Regulatory Activities of Dopamine and Its Derivatives towards Met- al

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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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ACS Chemical Neuroscience

Regulatory Activities of Dopamine and Its Derivatives towards Metal-Free and Metal-Induced Amyloid-β β 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*,‡ †

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 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 could 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.

INTRODUCTION Dysregulated neurotransmission has been widely observed in dementia, including Alzheimer’s disease and Parkinson’s disease (AD and PD).1-4 One of the critical neurotransmitters involved in dementia is dopamine (DA; Figure 1a), a catecholamine responsible for long-term memory and motor activity.2,3,5 DA is known to be oxidized under physiological conditions.2,6-8 As depicted in Figure 1b, DA first undergoes two-electron oxidation to dopamine-o-quinone (DQ).2,6-10 The unstable quinone further cyclizes and transforms to leukoaminochrome (LC).2,6-9 Through subsequent oxidation and tautomerization, LC is converted to aminochrome (AC), 5,6dihydroxyindole (DHI), and 5,6-indolequinone (IQ) that are capable of further polymerizing into neuromelanin.2,6-9 When DA is oxidatively transformed with consequent reduction of dioxygen (O2), reactive oxygen species (ROS) [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

functions of enzymes) and altering neuronal homeostasis in the brain.2,11 Since DA is abundant in the substantia nigra pars compacta, the brain region responsible for the motor activity, multiple studies have been carried out to reveal the impact of DA on PD pathology.2,7,11-18 The toxic effects of DA’s oxidation on mitochondrial dysfunction, oxidative stress, and protein degradation, found in PD, have been well discussed at the molecular level.2,7,11-18 In AD, the most common form of dementia, the involvement of DA in AD pathology has been suggested by several studies employing AD transgenic mice (e.g., Tg2576 mice).3,19 In the AD-affected brain, (i) Aβ plaque-induced dopaminergic dysfunction was identified;3 (ii) the selective degeneration of dopaminergic neurons in the ventral tegmental area (VTA) region, responsible for release of DA to hippocampus, was indicated;19 and (iii) the restoration of synaptic plasticity and memory was observed upon treatment with DA.3,19 Despite such suggested association of DA with AD, it has been relatively unexplored how DA’s chemistry is linked to the pathogenesis of AD.

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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 aggregation of both metal-free Aβ and metal-bound Aβ, as well as Aβ-triggered 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, amyloid-β (Aβ) aggregation, oxidative stress, and inflammation] are identified, indicating metal ions, Aβ, and ROS as potential pathological factors.1,2028 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 redox-active metal-bound Aβ (metal–Aβ) complexes.20-28 Although these pathological factors could individually or mutually facilitate DA’s oxidative transformation, proposing a relationship between DA and AD pathology,7,9,26 molecular-level interactions and reactivities of DA and its structural analogues with such multiple pathological components found in AD have not been well documented. The influence of DA’s oxidation on the modulation of αsynuclein (α-syn) aggregation observed in the PD-affected brain has been studied with proposed mechanisms, including stabilization of protofibrils, formation of soluble oligomers rather than fibrillation, and oxidation of α-syn at the methionine residues.2,8,12-18 Relative to α-syn, very little research was carried out to elucidate the interaction between metal-free or metal-bound Aβ and DA or its oxidized form.12,29 DA’s effects on the aggregation of metal-bound Aβ and modifications of both metal-free and metal-treated Aβ, however, have not been reported. Moreover, the impact of DA on the toxicity triggered under the AD-afflicted conditions is still controversial. For example, DA’s involvement in alleviation of cognitive impairment in vivo, along with its protective activity against Aβinduced toxicity, was observed; in contrast, cell viability upon incubation of DA with Aβ was diminished.30-32 Thus, in order

to identify the roles of DA and its structural analogues in AD, molecular-level studies of their interactions, reactivities, and effects towards multiple pathological factors found in the disease, 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 metal-free Aβ and metal-bound Aβ as well as inflammation and oxidative stress mediated by Aβ, through multiple mechanisms (Figure 1a). Our multidisciplinary investigations employing DA and its derivatives validate that the molecules, shown to be oxidatively transformed, trigger the oxidation of metal-free and metal-bound Aβ and subsequently regulate Aβ aggregation pathways. Moreover, in primary pan-microglial marker (CD11b)-positive cells, DA and its derivatives could lower the generation of inflammatory mediators and/or overexpress heme oxygenase-1 (HO-1), 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 towards multiple pathological features found in AD. Our overall findings regarding the effects of a signaling molecule on multiple 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 molecular-level approaches, including detailed mechanistic studies and utilization of chemical reagents, could further elucidate complex biological networks.20,35-45

RESULTS AND DISCUSSION


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Figure 2. Effects of DA and its derivatives on the aggregation of both metal-free Aβ and metal–Aβ in vitro. (a) Scheme of the inhibition experiments. (b) Analysis of the MW distribution of the resultant Aβ species generated under anaerobic (left) and aerobic (middle and right) conditions by gel/Western blot using an anti-Aβ 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 µM; [CuCl2 or ZnCl2] = 25 µM; [compound] = 50 µM; pH 6.6 (for Cu(II)-treated Aβ samples) or pH 7.4 (for metal-free and Zn(II)-added Aβ samples); 37 °C; 24 h incubation; constant agitation; experiments were carried out in triplicate.

