advanced techniques in confocal
imaging, EM, proteomics, transcriptomic, and biochemistry to investigate, at the spatio-
temporal resolution (from D7 to D21), how the seeded intracellular aggregates recapitulate
the Lewy body-like inclusion pathology at the morphological, biochemical, structural, and
biological levels.
Figure 2. Formation and maturation of LB-like inclusions require the lateral association
and the fragmentation of the newly formed α-syn fibrils over time.
PFFs were added for 7, 14, and 21 days to the extracellular media of primary hippocampal
neurons plated on dishes with alpha-numerical searching grids imprinted on the bottom,
which allowed easy localization of the cells. At the indicated time, neurons were fixed, ICC
against pS129-α-syn was performed and imaged by confocal microscopy (A, B, D, F top
images; scale bar = 10 μm), and the selected neurons were embedded and cut using a
ultramicrotome. Serial sections were examined by EM (A, B, D, F bottom images). Aa. Neurite
with pS129-positive newly formed α-syn fibrils. Ab. Neurite negative for pS129 staining. Ab,
inset. Graph representing the mean ± SD of the width of the microtubules compared to the
newly formed fibrils at D7 (a minimum of 120 microtubules or newly formed fibrils were
counted). Measurements confirmed that the width of newly formed α-syn fibrils of 11.94±3.97
nm (SD) is significantly smaller than the average width of the microtubules of 18.67±2.94 nm
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54
(SD). p<0.001=*** (student t-test for unpaired data with equal variance), indicating that this
parameter can be used to discriminate the newly formed fibrils from the cytoskeletal proteins.
Autophagolysosomal-like vesicles are indicated by a yellow asterisk (D) or colored in yellow
(F), and mitochondrial compartments are indicated by a green asterisk (D) or colored in
green (F). Accumulation of endomembranous compartment at a late stage (D21) are
colored in pink (endosomes-like vesicles) and purple (lysosomes-like vesicles),
respectively. Nuclei are highlighted in blue. C-G. Representative images at higher
magnifications corresponding to the area indicated by a yellow rectangle in EM images
shown in B, D, and F respectively.
A, B, D, F. Scale bar = 1 μm; Aa and Ab. Scale bar = 500 nm; C-G. Scale bars = 300 nm.
Figure 3. Maturation of α-syn-seeded aggregates into LB-like inclusions is
accompanied by the sequestration of organelles and the endomembrane system over
time.
A-D. At D21, PFF-treated neurons were fixed, ICC against pS129-α-syn was performed and
imaged by confocal microscopy (A-D, inset images; scale bar = 5 μm), and serial sections of
the selected neurons were examined by EM. Representative images of LB-like inclusions
which exhibited a ribbon-like morphology (A-B) or compact and rounded structures (C-D). A
high number of mitochondria (highlighted in green) and several types of vesicles, such as
autophagosomes, endosome, and lysosome-like vesicles (highlighted respectively in yellow,
pink and purple) were detected inside or at the edge of these inclusions. A, C-D. Scale bar =
1 μm; B. Scale bar = 2 μm.
E-H. LB-like inclusions were detected by ICC using pS129 (MJFR13 or 81a) in combination
with LAMP1 (late endosome/lysosome marker) (E), LAMP2A (autophagolysosome marker)
(F), Tom20 (mitochondria marker) (G), or BiP (ER marker) (H) antibodies. Neurons were
stained with microtubule-associated protein (MAP2) antibody, and the nucleus was
counterstained with DAPI. Scale bars = 10 μm.
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55
Figure 4. Temporal proteomic analyses of the protein contents found in the insoluble
fraction of the PFF-treated neurons reveals a high increase in proteins related to the
endomembrane system.
A. Insoluble proteins from neurons treated with PBS and PFFs for 7, 14, and 21 days were
extracted and analyzed using LC-MS/MS. Identified proteins were plotted using volcano plot.
