Detection of α-synuclein amyloidogenic aggregates in vitro and in cells using light-switching dipyridophenazine ruthenium(II) complexes

Article abstract of DOI:10.1021/ja3100287

Protein aggregation is the hallmark of a number of neurodegenerative diseases including Parkinson's and Huntington's diseases. There is a significant interest in understanding the molecular mechanisms involved in the self-association and fibrillization of monomeric soluble proteins into insoluble deposits in vivo and in vitro. Probes with novel properties, such as red-shifted emission, large Stokes shifts, and high photostability, are desirable for a variety of protein aggregation studies. To respond to the increasing need for aggregation-responsive compounds suitable to cellular studies, we present a ruthenium(II) dipyridophenazine derivative, [Ru(phen) ₂dppz]²⁺ (phen =1,10-phenanthroline, dppz = dipyrido[3,2-a:2′.3′-c]phenazine), to study aggregation of α-synuclein (αS), which is associated with the development of Parkinson's disease. We demonstrated the use of [Ru(phen)₂dppz] ²⁺ to monitor αS fibril formation in real-time and to detect and quantify αS aggregates in neuroglioma cells, thereby providing a novel molecular tool to study protein deposition diseases in vitro and in vivo.

Full text of DOI:10.1021/ja3100287

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Article
pubs.acs.org/JACS
Detection of α‑Synuclein Amyloidogenic Aggregates in Vitro and in
Cells using Light-Switching Dipyridophenazine Ruthenium(II)
Complexes
Nathan P. Cook,†,⊥ Kiri Kilpatrick,‡,⊥ Laura Segatori,*,‡,§,# and Angel A. Martí*,†,§
‡
§
#
†Departments of Chemistry, Chemical and Biomolecular Engineering, Bioengineering, and Biochemistry and Cell Biology, Rice
University, Houston Texas 77005, United States
S
* Supporting Information
ABSTRACT: Protein aggregation is the hallmark of a number of neurodegenerative diseases including Parkinson’s and
Huntington’s diseases. There is a significant interest in understanding the molecular mechanisms involved in the self-association
and fibrillization of monomeric soluble proteins into insoluble deposits in vivo and in vitro. Probes with novel properties, such as
red-shifted emission, large Stokes shifts, and high photostability, are desirable for a variety of protein aggregation studies. To
respond to the increasing need for aggregation−responsive compounds suitable to cellular studies, we present a ruthenium(II)
dipyridophenazine derivative, [Ru(phen)2dppz]2+ (phen =1,10-phenanthroline, dppz = dipyrido[3,2-a:2′.3′-c]phenazine), to
study aggregation of α-synuclein (αS), which is associated with the development of Parkinson’s disease. We demonstrated the
use of [Ru(phen)2dppz]2+ to monitor αS fibril formation in real-time and to detect and quantify αS aggregates in neuroglioma
cells, thereby providing a novel molecular tool to study protein deposition diseases in vitro and in vivo.
the structure of αS fibrils and to investigate the aggregation
kinetics of different αS mutants in vitro,13,14 which is crucial to
elucidate the molecular mechanisms of PD pathogenesis.
The main drawbacks associated with the use of ThT to
detect protein aggregates in cells are its small Stokes shift and
green fluorescence emission, which overlaps with intrinsic
fluorescence properties of other cellular components, such as
flavins or reduced NAD(P)H.15 Aldehyde-containing reagents
typically used to fix cell and tissue samples generate an
autofluorescent background signal with spectral properties
similar to ThT.16 Furthermore, polyphenols, such as curcumin
and quercetin, which inhibit aggregation in vitro, present strong
absorptive and fluorescent properties that overlap with ThT
photoluminescence and may compete with ThT for binding to
fibrillar binding sites.17 In summary, there is an urgent need for
fibrillization responsive probes with large Stokes shifts and red-
shifted fluorescence emissions, which display low background
signal and could be used as alternatives to ThT when other
commonly used green fluorescent reporters are present. The
development of tools to monitor cellular aggregation of
proteins, in turn, will provide new avenues to study the cellular
pathogenesis of numerous human diseases that result from
deposition of proteinaceous aggregates.18
INTRODUCTION
■
Parkinson’s disease (PD) is the most prevalent neuro-
degenerative movement disorder. It is characterized by the
accumulation of proteinaceous cytoplasmic inclusions (Lewy
bodies) in dopaminergic neurons.1 The major component of
Lewy bodies is α-synuclein (αS),2 a natively unfolded 140
amino acid protein with high propensity to misfold and
aggregate.3 The role of αS in the development of PD has been
extensively investigated and evidence points to a correlation
between αS misfolding and aggregation and the progression of
PD pathogenesis.4−6 However, the molecular mechanisms
underlying αS misfolding and aggregation and the role of αS
inclusions in the development of PD remain elusive.