Rational Design of DA Derivatives. To determine a structure-interaction-reactivity relationship of DA and its structural analogues with various pathogenic features found in AD (Figure 1a), a series of DA derivatives (1–6) was rationally designed through systematic structural variations of DA’s framework (i.e., catechol and amino groups responsible for DA’s oxidative transformation;2,6-9 Figure 1c). First, to tune the initial two-electron 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 dimethylamino (4) functionality. Lastly, the structural portions of DA at both Site I and Site II were changed to dimethoxy/methylamino (5) or dimethoxy/dimethylamino (6) moieties to simultaneously modify both oxidation and cyclization 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 β Aggregation In Vitro. To illuminate the effects of DA and its derivatives on the aggregation of both metal-free Aβ and metal–Aβ, two main isoforms of Aβ, i.e., Aβ40 and Aβ42,20,23,24 were employed. During the aggregation of Aβ, self-assembled Aβ monomers generate polymorphic

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 metal-free and metal-bound Aβ species upon incubation with DA and its derivatives were analyzed by gel electrophoresis with Western blotting (gel/Western blot) and transmission electron microscopy (TEM) to monitor their molecular 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 aggregates are hard to be observed on the gel/Western blot; however, they can be visualized by TEM. Smaller Aβ species, produced upon administration with compounds capable of modulating Aβ aggregation, induce significant smearing on the gel/Western blot, relative to compound-untreated peptides. Following a 24 h incubation period of DA and its derivatives with freshly prepared metal-free and metal-added 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 metal-free 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 metal-free and metal-induced Aβ40 aggregation. Note that DA’s effect on metal-free Aβ aggregation was previously ob-



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 metalfree Aβ40 and/or metal–Aβ40 (Figure 2b, middle). Interestingly, while DA and 3 showed their effects on both metal-free and metal-bound Aβ40, 4 could control Cu(II)–Aβ40 aggregation over metal-free Aβ and Zn(II)–Aβ40 analogue. Similar with the results from Aβ40 studies, DA, 3, and 4 displayed their anti-am yloidogenic activity towards metal-free Aβ42 and/or metal–Aβ42 (Figure 2b, right). TEM images of the resultant metal-free and metal-bound Aβ42 aggregates, formed

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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 DA-untreated samples (Figure 2c). Aβ42 incubated with 4 generated off-pathway 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 metal-free Aβ and metal–Aβ was exhibited (Figure 2b, left). This implies the importance of O2 in their anti-amyloidogenic activity (in detail, vide infra). The ability of DA and its derivatives to disassemble pre-

Figure 3. Interactions of DA and its derivatives, 3 and 4, with metal-free 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 µM; [CuCl2] = 100 µM; [compound] = 500 µM; 100 mM ammonium acetate, pH 7.5; 37 °C; 6 h incubation (for metal-free samples) or 1 h incubation (for Cu(II)-containing samples); 10-fold diluted samples were injected to the mass spectrometer; experiments were conducted in triplicate.


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formed metal-free and metal-bound Aβ aggregates was also evaluated (disaggregation experiments; Figures S9 and S10). In the disaggregation studies, following 24 h pre-incubation of metal-free 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 metal-free and metaltreated Aβ samples incubated with DA and 3. The derivative 4 could disaggregate preformed Aβ aggregates only in the presence of Cu(II) (Figure S8). From both inhibition and disaggregation experiments, the derivatives, 1, 2, 5, and 6, which contain modified catechol groups (Figure 1c) indicated no significant reactivity towards the aggregation of either metal-free 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 metal-free and/or metal-induced Aβ aggregates under aerobic conditions. In addition, DA, 3, and 4 disassemble the preformed aggregates. Moreover, through their noticeable anti-amyloidogenic effects, amorphous and off-pathway Aβ aggregates are shown to be produced. Direct Interactions of DA and Its Derivatives with MetalFree and Metal-Treated Aβ β. To elucidate how DA and its derivatives (i.e., 3 and 4) could affect the aggregation of both metal-free Aβ and metal–Aβ, the Aβ species resulted from incubation with DA and its derivatives were monitored by electrospray ionization mass spectrometry (ESI–MS) combined with ion mobility mass spectrometry (IM–MS), a method that can characterize interactions between proteins and small molecules (Figure 3).46,47 When DA was incubated with metal-free Aβ40, a new peak at 1087 m/z, indicative of the addition of an oxygen atom to the Aβ404+ 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 qualitative structure and distribution of Aβ species.46,47 Shorter arrival time distributions (ATDs) and the decreased collision cross section (CCS) of the oxidized Aβ40 monomer were observed compared to those of the non-oxidized Aβ40 monomer (Figure 3b; CCS data in Table S1), suggesting conformational compaction of the oxidized monomeric peptide. Such structural compaction could alter Aβ aggregation to form amorphous species instead of on-pathway structured Aβ aggregates, as previously reported.24,45 Thus, our mass spectrometric studies present that DA is able to cause Aβ oxidation and subsequently trigger the structural compaction of the peptide, which could be a mechanistic route for modulation of Aβ aggregation.45,47 Based on the previous results showing the oxidation of amino acid residues (e.g., methionine, cysteine, and histidine) by O2– and H2O2,48-50 ROS concomitantly produced upon oxidative transformation of DA, 3, and 4 could be the key agents for oxidation of Aβ and consequent modulation of peptide aggregation. This could be also supported by another previous study that reported the oxidation of Aβ fragments by 4methylcatechol.51 Moreover, since intracellular redox-active metal ions have been observed to accelerate ROS production with either DA or Aβ,2,7,9,20-24,26 the interaction of DA with Cu(II)-added Aβ40 was also investigated by ESI–MS (Figure 3c). Note that oxidative transformation of DA mediated by Cu(II)-added Aβ was

previously reported26,51 but the analysis of Cu(II)–Aβ species 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 +4-charged oxidized Aβ monomers were indicated 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 collision-induced dissociation for the singly oxidized Aβ40 was conducted (Figure 3d). The singly oxidized fragments were reported in b fragments cleaved from the N-terminus. The oxidized fragments larger than b34 in the oxidized metal-free 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 oxidation at M35 by DA is associated with its anti-amyloidogenic activity towards metal-free and metal-bound Aβ. This is supported by previous studies that present controlled Aβ aggregation upon oxidation of the M35 residue in Aβ by some chemical reagents.45,47,52,53 In addition to DA, the distinguishable amyloidogenic activity of 3 and 4 with metal-free 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 oxygen atom(s) into both metal-free and Cu(II)-bound Aβ40 (Figure 3e). Tandem MS data of the oxidized Aβ40 monomer, generated by incubation with 3, revealed the oxidation of M35, consistent with the results using DA (Figure S11). Furthermore, 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 metal-free and/or metal-induced Aβ aggregation 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 anti-amyloidogenic 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 oxidation sites. 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 subsequent control of Aβ aggregation, the electrochemical properties 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) spectroelectrochemistry 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),2,6-8 were presented within 60 min. The compounds equipped with a catechol moiety, which have a reactivity towards metal-free and/or metal-bound Aβ (i.e., 3 and 4), showed the redox be-