Mean difference (Log2 (Fold-Change) on the X-axis) between the insoluble fractions of PFF-
treated neurons and PBS neurons treated for 14 (left panel) or 21 days (right panel) were
plotted against Log10 (p-Value) on the Y-axis (T-Test). Dotted lines represent the FDR <0.05
(False Discovery Rate) and threshold of significance SO=1 assigned for the subsequent
analysis. A detailed list of the hits shown in the volcano plot is available in Table 1.
B-C. Classification of the proteins significantly enriched in the insoluble fractions of the PFF-
treated neurons at D14 and D21 by cellular component (B) and biological processes (C) using
Gene Ontology (GO) enrichment analyses determined by DAVID analysis.
D. Heat map representing color-coded fold change levels of mitochondrial (right) and synaptic
(left) proteins present in the insoluble fraction in neurons treated with PFFs 7, 14, and 21 days.
E. Cytoscape visualization of the proteins shared between 14 and 21 days PFFs treated
insoluble fraction based on cellular compartment related to mitochondrial and synaptic
function. The nodes (round shape) represent gene ontology terms; node color represents the
group and node size represent the significance of each gene ontology term.
Figure 5. Gene expression level changes during the formation of the newly formed
fibrils and their maturation into LB-like inclusions.
Temporal transcriptomic analysis of the gene expression level in PBS-treated neurons vs.
PFF-treated neurons treated for 7, 14, or 21 days. Genes with an absolute log2 fold-change
greater than 1 and an FDR less than 0.01 were considered as significantly differentially
expressed. Significant GO terms enrichment associated with cellular components (A) and
biological processes (B) from differentially regulated genes were plotted against –log10 P value
(t-test, PBS-treated neurons vs. PFF-treated neurons, Table 2). Log2 fold changes of the
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56
mitochondrial genes (C and Table 3), and the synaptic genes (D and Table 4) expression
levels are represented over time. “+” and “-” indicate a significant upregulation or
downregulation in the gene expression level respectively.
Figure 6. Dynamics of Lewy body formation and maturation induce mitochondrial
alterations.
Primary hippocampal neurons were treated with 70 nM of PFFs for 7, 14, or 21 days before
assessment of mitochondrial parameters.
A. ICC co-stainings of α-syn pS129 with VDAC1, TOM20, and TIM23 show increasing
colocalization of mitochondrial components with pS129-positive aggregates over time. Scale
bars = 5 μm. B. High-resolution respirometry revealed significant differences between PBS-
and PFF- treated groups at D21 in several respirational states (B, upper panel). Concurrently
assessed mitochondrial ROS-production was exclusively reduced at D7 (B, lower panel).
Statistical results in B were obtained from 2-way repeated measurement ANOVAs based on
a minimum of four independent experiments. C-D. Proteomic data on proteins implicated in
mitochondrial dysfunction suggest proteins related to oxidative phosphorylation (C) and
oxidative stress pathways (D) to be sequestered into aggregates only after D7 days.
Mitochondrial membrane potential was assessed fluorometrically from cells loaded with TMRE
(E); reduction in TMRE fluorescence intensity was observed at D21. Mitotracker green,
labeling mitochondrial proteins independent of mitochondrial membrane potential,
fluorescence (F) was moderately decreased at D21. G. WB analyses of total fractions derived
from neuronal cultures. Components of the oxidative phosphorylation (OXPHOS) system
(most notably the NDUF B8 subunit of complex I) are reduced at D21. A primary antibody mix
for subunits of OXPHOS complexes I-V was used, the band indicated by an orange arrow
represents CI-NDUF B8. Higher bands (from bottom to top) represent CII-SDHB, CIII-
UQCRC2, and CV-ATP5A, respectively. Also, levels of the mitochondrial shape changing
proteins mitofusin 2 and OPA1 were significantly decreased at D21. No significant changes in
levels of citrate synthase or VDAC1 proteins were observed at any time. The graphs (E-G),
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57
represent the mean +/- SD of a minimum of three independent experiments. p<0.05 = *,
p<0.005=**, p<0.0005=*** (ANOVA followed by Tukey HSD post-hoc test, PBS vs. PFF-
treated neurons). p<0.05 = # p<0.05 = ### (ANOVA followed by Tukey HSD post-hoc test,
D14 vs. D21 PFF-treated neurons).
