Regulatory Activities of Dopamine and Its Derivatives towards Metal-Free and Metal-Induced Amyloid-β Aggregation, Oxidative Stress, and Inflammation in Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.8b00122

A catecholamine neurotransmitter, dopamine (DA), is suggested to be linked to the pathology of dementia; however, the involvement of DA and its structural analogues in the pathogenesis of Alzheimer's disease (AD), the most common form of dementia, composed of multiple pathogenic factors has not been clear. Herein, we report that DA and its rationally designed structural derivatives (1-6) based on DA's oxidative transformation are able to modulate multiple pathological elements found in AD [i.e., metal ions, metal-free amyloid-β (Aβ), metal-bound Aβ (metal-Aβ), and reactive oxygen species (ROS)], with demonstration of detailed molecular-level mechanisms. Our multidisciplinary studies validate that the protective effects of DA and its derivatives on Aβ aggregation and Aβ-mediated toxicity are induced by their oxidative transformation with concomitant ROS generation under aerobic conditions. In particular, DA and the derivatives (i.e., 3 and 4) show their noticeable anti-amyloidogenic ability towards metal-free Aβ and/or metal-Aβ, verified to occur via their oxidative transformation that facilitates Aβ oxidation. Moreover, in primary pan-microglial marker (CD11b)-positive cells, the major producers of inflammatory mediators in the brain, DA and its derivatives significantly diminish inflammation and oxidative stress triggered by lipopolysaccharides and Aβ through the reduced induction of inflammatory mediators as well as upregulated expression of heme oxygenase-1, the enzyme responsible for production of antioxidants. Collectively, we illuminate how DA and its derivatives can prevent multiple pathological features found in AD with identification of molecular-level mechanisms. The overall studies could advance our understanding regarding distinct roles of neurotransmitters in AD and identify key interactions for alleviation of AD pathology.

Full text of DOI:10.1021/acschemneuro.8b00122

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Article
Regulatory Activities of Dopamine and Its Derivatives towards
Metal-Free and Metal-Induced Amyloid-# Aggregation,
Oxidative Stress, and Inflammation in Alzheimer’s Disease
Eunju Nam, Jeffrey S. Derrick, Seunghee Lee, Juhye Kang,
Jiyeon Han, Shin Jung C. Lee, Su Wol Chung, and Mi Hee Lim
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00122 • Publication Date (Web): 21 May 2018
Downloaded from http://pubs.acs.org on May 22, 2018
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Regulatory Activities of Dopamine and Its Derivatives towards Met-
al-Free and Metal-Induced Amyloid-β Aggregation, Oxidative Stress,
and Inflammation in Alzheimer’s Disease
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Eunju Nam,†,‡,§ Jeffrey S. Derrick,†,¶,§ Seunghee Lee,⊥,§ Juhye Kang,†,‡ Jiyeon Han,‡ Shin Jung C. Lee,†
Su Wol Chung,*,⊥ and Mi Hee Lim*,‡
†Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
‡Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
⊥School of Biological Sciences, College of Natural Sciences, University of Ulsan, Ulsan 44610, Republic of Korea
KEYWORDS: Alzheimer’s disease, dopamine and its structural derivatives, aggregation of metal-free and metal-bound
amyloid-β, amyloid-β-mediated toxicity, inflammation
ABSTRACT: A catecholamine neurotransmitter, dopamine (DA), is suggested to be linked to the pathology of dementia; however,
the involvement of DA and its structural analogues in the pathogenesis of Alzheimer’s disease (AD), the most common form of
dementia, composed of multiple pathogenic factors has not been clear. Herein, we report that DA and its rationally designed strucꢀ
tural derivatives (1–6) based on DA’s oxidative transformation are able to modulate multiple pathological elements found in AD
[i.e., metal ions, metalꢀfree amyloidꢀβ (Aβ), metalꢀbound Aβ (metal–Aβ), and reactive oxygen species (ROS)], with demonstration
of detailed molecularꢀlevel mechanisms. Our multidisciplinary studies validate that the protective effects of DA and its derivatives
on Aβ aggregation and Aβꢀmediated toxicity are induced by their oxidative transformation with concomitant ROS generation under
aerobic conditions. In particular, DA and the derivatives (i.e., 3 and 4) show their noticeable antiꢀamyloidogenic ability towards
metalꢀfree Aβ and/or metal–Aβ, verified to occur via their oxidative transformation that facilitates Aβ oxidation. Moreover, in priꢀ
mary panꢀmicroglial marker (CD11b)ꢀpositive cells, the major producers of inflammatory mediators in the brain, DA and its derivaꢀ
tives significantly diminish inflammation and oxidative stress triggered by lipopolysaccharides and Aβ through the reduced inducꢀ
tion of inflammatory mediators as well as upregulated expression of heme oxygenaseꢀ1, the enzyme responsible for production of
antioxidants. Collectively, we illuminate how DA and its derivatives could prevent multiple pathological features found in AD with
identification of molecular levelꢀmechanisms. The overall studies could advance our understanding regarding distinct roles of neuꢀ
rotransmitters in AD and identify key interactions for alleviation of AD pathology.
functions of enzymes) and altering neuronal homeostasis in
INTRODUCTION
Dysregulated neurotransmission has been widely observed in
dementia, including Alzheimer’s disease and Parkinson’s disꢀ
the brain.2,11 Since DA is abundant in the substantia nigra pars
compacta, the brain region responsible for the motor activity,
ease (AD and PD).1ꢀ4 One of the critical neurotransmitters
multiple studies have been carried out to reveal the impact of
DA on PD pathology.2,7,11ꢀ18 The toxic effects of DA’s
involved in dementia is dopamine (DA; Figure 1a), a catecholꢀ
oxidation on mitochondrial dysfunction, oxidative stress, and
amine responsible for longꢀterm memory and motor activiꢀ
ty.2,3,5 DA is known to be oxidized under physiological
protein degradation, found in PD, have been well discussed at
the molecular level.2,7,11ꢀ18
conditions.2,6ꢀ8 As depicted in Figure 1b, DA first undergoes
twoꢀelectron oxidation to dopamineꢀoꢀquinone (DQ).2,6ꢀ10 The
In AD, the most common form of dementia, the involveꢀ
ment of DA in AD pathology has been suggested by several
unstable quinone further cyclizes and transforms to leukoamꢀ
studies employing AD transgenic mice (e.g., Tg2576 mice).3,19
inochrome (LC).2,6ꢀ9 Through subsequent oxidation and tauꢀ
In the ADꢀaffected brain, (i) Aβ plaqueꢀinduced dopaminergic
tomerization, LC is converted to aminochrome (AC), 5,6ꢀ
dysfunction was identified;3 (ii) the selective degeneration of
dihydroxyindole (DHI), and 5,6ꢀindolequinone (IQ) that are
capable of further polymerizing into neuromelanin.2,6ꢀ9
dopaminergic neurons in the ventral tegmental area (VTA)
region, responsible for release of DA to hippocampus, was
When DA is oxidatively transformed with consequent
indicated;19 and (iii) the restoration of synaptic plasticity and
reduction of dioxygen (O2), reactive oxygen species (ROS)
ꢀ
memory was observed upon treatment with DA.3,19 Despite
such suggested association of DA with AD, it has been relaꢀ
tively unexplored how DA’s chemistry is linked to the
pathogenesis of AD.
[e.g., superoxide anion radical (O2 –) and hydrogen peroxide
(H2O2)] are also generated.2,7 Moreover, such an oxidative
transformation of DA has been implicated in modifying the
activities of biomolecules (e.g., lysosomal proteolysis and the
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Figure 1. DA, its derivatives, and multiple AD pathogenic factors investigated in this study. (a) Effects of DA and its derivatives on aggregaꢀ
tion of both metalꢀfree Aβ and metalꢀbound Aβ, as well as Aβꢀtriggered inflammation and oxidative stress. (b) DA’s oxidative transformation
in the presence of O2. (c) Structures of DA and its derivatives.