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). Second, the transformation of DA and its derivatives was identified in the presence of metal-free 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 observed 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 metal-free Aβ40. Such transformation of DA was also further supported by ESI–MS (Figure 4b, left). When DA was co-incubated with Aβ40 and Cu(II) for 60 min, in addition 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). Consistent with the results from the electrochemical studies, among DA derivatives, only 3 and 4, able to modulate Aβ aggregation via peptide oxidation, exhibited their distinct oxidative transformation upon incubation with or without Aβ and/or Cu(II) (Figures 4, S13, and S14). UV–Vis spectroscopic experiments identified that 3 containing both catechol and monomethylamino groups displayed optical bands at ca. 308

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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 metal-free Aβ40. The new peak at 164 m/z, observed by ESI–MS, also confirmed the formation of 3b or 3c in the presence of metal-free 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 semi-quinone species,55 in the presence of Cu(II)-bound Aβ (Figure 4b, right). 4, however, did not present the optical features consistent with the production of cyclized products, as expected for the tertiary amine-containing derivatives. ESI–MS analyses also detected a new peak at 180 m/z corresponding to the two-electron 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. Taken together, our electrochemical, UV–Vis spectroscopic, and MS studies validate that DA and its derivatives with anti-amyloidogenic effects are shown to interact with the redox 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 aggregation, is dependent on the degree of the oxidation of DA and its

Figure 4. Transformation of DA and its derivatives in the presence of Aβ40 with or without Cu(II). (a) Oxidative transformation of DA and its derivatives. (b) Transformation of DA, 3, and 4 in the absence and presence of Aβ40 and/or Cu(II), monitored by UV–Vis spectroscopy and MS. Conditions (for UV–Vis studies): [Aβ40] = 50 µM; [CuCl2] = 50 µM; [compound] = 100 µM, pH 6.6 (for Cu(II)-containing samples) or pH 7.4 (for metal-free samples); 37 °C; 1 h incubation; no agitation. Conditions (for mass spectrometric investigations): [Aβ40] = 100 µM; Paragon Plus pH Environment [CuCl2] = 100 µM; [compound] = 500 µM; 100 mMACS ammonium acetate, 7.5; 37 °C; 1 h incubation; 10-fold diluted samples were injected to the mass spectrometer; experiments were performed in triplicate.

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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 panmicroglial marker (CD11b)-positive cells. (a) Analysis of the expression of the pan-microglia 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 = 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. *P < 0.05 (versus LPS + Aβ42). (c) Expression of mRNA levels of HO-1. *P < 0.05 (versus LPS + Aβ42). (d) Protein levels of HO-1, 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 HO-1 were assessed by quantitative real-time RT-PCR 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 HO-1 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 HO-1 protein levels, analyzed in primary microglial cells upon treatment with DA or 3 in the presence of various inhibitors (for 12 h incubation) [NAC (N-acetyl-L-cysteine; a ROS inhibitor); Bay11-7085 (an inhibitor of NF-kB activation); SB203580 (p38 MAPK inhibitor); SP600125 (JNK MAPK inhibitor); LY294002 (phosphatidylinositol 3-kinase inhibitor)]. (h and i) mRNA levels of the pro-oxidant genes, iNOS (inducible nitric oxide synthase) and COX2 (cyclooxygenase 2), assessed by the quantitative real-time RT-PCR 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 protection of DA, 3, and 4 against Aβ-induced toxicity in primary microglial cells. The detailed procedures and conditions are presented in the Supporting Information.

derivatives; (ii) ROS, produced upon compounds’ oxidative modifications, are required for oxidation of both metal-free Aβ and metal–Aβ as well as subsequent modulation of their aggregation pathways. Effects of DA and Its Derivatives on Aβ β -Induced Toxicity Associated with Inflammation and Oxidative Stress. To determine whether and how DA and its derivatives (i.e., 3 and 4) could alter Aβ-mediated toxicity (e.g., inflammation and oxidative stress),56-58 their effects were monitored employing primary CD11b-positive cells, the major producers of inflammatory mediators in the brain.56,59,60 The primary CD11bpositive cells were isolated from postnatal day 1 (P1) C57BL/6 wild-type pups and identified by flow cytometry. Over 99% of the cells showed positive responses to CD11b, a well-known pan-microglial marker (Figure 5a). The systemic inflammation leads to the induction of neuroinflammation and

the increase of Aβ after lipopolysaccharides (LPS) administration.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 verify the ability of DA and its derivatives (i.e., 3, and 4) to improve 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 responsible for inflammation and oxidative stress,56,59,60,62 influence of DA and its derivatives (i.e., 3, and 4) on the gene expression induced by LPS and Aβ42 was monitored. After administration of LPS and Aβ42, the enhanced mRNA levels and protein secretion of proinflammatory cytokines, such as inter-



leukin-1β (IL-1β), interleukin-6 (IL-6), and the tumor necrosis factor α (TNF-α), were not altered by addition of DA and its derivatives (data not shown). Noticeably, however, the levels of mRNA and protein of HO-1, 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 HO-1 expression was not indicated in the case of 4. To correlate between neuroprotection and the HO-1 expression induced by DA and 3, cell viability was measured in the absence or presence of a selective HO-1 inhibitor, Zn(II) protoporphyrin IX [Zn(II)(PP)] (Figure 5e; bars marked with blue stripes).63 The inhibition of HO-1 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 HO1. To further delineate a molecular-level mechanism underlying the activation of HO-1 by DA and 3, the induction of HO1 was monitored upon addition of the compounds in primary CD11b-positive cells with different signaling inhibitors [Figures 5f (for DA) and 5g (for 3)]. The HO-1 protein level was noticeably reduced when NAC (N-acetyl-L-cystein; a ROS inhibitor)64 was co-incubated with DA and 3, suggesting that an increase in cellular ROS followed by induction of HO-1 could be a mechanism of DA for cell survival (Figure 5k, left). In addition to HO-1 expression, DA and its derivatives (i.e., 3 and 4) were indicated to decrease the levels of inflammatory mediators [i.e., inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), nitric oxide (NO)], provoked by treatment with LPS and Aβ (Figures 5h-j and 5k, right).65 Taken together, employing primary CD11b-positive cells, our investigations suggest that DA and its derivatives (i.e., 3 and 4) are able to protect the cells against LPS/Aβ-induced toxicity via a proposed mechanism (Figure 5k). Against LPS/Aβinduced toxicity, DA and the derivative 3 could upregulate the expression of HO-1, 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/Aβ-induced toxicity could be diminished upon incubation with the derivative via regulation of inflammatory mediators with no significant association with HO-1 expression (Figure 5k, right).