Figure 7. Synaptic dysfunctions were associated with the formation and maturation of
the LB-like inclusions.
Primary hippocampal neurons were treated with 70 nM of PFFs for 7, 14, or 21 days before
assessment of synaptic parameters.
A. After total cell extraction, cells lysates were analyzed by WB. The levels of Synapsin I (pre-
synaptic protein), PSD95 (post-synaptic protein), and ERK 1/2 (synaptic plasticity marker)
were significantly decreased at D21. The phosphorylation level of ERK 1/2 (p-ERK 1/2)
significantly increased over time. The graphs represent the mean ± SD of a minimum of three
independent experiments. p<0.05 = *, p<0.005=** (ANOVA followed by Tukey HSD post-hoc
test, PBS vs. PFF-treated neurons). p<0.05 = ### (ANOVA followed by Tukey HSD post-hoc
test, D14 vs. D21 PFF-treated neurons).
B-C. Synaptic area decreases in PFF-treated neurons from D14. B. Aggregates were detected
by ICC using pS129 (MJFR13) in combination with Synapsin I antibodies. Neurons were
counterstained with microtubule-associated protein (MAP2) antibody, and the nucleus was
counterstained with DAPI staining. Scale bars = 5 μm. C. Measurement of the synaptic area
was performed over time. For each time-point, 24 neurons were imaged (z-step of 400 nm).
The image analysis was performed in Fiji using a custom script to automatically analyze the
z-stack images and measures the area of the synapse that was normalized to the perimeter
of the neuron of interest. p<0.05 = *, p<0.005=** (ANOVA followed by Tukey HSD post-hoc
test, PBS vs. PFF-treated neurons).
D. Aggregates were detected by ICC using pS129 (MJFR13) in combination with p-ERK 1/2
antibodies. ICC revealed the presence of p-ERK 1/2 associated with the newly formed fibrils
at D14 before being sequestered in the LB-like inclusions at D21. Scale bars = 5 μm.
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58
Figure 8. At late stages, the processes of formation and maturation of the LB-like
inclusions are associated with neuronal toxicity.
A-F. Addition of α-syn PFFs to primary neurons induces cell death in a time-dependent
manner in WT neurons. A-B. Cell death levels were assessed in WT neurons treated with
PFFs (70 nm) for up to D21 using complementary assays. Caspase 3 activation (A) was
measured as an early event of apoptotic cell death, while late cell death events were assessed
based on lactate dehydrogenase (LDH) release (B-C). For each independent experiment,
triplicate wells were measured per condition. D-F. Neuronal loss was also assessed by high
content image analysis (HCA) that allows us to determine the total number of neuronal cell
bodies (D), neurites (E) and the total level of pS129 in MAP2 positive neurons (F) over time in
PFF-treated primary culture. For each independent experiment, duplicated wells were
acquired per condition, and nine fields of view were imaged for each well. Images were then
analyzed using Cell Profile software to identify and quantify the number of neuronal cell bodies
(DAPI and MAP2-positive areas) and the number of neurites (MAP2-positiveareas).
The graphs (A-F) represent the mean ± SD of three independent experiments. p<0.01=*,
p<0.0001=*** (ANOVA followed by Tukey HSD post-hoc test, PBS vs. PFF-treated neurons).
p<0.01= #, p<0.001= ## (ANOVA followed by Tukey HSD post-hoc test, PFF-treated neurons
D14 vs. D21).