Currently available techniques to monitor the accumulation
of protein aggregates in cell cultures present a number of
limitations. Analytical methods, including transmission electron
microscopy and polyacrylamide gel electrophoresis followed by
Western blotting,7 are time-consuming and not amenable to
high-throughput applications. Fluorescence-based techniques
have been widely used to monitor αS aggregation in vitro and in
vivo and are typically based on the fusion of αS to reporter
proteins, such as the green fluorescent protein (GFP),8 or
covalent binding of αS to small fluorophores, such as biarsenical
labeling reagents9 and Alexa dyes.10 To avoid altering the
peptide backbone of the target protein or inducing covalent
modifications that could potentially influence its folding
landscape, significant focus has been devoted to the design of
compounds that display high affinity for specific protein
conformations, such as the fibrillar aggregates characteristic of
amyloidogenic proteins. Thioflavin T (ThT), a benzothiazole
molecule that displays minimal fluorescence in aqueous media
and enhanced fluorescence when bound to amyloid aggregates,
has been extensively used to probe and quantify fibril
formation.11,12 Particularly, ThT has been used to characterize
In this work, we demonstrate the use of the dipyridophe-
nazine derivative, [Ru(phen)2dppz]2+ (phen =1,10-phenanthro-
line, dppz = dipyrido[3,2-a:2′.3′-c]phenazine), to monitor the
formation of αS fibrils in vitro and to detect αS aggregation in
cell cultures. [Ru(phen)2dppz]2+ and related compounds are
commonly referred to as “light switch molecules” because of
their on−off photoluminescent behavior. These complexes are
generally nonemissive in aqueous solution. However, in the
Received: October 10, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja3100287 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Journal of the American Chemical Society
Article
In vitro Aggregation Experiments. The αS fibrils were prepared
as described by Antony et al.11 Briefly, a 100 μM solution of αS
(rPeptide) was prepared in PBS (pH 7.4, 300 mM NaCl, 100 mM
sodium phosphate). The concentration was verified using an
extinction coefficient of 5600 L·M−1 cm−1. Spermine was then added
to a final concentration of 100 μM, and the solution was incubated
with 10 μM [Ru(phen)2dppz]2+ or [Ru(bpy)2dppz]2+ in glass vials.
Fibrillization reactions were incubated at 37 °C and stirred at 550 rpm,
and photoluminescence spectra taken every 30 min with a Horiba-
Jobin Yvon Fluorolog 3. [Ru(phen)2dppz]2+ and [Ru(bpy)2dppz]2+
were excited at 440 nm, and front face emission was measured from
550 to 700 nm with 2 nm slits. Both emission and excitation were
corrected for instrument-dependent inefficiencies. The solution
photoluminescence intensity at 640 nm was monitored to quantify
the monomer to fibril transition. The photoluminescence intensity of
10 μM [Ru(phen)2dppz]2+ and [Ru(bpy)2dppz]2+ solutions in PBS
was used as blanks.
AFM samples were prepared by dropping 20 μL of αS solution onto
a freshly cleaved mica surface. The protein was allowed to adhere to
the mica for 5 min then washed with 20 μL H2O three times while
being spun dry for 10 min. At 1.0 Hz, 5 × 5 μm scans were taken with
512 lines of resolution. TEM samples were prepared by dropping 10
μL of αS solution onto a glow discharged 200 mesh carbon type B
coated copper grid (Ted Pella 01811). The fibrils were allowed to
adhere for 5 min, then buffer solution was wicked away with filter
paper. The grid was washed 3 times with 10 μL H2O and stained with
10 μL of a 2% w/v phosphotungstic acid solution for 30 s. Samples
were imaged on a JEOL 2010 transmission electron microscope
operating at 100 kV.
Aggregation Studies in Cell Cultures. The cDNA encoding
human wild-type α-syn (P37840) was generated by assembly PCR.