CONCLUSIONS 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 aggregation of both metal-free Aβ and metal–Aβ as well as the inflammation and oxidative stress triggered by Aβ were evaluated. DA’s derivatives were prepared by rational structural modifications of DA on the basis of its oxidative transformation. First, DA and its derivatives (i.e., 3 and 4) modulate metal-free and/or metal-induced Aβ aggregation, generating nontoxic, amorphous aggregates. Such anti-amyloidogenic 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 Aβ-induced inflammation and oxidative stress in primary CD11b-positive cells, the major supplier of inflamma-

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tory mediators in the brain, through induction of HO-1 and/or downregulation of inflammatory mediators. Overall, our multidisciplinary 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 molecular-level 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.

EXPERIMENTAL SECTION Materials and Methods. All reagents were purchased from commercial suppliers and used as received unless otherwise stated. 3HBr [4-(2-(methylamino)ethyl)benzene-1,2-diol hydrobromide] (purity 97%) was prepared following a previously reported method.66 DA (purity 98%), 1 [5-(2-aminoethyl)-2methoxyphenol] (purity 98%), and 5 [2-(3,4dimethoxyphenyl)-N-methylethan-1-amine] (purity 97%) were purchased from Sigma-Aldrich (St Louis, MO, USA). 2 [2(3,4-dimethoxyphenyl)ethan-1-amine] (purity 98%) was obtained from Acros Organics (Morris Plains, NJ, USA). 4HBr [4-(2-(dimethylamino)ethyl)benzene-1,2-diol hydrobromide] (purity 95%) and 6 [2-(3,4-dimethoxyphenyl)-N,Ndimethylethan-1-amine] (purity 95%) were acquired from Enamine (Monmouth Junction, NJ, USA). Trace metal contamination was removed from all solutions used for Aβ experiments by treating with Chelex (Sigma-Aldrich) overnight. Aβ40 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and Aβ42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) were obtained from Anygen (Nam-myun, Jangseong-gun, Republic of Korea) and Anaspec (Fremont, CA, USA). Double distilled water (ddH2O) used for all experiments was obtained from a Milli-Q Direct 16 system (Merck KGaA, Darmstadt, 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 JEM-1400 transmission electron microscope [Ulsan National Institute of Science and Technology (UNIST) Central Research Facilities, Ulsan, Republic of Korea]. Mass spectrometric analyses were performed by a Waters Synapt G2-Si quadrupole time-of-flight 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 Research Facilities, Ulsan, Republic of Korea). 1H and 13C NMR spectra of compounds were recorded on an Agilent 400MR DD2 NMR spectrometer (UNIST Central Research Facilities, Ulsan, Republic of Korea) and a Bruker AVHD400 NMR spectroscopy (KAIST Analysis Center for Research Advancement, Daejeon, Republic of Korea). Mass spectrometric analysis of molecules was carried out by a Bruker maXisTM HD Ultra-high resolution Q-TOF LC MS/MS system (HRMS; the Cooperative Laboratory Center of Pukyong National University, Busan, Republic of Korea). Preparation of 3. 3 was synthesized following a previously reported method with modifications.66 2-(3,4-Dimethoxyphenyl)-N-methylethan-1-amine (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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reaction mixture was concentrated under vacuum and dried. The final product was obtained without further purification (brown powder; 483.0 mg; 1.9 mmol; 72%). 1H NMR [400 MHz, D2O, δ (ppm)]: 6.79 (d, J = 8.0 Hz, 1H), 6.73 (s, 1H), 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)]: 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. Aβ β Aggregation Experiments. All experiments were conducted 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 re-dissolving Aβ with NH4OH (1% w/v, aq; 10 µL) followed by dilution with ddH2O. The concentration of Aβ was determined by measuring the absorbance of the solution at 280 nm (ε = 1450 M−1cm−1 for Aβ40; ε = 1490 M−1cm−1 for Aβ42) by UV−Vis spectroscopy. The peptide stock solution was diluted to a final concentration of 25 µM in the Chelex-treated buffered solution [20 µM HEPES (4(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4 (for metal-free and Zn(II)-added samples) or pH 6.6 (for Cu(II)treated samples), and 150 µM NaCl]. For the inhibition experiments, Aβ (25 µM) was first treated with or without a metal chloride salt (CuCl2 or ZnCl2; 25 µM) for 2 min followed by addition of compounds (50 µM; 1% v/v final DMSO concentration). 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 constant 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 N2-filled glove box (Korea Kiyon, Bucheon-si, Gyeonggi-do, Republic of Korea). Gel/Western Blot. Aβ samples from the inhibition and disaggregation experiments in vitro were analyzed by gel electrophoresis with Western blotting (gel/Western blot) using a primary anti-Aβ antibody (6E10; 1:2,000, Covance, Princeton, NJ, USA).24,39 Samples (10 µL) were separated on a 10−20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA). Following separation, the proteins were transferred onto nitrocellulose membranes and blocked with bovine serum albumin (BSA, 3% w/v, RMBIO, Missoula, MT, USA) in Trisbuffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 3 h at room temperature. Experiments were performed in triplicate. For biological experiments, the membranes were incubated with primary antibodies [an anti-HO-1 (1:4,000, Enzo Life Sciences, Inc., Farmingdale, NY, USA); an anti-β-actin (1:5,000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA)] in a solution of 2% BSA (w/v in TBS-T) for 4 h at room temperature or overnight at 4 °C. After washing with TBS-T three times (8 min each), the horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5,000) or anti-rabbit secondary antibody (1:5,000) in 2% BSA (w/v in TBS-T) 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 SuperSig-