G-H, J. Temporal transcriptomic analysis of the gene expression level in PBS-treated neurons
vs. PFF-treated neurons treated for 7, 14, or 21 days. Genes with an absolute log2 fold-change
greater than 1 and an FDR less than 0.01 were considered as significantly differentially
expressed. G. Log2 fold changes of the cell death genes at D7, D14, and D21 (G: PBS vs.
PFF-treated neurons; H. Expression level of cell death genes D14 vs. D21. J. Fold changes
of SNCA and PLK2 genes (PBS vs. PFF-treated neurons). “+” and “-” indicate respectively a
significant upregulation or downregulation in the gene expression level.
I. Quantitative RT-qPCR was performed with primers specific for α-syn at the indicated time-
points after adding PFFs to WT neurons. Results are presented as fold change in comparison
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59
to the respective level in control neurons treated with PBS buffer. mRNA levels were
normalized to the relative transcriptional levels of GAPDH and actin housekeeping genes. The
graphs represent the mean ± SD of three independent experiments. p<0.005=** (ANOVA
followed by Tukey HSD post-hoc test, PBS vs. PFF-treated neurons).
Figure 9. The dynamics of Lewy body formation, rather than simply α-syn fibrillization,
is the primary cause of mitochondrial alterations, synaptic dysfunction, and
neurodegeneration.
Formation of α-syn inclusions in the context of the neuronal seeding requires a sequence of
events starting with 1) the internalization and cleavage of PFF seeds (D0-D1); followed by 2)
the initiation of the seeding by the recruitment of endogenous α-syn (D0-D4); 3) fibril
elongation along with the incorporation of PTMs, such as phosphorylation at residue S129
(D4-D14); 4) formation of seeded filamentous aggregates (D7) that are fragmented and
laterally associated over time (D7-D21); 5–6) the packing of the fibrils into LB-like inclusions
(D14-D21) that have a morphology similar to the bona fide LB detected in human
synucleinopathies (D21) is accompanied by the sequestration of organelles, endomembranes,
proteins and lipids. Our integrated approach using advanced techniques in EM, proteomics,
transcriptomic, and biochemistry clearly demonstrate that LB formation and maturation
impaired the normal functions and biological processes in PFF-treated neurons causing
mitochondrial alterations and synaptic dysfunctions that result in a progressive
neurodegeneration.
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Figure 1
A
PFFs(seeds)
Day 14 Day 21Day 7
α-synuclein monomer
Newlyformed fibrils
+ pS129
+ Interactors
Lewy body-like inclusion
Seeding
PFFs(seeds)
B
Elongation
Spatio-Temporal resolution of the LB-like inclusions
LC-MS/MS-based quantitative proteomic
Cell deathQuantitative transcriptomic analysis
Ultrastructure (CLEM)
Biochemistry - Candidate validation
Confocal imagingMorphology
Inclusion Composition
Confocal imaging - Candidate validation
Neuronal dysfunctions
Cellular dysfunctions
K
D21
D21
D21
D7
D7
D7
Q
Merged Total α-syn (1-20)pS129 (81a)
PFFs–treated neurons (D7) PFFs–treated neurons (D14) PFFs–treated neurons (D21)
C
E
G
10 μm10 μm
Merged pS129 (MJFR13) p62 Merged pS129 (MJFR13) p62 Merged pS129 (MJFR13) p62
Merged pS129 (MJFR13) ub Merged pS129 (MJFR13) ub Merged pS129 (MJFR13) ub