The PCR product was first cloned into pENTR11 and then transferred
into pcDNA6.2/C-EmGFP-DEST using Gateway recombination
cloning technology (Invitrogen) according to the manufacture’s
protocol. Human H4 neuroglioma cells (HTB-148, ATCC) were
cultured in high glucose DMEM (Fisher) supplemented with 10% fetal
bovine serum, 1% PSQ, 4 mM L-Glutamine, and 1 mM sodium
pyruvate and maintained at 37 °C and 5% CO2. Cells were transfected
with pcDNA6.2/α-syn-EmGFP using lipofectamine 2000 according to
the manufacturer’s instructions (Invitrogen). Stably transfected cells
were selected by culturing cells with 5 μg/mL blasticidin S HCl, and
monoclonal populations of blasticidin-resistant cells were isolated.
H4 and H4/α-syn-GFP cells were cultured on poly L-lysine (Sigma)
coated glass coverslips and treated with MG-132 (2 μM) for 16 h at 37
°C. After treatment with MG-132, cells were fixed for 30 min using 4%
paraformaldehyde, permeabilized for 30 min on ice with 0.5% Triton
X-100, and incubated for 30 min with [Ru(phen)2dppz]2+ or with the
ProteoStat Aggregation detection dye (Enzo Life Sciences) according
to the manufacturer’s protocol.
Protein aggregation in H4/α-syn-GFP cells was analyzed by
fluorescence microscopy (Olympus Fluoview 1000) using a 458-nm
laser and 560−660 nm band-pass filter to detect [Ru(phen)2dppz]2+
photoluminescence. Colocalization of αS-GFP and [Ru-
(phen)2dppz]2+ in H4/α-syn-GFP cells was evaluated using the
Colocalization Colormap script, an ImageJ plugin that calculates the
correlation of intensity between complementary fluorescent signals.
The results are presented as a colormap, where hot colors represent
positive correlation and cold colors represent negative correlation.34
Colormaps were analyzed using the ImageJ plugin Threshold Color,
which allows RGB images to be filtered based on hue, saturation, and
brightness (http://www.dentistry.bham.ac.uk/landinig/software/
software.html). To indicate high colocalization, the hue was filtered
to display pixel intensities from 0 to 35 and designated as red pixels.
To indicate low colocalization, the hue was filtered to display pixel
intensities from 35 to 60 and designated as yellow pixels. Pixels in the
hue range from 60 to 255 were considered negative correlation and
not evaluated in this study. To quantify aggregation in H4 and H4/α-
syn-GFP cells, the average pixel intensity of images from cells stained
with aggregate dye was evaluated by determining the brightness of
presence of biomolecules, such as DNA, which have a fibril-like
structure, they display a dramatic increase in photolumines-
cence intensity.19 We previously reported the use of the
dipyridophenazine probe [Ru(bpy)2dppz]2+ (bpy =2,2′-bipyr-
idine; dppz = dipyrido[3,2-a:2′.3′-c]phenazine) for real-time
monitoring of Aβ aggregation in vitro.20 These complexes have
been used in a wide variety of applications including DNA
detection,21 photoinduced electron transport,22 carbon nano-
tubes,23 and cell imaging.24,25 To the best of our knowledge,
while ruthenium(II) complexes and related metal complexes
have been extensively studied in cells,26−28 they have never
been used to characterize αS fibrilization or to measure
intracellular protein aggregation. We demonstrate here the use
of the metal complex [Ru(phen)2dppz]2+, which presents light
switching properties and a significantly stronger photo-
luminescence intensity than [Ru(bpy)2dppz]2+,29 as a real-
time probe for αS fibrillization. Furthermore, we investigated
the use of [Ru(phen)2dppz]2+ complexes to detect αS
aggregation in human neuroglioma cells that overexpress αS
fused to GFP and accumulate αS-GFP aggregates. We observed
an increase in [Ru(phen)2dppz]2+ photoluminescence under
conditions that induce protein aggregation, such as inhibition of
proteasomal degradation. We also demonstrated colocalization
of αS-GFP and [Ru(phen)2dppz]2+ photoluminescence in cells,
indicating a correlation between the intensity of [Ru-
(phen)2dppz]2+ photoluminescence and the formation of αS
aggregates. In summary, we demonstrated the use of [Ru-
(phen)2dppz]2+ as a molecular probe to detect αS aggregation
in vitro and in cells, thereby providing a novel and much needed
tool to quantify the aggregation of amyloidogenic proteins and
to study the cellular pathogenesis of protein deposition
diseases.