Upon progression of AD, multiple pathogenic features
[e.g., metal ion dyshomeostasis, amyloidꢀβ (Aβ) aggregation,
oxidative stress, and inflammation] are identified, indicating
metal ions, Aβ, and ROS as potential pathological factors.1,20ꢀ
to identify the roles of DA and its structural analogues in AD,
molecularꢀlevel studies of their interactions, reactivities, and
effects towards multiple pathological factors found in the disꢀ
ease, along with detailed mechanisms, would be valuable.
Herein, we report that DA and its rationally designed
structural derivatives (1–6) (Figure 1c) are demonstrated to
regulate aggregation of both metalꢀfree Aβ and metalꢀbound
Aβ as well as inflammation and oxidative stress mediated by
Aβ, through multiple mechanisms (Figure 1a). Our multidisciꢀ
plinary investigations employing DA and its derivatives valiꢀ
date that the molecules, shown to be oxidatively transformed,
trigger the oxidation of metalꢀfree and metalꢀbound Aβ and
subsequently regulate Aβ aggregation pathways. Moreover, in
primary panꢀmicroglial marker (CD11b)ꢀpositive cells, DA
and its derivatives could lower the generation of inflammatory
mediators and/or overexpress heme oxygenaseꢀ1 (HOꢀ1), the
enzyme involved in cellular antioxidative defense.33,34 Taken
together, our functional and mechanistic studies illustrate that
oxidative transformation of DA and its structural derivatives
could be key for their noticeable modulating reactivities toꢀ
wards multiple pathological features found in AD. Our overall
findings regarding the effects of a signaling molecule on mulꢀ
tiple pathogenic features found in AD, along with detailed
mechanisms, could provide a novel and broad insight into
distinct chemistry of neurotransmitters under physiological
and pathogenic conditions. Such molecularꢀlevel approaches,
including detailed mechanistic studies and utilization of chemꢀ
ical reagents, could further elucidate complex biological netꢀ
works.20,35ꢀ45
28
These three factors are observed to mutually interact with
each other through various pathways: (i) binding of Aβ to
metal ions, such as Cu(I/II) and Zn(II), followed by facilitation
of Aβ aggregation and generation of toxic Aβ oligomers; (ii)
overproduction of ROS by redoxꢀactive metalꢀbound Aβ (metꢀ
al–Aβ) complexes.20ꢀ28 Although these pathological factors
could individually or mutually facilitate DA’s oxidative transꢀ
formation, proposing a relationship between DA and AD paꢀ
thology,7,9,26 molecularꢀlevel interactions and reactivities of
DA and its structural analogues with such multiple pathologiꢀ
cal components found in AD have not been well documented.
The influence of DA’s oxidation on the modulation of αꢀ
synuclein (αꢀsyn) aggregation observed in the PDꢀaffected
brain has been studied with proposed mechanisms, including
stabilization of protofibrils, formation of soluble oligomers
rather than fibrillation, and oxidation of αꢀsyn at the methioꢀ
nine residues.2,8,12ꢀ18 Relative to αꢀsyn, very little research was
carried out to elucidate the interaction between metalꢀfree or
metalꢀbound Aβ and DA or its oxidized form.12,29 DA’s effects
on the aggregation of metalꢀbound Aβ and modifications of
both metalꢀfree and metalꢀtreated Aβ, however, have not been
reported. Moreover, the impact of DA on the toxicity triggered
under the ADꢀafflicted conditions is still controversial. For
example, DA’s involvement in alleviation of cognitive imꢀ
pairment in vivo, along with its protective activity against Aβꢀ
induced toxicity, was observed; in contrast, cell viability upon
incubation of DA with Aβ was diminished.30ꢀ32 Thus, in order
RESULTS AND DISCUSSION
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Figure 2. Effects of DA and its derivatives on the aggregation of both metalꢀfree Aβ and metal–Aβ in vitro. (a) Scheme of the inhibition experꢀ
iments. (b) Analysis of the MW distribution of the resultant Aβ species generated under anaerobic (left) and aerobic (middle and right) condiꢀ
tions by gel/Western blot using an antiꢀAβ antibody (6E10). The original gel images were presented in Figure S8. (c) TEM images of the Aβ42
aggregates formed under aerobic conditions (from b, right). Scale bar = 500 nm. Conditions: [Aβ] = 25 ꢀM; [CuCl2 or ZnCl2] = 25 ꢀM; [comꢀ
pound] = 50 ꢀM; pH 6.6 (for Cu(II)ꢀtreated Aβ samples) or pH 7.4 (for metalꢀfree and Zn(II)ꢀadded Aβ samples); 37 °C; 24 h incubation; conꢀ
stant agitation; experiments were carried out in triplicate.
Rational Design of DA Derivatives. To determine a
oligomers, protofibrils, as well as large and insoluble fibrils. In
the presence of metal ions, the aggregation of Aβ could be
promoted showing different conformations of Aβ aggregates
or be modified thermodynamically or kinetically.20,21,23 The
resultant metalꢀfree and metalꢀbound Aβ species upon incubaꢀ
tion with DA and its derivatives were analyzed by gel electroꢀ
phoresis with Western blotting (gel/Western blot) and transꢀ
mission electron microscopy (TEM) to monitor their molecuꢀ
lar weight (MW) distribution and morphology, respectively.
Typically, large and insoluble Aβ aggregates are too large to
penetrate into the gel matrix.20,21,23,24,39,45 These large aggreꢀ
gates are hard to be observed on the gel/Western blot; howevꢀ
er, they can be visualized by TEM. Smaller Aβ species, proꢀ
duced upon administration with compounds capable of moduꢀ
lating Aβ aggregation, induce significant smearing on the
gel/Western blot, relative to compoundꢀuntreated peptides.
Following a 24 h incubation period of DA and its derivaꢀ
tives with freshly prepared metalꢀfree and metalꢀadded Aβ40
and Aβ42, the modified aggregation of both Aβ40 and Aβ42 was
observed from the gel/Western blots (inhibition experiments;
Figure 2b). Under aerobic conditions, when DA was treated
with metalꢀfree Aβ40 and Cu(II)–/Zn(II)–Aβ40, noticeable
smearing in the MW range from 4 to 260 kDa was exhibited in
the gel/Western blots, indicative of its inhibitory effect on
metalꢀfree and metalꢀinduced Aβ40 aggregation. Note that
DA’s effect on metalꢀfree Aβ aggregation was previously obꢀ
structureꢀinteractionꢀreactivity relationship of DA and its
structural analogues with various pathogenic features found in
AD (Figure 1a), a series of DA derivatives (1–6) was rationalꢀ
ly designed through systematic structural variations of DA’s
framework (i.e., catechol and amino groups responsible for
DA’s oxidative transformation;2,6ꢀ9 Figure 1c). First, to tune
the initial twoꢀelectron oxidation of compounds to generate a
quinone (as described in Figure 1b), the catechol moiety (Site
I) was altered to a monomethoxy (1) or dimethoxy (2) group.
Second, to control the oxidative cyclization of DA, the amino
group (Site II) was substituted to the methylamino (3) or dimeꢀ
thylamino (4) functionality. Lastly, the structural portions of
DA at both Site I and Site II were changed to diꢀ
methoxy/methylamino (5) or dimethoxy/dimethylamino (6)
moieties to simultaneously modify both oxidation and cyclizaꢀ
tion of the molecule. The characterization of the compounds is
summarized in the Figures S1–S7.