Merged pS129 (81a) Amytracker Merged pS129 (81a) Amytracker Merged pS129 (81a) Amytracker
Merged Total α-syn (1-20)pS129 (81a) Merged Total α-syn (1-20)pS129 (81a)
H
D
F
I J
O
M
N
P
C
500 nm
pS129 pS129/MAP2/DAPIL
75-
PBS PFFs
IB: SYN-1
IB: actin
IB: pS129 (MJFR13)
Inso
lubl
e Fr
actio
n –
D14
15-
10-
50-40-
40-
15-
35-25-
100-
Accumulation Maturation
***
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Figure 2
pS129 (81a) pS129// MAP2
300 nm
C
PFFs–treated neurons (D7) PFFs–treated neurons (D14)
pS129 (81a)
D F
pS129 (81a) pS129/ // MAP2 // DAPI
*
*
*
*
*
*
*
*
**
*
**
**
*
*
**
*
*
*
300 nm
E G 1 μm 1 μm
Nucleus
300 nm
PFFs–treated neurons (D21)
pS129/ // MAP2 // DAPI
pS129// MAP2
PFFs–treated neurons (D7)
1 μm
*
*
***
*
*
**
*
Nucleus
A
a
b
b
a
pS129 (81a)
a
1 μm
baNewly formed fibrils in pS129-positive neurite pS129-negative neurite
500 nm 500 nm
B
pS129// MAP2
0
5
10
15
20
25
1 2
Wid
th (n
m)
***
Newly formed fibrilsMicrotubules
c
*
*
*
*
*
** *
**
*
*
*
* * *
* **
**
*
*
*
*
**
*
* *
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
1 μm
Figure 3
LAMP1 LAMP2A
BiPTom20
pS129 (81a)
pS129 (MJFR13)
pS129 (81a)
pS129 (81a)
Merged
Merged
Merged
Merged G
E
A
F
H
B
C D
10 µM
Nucleus
NucleusNu
cleu
s
pS129 pS129
pS129 pS129
Nucleus
Nucleus
2 μm
1 μm 1 μm
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
0
20
40
60
80
100
0
15
30
45
-log1
0(P
valu
e) %
GO-term: Cellular Component
Day 21Day 14
Nucleocytoplasmic transport
Protein transport
0 5 10 15 20 25
TransportIntracellular protein transport
ER to Golgi vesicle-mediated transportVesicle-mediated transport
Protein export from nucleusNucleocytoplasmic transport
Protein import into nucleus
Protein foldingChaperone-mediated protein folding
Protein folding in endoplasmic reticulum
Tricarboxylic acid cycleATP metabolic process
NADH metabolic processATP biosynthetic process
Protein targeting to mitochondrionApoptotic mitochondrial changes
Mitochondrial transportMitochondrial fission
Apoptotic processProtein localization to pre-autophagosome
Response to endoplasmic reticulum stressResponse to oxidative stress
Regulation of stress-activated MAPK cascade
Synapse assemblyNeuron-neuron synaptic transmission
Neurotransmitter secretionSynaptic vesicle endocytosis
Synapse maturation
Actin cytoskeleton organizationAxonogenesis
Regulation of protein ubiquitinationUbiquitin-dependent protein catabolic process
Negative regulation of protein ubiquitination
Small GTPase mediated signal transduction
Dendritic spine development
Apoptosis
Mitochondria
Energymetabolism
Protein folding
-log10 (Pvalue)
Response tostress
Cytoskeleton
Ubiquitination
GO-term: Biological Processes Day 14Day 21
Ubiquitination
Cytoskeleton
Synapse
Response to Stress
Apoptosis
Mitochondria
Energy metabolism
Protein folding
Figure 4 Temporal protein expression profileLB-like inclusion specific components
Proteins extraction (Insoluble fraction) and Sample preparation (Trypsin digestion)
LC-MS/MS analysis(MS and data processing)
Day 14 Day 21
A
D
B
C
E
PFFs–treated neurons
D7 D14 D21PFFs–treated neurons
D7 D14 D21
Mito
chon
dria
l pro
tein
s in
inso
lubl
e fr
actio
n (fo
ld c
hang
e)
(PB
S-tr
eate
d ne
uron
s vs
PFF
s–tr
eate
d ne
uron
s)
Syna
ptic
pro
tein
s in
inso
lubl
e fr
actio
n (fo
ld c
hang
e)
(PB
S-tr
eate
d ne
uron
s vs
PFF
s–tr
eate
d ne
uron
s)