nalTM West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA) after exposure to the Xray film. TEM. Samples for TEM were prepared following previously reported methods using Glow-discharged grids (Formar/Carbon 300-mesh, Electron Microscopy Sciences, Hatfield, PA, USA).24,39 Images from Aβ42 samples were taken on a JEOL JEM-1400 (120 kV; 25,000× magnification) TEM. ESI–IM–MS. Aβ40 (100 µM) was incubated with DA, 3, and 4 (500 µM) in the absence or presence of CuCl2 (100 µM) in 100 mM ammonium acetate (pH 7.5) at 37 °C without agitation. Metal-free and Cu(II)-treated samples were incubated for 6 h and 1 h, respectively. Before injection into the mass spectrometer, Aβ samples were diluted by 10 fold. ESI–MS analyses were performed using a Synapt G2-Si quadrupole timeof-flight 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 measure 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 proteins that bracket the likely CCS and ion mobility values of the unknown ions.68,69 MS Analyses for Characterization of Oxidized Compounds. DA and its derivatives (500 µM) were incubated with or without Aβ40 (100 µM) in the absence or presence of CuCl2 (100 µM) in 100 mM ammonium acetate (pH 7.5) at 37 °C without agitation. After 1 h incubation, the samples were diluted by 10 fold before injection into the mass spectrometer. The spectra of DA, 3, and 4 were obtained from a Synapt G2Si quadrupole time-of-flight mass spectrometer (Waters, Manchester, 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. Cyclic Voltammetry. Cyclic voltammograms of all compounds were recorded under N2 (g) with a CHI620E model potentiostat (Qrins, Seoul, Republic of Korea). A threeelectrode setup, composed of an Ag/AgCl reference electrode (RE-1B Reference electrode; Qrins, Seoul, Republic of Korea), 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 electrolyte at various scan rates (5, 10, 25, 50, 100, 150, 200, 250,



and 300 mV/s) at room temperature.

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tive to the vehicle control (DMSO). Data were the mean of three independent experiments (n = 9–12).

Spectroelectrochemical Analysis. Spectroelectrochemical data for DA (1 mM) were recorded under N2 (g) with a potentiostat and an UV−Vis spectrophotometer. A spectroelectrochemical cell kit was utilized containing a thin layer quartz glass cell, a Pt gauze working electrode, a Pt wire auxiliary electrode [SEC-C Spectroelectrochemical cell kit (Pt); Qrins, Seoul, Republic of Korea], and an Ag/AgCl reference electrode (RE-1B 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. UV–Vis Spectroscopy. Optical spectra of DA and its derivatives (100 µM) with or without CuCl2 (50 µM) in the absence or presence of Aβ40 (50 µM) were obtained by an UV−Vis spectrophotometer. Chelex-treated buffered solutions [20 µM HEPES, pH 7.4 (for metal-free samples) or pH 6.6 (for Cu(II)treated samples), 150 µM 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 CD11b-positive cells were isolated from C57BL/6 mice at 1 day old as described.70 Mice were sacrificed and mesencephalons were dissected and collected in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Rockford, IL, USA). Mesencephalons were homogenized 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 centrifuged for 5 min at 300 g. The supernatant was resuspended in DMEM supplemented with 10% fetal bovine serum (FBS), 1% streptomycin/penicillin, and 1% L-glutamine (Thermo Fisher Scientific, Rockford, IL, USA). Cells were seeded on poly-D-lysine-coated plates and cultivated at 37 °C with 5% CO2 in a humidified atmosphere. After 11-13 days, cells were sub-plated and used for further experiments. For experiments, cell treatments were performed in 24 h after seeding. Flow Cytometry. Cultured microglia cells were counted and 1×105 cells were washed with PBS followed by an incubation of FITC-conjugated anti-mouse 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 (Canto-II, 8-color, flow cytometric analysis; BD Biosciences, San Jose, CA, USA). 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 96-well plates. After treatment of reagents, the MTS solution (20 µL) 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 rela-

RT-PCR. Total RNA was isolated by TRIzol reagent (Thermo Fisher Scientific, Rockford, IL, USA). Reverse transcription was performed using SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific, Rockford, IL, USA). Realtime quantitative PCR was conducted using iQ SYBR Green Supermix (BioRad, Hercules, CA, USA). Triplicate samples per condition were analyzed on an Applied Biosystems StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific, Rockford, IL, USA) using absolute quantification settings. 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 β-actin (forward: 5’-GATCTGGCACCACACCTTCT-3’ and reverse: 5’-GGGGTGTTGAAGGTCTCAAA-3’); mouse COX2 (forward: 5’CAAGGGAGTCTGGAACATTG-3’ and reverse: 5’-ACCCAGGTCCTCGCTTATGA-3’); mouse HO-1 (forward: 5’CGCCTTCCTGCTCAACATT-3’ and reverse: 5’-TGTGTTCCTCTGTCAGCATCAC-3’); mouse iNOS (forward: 5’AACGGAGAACGTTGGATTTG-3’ and reverse: 5’-CAGCACAAGGGGTTTTCTTC-3’). Nitrite Assay. NO production was determined by measuring the amount of nitrite, a stable breakdown product of NO metabolism, in cell supernatants. Supernatants were combined with an equal volume of 1% sulphanilamide (Sigma-Aldrich, St Louis, MO, USA) in 5% H3PO4 (Sigma-Aldrich, St Louis, MO, USA) and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma-Aldrich, St Louis, MO, USA) to convert nitrite into a magenta colored azo compound showing absorbance 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 comparisons between two groups, Student’s two-tailed 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)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (M.H.L.) *E-mail: [email protected] (S.W.C.) §

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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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 manuscript.