Influence of DA and Its Derivatives on Metal-Free and
Metal-Induced Aβ Aggregation In Vitro. To illuminate the
effects of DA and its derivatives on the aggregation of both
metalꢀfree Aβ and metal–Aβ, two main isoforms of Aβ, i.e.,
Aβ40 and Aβ42,20,23,24 were employed. During the aggregation
of Aβ, selfꢀassembled Aβ monomers generate polymorphic
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served;12,29 however, its impact on aggregation of Cu(II)ꢀ or
Zn(II)ꢀbound Aβ has not been reported. In addition to DA, its
derivatives containing a catechol moiety (i.e., 3 and 4) also
revealed the capacity to modulate the aggregation of metalꢀ
free Aβ40 and/or metal–Aβ40 (Figure 2b, middle). Interestingꢀ
ly, while DA and 3 showed their effects on both metalꢀfree
and metalꢀbound Aβ40, 4 could control Cu(II)–Aβ40 aggregaꢀ
tion over metalꢀfree Aβ and Zn(II)–Aβ40 analogue. Similar
with the results from Aβ40 studies, DA, 3, and 4 displayed
their antiꢀam yloidogenic activity towards metalꢀfree Aβ42
and/or metal–Aβ42 (Figure 2b, right). TEM images of the reꢀ
sultant metalꢀfree and metalꢀbound Aβ42 aggregates, formed
after addition of DA or 3, revealed smaller and amorphous
aggregates, reported to be less toxic,24,39,45 instead of the large
fibrils observed from DAꢀuntreated samples (Figure 2c). Aβ42
incubated with 4 generated offꢀpathway Aβ species only in the
presence of Cu(II). Distinguished from the samples prepared
under aerobic conditions, when the aggregation studies were
conducted in the absence of O2, no significant reactivity of DA
and its derivatives towards the aggregation of both metalꢀfree
Aβ and metal–Aβ was exhibited (Figure 2b, left). This implies
the importance of O2 in their antiꢀamyloidogenic activity (in
detail, vide infra).
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The ability of DA and its derivatives to disassemble preꢀ
Figure 3. Interactions of DA and its derivatives, 3 and 4, with metalꢀfree Aβ40 and Cu(II)ꢀbound Aβ40. (a) MS analyses of monomeric Aβ40 in
the absence and presence of DA. Addition of the oxygen atoms to the peptide is marked in red dots. (b) Arrival time distribution (ATD) of the
singly oxidized Aβ40 from (a) (i.e., [Aβ40 + O + 4H]4+). (c) Mass spectrometric analyses of Cu(II)ꢀtreated Aβ40 monomers in the absence or
presence of DA. (d) Tandem MS sequencing of the oxidized Aβ40 peak from (a). (e) Mass spectrometric analyses of Aβ40 monomers incubated
with 3 and 4 in the absence and presence of Cu(II). Conditions: [Aβ40] = 100 ꢀM; [CuCl2] = 100 ꢀM; [compound] = 500 ꢀM; 100 mM ammoꢀ
nium acetate, pH 7.5; 37 °C; 6 h incubation (for metalꢀfree samples) or 1 h incubation (for Cu(II)ꢀcontaining samples); 10ꢀfold diluted samples
were injected to the mass spectrometer; experiments were conducted in triplicate.
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formed metalꢀfree and metalꢀbound Aβ aggregates was also
previously reported26,51 but the analysis of Cu(II)–Aβ species
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evaluated (disaggregation experiments; Figures S9 and S10).
In the disaggregation studies, following 24 h preꢀincubation of
metalꢀfree and metal [Cu(II) or Zn(II)]ꢀtreated Aβ40 or Aβ42,
the samples of peptides were further treated for additional 24 h
with DA or its derivatives (Figure S8a). Diverse sizes of Aβ
aggregates were monitored for both metalꢀfree and metalꢀ
treated Aβ samples incubated with DA and 3. The derivative 4
could disaggregate preformed Aβ aggregates only in the presꢀ
ence of Cu(II) (Figure S8). From both inhibition and disaggreꢀ
gation experiments, the derivatives, 1, 2, 5, and 6, which conꢀ
tain modified catechol groups (Figure 1c) indicated no signifiꢀ
cant reactivity towards the aggregation of either metalꢀfree Aβ
or Cu(II)–/Zn(II)–Aβ (Figures 2 and S8). Collectively, our in
vitro investigations demonstrate that DA and its derivatives
(i.e., 3 and 4) with a catechol moiety, which are oxidatively
transformed, are able to control the formation of metalꢀfree
and/or metalꢀinduced Aβ aggregates under aerobic conditions.
In addition, DA, 3, and 4 disassemble the preformed aggreꢀ
gates. Moreover, through their noticeable antiꢀamyloidogenic
effects, amorphous and offꢀpathway Aβ aggregates are shown
to be produced.
after DA treatment has not been carried out. In the presence of
Cu(II), Aβ oxidation rapidly occurred even after 1 h treatment
with DA. The +4ꢀcharged oxidized Aβ monomers were indiꢀ
cated displaying three new peaks (ca. 1087, 1091, and 1095
m/z; marked in red) corresponding to the incorporation of one,
two, and three oxygen atoms into the peptide, respectively
(Figure 3c).
To further determine the oxidation site of Aβ40, tandem
MS in conjunction with collisionꢀinduced dissociation for the
singly oxidized Aβ40 was conducted (Figure 3d). The singly
oxidized fragments were reported in b fragments cleaved from
the Nꢀterminus. The oxidized fragments larger than b34 in the
oxidized metalꢀfree Aβ40 monomer were only shown, implying
the addition of an oxygen atom to the methionine 35 residue
(M35) of Aβ (Figure 3d; marked with red). Thus, Aβ oxidaꢀ
tion at M35 by DA is associated with its antiꢀamyloidogenic
activity towards metalꢀfree and metalꢀbound Aβ. This is supꢀ
ported by previous studies that present controlled Aβ aggregaꢀ
tion upon oxidation of the M35 residue in Aβ by some chemiꢀ
cal reagents.45,47,52,53
In addition to DA, the distinguishable amyloidogenic activiꢀ
ty of 3 and 4 with metalꢀfree Aβ40 and Cu(II)–Aβ40 was also
studied in detail by ESI–MS. Similar to DA, in the presence of
3, Aβ oxidation was observed indicating the addition of oxyꢀ
gen atom(s) into both metalꢀfree and Cu(II)ꢀbound Aβ40 (Figꢀ
ure 3e). Tandem MS data of the oxidized Aβ40 monomer, genꢀ
erated by incubation with 3, revealed the oxidation of M35,
consistent with the results using DA (Figure S11). Furtherꢀ
more, 4 was able to induce Aβ oxidation only in the presence
of Cu(II) (Figure 3e). Such peptide oxidation could lead to
noticeable control of metalꢀfree and/or metalꢀinduced Aβ agꢀ
gregation by the derivatives (i.e., 3 and 4). Overall, our mass
spectrometric studies confirm that DA and its derivatives are
capable of oxidizing Aβ (especially, at M35) with and/or
without Cu(II) under aerobic conditions via their oxidative
transformation, which could be the source of their observed
antiꢀamyloidogenic activity. Note that (i) our results did not
exhibit the formation of transformed DQ–protein adducts, as
proposed in the previous studies (structure of DQ is shown in
Figure 1b);2,15,51 (ii) histidine residues (i.e., H13 and
H14),45,49,51 along with M35, could not be ruled out as oxidaꢀ
tion sites.