Primary Neurons
Day 14Day 7 Day 21PFFs(seeds)
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
0 10 20
cleavage furrow
transport vesicleexocytic vesicle
secretory vesicle
presynapsesynaptic vesicleterminal bouton
postsynapsesynaptic membraneexcitatory synapse
postsynaptic density
axon partaxon terminusdendritic shaft
actin cytoskeletondendritic spine
neuron spine
focal adhesionadherens junction
cell-cell junction
ion channel complexcation channel complex
potassium channel complex
extracellular matrixproteinaceous extracellular matrix
transmembrane transporter complexplasma membrane protein complex
-log10(PValue)
Primary Neurons
Day 14Day 7 Day 21PFFs(seeds)
Mito
chon
dria
l gen
es (3
5 ge
nes)
exp
ress
ion
(fold
cha
nge)
(P
BS-
trea
ted
neur
ons
vs P
FFs–
trea
ted
neur
ons)
0 10 20dendrite extension
positive regulation of neuron projection developmentpositive regulation of neuron differentiation
axon developmentglial cell differentiation
positive regulation of neurogenesis
positive regulation of cell projection organizationpositive regulation of cell growthcellular component disassembly
response to calcium ionpositive regulation of ion transmembrane transport
calcium ion transportcellular calcium ion homeostasis
negative regulation of MAP kinase activitynegative regulation of protein serine/threonine kinase…
modulation of synaptic transmissionregulation of membrane potential
regulation of synapse structure or activityneuronal action potential
regulation of synaptic plasticitysynapse organization
neurotransmitter transportneurotransmitter secretion
learning or memorycognition
regulation of actin filament-based processregulation of cytoskeleton organization
neuron deathregulation of neuron deathneuron apoptotic process
mitophagy in response to mitochondrial depolarizationresponse to mitochondrial depolarisation
macromitophagymitophagy
mitochondrion disassembly
multicellular organismal response to stressresponse to oxidative stress
small GTPase mediated signal transductionRas protein signal transduction
gamma-aminobutyric acid signaling pathwayextracellular matrix organization
Stre
ssM
itoch
ondr
iaD
eath
Cyt
oske
leto
nSy
naps
eC
alci
umio
nN
euro
gene
sis
-log10(PValue)
D14D21
D7
PFFs–treated neurons
Syna
ptic
gen
es (2
17) e
xpre
ssio
n (fo
ld c
hang
e)
(PB
S-tr
eate
d ne
uron
s vs
PFF
s–tr
eate
d ne
uron
s)
PFFs–treated neurons
Temporal transcriptomic analysis(Sequencing nextseq Illumina)
Gene expression changes
GO-term: Cellular Component GO-term: Biological processes
D14D21
D7
Heatmap key
D7 D14 D21
Figure 5
A B
C D
D7 D14 D21
Heatmap keyGrhprPcxBaxAcadsPink1Isoc2aMpstGpx1Ifi27Cmpk2TspoIdh2Sept4DmpkNdufb7Akr7a5BckdhaEtfbBloc1s1Ndufa11Slc25a18TstAifm3Acsl4Ttc19Cyb5r1PnpoGlsPak7Ndufaf5Prss35MsraBnip3Oxr1Nt5dc3
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
α-s
yn/a
ctin
a.u
0
0.5
1
1.5
*
Merged Merged
routine NL NP NSP
0.00
0.05
0.10
0.15
mito
H2O
2 &
O2- (p
mol
/(s*u
nit)) PBS
PFF 7 daysPFF 14 daysPFF 21 days
*
routine NL NP NSP NSE SE
0
20
40
60
Oxy
gen
flux
[pm
ol/(s
*uni
t prim
ary
cultu
res)
]
**
** *
* D7 D14 D14-D21 D21
Sequestration of mitochondrial proteinsinto aggregates
Figure 6
Primary Neurons
Day 14Day 7 Day 21
MitochondrialDysfunction
01000020000300004000050000
Tris WT
Mito
trac
ker
a.u
01000020000300004000050000
Tris WT
TMR
E a.