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Notes The authors declare no competing financial interest. (15)

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government [NRF2017R1A2B3002585 and NRF-2016R1A5A1009405 (to M.H.L.); NRF-2014R1A6A1030318 (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 (NRF2015HIA2A1030823). We thank Dr. Kyle Korshavn and Yelim Yi for the assistance with the synthesis of 3 and the characterization of compounds, resepectively.

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REFERENCES (1)

(2)

(3) (4) (5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

Palop, J. J., Chin, J., and Mucke, L. (2006) A network dysfunction perspective on neurodegenerative diseases. Nature 443, 768–773. Segura-Aguilar, J., Paris, I., Muñoz, P., Ferrari, E., Zecca, L., and Zucca, F. A. (2014) Protective and toxic roles of dopamine in Parkinson's disease. J. Neurochem. 129, 898–915. Martorana, A., and Koch, G. (2014) Is dopamine involved in Alzheimer's disease? Front. Aging. Neurosci. 6, 252. Selkoe, D. J. (2002) Alzheimer’s disease is a synaptic failure. Science 298, 789–791. Rossato, J. I., Bevilaqua, L. R. M., Izquierdo, I., Medina, J. H., and Cammarota, M. (2009) Dopamine controls persistence of long-term memory storage. Science 325, 1017–1020. Graham, G. D. (1978) Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol. Pharmacol. 14, 633–643. Linert, W., Herlinger, E., Jameson, R. F., Kienzl, E., Jellinger, K., and Youdim, M. B. (1996) Dopamine, 6-hydroxydopamine, iron, and dioxygen–their mutual interactions and possible implication in the development of Parkinson's disease. Biochim. Biophys. Acta 1316, 160–168. Bisaglia, M., Mammi, S., and Bubacco, L. (2007) Kinetic and structural analysis of the early oxidation products of dopamine: Analysis of the interactions with α-synuclein. J. Biol. Chem. 282, 15597–15605. Pham, A. N. and Waite, T. D. (2014) Cu(II)-catalyzed oxidation of dopamine in aqueous solutions: Mechanism and kinetics. J. Inorg. Biochem. 137, 74–84. Iuga, C., Alvarez-Idaboy, J. R., and Vivier-Bunge, A. (2011) ROS initiated oxidation of dopamine under oxidative stress conditions in aqueous and lipidic environments. J. Phys. Chem. B 115, 12234–12246. Burbulla, L. F., Song, P., Mazzulli, J. R., Zampese, E., Wong, Y. C., Jeon, S., Santos, D. P., Blanz, J., Obermaier, C. D., Strojny, C., Savas, J. N., Kiskinis, E., Zhuang, X., Krüger, R., Surmeier, D. J., and Krainc, D. (2017) Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson's disease. Science 357, 1255–1261. Li, J., Zhu, M., Manning-Bog, A. B., Di Monte, D. A., and Fink, A. L. (2004) Dopamine and L-dopa disaggregate amyloid fibrils: Implications for Parkinson's and Alzheimer's disease. FASEB J. 18, 962–964. Norris, E. H., Giasson, B. I., Hodara, R., Xu, S., Trojanowski, J. Q., Ischiropoulos, H., and Lee, V. M.-Y. (2005) Reversible inhibition of α-synuclein fibrillization by dopaminochrome-

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

mediated conformational alterations. J. Biol. Chem. 280, 21212–21219. Leong, S. L., Pham, C. L. L., Galatis, D., Fodero-Tavoletti, M. T., Perez, K., Hill, A. F., Masters, C. L., Ali, F. E., Barnham, K. J., and Cappai, R. (2009) Formation of dopamine-mediated αsynuclein-soluble oligomers requires methionine oxidation. Free Radic. Biol. Med. 46, 1328–1337. Conway, K. A., Rochet, J.-C., Bieganski, R. M., and Lansbury, P. T. (2001) Kinetic stabilization of the α-synuclein protofibril by a dopamine –α-synuclein adduct. Science 294, 1346–1349. 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 αsynuclein in the absence of oxygen: Two types of oligomerization mechanisms for α-synuclein and related cell toxicity studies. J. Neurosci. Res. 92, 359–368. Tavassoly, O., Nokhrin, S., Dmitriev, O. Y., and Lee, J. S. (2014) Cu(II) and dopamine bind to α-synuclein and cause large conformational changes. FEBS J. 281, 2738–2753. 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 αsynuclein oligomers and nigrostriatal degeneration. Nat. Neurosci. 20, 1560–1568. 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., PuglisiAllegra, 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. Que, E. L., Domaille, D. W., and Chang, C. J. (2008) Metals in neurobiology: Probing their chemistry and biology. Chem. Rev. 108, 1517–1549. Faller, P., Hureau, C., and Berthoumieu, O. (2013) Role of metal ions in the self-assembly of the Alzheimer’s amyloid-β peptide. Inorg. Chem. 52, 12193–12206. Barnham, K. J. and Bush, A. I. (2014) Biological metals and metal-targeting compounds in major neurodegenerative diseases. Chem. Soc. Rev. 43, 6727–6749. Savelieff, M. G., Lee, S., Liu, Y., and Lim, M. H. (2013) Untangling amyloid-β, tau, and metals in Alzheimer’s disease. ACS Chem. Biol. 8, 856–865. Beck, M. W., Derrick, J. S., Kerr, R. A., Oh, S. B., Cho, W. J., Lee, S. J. C., Ji, Y., Han, J., Tehrani, Z. A., Suh, N., Kim, S., Larsen, S. D., Kim, K. S., Lee, J.-Y., Ruotolo, B. T., and Lim, M. H. (2016) Structure-mechanism-based engineering of chemical regulators targeting distinct pathological factors in Alzheimer’s disease. Nat. Commun. 7, 13115. Peck, K. L., Clewett, H. S., Schmitt, J. C., and Shearer, J. (2013) Copper ligation to soluble oligomers of the english mutant of the amyloid-β peptide yields a linear Cu(I) site that is resistant to O2 oxidation. Chem. Commun. 49, 4797–4799. da Silva, G. F. Z. and Ming, L.-J. (2007) Metallo-ROS in Alzheimer's disease: Oxidation of neurotransmitters by Cu(II)– β-amyloid and neuropathology of the disease. Angew. Chem. Int. Ed. 46, 3337–3341. Gonzalez, P., da Costa, V. C. P., Hyde, K., Wu, Q., Annunziata, O., Rizo, J., Akkaraju, G., and Green, K. N. (2014) Bimodalhybrid heterocyclic amine targeting oxidative pathways and copper mis-regulation in Alzheimer's disease. Metallomics 6, 2072−2082. Lincoln, K. M., Richardson, T. E., Rutter, L., Gonzalez, P., Simpkins, J. W., and Green, K. N. (2012) An N-heterocyclic amine chelate capable of antioxidant capacity and amyloid disaggregation. ACS Chem. Neurosci. 3, 919−927.