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Direct Interactions of DA and Its Derivatives with Metal-
Free and Metal-Treated Aβ. To elucidate how DA and its
derivatives (i.e., 3 and 4) could affect the aggregation of both
metalꢀfree Aβ and metal–Aβ, the Aβ species resulted from
incubation with DA and its derivatives were monitored by
electrospray ionization mass spectrometry (ESI–MS) comꢀ
bined with ion mobility mass spectrometry (IM–MS), a methꢀ
od that can characterize interactions between proteins and
small molecules (Figure 3).46,47 When DA was incubated with
metalꢀfree Aβ40, a new peak at 1087 m/z, indicative of the
4+
addition of an oxygen atom to the Aβ40 monomer, was
shown (Figure 3a; marked with a red dot). Conformational
changes of the oxidized Aβ40 monomer were analyzed by IM–
MS, an analytical technique capable of identifying the qualitaꢀ
tive structure and distribution of Aβ species.46,47 Shorter arriꢀ
val time distributions (ATDs) and the decreased collision cross
section (CCS) of the oxidized Aβ40 monomer were observed
compared to those of the nonꢀoxidized Aβ40 monomer (Figure
3b; CCS data in Table S1), suggesting conformational comꢀ
paction of the oxidized monomeric peptide. Such structural
compaction could alter Aβ aggregation to form amorphous
species instead of onꢀpathway structured Aβ aggregates, as
previously reported.24,45 Thus, our mass spectrometric studies
present that DA is able to cause Aβ oxidation and subsequentꢀ
ly trigger the structural compaction of the peptide, which
could be a mechanistic route for modulation of Aβ aggregaꢀ
tion.45,47 Based on the previous results showing the oxidation
of amino acid residues (e.g., methionine, cysteine, and histiꢀ
Redox Property and Oxidative Transformation of DA and
Its Derivatives. To verify how oxidative transformation of
DA and its derivatives is linked to Aβ oxidation and subseꢀ
quent control of Aβ aggregation, the electrochemical properꢀ
ties of DA and its derivatives were first investigated by cyclic
voltammetry (CV) in H2O (Figure S12 and Table S2). In the
case of DA, a redox pair with a peak anodic potential (Epa) at
ca. 0.627 V and a peak cathodic potential (Epc) at ca. 0.176 V
versus Ag/AgCl was detected, consistent with the previously
reported values.54 UV–Visible (UV–Vis) spectroelectrochemꢀ
istry of DA was further conducted at the Epa of 0.627 V versus
Ag/AgCl to monitor its oxidative transformation via the
change in the optical spectra over time (Figure S12). New
optical bands at ca. 308 nm and 475 nm, previously assigned
to the oxidative transformation of DA to AC (Figure 4a),2,6ꢀ8
were presented within 60 min. The compounds equipped with
a catechol moiety, which have a reactivity towards metalꢀfree
and/or metalꢀbound Aβ (i.e., 3 and 4), showed the redox beꢀ
–
ꢀ
dine) by O2 and H2O2,48ꢀ50 ROS concomitantly produced upꢀ
on oxidative transformation of DA, 3, and 4 could be the key
agents for oxidation of Aβ and consequent modulation of pepꢀ
tide aggregation. This could be also supported by another preꢀ
vious study that reported the oxidation of Aβ fragments by 4ꢀ
methylcatechol.51
Moreover, since intracellular redoxꢀactive metal ions have
been observed to accelerate ROS production with either DA or
Aβ,2,7,9,20ꢀ24,26 the interaction of DA with Cu(II)ꢀadded Aβ40
was also investigated by ESI–MS (Figure 3c). Note that oxidaꢀ
tive transformation of DA mediated by Cu(II)ꢀadded Aβ was
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havior similar to DA (Epa at ca. 0.657 V and Epc at ca. 0.160 V
for 3; Epa at ca. 0.647 V and Epc at ca. 0.179 V for 4).
and 475 nm (Figure 4b, middle), suggesting the formation of
the oxidatively cyclized product (i.e., 3b or 3c) (Figure 4b,
middle), similar to those detected for DA (Figure 4b, left). The
spectral change of 3 to 3b or 3c was more accelerated in the
presence of Cu(II)ꢀbound Aβ40 over metalꢀfree Aβ40. The new
peak at 164 m/z, observed by ESI–MS, also confirmed the
formation of 3b or 3c in the presence of metalꢀfree and Cu(II)ꢀ
treated Aβ (Figure 4b, middle).
Furthermore, the derivative 4 containing both catechol and
dimethylamino moieties showed a rapid spectral shift from ca.
280 to 308 nm, indicative of the generation of a semiꢀquinone
species,55 in the presence of Cu(II)ꢀbound Aβ (Figure 4b,
right). 4, however, did not present the optical features conꢀ
sistent with the production of cyclized products, as expected
for the tertiary amineꢀcontaining derivatives. ESI–MS analꢀ
yses also detected a new peak at 180 m/z corresponding to the
twoꢀelectron oxidized quinone product, 4a (Figure 4b, right).
When the derivatives without a catechol moiety (i.e., 1, 2, 5,
and 6) were used under all conditions (Figures S14 and S15),
no optical bands corresponding to transformed products were
indicated by UV–Vis spectroscopy and MS.
Second, the transformation of DA and its derivatives was
identified in the presence of metalꢀfree Aβ, Cu(II), or Cu(II)ꢀ
treated Aβ, by UV–Vis spectroscopy (Figures 4, S13, and
S14). In the case of DA, similar to previous studies, Cu(II) or
Cu(II)–Aβ induced DA’s oxidation.9,26 Upon incubation of DA
in a buffer solution, no noticeable spectral change was obꢀ
served over 60 min; however, the addition of Cu(II) was
shown to trigger DA’s oxidation displaying the conversion to
AC (ca. 308 and 475 nm) (Figures 4b and S13). Particularly,
the introduction of Cu(II)ꢀbound Aβ40 into DA facilitated its
transformation to AC within 10 min. Compared to DA treated
with Cu(II)–Aβ, its slower oxidation was indicated with being
incubated with metalꢀfree Aβ40. Such transformation of DA
was also further supported by ESI–MS (Figure 4b, left). When
DA was coꢀincubated with Aβ40 and Cu(II) for 60 min, in adꢀ
dition to the peaks assigned to [DA + H]+ at 154 m/z, a new
peak at 150 m/z consistent with the parent ion of either AC or
DHI (i.e., [AC + H]+ or [DHI + H]+) was detected (structures
of AC and DHI are depicted in Figure 4a).
Taken together, our electrochemical, UV–Vis spectroscopꢀ
ic, and MS studies validate that DA and its derivatives with
antiꢀamyloidogenic effects are shown to interact with the reꢀ
dox partners, Aβ and Cu(II), followed by promotion of their
oxidative transformation. Moreover, our results support that (i)
peptide oxidation, which results in alteration of Aβ aggregaꢀ
tion, is dependent on the degree of the oxidation of DA and its
Consistent with the results from the electrochemical studꢀ
ies, among DA derivatives, only 3 and 4, able to modulate Aβ
aggregation via peptide oxidation, exhibited their distinct oxiꢀ
dative transformation upon incubation with or without Aβ
and/or Cu(II) (Figures 4, S13, and S14). UV–Vis spectroscopꢀ
ic experiments identified that 3 containing both catechol and
monomethylamino groups displayed optical bands at ca. 308

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Figure 5. Capability of DA, 3, and 4 to regulate cell death, inflammation, and oxidative stress induced by LPS and Aβ42 in primary panꢀ
microglial marker (CD11b)ꢀpositive cells. (a) Analysis of the expression of the panꢀmicroglia marker, CD11b, by flow cytometry. (b) Viability
of cells pretreated with LPS and Aβ42 upon incubation with DA, 3, and 4, monitored by the MTS assay [MTS = 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ
(3ꢀcarboxymethoxyꢀphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium]. *P < 0.05 (versus LPS + Aβ42). (c) Expression of mRNA levels of HOꢀ1. *P <
0.05 (versus LPS + Aβ42). (d) Protein levels of HOꢀ1, quantitated as signal intensities corrected for loading in control cells (white bar) or cells
exposed to LPS + Aβ42, LPS + Aβ42 + DA, LPS + Aβ42 + 3, or LPS + Aβ42 + 4. The values represent mean ± SD (n = 3). *P < 0.05 (versus
LPS + Aβ42). Both (c) mRNA and (d) protein levels of HOꢀ1 were assessed by quantitative realꢀtime RTꢀPCR and gel/Western blot analyses
(for 1 h incubation). (e) Viability of cells pretreated with LPS and Aβ42 upon treatment of DA and 3 in the absence and presence of a HOꢀ1
inhibitor [Zn(II) protoporphyrin IX; Zn(II)(PP)], monitored by the MTS assay. *P < 0.05 (versus LPS + Aβ42); †P < 0.05 [LPS + Aβ42 + DA or
3 versus with Zn(II)(PP)]. (f and g) Expression of HOꢀ1 protein levels, analyzed in primary microglial cells upon treatment with DA or 3 in the
presence of various inhibitors (for 12 h incubation) [NAC (NꢀacetylꢀLꢀcysteine; a ROS inhibitor); Bay11ꢀ7085 (an inhibitor of NFꢀkB activaꢀ
tion); SB203580 (p38 MAPK inhibitor); SP600125 (JNK MAPK inhibitor); LY294002 (phosphatidylinositol 3ꢀkinase inhibitor)]. (h and i)
mRNA levels of the proꢀoxidant genes, iNOS (inducible nitric oxide synthase) and COX2 (cyclooxygenase 2), assessed by the quantitative
†
realꢀtime RTꢀPCR analysis. Values are mean ± SD (n = 3). P < 0.05 (versus LPS + Aβ42). (j) Concentration of NO in the culture medium,
measured by the Griess method. The data represent the means ± SD (n = 9). †P < 0.05 (versus LPS + Aβ42). (k) Proposed mechanism for proꢀ
tection of DA, 3, and 4 against Aβꢀinduced toxicity in primary microglial cells. The detailed procedures and conditions are presented in the
Supporting Information.