u
###
***
*
E
A
RespirometryMitochondrial membrane potential (TMRE)Total mitochondria mass (Mitotracker)Biochemistry
PFFs(seeds)
IB: OPA-1
PFFs–treated neurons (Day 7) PFFs–treated neurons (Day 14) PFFs–treated neurons (Day 21)
Tom20
pS129 (MJFR13) Merged pS129 (MJFR13) pS129 (MJFR13)Mitochondria
Tim23
Mitochondria Mitochondria
VDAC1
Confocal imaging
00.5
11.5
2
#
00.5
11.5
2**
*
#
pS12
9/ac
tin a
.u
Mito
fusi
n2/a
ctin
a.u
Citr
ate
synt
hase
/act
in a
.u
*
OPA
1/ac
tin a
.u
0
0.5
1
1.5 #
**
00.5
11.5
2
00.5
11.5
2
CI-N
DU
FB8/
actin
a.u
#
*0
0.5
1
1.5
VDAC
1/ac
tin a
.u
IB: SYN-1
+ PFFs
10-
15-
25-
40-35-
50-
IB: Actin
40-
IB: pS129
15-
25-
40-35-
50-
IB: Mitofusin 2
86-
IB: OXPHOS
50-
25-
15-
35-
Complex I
Q
Complex II
Complex IIIComplex IV
C
ADP ATP
F0
F1
Complex V
SDHASDHB
NDUF B11NDUF A4
SDHC
COX6B
COX1
ATP5O
ATP5JATP5J2ATP5L
Cat
PRX3
PRX5
GPX4GPD2
JNK
MKK4
mtSOD
DHOH
C D
F
G
IB: Citrate Synthase
40-
IB: VDAC-1
31-
+ PFFs
100-
Mito
H20
2 an
d 0- 2
[pm
ol/(s
*Uni
t)]
B
Oxy
gen
flux
[pm
ol/(s
*Uni
t prim
ary
cultu
res)
]
**
**
*D7D14D21
PBS
Tom20
Tim23
VDAC1
Tom20
Tim23
VDAC1
+ PFFs
PBS PFFs
PBS PFFs
PBS PFFs
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
00.5
11.5
2
PBS D7 D14 D21
**
Total protein fractions
Figure 7
Primary Neurons
Synaptic dysfunctions
Biochemistry Confocal Imaging
A
IB: PSD95
IB: Synapsin I
IB: p-ERK 1/2
IB: ERK 1/2
IB: Actin
00.5
11.5
2
PBS D7 D14 D21
**
PSD
95/actin
a.u
##
00.5
11.5
2
PBS D7 D14 D21
*
##Synap
sinI/actin
a.u
pER
K/ERK
a.u
##
+ PFFs
75-
95-
40-
35-
35-
Day 14Day 7 Day 21PFFs(seeds)
PFFs
–tr
eate
d ne
uron
s
pS129 (81a) Merged p-ERK 1/2
D7
D21
D14
D21
PBS
D
PFFs
–tr
eate
d ne
uron
s
pS129 (MJFR13) Merged Synapsin I
D7
D21
D14
D21
PBS
B
Area
of t
he s
ynpa
se/ p
erim
eter
of
the
neur
ons
(μm
2 )
C
PBS PFFs
**
*
D7 D14 D21PBS
40
30
20
10
0
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
0
100
200
300
400
500
D7 D14 D21
Transcriptomic analysisExpression level of cell death Genes
00.05
0.10.15
0.20.25
0.30.35
D7 D14 D21
LDH
rele
ase
(Abs
490
nm
) ***
A
0
500
1000
1500
2000
2500
D7 D14 D21
D
Cou
ntof
cel
l bod
ies
Cou
ntof
cel
l Neu
rites
*
#
*
#E
B
PBS PFFs
Temporal Toxicity analysisand
0
0.1
0.2
0.3
0.4
0.5
0.6
PBS D14 D16 D19 D21
LDH
rele
ase
(Abs
490
nm
) ***
***
###
C
G
I
Figure 8
01000200030004000500060007000