(29) Huong, V. T., Shimanouchi, T., Shimauchi, N., Yagi, H., Umakoshi, H., Goto, Y., and Kuboi, R. (2010) Catechol derivatives inhibit the fibril formation of amyloid-β peptides. J. Biosci. Bioeng. 6, 629−634. (30) Di Giovanni, S., Eleuteri, S.; Paleologou, K. E., Yin, G., Zweckstetter, M., Carrupt, P.-A., and Lashuel, H. A. (2010) Entacapone and tolcapone, two catechol O-methyltransferase inhibitors, block fibril formation of α-synuclein and β-amyloid and protect against amyloid-induced toxicity. J. Biol. Chem. 285, 14941–14954. (31) Ambrée, O., Richter, H., Sachser, N., Lewejohann, L., Dere, E., de Souza Silva, M. A., Herring, A., Keyvani, K., Paulus, W., and Schäbitz, W.-R. (2009) Levodopa ameliorates learning and memory deficits in a murine model of Alzheimer’s disease. Neurobiol. Aging 30, 1192–1204. (32) Liu, M., Kou, L., Bin, Y., Wan, L., and Xiang, J. (2016) Complicated function of dopamine in Aβ-related neurotoxicity: Dual interactions with Tyr10 and SNK(26–28) of Aβ. J. Inorg. Biochem. 164, 119–128. (33) Chen, K., Gunter, K., and Maines, M. D. (2000) Neurons overexpressing heme oxygenase-1 resist oxidative stressmediated cell death. J. Neurochem. 75, 304–313. (34) Stocker, R., Yamamoto, Y., McDonagh, A., Glazer, A., and Ames, B. (1987) Bilirubin is an antioxidant of possible physiological importance. Science 235, 1043–1046. (35) Vellaisamy, K., Li, G., Ko, C.-N., Zhong, H.-J., Fatima, S., Kwan, H.-Y., Wong, C.-Y., Kwong, W.-J., Tan, W., Leung, C.H., and Ma, D.-L. (2018) Cell imaging of dopamine receptor using agonist labeling iridium(III) complex. Chem. Sci. 9, 1119–1125. (36) Wang, L., Su, D., Berry, S. N., Lee, J., and Chang, Y.-T. (2017) A new approach for turn-on fluorescence sensing of L-DOPA. Chem. Commun. 53, 12465–12468. (37) Lin, S., Yang, X., Jia, S., Weeks, A. M., Hornsby, M., Lee, P. S., Nichiporuk, R. V., Iavarone, A. T., Wells, J. A., Toste, F. D., and Chang, C. J. (2017) Redox-based reagents for chemoselective methionine bioconjugation. Science 355, 597– 602. (38) Verwilst, P., Kim, H.-R., Seo, J., Sohn, N.-W., Cha, S.-Y., Kim, Y., Maeng, S., Shin, J.-W., Kwak, J. H., Kang, C., and Kim, J. S. (2017) Rational design of in vivo tau tangle-selective nearinfrared fluorophores: expanding the BODIPY universe. J. Am. Chem. Soc. 139, 13393–13403. (39) Han, J., Lee, H. J., Kim, K. Y., Lee, S. J. C., Suh, J.-M., Cho, J., Chae, J., and Lim, M. H. (2018) Tuning structures and properties for developing novel chemical tools toward distinct pathogenic elements in Alzheimer’s disease. ACS Chem. Neurosci. 9, 800–808. (40) Liu, L.-J., Wang, W., Huang, S.-Y., Hong, Y., Li, G., Lin, S., Tian, J., Cai, Z., Wang, H.-M. D., Ma, D.-L., and Leung, C.-H. (2017) Inhibition of the Ras/Raf interaction and repression of renal cancer xenografts in vivo by an enantiomeric iridium(III) metal-based compound. Chem. Sci. 8, 4756–4763. (41) Wang, W., Vellaisamy, K., Li, G., Wu, C., Ko, C.-N., Leung, C.-H., and Ma, D.-L. (2017) Development of a long-lived luminescence probe for visualizing β-galactosidase in ovarian carcinoma cells. Anal. Chem. 89, 11679–11684. (42) Hori, Y., Otomura, N., Nishida, A., Nishiura, M., Umeno, M., Suetake, I., and Kikuchi, K. (2018) Synthetic-molecule/protein hybrid probe with fluorogenic switch for live-cell imaging of DNA methylation. J. Am. Chem. Soc. 140, 1686–1690. (43) Wang, H., Feng, Z., Del Signore, S. J., Rodal, A. A., and Xu, B. (2018) Active probes for imaging membrane dynamics of live cells with high spatial and temporal resolution over extended time scales and areas. J. Am. Chem. Soc. 140, 3505–3509. (44) Liu, J.-B., Yang, C., Ko, C.-N., Vellaisamy, K., Yang, B., Lee, M.-Y., Leung, C.-H., and Ma, D.-L. (2017) A long lifetime iridium(III) complex as a sensitive luminescent probe for

(45)

(46)

(47)

(48)

(49)

(50) (51)

(52)

(53)

(54)

(55)

(56) (57)

(58)

(59) (60)

(61)

(62)