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derivatives; (ii) ROS, produced upon compounds’ oxidative
modifications, are required for oxidation of both metalꢀfree Aβ
and metal–Aβ as well as subsequent modulation of their agꢀ
gregation pathways.
the increase of Aβ after lipopolysaccharides (LPS) administraꢀ
tion.61 Furthermore, treatment of LPS and Aβ to the primary
microglial cells could trigger microglial activation, causing
noticeable inflammation and oxidative stress.57,59 First, to veriꢀ
fy the ability of DA and its derivatives (i.e., 3, and 4) to imꢀ
prove the survival of cells treated with LPS and Aβ42, the MTS
assay was carried out (Figure 5b). Upon treatment of DA and
its derivatives for 48 h in the presence of LPS and Aβ42, cell
viability was significantly enhanced [DA (85 ± 4%), 3 (107 ±
7%), and 4 (83 ± 6%)], compared with that treated only with
LPS and Aβ (49 ± 6%) (Figure 5b).
Since microglial activation has been considered to be reꢀ
sponsible for inflammation and oxidative stress,56,59,60,62 influꢀ
ence of DA and its derivatives (i.e., 3, and 4) on the gene exꢀ
pression induced by LPS and Aβ42 was monitored. After adꢀ
ministration of LPS and Aβ42, the enhanced mRNA levels and
protein secretion of proinflammatory cytokines, such as interꢀ
Effects of DA and Its Derivatives on Aβ-Induced Toxicity
Associated with Inflammation and Oxidative Stress. To
determine whether and how DA and its derivatives (i.e., 3 and
4) could alter Aβꢀmediated toxicity (e.g., inflammation and
oxidative stress),56ꢀ58 their effects were monitored employing
primary CD11bꢀpositive cells, the major producers of inflamꢀ
matory mediators in the brain.56,59,60 The primary CD11bꢀ
positive cells were isolated from postnatal day 1 (P1)
C57BL/6 wildꢀtype pups and identified by flow cytometry.
Over 99% of the cells showed positive responses to CD11b, a
wellꢀknown panꢀmicroglial marker (Figure 5a). The systemic
inflammation leads to the induction of neuroinflammation and
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tory mediators in the brain, through induction of HOꢀ1 and/or
leukinꢀ1β (ILꢀ1β), interleukinꢀ6 (ILꢀ6), and the tumor necrosis
factor α (TNFꢀα), were not altered by addition of DA and its
derivatives (data not shown). Noticeably, however, the levels
of mRNA and protein of HOꢀ1, a key neuroprotective enzyme
for antioxidative defense in the brain,33,34 were increased in the
presence of DA and 3 (Figures 5c, 5d, and 5k, left). Note that
the noticeable HOꢀ1 expression was not indicated in the case
of 4.
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downregulation of inflammatory mediators. Overall, our mulꢀ
tidisciplinary results and observations demonstrate that the
regulatory activities of DA and its derivatives against multiple
AD pathological factors are directed via their redox chemistry.
Our molecularꢀlevel identification regarding effects of DA and
its derivatives on multiple pathological features found in AD,
presented in this work, would open our new views into the
actions of neurotransmitters in the diseased brain.
To correlate between neuroprotection and the HOꢀ1 exꢀ
pression induced by DA and 3, cell viability was measured in
the absence or presence of a selective HOꢀ1 inhibitor, Zn(II)
protoporphyrin IX [Zn(II)(PP)] (Figure 5e; bars marked with
blue stripes).63 The inhibition of HOꢀ1 by Zn(II)(PP) could not
recover the toxicity from LPS and Aβ42 even in the presence
of DA and 3. Therefore, our studies support that DA and 3 are
able to ameliorate the toxicity triggered by LPS and Aβ via the
elevated expression of both mRNA and protein levels of HOꢀ
1. To further delineate a molecularꢀlevel mechanism underlyꢀ
ing the activation of HOꢀ1 by DA and 3, the induction of HOꢀ
1 was monitored upon addition of the compounds in primary
CD11bꢀpositive cells with different signaling inhibitors [Figꢀ
ures 5f (for DA) and 5g (for 3)]. The HOꢀ1 protein level was
noticeably reduced when NAC (NꢀacetylꢀLꢀcystein; a ROS
inhibitor)64 was coꢀincubated with DA and 3, suggesting that
an increase in cellular ROS followed by induction of HOꢀ1
could be a mechanism of DA for cell survival (Figure 5k, left).
In addition to HOꢀ1 expression, DA and its derivatives
(i.e., 3 and 4) were indicated to decrease the levels of inflamꢀ
matory mediators [i.e., inducible nitric oxide synthase (iNOS),
cyclooxygenaseꢀ2 (COX2), nitric oxide (NO)], provoked by
treatment with LPS and Aβ (Figures 5hꢀj and 5k, right).65 Takꢀ
en together, employing primary CD11bꢀpositive cells, our
investigations suggest that DA and its derivatives (i.e., 3 and
4) are able to protect the cells against LPS/Aβꢀinduced toxicity
via a proposed mechanism (Figure 5k). Against LPS/Aβꢀ
induced toxicity, DA and the derivative 3 could upregulate the
expression of HOꢀ1, an enzyme responsible for antioxidative
defense in the brain, via ROS signaling (Figure 5k, left). In
addition, they could decrease the induction of inflammatory
mediators, suggesting their activities against inflammation and
oxidative stress (Figure 5k, right). In the case of 4, oxidatively
transformed different from DA and 3, LPS/Aβꢀinduced toxiciꢀ
ty could be diminished upon incubation with the derivative via
regulation of inflammatory mediators with no significant assoꢀ
ciation with HOꢀ1 expression (Figure 5k, right).
9
EXPERIMENTAL SECTION
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Materials and Methods. All reagents were purchased from
commercial suppliers and used as received unless otherwise
stated. 3ꢁHBr [4ꢀ(2ꢀ(methylamino)ethyl)benzeneꢀ1,2ꢀdiol hyꢀ
drobromide] (purity 97%) was prepared following a previousꢀ
ly reported method.66 DA (purity 98%), 1 [5ꢀ(2ꢀaminoethyl)ꢀ2ꢀ
methoxyphenol]
(purity
98%),
and
5
[2ꢀ(3,4ꢀ
dimethoxyphenyl)ꢀNꢀmethylethanꢀ1ꢀamine] (purity 97%) were
purchased from SigmaꢀAldrich (St Louis, MO, USA). 2 [2ꢀ
(3,4ꢀdimethoxyphenyl)ethanꢀ1ꢀamine] (purity 98%) was obꢀ
tained from Acros Organics (Morris Plains, NJ, USA). 4ꢁHBr
[4ꢀ(2ꢀ(dimethylamino)ethyl)benzeneꢀ1,2ꢀdiol hydrobromide]
(purity 95%) and
6
[2ꢀ(3,4ꢀdimethoxyphenyl)ꢀN,Nꢀ
dimethylethanꢀ1ꢀamine] (purity 95%) were acquired from
Enamine (Monmouth Junction, NJ, USA). Trace metal conꢀ
tamination was removed from all solutions used for Aβ experꢀ
iments by treating with Chelex (SigmaꢀAldrich) overnight.
Aβ40
(DAEFRHDSGYEVHHQKꢀ
LVFFAEDVGSNKGAIIGLMVGGVV) and Aβ42 (DAEFRHꢀ
DSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA)
were obtained from Anygen (Namꢀmyun, Jangseongꢀgun,
Republic of Korea) and Anaspec (Fremont, CA, USA). Douꢀ
ble distilled water (ddH2O) used for all experiments was obꢀ
tained from a MilliꢀQ Direct 16 system (Merck KGaA, Darmꢀ
stadt, Germany). The concentrations of Aβ were determined
by an Agilent 8453 UV–Visible spectrophotometer (Santa
Clara, CA, USA). TEM images were collected on a JEOL
JEMꢀ1400 transmission electron microscope [Ulsan National
Institute of Science and Technology (UNIST) Central Reꢀ
search Facilities, Ulsan, Republic of Korea]. Mass spectrometꢀ
ric analyses were performed by a Waters Synapt G2ꢀSi quadꢀ
rupole timeꢀofꢀflight ion mobility mass spectrometer equipped
with an electrospray ionization source (Daegu Gyeongbuk
Institute of Science and Technology (DGIST) Center for Core
Research Facilities, Daegu, Republic of Korea) and a High
Capacity Ion Trap (HCT) mass spectrometer (UNIST Central
CONCLUSIONS
Research Facilities, Ulsan, Republic of Korea). H and 13C
1
To provide functional and mechanistic insights of DA and its
structural derivatives towards multiple pathological factors
found in AD at the molecular level, their effects on the aggreꢀ
gation of both metalꢀfree Aβ and metal–Aβ as well as the inꢀ
flammation and oxidative stress triggered by Aβ were evaluatꢀ
ed. DA’s derivatives were prepared by rational structural modꢀ
ifications of DA on the basis of its oxidative transformation.
First, DA and its derivatives (i.e., 3 and 4) modulate metalꢀfree
and/or metalꢀinduced Aβ aggregation, generating nontoxic,
amorphous aggregates. Such antiꢀamyloidogenic activity is
observed to be directed by compounds’ oxidation that can
produce ROS and consequently trigger Aβ oxidation. Second,
DA, 3, and 4, which could be oxidatively transformed, appear
to mitigate Aβꢀinduced inflammation and oxidative stress in
primary CD11bꢀpositive cells, the major supplier of inflammaꢀ
NMR spectra of compounds were recorded on an Agilent 400ꢀ
MR DD2 NMR spectrometer (UNIST Central Research Faciliꢀ
ties, Ulsan, Republic of Korea) and a Bruker AVHD400 NMR
spectroscopy (KAIST Analysis Center for Research Adꢀ
vancement, Daejeon, Republic of Korea). Mass spectrometric
analysis of molecules was carried out by a Bruker maXisTM
HD Ultraꢀhigh resolution QꢀTOF LC MS/MS system (HRMS;
the Cooperative Laboratory Center of Pukyong National Uniꢀ
versity, Busan, Republic of Korea).
Preparation of 3. 3 was synthesized following a previously
reported method with modifications.66 2ꢀ(3,4ꢀDimethoxyꢀ
phenyl)ꢀNꢀmethylethanꢀ1ꢀamine (5) (529.5 mg, 2.7 mmol) was
slowly added to HBr (3.1 mL, 27.1 mmol). The system was
flushed with N2 for 0.5 h and refluxed at 130 °C. After 2 h, the
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nalTM West Pico Chemiluminescent Substrate (Thermo Fisher
reaction mixture was concentrated under vacuum and dried.
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The final product was obtained without further purification
(brown powder; 483.0 mg; 1.9 mmol; 72%). H NMR [400
Scientific, Inc., Waltham, MA, USA) after exposure to the Xꢀ
ray film.
1
MHz, D2O,
6.65 (d, J = 8.4 Hz, 1H), 3.16 (t, J = 7.2 Hz, 2H), 2.80 (t, J =
7.2 Hz, 2H), 2.59 (s, 3H). 13C NMR [100 MHz, D2O,
δ (ppm)]: 6.79 (d, J = 8.0 Hz, 1H), 6.73 (s, 1H),
TEM. Samples for TEM were prepared following previously
reported methods using Glowꢀdischarged grids (Forꢀ
mar/Carbon 300ꢀmesh, Electron Microscopy Sciences,
Hatfield, PA, USA).24,39 Images from Aβ42 samples were taken
on a JEOL JEMꢀ1400 (120 kV; 25,000× magnification) TEM.
δ
(ppm)]: 144.2, 143.0, 128.9, 121.1, 116.5, 116.4, 50.1, 32.7,
30.9. HRMS (m/z): [M+H]+ Calcd. for C9H14NO2, 168.1025;
found, 168.1016.
9
ESI–IM–MS. Aβ40 (100 ꢀM) was incubated with DA, 3, and
4 (500 ꢀM) in the absence or presence of CuCl2 (100 ꢀM) in
100 mM ammonium acetate (pH 7.5) at 37 °C without agitaꢀ
tion. Metalꢀfree and Cu(II)ꢀtreated samples were incubated for
6 h and 1 h, respectively. Before injection into the mass specꢀ
trometer, Aβ samples were diluted by 10 fold. ESI–MS analꢀ
yses were performed using a Synapt G2ꢀSi quadrupole timeꢀ
ofꢀflight mass spectrometer (Waters, Manchester, UK)
equipped with electrospray ionization source. The capillary
voltage, sampling cone voltage, and source temperature were
adjusted to 2.8 kV, 70 V, and 40 °C, respectively. The backing
pressure was adjusted to 3.2 mbar. For IM−MS experiments,
all mass spectra were obtained under the IMS mode to measꢀ
ure the drift time for each ion. Ion mobility wave velocity and
height were set to 450 m/s and 10 V, respectively. Trap gas
flow (Ar), helium gas flow, and IMS gas flow (N2) were set as
4 mL/min, 120 mL/min, and 30 mL/min, respectively. Tandem
MS experiments were conducted for the 4+ꢀcharged singly
oxidized Aβ40 with 1087 m/z (monoisotopic mass 1086 m/z)
by setting the trap collision energy, LM resolution, and HM
resolution as 55 eV, 10, and 15, respectively. Collision cross
section (CCS) measurements were externally calibrated using
a database of known values in helium, using values for proꢀ
teins that bracket the likely CCS and ion mobility values of the
unknown ions.68,69
Aβ Aggregation Experiments. All experiments were conꢀ
ducted according to previously published methods.24,39 Aβ
peptides were dissolved with ammonium hydroxide (NH4OH,
1% v/v, aq), aliquoted, lyophilized, and stored at −80 °C. A
stock solution was prepared by reꢀdissolving Aβ with NH4OH
(1% w/v, aq; 10 ꢀL) followed by dilution with ddH2O. The
concentration of Aβ was determined by measuring the absorbꢀ
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ance of the solution at 280 nm (ε ε
= 1450 M−1cm−1 for Aβ40;
= 1490 M−1cm−1 for Aβ42) by UV−Vis spectroscopy. The pepꢀ
tide stock solution was diluted to a final concentration of 25
ꢁM in the Chelexꢀtreated buffered solution [20 ꢀM HEPES (4ꢀ
(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid), pH 7.4 (for
metalꢀfree and Zn(II)ꢀadded samples) or pH 6.6 (for Cu(II)ꢀ
treated samples), and 150 ꢀM NaCl]. For the inhibition experꢀ
iments, Aβ (25 ꢀM) was first treated with or without a metal
chloride salt (CuCl2 or ZnCl2; 25 ꢀM) for 2 min followed by
addition of compounds (50 ꢁM; 1% v/v final DMSO concenꢀ
tration). The resultant samples were incubated at 37 °C for 24
h with constant agitation. For the disaggregation experiments,
Aβ in the absence or presence of a metal chloride salt (CuCl2
or ZnCl2) was initially incubated at 37 °C for 24 h with conꢀ
stant agitation. Compounds were added to the preincubated Aβ
aggregates followed by an additional 24 h of incubation at 37
°C with constant agitation. The preparation of samples under
anaerobic conditions was carried out in a N2ꢀfilled glove box
(Korea Kiyon, Bucheonꢀsi, Gyeonggiꢀdo, Republic of Korea).
MS Analyses for Characterization of Oxidized Com-
pounds. DA and its derivatives (500 ꢀM) were incubated with
or without Aβ40 (100 ꢀM) in the absence or presence of CuCl2
(100 ꢀM) in 100 mM ammonium acetate (pH 7.5) at 37 °C
without agitation. After 1 h incubation, the samples were diꢀ
luted by 10 fold before injection into the mass spectrometer.
The spectra of DA, 3, and 4 were obtained from a Synapt G2ꢀ
Si quadrupole timeꢀofꢀflight mass spectrometer (Waters, Manꢀ
chester, UK); the spectra of 1, 2, 5, and 6 were observed using
a HCT mass spectrometer (Bruker Daltonics, Billerica, MA,
USA). The capillary voltage, sampling cone voltage, and
source temperature were adjusted to 2.8 kV, 70 V, and 40 °C,
respectively.
Gel/Western Blot. Aβ samples from the inhibition and disꢀ
aggregation experiments in vitro were analyzed by gel electroꢀ
phoresis with Western blotting (gel/Western blot) using a priꢀ
mary antiꢀAβ antibody (6E10; 1:2,000, Covance, Princeton,
NJ, USA).24,39 Samples (10 ꢀL) were separated on a 10−20%
Trisꢀtricine gel (Invitrogen, Grand Island, NY, USA). Followꢀ
ing separation, the proteins were transferred onto nitrocelluꢀ
lose membranes and blocked with bovine serum albumin
(BSA, 3% w/v, RMBIO, Missoula, MT, USA) in Trisꢀ
buffered saline (TBS) containing 0.1% Tweenꢀ20 (TBSꢀT) for
3 h at room temperature. Experiments were performed in triplicate.
For biological experiments, the membranes were incubated
with primary antibodies [an antiꢀHOꢀ1 (1:4,000, Enzo Life
Sciences, Inc., Farmingdale, NY, USA); an antiꢀβꢀactin
(1:5,000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA)]
in a solution of 2% BSA (w/v in TBSꢀT) for 4 h at room temꢀ
perature or overnight at 4 °C. After washing with TBSꢀT three
times (8 min each), the horseradish peroxidaseꢀconjugated
goat antiꢀmouse secondary antibody (1:5,000) or antiꢀrabbit
secondary antibody (1:5,000) in 2% BSA (w/v in TBSꢀT) was
added for 1 h at room temperature. A homemade ECL kit67
was used to visualize the gel/Western data (for 6E10) on a
ChemiDoc MP Imaging System (BioRad, Hercules, CA,
USA). The other immunoblots were detected by SuperSigꢀ
Cyclic Voltammetry. Cyclic voltammograms of all comꢀ
pounds were recorded under N2 (g) with a CHI620E model
potentiostat (Qrins, Seoul, Republic of Korea). A threeꢀ
electrode setup, composed of an Ag/AgCl reference electrode
(REꢀ1B Reference electrode; Qrins, Seoul, Republic of Koꢀ
rea), a Pt wire auxiliary electrode (SPTE Platinum electrode;
Qrins, Seoul, Republic of Korea), and a glassy carbon working
electrode (Qrins, Seoul, Republic of Korea), was utilized.
Electrochemical analyses of DA and its derivatives (1–6) (1
mM) were recorded in 1 M NaCl (aq) as a supporting electroꢀ
lyte at various scan rates (5, 10, 25, 50, 100, 150, 200, 250,
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and 300 mV/s) at room temperature.
tive to the vehicle control (DMSO). Data were the mean of
three independent experiments (n = 9–12).
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Spectroelectrochemical Analysis. Spectroelectrochemical
data for DA (1 mM) were recorded under N2 (g) with a potenꢀ
tiostat and an UV−Vis spectrophotometer. A spectroelectroꢀ
chemical cell kit was utilized containing a thin layer quartz
glass cell, a Pt gauze working electrode, a Pt wire auxiliary
electrode [SECꢀC Spectroelectrochemical cell kit (Pt); Qrins,
Seoul, Republic of Korea], and an Ag/AgCl reference elecꢀ
trode (REꢀ1B Reference electrode; Qrins, Seoul, Republic of
Korea). The optical spectra of DA were monitored at 0.627 V
in 1 M NaCl (aq) as a supporting electrolyte at 37 °C over 60
min.
RT-PCR. Total RNA was isolated by TRIzol reagent (Thermo
Fisher Scientific, Rockford, IL, USA). Reverse transcription
was performed using SuperScript™ III FirstꢀStrand Synthesis
System (Thermo Fisher Scientific, Rockford, IL, USA). Realꢀ
time quantitative PCR was conducted using iQ SYBR Green
Supermix (BioRad, Hercules, CA, USA). Triplicate samples
per condition were analyzed on an Applied Biosystems
StepOnePlusTM RealꢀTime PCR System (Thermo Fisher Sciꢀ
entific, Rockford, IL, USA) using absolute quantification setꢀ
tings. Amplification of cDNA started with 10 min at 95 °C
followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The
primers’ sequences were as follows: mouse βꢀactin (forward:
5’ꢀGATCTGGCACCACACCTTCTꢀ3’ and reverse: 5’ꢀGGGꢀ
GTGTTGAAGGTCTCAAAꢀ3’); mouse COX2 (forward: 5’ꢀ
CAAGGGAGTCTGGAACATTGꢀ3’ and reverse: 5’ꢀACCCꢀ
AGGTCCTCGCTTATGAꢀ3’); mouse HOꢀ1 (forward: 5’ꢀ
CGCCTTCCTGCTCAACATTꢀ3’ and reverse: 5’ꢀTGTGTTꢀ
CCTCTGTCAGCATCACꢀ3’); mouse iNOS (forward: 5’ꢀ
AACGGAGAACGTTGGATTTGꢀ3’ and reverse: 5’ꢀCAGꢀ
CACAAGGGGTTTTCTTCꢀ3’).
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UV–Vis Spectroscopy. Optical spectra of DA and its derivaꢀ
tives (100 ꢀM) with or without CuCl2 (50 ꢀM) in the absence
or presence of Aβ40 (50 ꢀM) were obtained by an UV−Vis
spectrophotometer. Chelexꢀtreated buffered solutions [20 ꢀM
HEPES, pH 7.4 (for metalꢀfree samples) or pH 6.6 (for Cu(II)ꢀ
treated samples), 150 ꢀM NaCl] were used for all UV−Vis
experiments.
Isolation and Culture of Primary CD11b-Positive Cells. All
experimental protocols were approved by the office of the
district president and the Institutional Animal Care and Use
Committee of the University of Ulsan (Ulsan, Republic of
Korea). Primary CD11bꢀpositive cells were isolated from
C57BL/6 mice at 1 day old as described.70 Mice were sacriꢀ
ficed and mesencephalons were dissected and collected in
Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fishꢀ
er Scientific, Rockford, IL, USA). Mesencephalons were hoꢀ
mogenized by 30 times of pipetting. After homogenization,
DMEM (5 mL) was added and left for 10 min to remove cell
debris. The medium was transferred into a new tube and cenꢀ
trifuged for 5 min at 300 g. The supernatant was resuspended
in DMEM supplemented with 10% fetal bovine serum (FBS),
1% streptomycin/penicillin, and 1% Lꢀglutamine (Thermo
Fisher Scientific, Rockford, IL, USA). Cells were seeded on
polyꢀDꢀlysineꢀcoated plates and cultivated at 37 °C with 5%
CO2 in a humidified atmosphere. After 11ꢀ13 days, cells were
subꢀplated and used for further experiments. For experiments,
cell treatments were performed in 24 h after seeding.
Nitrite Assay. NO production was determined by measuring
the amount of nitrite, a stable breakdown product of NO meꢀ
tabolism, in cell supernatants. Supernatants were combined
with an equal volume of 1% sulphanilamide (SigmaꢀAldrich,
St Louis, MO, USA) in 5% H3PO4 (SigmaꢀAldrich, St Louis,
MO, USA) and 0.1% Nꢀ(1ꢀnaphthyl)ethylenediamine dihydroꢀ
chloride (SigmaꢀAldrich, St Louis, MO, USA) to convert niꢀ
trite into a magenta colored azo compound showing absorbꢀ
ance at 550 nm. Nitrite levels were monitored based on a
standard curve of known sodium nitrite concentrations.
Statistical Analysis. All data represent mean ± SD. For comꢀ
parisons between two groups, Student’s twoꢀtailed unpaired t
test was employed. For comparison between more than two
groups and multiple comparisons, an ANOVA test was used.
Statistically significant differences were accepted at P < 0.05.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Tables S1 and S2 and Figures S1–
S15 (PDF)
Flow Cytometry. Cultured microglia cells were counted and
1×105 cells were washed with PBS followed by an incubation
of FITCꢀconjugated antiꢀmouse CD11b and rat IgG antibodies
(BioLegend, San Diego, CA, USA) in FACS buffer (1% BSA
in PBS) at 4 °C for 30 min. All samples were analyzed by flow
cytometry using BD FACS (CantoꢀII, 8ꢀcolor, flow cytometric
analysis; BD Biosciences, San Jose, CA, USA).
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail: miheelim@kaist.ac.kr (M.H.L.)
*Eꢀmail: swchung@ulsan.ac.kr (S.W.C.)
Cell Viability Assay. Cell survival was determined by the
MTS assay using the CellTiter 96® AQueous One Solution
Cell Proliferation Assay kit (Promega, Madison, WI, USA).
Cells were seeded at 3.7×104 cells per well in 96ꢀwell plates.
After treatment of reagents, the MTS solution (20 ꢀL) was
added to each well. Plates were incubated for an additional 2–
4 h at 37 °C. The absorbance was measured at 490 nm using a
SpectraMax M2 microplate reader (Molecular Devices, LLC,
Sunnyvale, CA, USA). Cell viability (%) was calculated relaꢀ
§E.N., J.S.D., and S.L. contributed equally to this work.
¶Present address (J.S.D.): Department of Chemistry, University of
California, Berkeley, CA 94720, USA
Author Contributions
M.H.L., E.N., and J.S.D. designed the research. E.N., J.S.D., J.K.,
S.J.C.L., and J.H. were fully or partially involved in the
gel/Western blot, TEM, UV–Vis, ESI–MS, and electrochemical
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ACS Chemical Neuroscience
mediated conformational alterations. J. Biol. Chem. 280,
21212 21219.
(14) Leong, S. L., Pham, C. L. L., Galatis, D., FoderoꢀTavoletti, M.
T., Perez, K., Hill, A. F., Masters, C. L., Ali, F. E., Barnham, K.
J., and Cappai, R. (2009) Formation of dopamineꢀmediated αꢀ
synucleinꢀsoluble oligomers requires methionine oxidation.
studies with data analyses. S.W.C., S.L., and J.K. conducted and
analyzed the biological experiments using primary microglia
cells. E.N., J.S.D., J.K., S.W.C., and M.H.L. wrote the manuꢀ
script.
–
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8
Notes
Free Radic. Biol. Med. 46, 1328–1337.
(15) Conway, K. A., Rochet, J.ꢀC., Bieganski, R. M., and Lansbury,
The authors declare no competing financial interest.
P. T. (2001) Kinetic stabilization of the αꢀsynuclein protofibril
by a dopamine –αꢀsynuclein adduct. Science 294, 1346–1349.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation of
Korea (NRF) grant funded by the Korean government [NRFꢀ
2017R1A2B3002585 and NRFꢀ2016R1A5A1009405 (to M.H.L.);
NRFꢀ2014R1A6A1030318 (to S.W.C.)]; the Korea Advanced
Institute of Science and Technology (KAIST) (to M.H.L.). J.K.
acknowledges the Global Ph.D. fellowship program through the
NRF funded by the Ministry of Education (NRFꢀ
2015HIA2A1030823). We thank Dr. Kyle Korshavn and Yelim
Yi for the assistance with the synthesis of 3 and the characterizaꢀ
tion of compounds, resepectively.
(16) Ha, Y., Yang, A., Lee, S., Kim, K., Liew, H., Lee, S. H., Lee, J.
E., Lee, H.ꢀI., Suh, Y.ꢀH., Park, H.ꢀS., and Churchill, D. G.
(2014) Dopamine and Cu(I/II) can induce oligomerization of αꢀ
synuclein in the absence of oxygen: Two types of
oligomerization mechanisms for αꢀsynuclein and related cell
toxicity studies. J. Neurosci. Res. 92, 359–368.
(17) Tavassoly, O., Nokhrin, S., Dmitriev, O. Y., and Lee, J. S.
(2014) Cu(II) and dopamine bind to αꢀsynuclein and cause
large conformational changes. FEBS J. 281, 2738–2753.
(18) Mor, D. E., Tsika, E., Mazzulli, J. R., Gould, N. S., Kim, H.,
Daniels, M. J., Doshi, S., Gupta, P., Grossman, J. L., Tan, V. X.,
Kalb, R. G., Caldwell, K. A., Caldwell, G. A., Wolfe, J. H., and
Ischiropoulos, H. (2017) Dopamine induces soluble αꢀ
synuclein oligomers and nigrostriatal degeneration. Nat.
Neurosci. 20, 1560–1568.
(19) Nobili, A., Latagliata, E. C., Viscomi, M. T., Cavallucci, V.,
Cutuli, D., Giacovazzo, G., Krashia, P., Rizzo, F. R., Marino,
R., Federici, M., De Bartolo, P., Aversa, D., Dell'Acqua, M. C.,
Cordella, A., Sancandi, M., Keller, F., Petrosini, L., Puglisiꢀ
Allegra, S., Mercuri, N. B., Coccurello, R., Berreta, N., and
D'Amelio, M. (2017) Dopamine neuronal loss contributes to
memory and reward dysfunction in a model of Alzheimer's
disease. Nat. Commun. 8, 14727.
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