D7 D14 D21
***
***
Cas
pase
3 a
ctiv
atio
n (a
.u)
##
Primary Neurons
Day 14Day 7 Day 21PFFs(seeds)
00.0050.01
0.0150.02
0.0250.03
0.0350.04
D7 D14 D21
F
***
pS12
9 le
vel (
mea
n in
tens
ity)
in M
AP2
posi
tive
neur
ons
*
***
00.20.40.60.8
11.21.4
1 2 3 4
α-s
yn m
RN
A Fo
ld in
crea
se
**
D7 D14 D21D21
J
Plk2
Snca
--
-
D14D7 D21
Profile of SNCA and Plk2 genes differential expressedin PFFs-treated neurons overtime
Diff
eren
tially
exp
ress
ed G
enes
invo
lved
in
cell
deat
h B
P -D
14 v
s D
21
(26
gene
s)
Diff
eren
tially
exp
ress
ed g
enes
invo
lved
in
cell
deat
h bi
olog
ical
pro
cess
es (B
P)PF
Fs-tr
eate
d ne
uron
s vs
PB
S (1
58 g
enes
)
D14D7 D21
Upregulated
Downregulated
D14 D21
D14 D21
KdrLpar1Emp1Nuak2Traf1Cyp26b1Xaf1Cxcl10Osr1HhipIgfbp3Fn1Thbs1Bmp6
PrkcgNptx1Gabra5CckSncaPtk2bCamk2aNtsr1Itga4Gpr37Gdf10Cd36
Pink1
Tspo1
HDAC7ApoeBaxRtknRapgef3Traf4Aifm3Wnt7a
Traf1Xaf1
PawrRnf112Nos1Ntsr1
4.5
4
3.5
3
2.5
2
Norm
.Exp
r(lo
g10)
2
Fold
Cha
nge
(log2
)
1.510.50
-2-1.5-1-0.5
H
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint
α-syn PFFs
Escape
PFFsinternalisation
C-ter Truncated fibrils and full-length fibrils
TruncationTruncated
seeds
C-terpeptide
+
+ PTMsSeeding
α-synMonomer
Elongation
Newly formed fibrils(p62, ub, ThS, pS129)
ub
ub
P
P
p62
Truncation
ub
ub
P
P
ub
P
P
Lateral association
Nucleus
Maturation
Lewy body-like inclusion α-syn aggregate
Interaction with
endomembranes,organelles,
proteins and lipids
ub
P
PLysosome
Endosome
Figure 9 Dynamics of Lewy body formation and maturation results in mitochondria alterations, synaptic dysfunctions and neurodegeneration
1 2 3
4
56
α-syn PFFs
α-syn Monomer
Newly formed fibrils
Truncated newly formed fibrils
Mitochondria
Vesicles(endosomes, lysosomes, autophagosomes)
EndoplasmicReticulum
Golgi apparatus
MicrotubuleActinNeurofilament
Lipid droplets
Synaptic Dysfunctions:
Synapse densityPre and post Synaptic markersDysregulation synaptic-gene relatedp-ERK 1/2 signalling pathway
Mitochondria Dysfunctions:SequestrationRespiration (Complexes I and II)BiomassPro-fusion markers
Cell death:
Caspase 3 activationLoss of plasma membrane integrityNeuritic atrophy Expression cell death gene-related
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted September 2, 2019. . https://doi.org/10.1101/751891doi: bioRxiv preprint