EXPERIMENTAL SECTION
■
Synthesis of Ruthenium(II) Complexes. cis-Ru(phen)2Cl2. This
complex is synthesized following Sullivan et al.30 In a typical synthesis,
RuCl3·xH2O (56 mmol, Strem chemicals), phen (112 mmol, Sigma-
Aldrich), and LiCl (3.7 mmol, VWR) were refluxed in 25 mL DMF for
8 h.30 Acetone was then added to the reaction mixture, which was
cooled overnight at 4 °C. The resulting complex was used without
further purification.
cis-Ru(bpy)2Cl2. cis-Ru(bpy)2Cl2 was purchased from Strem
chemicals and used as received.
Dipyrido[3,2-a:2′.3′-c]phenazine (dppz). The dppz ligand was
synthesized in two parts, following Dickeson et al.31 1,10-phenanthro-
line-5,6-dione was synthesized by adding an ice cold mixture of H2SO4
(10 mL) and HNO3 (5 mL) to 1g of phen and 1g KBr. The reaction
was refluxed for 3 h, poured onto crushed ice, then carefully
neutralized with NaOH to slightly acidic pH and extracted with
dichloromethane. The 1,10-phenanthroline-5,6-dione (1.42 mmol)
was subsequently reacted with diaminobenzene (1.71 mmol) in 30 mL
of ethanol for 2 h under reflux.31
[Ru(phen)2dppz]2+. cis-Ru(phen)2Cl2·2H2O was refluxed in 1:1
methanol and water with vigorous stirring for 3 h with dppz as
described by Amouyal et al.32 Upon cooling, the product was
precipitated from solution by the addition of NH4PF6 and filtered. The
reddish-orange crystals were purified by column chromatography (4:1
dichloromethane and acetonitrile) and recrystallization (90:10 ethanol
and water). An extinction coefficient of 20 000 M−1 cm−1 at 440 nm
was used to adjust the compound concentration.33
[Ru(bpy)2dppz]2+. This complex was synthesized and purified
following the procedure described above with the exception that cis-
Ru(bpy)2Cl2 was used as starting reagent. An extinction coefficient of
15 700 M−1 cm−1 at 448 nm was used to verify the compound
concentrations.32
B
dx.doi.org/10.1021/ja3100287 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Journal of the American Chemical Society
Article
Figure 1. Detection of αS fibrillization using [Ru(phen)2dppz]2+. (a) Schematic (left) and 3-D (right) structures of [Ru(bpy)2dppz]2+ and
[Ru(phen)2dppz]2+ (hydrogen atoms have been omitted for clarity). (b) Change in photoluminescence intensity of [Ru(bpy)2dppz]2+ (blue) and
[Ru(phen)2dppz]2+ (red) in the presence of αS upon transition from monomeric to fibrillar state. (c) Comparison of the absolute
photoluminescence intensity of [Ru(bpy)2dppz]2+ (blue) and [Ru(phen)2dppz]2+ (red) in the presence of aggregated αS. (d) Fibrillization of αS in
real-time detected by monitoring [Ru(phen)2dppz]2+ photoluminescence (λem = 640 nm). (e) AFM (top) and TEM (bottom) of mature αS fibrils
(scale bars AFM: 1 μm; TEM = 200 nm). The experiments were performed in PBS (pH 7.4, 300 mM NaCl, 100 mM sodium phosphate), with 100
μM αS, 100 μM spermine, and 10 μM of the ruthenium complex.
each pixel on a scale of 0−255, where 0 is black and 255 is white, and
calculating the average pixel brightness across the entire image.
Protein aggregation was evaluated by measuring fluorescence
intensity by flow cytometry (FACSCanto II, BD Biosciences) using
a 488 nm argon laser and 585/42 band-pass filter. H4 and H4/α-syn-
GFP cells were treated with MG-132 and incubated with [Ru-
(phen)2dppz]2+ or the ProteoStat dye as described above. The
aggregation propensity factor (APF) was calculated using the following
formula: APF = 100 × (MFItreated − MFIcontrol)/MFItreated, where MFI
is the mean fluorescence intensity of the aggregation dye and
untreated H4 cells were used as the control.