Page 12 of 14

bisulfite detection in living zebrafish. Sens. Actuators B Chem. 243, 971–976. Kang, J., Lee, S. J. C., Nam, J. S., Lee, H. J., Kang, M.-G., Korshavn, K. J., Kim, H.-T., Cho, J., Ramamoorthy, A., Rhee, H.-W., Kwon, T.-H., and Lim, M. H. (2017) An iridium(III) complex as a photoactivatable tool for oxidation of amyloidogenic peptides with subsequent modulation of peptide aggregation. Chem.-Eur. J. 23, 1645–1653. Bernstein, S. L., Dupuis, N. F., Lazo, N. D., Wyttenbach, T., Condron, M. M., Bitan, G., Teplow, D. B., Shea, J.-E., Ruotolo, B. T., Robinson, C. V., and Bowers, M. T. (2009) Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease. Nat. Chem. 1, 326–331. Young, L. M., Saunders, J. C., Mahood, R. A., Revill, C. H., Foster, R. J., Tu, L.-H., Raleigh, D. P., Radford, S. E., and Ashcroft, A. E. (2015) Screening and classifying smallmolecule inhibitors of amyloid formation using ion mobility spectrometry–mass spectrometry. Nat. Chem. 7, 73–81. Stadtman, E. R. and Levine, R. L. (2003) Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25, 207–218. Cardey, B. and Enescu, M. (2009) Cysteine oxidation by the superoxide radical: A theoretical study. ChemPhysChem. 10, 1642–1648. Vogt, W. (1995) Oxidation of methionyl residues in proteins: Tools, targets, and reversal. Free Radic. Biol. Med. 18, 93–105. Pirota, V., Dell'Acqua, S., Monzani, E., Nicolis, S., and Casella, L. (2016) Copper–Aβ peptides and oxidation of catecholic substrates: Reactivity and endogenous peptide damage. Chem.-Eur. J. 22, 16964–16973. Bitan, G., Tarus, B., Vollers, S. S., Lashuel, H. A., Condron, M. M., Straub, J. E., and Teplow, D. B. (2003) A molecular switch in amyloid assembly: Met35 and amyloid β-protein oligomerization. J. Am. Chem. Soc. 125, 15359–15365. Palmblad, M., Westlind-Danielsson, A., and Bergquist, J. (2002) Oxidation of methionine 35 attenuates formation of amyloid β-peptide 1–40 oligomers. J. Biol. Chem. 277, 19506– 19510. Corona-Avendaño, S., Alarcón-Angeles, G., Ramírez-Silva, M. T., Rosquete-Pina, G., Romero-Romo, M., and PalomarPardavé, M. (2007) On the electrochemistry of dopamine in aqueous solution. Part I: The role of [SDS] on the voltammetric behavior of dopamine on a carbon paste electrode. J. Electroanal. Chem. 609, 17–26. Barreto, W. J., Barreto, S. R. G., Ando, R. A., Santos, P. S., DiMauro, E., and Jorge, T. (2008) Raman, IR, UV–Vis and EPR characterization of two copper dioxolene complexes derived from L-dopa and dopamine. Spectrochim. Acta. A Mol. Biomol. Spectrosc. 71, 1419–1424. Andersen, J. K. (2004) Oxidative stress in neurodegeneration: Cause or consequence? Nat. Med. 5, S18–S25. Mandrekar, S. and Landreth, G. E. (2010) Microglia and inflammation in Alzheimer’s disease. CNS Neurol. Disord. Drug Targets 9, 156–167. Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C., and Gage, F. H. (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934. Lull, M. E. and Block, M. L. (2010) Microglial activation and chronic neurodegeneration. Neurotherapeutics 7, 354–365. Amor, S., Puentes, F., Baker, D., and Van Der Valk, P. (2010) Inflammation in neurodegenerative diseases. Immunology 129, 154–169. Sheng, J. G., Bora, S. H., Xu, G., Borchelt, D. R., Price, D. L., and Koliatsos, V. E. (2003) Lipopolysaccharide-induced neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice. Neurobiol. Dis. 14, 133–145. Von Bernhardi, R. (2007) Glial cell dysregulation: A new perspective on Alzheimer disease. Neurotox. Res. 12, 215–232.


Page 13 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(63) Nowis, D., Bugajski, M., Winiarska, M., Bil, J., Szokalska, A., Salwa, P., Issat, T., Was, H., Jozkowicz, A., Dulak, J., Stoklosa, T., and Golab, J. (2008) Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice. BMC Cancer 8, 197. (64) Halasi, M., Wang, M., Chavan, T. S., Gaponenko, V., Hay, N., and Gartel, A. L. (2013) ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors. Biochem. J. 454, 201–208. (65) Song, S.-Y., Jung, Y. Y.; Hwang, C. J., Lee, H. P., Sok, C. H., Kim, J. H., Lee, S. M., Seo, H. O., Hyun, B. K., Choi, D. Y., Han, S. B., Ham, Y. W., Hwang, B. Y., and Hong, J. T. (2014) Inhibitory effect of ent-Sauchinone on amyloidogenesis via inhibition of STAT3-mediated NF-κB activation in cultured astrocytes and microglial BV-2 cells. J. Neuroinflammation 11, 118. (66) Borgman, R. J., McPhillips, J. J., Stitzel, R. E., and Goodman, I. J. (1973) Synthesis and pharmacology of centrally acting dopamine derivatives and analogs in relation to Parkinson's disease. J. Med. Chem. 16, 630–633. (67) Mruk, D. D. and Cheng, C. Y. (2011) Enhanced chemiluminescence (ECL) for routine immunoblotting: An inexpensive alternative to commercially available kits. Spermatogenesis 1, 121–122. (68) Ruotolo, B. T., Benesch, J. L. P., Sandercock, A. M., Hyung, S.-J., and Robinson, C. V. (2008) Ion mobility–mass spectrometry analysis of large protein complexes. Nat. Protoc. 3, 1139–1152. (69) Bush, M. F., Hall, Z., Giles, K., Hoyes, J., Robinson, C. V., and Ruotolo, B. T. (2010) Collision cross sections of proteins and their complexes: A calibration framework and database for gas-phase structural biology. Anal. Chem. 82, 9557–9565. (70) Chen, X., Zhang, Y.; Sadadcharam, G., Cui, W., and Wang, J. H. (2013) Isolation, purification, and culture of primary murine microglia cells. Bio-protocol 3, e314.




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