phenazine derivatives can be used to detect fibrillar aggregates
of other amyloidogenic proteins. To investigate this question,
we monitored the photoluminescence intensity of ruthenium-
(II) dipyridophenazine derivatives incubated with purified αS as
described previously by Antony et al.11 We observed a 9-fold
increase in [Ru(bpy)2dppz]2+ photoluminescence when αS
transitions from monomeric to fibrillar state (Figure 1b),
suggesting high affinity of this compound for amyloidogenic
protein aggregates. Previous studies showed that [Ru-
(phen)2dppz]2+ presents light switching characteristics but
displays stronger photoluminescence intensity (higher quantum
yield) than [Ru(bpy)2dppz]2+ when bound to DNA.29
Interestingly, [Ru(bpy)2dppz]2+ and [Ru(phen)2dppz]2+ have
related chemical structures (Figure 1a) and similar UV−vis and
photoluminescence spectra with far-red emissions at ca. 640 nm
and large Stokes shifts36 of about 190 nm (Figure S1). We
observed an 18-fold increase in [Ru(phen)2dppz]2+ photo-
luminescence in the presence of fibrillar αS, which is twice the
increase in photoluminescence signal obtained using [Ru-
(bpy)2dppz]2+ under the same conditions (Figure 1b). To
further evaluate the use of these two metal complexes to
quantify αS aggregation, we compared their absolute photo-
luminescence intensity. As shown in Figure 1c, the absolute
photoluminescence of [Ru(phen)2dppz]2+ is 3.3-fold higher
than that of [Ru(bpy)2dppz]2+ in the presence of equal
concentrations of aggregated αS and obtained under the same
RESULTS AND DISCUSSION
■
Real-Time Detection of the Formation of αS Fibrillar
Aggregates. Most amyloid responsive ligands, such as ThT,
are widely used to monitor fibril formation in real-time but are
of limited utility for the detection of protein aggregates in cell
cultures and in vivo. In an attempt to develop a reliable
molecular probe to assess protein aggregation in cells, we
investigated the use of ruthenium(II) dipyridophenazine
complexes to monitor the fibrillization and intracellular
aggregation of αS, a misfolding- and aggregation-prone protein
associated with the development of PD.35 We previously
reported that [Ru(bpy)2dppz]2+ (Figure 1a) presents high
affinity for Aβ1−40 fibrils and can be used to monitor in real-
time the transition of Aβ1−40 monomers into fibrils.20 Based on
this evidence, we hypothesized that ruthenium(II) dipyrido-
C
dx.doi.org/10.1021/ja3100287 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Journal of the American Chemical Society
Article
experimental conditions. Since [Ru(phen)2dppz]2+ presented
the largest change in photoluminescence and the strongest
photoluminescence intensity, it was selected to further
investigate αS aggregation.
are consistent with those obtained by Antony et al.11 using ThT
as probe.43
Detection of αS Aggregates in Neuroglioma Cells. To
investigate the use of [Ru(phen)2dppz]2+ as a probe to monitor
αS aggregation in living cells, we used human neuroglioma cells
(H4) stably transfected for the overexpression of αS fused to
GFP (H4/α-syn-GFP). The use of αS-GFP fusion as a reliable
reporter for disease-associated phenotypes has been previously
established.44−46 To evaluate binding of [Ru(phen)2dppz]2+ to
αS aggregates, we first quantified [Ru(phen)2dppz]2+ photo-
luminescence in H4 and H4/α-syn-GFP cells. Cells were
incubated with a range of [Ru(phen)2dppz]2+ concentrations,
and [Ru(phen)2dppz]2+ photoluminescence intensity was
quantified by flow cytometry. As expected, untreated H4 cells
(that do not overexpress αS) did not display significant
[Ru(phen)2dppz]2+ photoluminescence. However, we did
observe an increase in [Ru(phen)2dppz]2+ photoluminescence
signal in H4 cells overexpressing αS (H4/α-syn-GFP; Figure
2a). MG-132, an inhibitor of proteasomal degradation,47 was
The more intense photoluminescence of [Ru(phen)2dppz]2+
in the presence of fibrillar αS is in agreement with a previous
study by Jenkins et al. reporting that [Ru(phen)2dppz]2+
photoluminescence is approximately 6 times higher than that
of the bipyridine derivative when bound to DNA.29
Interestingly, when both complexes are dissolved in acetonitrile,
they display similar quantum yields.29 Jenkins et al.
hypothesized that the phenanthroline complex, being more
hydrophobic, shields better the dppz ligand from water, thus
explaining the superior photoluminescence properties of the
phenanthroline complex compared to the bipyridine complex.
In agreement with this previous report, our study suggests that
the higher photoluminescence signal measured upon [Ru-
(phen)2dppz]2+ binding to fibrillar αS is due to the stronger
interaction between [Ru(phen)2dppz]2+ and the fibrillar
aggregates than the parent compound, [Ru(bpy)2dppz]2+.
This is consistent with the availability of exposed hydrophobic
domains in the fibril structure that allow stronger binding of the
more hydrophobic [Ru(phen)2dppz]2+ compound. This bind-
ing results in a larger change in the microenvironment
surrounding [Ru(phen)2dppz]2+, and consequently, a larger
increase in photoluminescence.
A number of αS alleles presenting point mutations that alter
the protein’s rate of aggregation have been characterized.37−40
Among the mutations linked to familial cases of PD, the A30P
αS variant was reported to aggregate at slower rate than wild-
type αS in vitro.41 In agreement with these observations, we
observed an 11-fold increase in the photoluminescence of
[Ru(phen)2dppz]2+ in the presence of A30P αS fibrils under
the same conditions used to test wild-type αS, confirming that
[Ru(phen)2dppz]2+ is a reliable probe to monitor αS protein
fibrillization (Figure S2). Furthermore, control studies
conducted to evaluate [Ru(phen)2dppz]2+ photoluminescence
in the presence of the globular protein bovine serum albumin
(BSA) revealed minimal changes in photoluminescence signal
under the same conditions used to test αS. In particular,
[Ru(phen)2dppz]2+ photoluminescence in the presence of
fibrillar αS was 8-fold higher than in the presence of BSA when
equal amounts of proteins (1250 μg/mL) were tested (Figure
S3).
Figure 2. [Ru(phen)2dppz]2+ photoluminescence intensity in H4 and
H4/α-syn-GFP cells. Flow cytometry analysis of [Ru(phen)2dppz]2+
photoluminescence intensity in H4 and H4/α-syn-GFP cells untreated
and treated with MG-132 (2 μM) for 16 h (*p < 0.05, **p < 0.005).
[Ru(phen)2dppz]2+ photoluminescence was measured using a 488 nm
laser and a 585/42 band-pass filter. The relative photoluminescence
was calculated by subtracting the background photoluminescence of
[Ru(phen)2dppz]2+ in untreated H4 cells. The experiments were
repeated three times, and the data are reported as mean SD.
Because [Ru(phen)2dppz]2+ photoluminescence is highly
dependent on changes in αS aggregation state, we asked
whether this probe can be used to monitor real-time formation
of αS fibrils in vitro. A solution of monomeric αS was incubated
with [Ru(phen)2dppz]2+, and αS aggregation was induced as
previously described.11 The complexes were excited at the
metal-to-ligand charge transfer (MLCT) band (440 nm), and
the emission was recorded at 640 nm (Figure S1). The
photoluminescence data depict the typical sigmoidal curve
(Figure 1d) observed when monitoring the formation of
amyloidogenic fibrils in real-time.42 The initial phase represents
a lag phase (approximately 2 h) where αS is predominantly in a
monomeric, soluble state, that is followed by an elongation
phase during which fibrillization occurs exponentially and then
by a plateau phase where fibrillar and monomeric αS are
present in equilibrium. The presence of fibril aggregates was
confirmed by AFM and TEM (Figure 1e). Furthermore, the
real-time fibrillization kinetics of αS with [Ru(phen)2dppz]2+
used to induce aggregation of misfolding-prone proteins,
including αS.44 MG-132 treatment resulted in dramatic increase
in [Ru(phen)2dppz]2+ photoluminescence in H4 cells suggest-
ing that [Ru(phen)2dppz]2+ binds to protein aggregates, since a
reasonable percentage of proteins are aggregation-prone18,48
and MG-132 treatment promotes aggregation.49 The increase
in [Ru(phen)2dppz]2+ photoluminescence was even more
dramatic when MG-132 treatment was applied to H4/α-syn-
GFP cells, which is expected to cause accumulation of αS
aggregates. Similarly to what we discussed for the [Ru-
(phen)2dppz]2+ in the presence of αS aggregates, we
hypothesized that the changes in photoluminescence due to
protein aggregation in H4 cells treated with MG-132 are a
D
dx.doi.org/10.1021/ja3100287 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX