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Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Conformational-Switch Based Strategy Triggered by [18] π Heteroannulenes toward Reduction of Alpha Synuclein Oligomer Toxicity Ritobrita Chakraborty,† Sumit Sahoo,‡ Nyancy Halder,‡ Harapriya Rath,*,‡ and Krishnananda Chattopadhyay*,†

ACS Chem. Neurosci. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/10/19. For personal use only.

†

Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India ‡ School of Chemical Sciences, Indian Association for the Cultivation of Science (IACS), 2A/2B Raja S. C. Mullick Road, Kolkata 700032, India S Supporting Information *

ABSTRACT: A water-soluble meso-carboxy aryl substituted [18] heteroannulene (porphyrin) and its Zn-complex have been found to be viable in targeting α-Syn aggregation at all its key microevents, namely, primary nucleation, fibril elongation, and secondary nucleation, by converting the highly heterogeneous and cytotoxic aggresome into a homogeneous population of minimally toxic off-pathway oligomers, that remained unexplored until recently. With the EC50 and dissociation constants in the low micromolar range, these heteroannulenes induce a switch in the secondary structure of toxic prefibrillar on-pathway oligomers of α-Syn, converting them into minimally toxic nonseeding off-pathway oligomers. The inhibition of the aggregation and the reduction of toxicity have been studied in vitro as well as inside neuroblastoma cells. KEYWORDS: Heteroannulenes, α-Syn, conformational switch, on-pathway oligomer, off-pathway oligomer, neuroblastoma cell

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96−140).4 The amino acid sequence of the NAC also occurs individually as a 35 residue long peptide fragment (nonamyloid β) along with the amyloid β peptide in the cytotoxic deposits of related neurodegenerative Alzheimer’s disease (AD).5,6 Interestingly, this NAC sequence is also found as a domain within the α-Syn protein. As a domain within the α-Syn protein, the NAC, due to its hydrophobicity, acts as the initiator of aggregation. Previous studies using paramagnetic relaxation enhancement and NMR dipolar coupling have reported that the native monomeric α-Syn forms an ensemble of fluctuating conformations that are stabilized by a network of long-range intramolecular interactions involving the N and C termini.7,8 These conformations are ringlike, compact, and relatively stable, and are thought to prevent oligomerization

INTRODUCTION Alpha synuclein (α-Syn) is a 140 amino acid residue long, natively unfolded protein associated with incurable synucleopathies such as Parkinson’s disease (PD), dementia with Lewy bodies, and multiple system atrophy.1,2 PD is characterized by neuronal loss accompanied by motor and cognitive deficits in patients.3 The molecular interpretation of PD entails a conformational alteration of the soluble protein α-Syn into misfolded monomers that self-coalesce into intermediate structures ultimately forming well-organized amyloid fibrils. The deposition of these cross-β sheet-rich amyloid fibrils in insoluble cytoplasmic inclusions (Lewy bodies) within the dopaminergic neurons of the substantia nigra pars compacta of the brain is the physiological hallmark of PD.1 α-Syn consists of an amphipathic N-terminal domain (residues 1−60), a hydrophobic core domain, also known as the NAC (nonamyloid β component) region (residues 61− 95), and an acidic, proline-rich C-terminal domain (residues © XXXX American Chemical Society

Received: August 27, 2018 Accepted: October 8, 2018 Published: October 8, 2018 A

DOI: 10.1021/acschemneuro.8b00436 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience Scheme 1. Synthesis of Macrocycles under Study

and fibrillogenesis by shielding the highly amyloidogenic NAC from the surrounding solvent. A promising approach to hinder aggregation resulting from protein misfolding is to use small molecules or chaperones to bind and stabilize these native autoinhibitory states of the protein.8,9 The development of such compounds has been successful in the case of globular proteins, such as transthyretin,10 a protein implicated in systemic amyloidosis, but the structural heterogeneity and transient nature of the early stage structural elements11 of intrinsically disordered proteins like α-Syn pose a major challenge in the discovery and design of small molecule inhibitors. Experimental evidence has also suggested that amyloid fibril formation is not linear but rather a competitive multipathway process that involves stable as well as metastable polymers, which show diverse degrees of cellular toxicity.12 The formation of amyloid fibrils of α-Syn typically follows the nucleation−conversion−polymerization model.13,14 This process initiates through an event called primary nucleation in which the misfolded monomers of α-Syn associate via diverse pathways to form toxic oligomers and protofilaments, which elongate to form fibrils.15 Although the fibrils are minimally toxic,16 they have been shown to dissociate, acting as reservoirs of toxic misfolded monomers and oligomeric species.17 These misfolded species can nucleate again or seed collateral fibrillization in a process known as secondary nucleation.18−20 Several studies have suggested the existence of a variety of intermediate oligomeric species that transiently populate the conformational landscape of α-Syn. The “on-pathway” oligomers consist of antiparallel β sheet conformation and eventually form parallel β sheet fibrils. These oligomers contain a high percentage of exposed hydrophobic surfaces and are cytotoxic.21 In contrast, dead-end “off-pathway” oligomers are those that contain parallel β sheet conformation with less exposed hydrophobic surfaces and do not form fibrils. These are not cytotoxic.12,22−24 The precise molecular underpinnings of the conformational switch between the on- and off-pathway oligomers remain unclear. Although several small molecules have been tested as therapeutics for PD, none of them have cleared clinical trials.25

There is an upsurge of demand for small molecules that can inhibit and/or terminate amyloid formation, and decrease PDassociated mitochondrial stress and cellular toxicity. In this paper, a novel mechanism of action of amyloid-inhibition involving a “conformational switch” in the oligomers of α-Syn has been explained, that might aid in developing neuroprotection against PD and other neurodegenerative diseases. We have explored the ability of two porphyrins (among a set of six porphyrins): TCPP (tetrakis (4-carboxyphenyl)porphyrin tetrasodium, 8) and ZnTCPP (zinc-tetrakis (4carboxyphenyl)porphyrin tetrasodium, 9), to interfere with the on-pathway intermediates (containing antiparallel β-sheet, formed via primary nucleation) of α-Syn leading to the formation of off-pathway species, with parallel β sheet content, that do not self-adhere to form amyloid fibrils. In contrast to the on-pathway oligomers, these off-pathway aggregates show reduced cytotoxicity to mammalian cells and are not capable of disrupting the integrity of synthetic liposomal membranes. We have also reported that 8 and 9 can block secondary processes such as the seeding capacity of α-Syn preformed aggregates. Furthermore, these porphyrins can also disintegrate preformed fibrils into smaller-sized oligomers which are rich in parallel β sheets and, more importantly, prevent these oligomers from seeding new aggregation reactions. 8 and 9 can also interrupt N-methyl-4-phenylpyridinium iodide (MPP+)-induced aggregation inside SH-SY5Y neuroblastoma cells. Additionally, according to our observations, sub-micromolar doses of the porphyrins are sufficient to interfere with the production of toxic oligomers of α-Syn.

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RESULTS Synthesis and Reaction of the Porphryrin Macrocycles with α-Syn: Macrocycles 8 and 9 Are the Most Effective Inhibitors of α-Syn Aggregation. The macrocycles under biological investigation described in the present article are shown in Scheme 1. We have taken into consideration the parent basic porphyrin, i.e., tetraphenyl porphyrin, and variations in its periphery with other mesosubstituents. Synthesis of porphyrins 1, 4, and 7 were carried B


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ACS Chemical Neuroscience out following the Adler method.26 Since these macrocycles (1, 4, and 7) were not water-soluble, they were made soluble following literature methods.27−29 Subsequently, these watersoluble macrocycles were metalated with zinc salt. The UV−vis absorption spectra, NMR spectra, and chemical analysis of all the macrocycles (both in free base forms and zinc-complexes) are well consistent with the structures. The viability of the macrocycles under study toward inhibition of α-Syn aggregation is based on the fact that tetrakis(4-carboxyphenyl)porphyrin tetrasodium) (8), Zn-tetrakis(4-carboxyphenyl)porphyrin tetrasodium) (9), tetrakis (4-sulfonatophenyl)porphyrin tetrasodium (2), and Zn-tetrakis (4sulfonatophenyl)porphyrin tetrasodium (3) are negatively charged at the periphery when water-soluble. On the other hand, tetra-(N-methylpyridyl porphyrin) (5) and the zinc complex of tetra-(N-methylpyridyl porphyrin) (6) are positively charged at the periphery in the pH range in which they are water-soluble. It is a well-known fact that the anionic porphyrins aggregate in water while the cationic porphyrins do not. It may be surmised that the positive centers on the periphery cause the delocalized π-electron cloud to be more diffuse over the surface of the molecule, while negative centers lead to a partial localization of electron density near the center. This localized electron density would make the center a more attractive site for protons, thereby increasing the basicity and decreasing the acidity of the species, and would lead to stronger van der Waals interactions for a stacking-type dimer. In other words, the extent of self-aggregation properties of all the water-soluble macrocycles under study has been considered as the key parameter in binding to the protein and inhibiting its aggregation. Initially, the binding between the six porphyrin molecules and monomeric α-Syn was investigated by titrating 0.1 μM of the porphyrins with α-Syn concentration ranging between 0.1 and 4 μM. The values of the dissociation constant Kd (Kd = 1/ Ka) are stated in Table 1. The dissociation constants (Kd) for

emission of thioflavin T (ThT), and the results are shown in Figure 1. ThT is a benzothiazole dye that exhibits enhanced

Figure 1. ThT fluorescence intensity of 200 μM α-Syn after 96 h of incubation in the absence and presence of 2 μM porphyrin macrocycles added from the beginning of the incubation period. Porphyrins 8 and 9 show maximum inhibition of amyloid fibrillization.

fluorescence upon binding to amyloid fibrils.32 Figure 1 clearly shows that the molecules 8 and 9 offered maximum inhibition of amyloid fibril formation. Because of their better binding (Table 1) and more potent aggregation inhibition (Figure 1), these two molecules (8 and 9) were chosen for further detailed investigations as discussed below. Figure 2a shows that the amyloid-formation kinetics of αSyn follows a sigmoidal behavior. In the absence of porphyrin, the kinetics was characterized by a lag phase of ∼16 h, in which a negligible change in ThT fluorescence was observed. This was followed by rapid exponential growth, until a saturation plateau phase of fibril maturation was achieved at ∼75 h (Figure 2a). This behavior is typical for the nucleation− conversion−polymerization13,14 model of protein aggregation as outlined in Scheme 2. In the presence of both 8 and 9 (protein/porphyrin 100:1), a large increase in the lag time accompanied by minimal increase in ThT fluorescence intensity was observed. (Figure 2a). The half maximal effective concentration (EC50) was calculated by plotting on the y-axis the ThT fluorescence intensity values of 200 μM α-Syn incubated (for 96 h with constant agitation at 37 °C) with various concentrations of 8 or 9. The log values of the porphyrin concentrations have been plotted on the x-axis. Figure 2b,c illustrate the representative sigmoidal semilog plots for porphyrins 8 and 9 obtained by fitting the data using the Dose Response fitting function. Along with the EC50, the values of EC20 and EC80, which are the effective concentrations of the porphyrins at which 20% and 80% response is achieved, were also calculated. Doses of 8 and 9 as low as 1.22 and 1.49 μM, respectively, were found adequate to cause 50% reduction in aggregation of 200 uM αSyn. Although both macrocycles were found effective in inhibiting the formation of fibrils, macrocycle 8 was found to be more potent than 9. Subsequently, we investigated if macrocycles 8 or 9 could show any ability to dissociate mature fibrils. When added at a molar ratio of 10:1 (α-Syn/porphyrin) to mature fibrils (at the stationary phase, after 96 h of aggregation), 8 and 9 caused disaggregation of the mature fibrils manifested by a sharp and permanent decrease in ThT fluorescence (Figure 2d). It was found that, at a ratio lesser

Table 1. Binding Data Derived for the Binding of the Six Water-Soluble Porphyrins under Study, with α-Syn porphyrin 2 3 5 6 8 9

dissociation constant (Kd), M 6.47 4.36 2.8 1 8.92 4.69

× × × × × ×

10−6 10−6 10−6 10−6 10−7 10−7

TCPP, sodium salt (porphyrin 8) and ZnTCPP, sodium salt (porphyrin 9) were found to be 8.92 × 10−7 M and 4.69 × 10−7 M, respectively, and were lower by 1 order of magnitude than the Kd values of the remaining porphyrin molecules (Table 1 and Supporting Information Figure S1). Subsequently, we investigated the amyloid fibril formation of the protein by incubating 200 μM α-Syn dissolved in sodium phosphate buffer, pH 7.4, at 37 °C with constant agitation at 180 rpm for 96 h in the absence and presence of 2 μM porphyrins (protein/porphyrin 100:1). Similar concentration of protein and experimental conditions has been shown by us to result in consistent and reproducible aggregation kinetics with defined lag time and saturation behavior.30,31 The extent of amyloid formation after 96 h of incubation with or without the porphyrins was measured by steady state fluorescence C


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Figure 2. ThT fluorescence of (a) 200 μM α-Syn incubated for 96 h in the absence (blue) and presence of 2 μM 8 (black) or 2 μM 9 (red). Figures (b) and (c) illustrate the dose−response curves and the calculated EC20, EC50, and EC80 (in μM) values of 8 and 9 when incubated with 200 μM α-Syn for 96 h. (d) Fragmentation of 200 μM mature α-Syn fibrils (saturation phase) upon addition of 20 μM 8 (black) and 9 (red). (e) Comparison of the seeding effect of porphyrin-treated (α-Syn/porphyrin 100:1; 8: black; 9: red) and untreated (green) oligomers on α-Syn; the aggregation profile of 200 μM α-Syn (blue) has been added to this figure for comparison. TEM and AFM micrographs of 200 μM α-Syn incubated for 96 h in the absence (f−h) or presence of 2 μM 8 (i−k) or 9 (l−n). TEM and AFM images of 200 μM saturation phase fibrils fragmented by 20 μM 8 (o−q) or 9 (r−t). The representative AFM height distribution profile is shown below each micrograph. Oligomers formed as a result of fibril disintegration are marked with black arrows in the TEM images. The white bar corresponds to 200 nm in the TEM and AFM images.

than 10:1, the defibrillization by 8 or 9 was significantly slower. Thus, the 10:1 ratio was used as the optimal concentration in these studies. In another experiment, “oligomer seeds” were prepared by incubating 200 μM monomeric α-Syn for 24 h in the absence or presence of 2 μM 8 or 9. These oligomer seeds were then added at the beginning of the aggregation assay consisting of 200 μM monomeric α-Syn. Figure 2e shows the aggregation profile of α-Syn as control (in the absence of seeds or porphyrins), which followed the typical sigmoidal behavior. In the presence of porphyrin-untreated oligomer seeds, the kinetics became hyperbolic, a behavior observed by other research groups12,24 in seeded aggregation. The oligomer seeds that had been incubated for 24 h in the presence of 8 and 9 (in the ratio of α-Syn/porphyrin 100:1) showed complete inhibition of seeding-induced aggregation, indicating that porphyrin-generated oligomers do not function as templates

for the conversion into amyloid and are therefore off-pathway structures. The ThT fluorescence measurements were substantiated by negative stain TEM and AFM. According to the TEM micrographs, the diameter of an individual mature fibril after 96 h of incubation in the absence of porphyrins measured between 6 and 7 nm (Figure 2f), while its AFM height ranged between 2.8 and 6 nm (Figure 2g,h). The length of each fibril measured between 0.5 and 2 μm. In contrast, the TEM images of 200 μM α-Syn coincubated from the lag phase with 2 μM 8 (Figure 2i) or 9 (Figure 2l) shows the absence of fibrillar networks even after 96 h. Instead, small-length fibrils clumped laterally along their longitudinal axes, with diameters ranging between 10 and 15 nm and lengths of approximately 0.1−0.3 μm, were observed. Spherical oligomers (depicted in the TEM micrographs using arrows) with diameters ranging between 15 and 30 nm were also present. AFM images (Figure 2j,k: 8 and D


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ACS Chemical Neuroscience Scheme 2. Nucleation−Conversion−Polymerization Model of α-Syn Fibrillogenesisa

a

Under pathogenic conditions, the monomeric protein is converted into and remains in equilibrium with an ensemble of misfolded conformations, which further self-associate via primary nucleation to form on-pathway oligomeric states and amyloid fibrils. Secondary processes of fibril fragmentation and secondary nucleation of these fragmented polymers onto the mature fibrils form a feedback loop that maintains the pathogenic amyloidogenesis cycle. Addition of porphyrins 8 or 9 to α-Syn leads to the formation off-pathway olgomers which do not lead to mitochondrial dysfunction or membrane perforation. The microevents that are inhibited by porphyrins 8 and 9 are represented by dotted lines.

Figure 3. Confocal microscopy images of SH-SY5Y cells: (a) White arrows indicate the intense punctate cytoplasmic aggregates of GFP-tagged αSyn induced by the exposure to 5 μM MPP+ for 6 h. Further incubation for 2 h of the MPP+-treated cells with 5 μM (b) 8 or (c) 9 reduces the presence of the high intensity cytoplasmic aggregates. (d) Control cells transfected with α-Syn-GFP, but without the addition of MPP+. The white scale bars denote 20 μm. The intensity profiles of the fluorescence signals along the yellow lines in the confocal images are shown on the right for comparison.

m,n: 9) report the height of individual “broken” fibrils to be ∼4 nm and those of oligomers to be between 2.5 and 15 nm. Figure 2o−q and r−t shows the disaggregation of 200 μM mature α-Syn fibrils (after 96 h of aggregation) into shorter length clumped fibrils and oligomers upon the addition of 20 μM 8 or 9, respectively. Under these circumstances, the TEM images (Figure 2o: 8 and r: 9) showed broken fibrils evidently clustered lengthwise, with the clumped fibrils having diameters

ranging between 10 and 15 nm and lengths between 0.1 and 0.3 μm. In addition, numerous oligomers with diameters varying between 10 and 20 nm were also present. AFM (Figure 2p,q: 8 and s,t: 9) images quantified the diameters of the clumped fibrils to be in the range between 7 and 20 nm and length between 0.3 and 1 μm. The diameters of the oligomers were found to be between 3 and 9 nm. E


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Figure 4. Uptake of porphyrins (a) 8 and (b) 9 inside SH-SY5Y cells. The superior fluorescence of 9 inside cells is presumably due to its lesser propensity to self-associate in aqueous medium. (c) Colocalization of 9 with LysoTracker Green DND-26 within lysosomes. (d) Porphyrin 9 does not colocalize within mitochondria with MitoTracker Green FM.

Macrocycles 8 and 9 Inhibit Aggregation of α-Syn Inside Live Neuroblastoma Cells. Further, we examined αSyn aggregation inside SH-SY5Y neuroblastoma cells in the absence and presence of 8 or 9. Figure 3a represents the confocal image of cells that were transiently transfected using an EGFP-α-Syn construct, and the aggregation of α-Syn was induced by treating these cells with 5 μM N-methyl-4phenylpyridinium iodide (MPP+) dissolved in DMEM for a period of 6 h. The intense green dots (marked by white arrows) within the cytoplasm represent α-Syn aggregates, as has been shown in previous literature.33,34 The incubation for 2 h with 5 μM of either 8 or 9 of the transfected MPP+-treated cells, represented by Figure 3b and c, respectively, prevented the formation of the aggregates marked by the absence of

intense green dots in the cell cytoplasm (only the outlines of the cells can be ascertained from the images). The fluorescence from these cells (Figure 3b and c) were comparable to the basal fluorescence of the control SH-SY5Y cells that contained only minute green aggregates showing diminished fluorescence, because these cells were transfected with the protein construct, but not treated with MPP+ (Figure 3d). The corresponding intensity profiles along the yellow lines drawn across the confocal images are presented in Figure 3 and show that the fluorescence emission of the MPP+-treated cells in Figure 3a is about 3-fold greater than that of those cells that were treated 8 or 9 (Figure 3b and c, respectively). We studied the colocalization with other cellular organelles of porphyrin 9, because of its higher fluorescence compared to F


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Figure 5. (a) Calcein-loaded SUV membrane permeation, (b) % of early apoptotic SH-SY5Y cells, and (c) % of SH-SY5Y cells showing mitochondrial membrane depolarization in the presence of following conditions, depicted along the x-axis: (1) 20 mM sodium phosphate buffer; (2) 20 μM 8; (3) 20 μM 9; (4) α-Syn oligomers incubated for 28 h at 37 °C with constant agitation; (5, 6) porphyrin-treated oligomers formed when 200 μM α-Syn monomers were incubated from the lag phase with (5) 2 μM 8 or (6) 2 μM 9 for 28 h; (7, 8) porphyrin-treated oligomers formed when 200 μM α-Syn fibrils (agitated for 96 h) were further incubated for 48 h with (7) 20 μM 8 or (8) 20 μM 9.

Figure 6. Deconvoluted FTIR spectra of (a) α-Syn oligomers formed after 28 h of aggregation; (b, c) α-Syn oligomers formed when the monomeric protein was incubated with porphyrins (b) 8 and (c) 9 (α-Syn/porphyrin 100:1) for 28 h; (d, e) α-Syn oligomers formed when α-Syn mature fibrils were treated for 48 h with (d) 8 or (e) 9 (α-Syn/porphyrin 10:1). Representative FTIR peaks denoting the secondary structures are color-coded, and percentage of secondary structural content is mentioned underneath each curve. (f, g) Fluorescence emission from ANS binding experiments for α-Syn oligomers aggregated for 28 h (black), and porphyrin treated oligomers (red: 8 added at the lag phase; blue: 8 added at the saturation phase; magenta: 9 added at the lag phase; green: 9 added at the saturation phase).

LysoTracker (Figure 4c). A lesser extent of 9 was also detected in the cell cytoplasm, but not at all in the mitochondria or the nucleus (Figure 4d). This is in accordance with previous studies that have reported that cationic porphyrins localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes.37 Porphyrins 8 and 9 Alleviate Membrane Perturbation Propensity and Cytotoxicity of α-Syn Oligomers. The aggregation pathway of α-Syn is extremely heterogeneous and comprises different microscopic events that may individually or collectively contribute to the generation of toxic oligomers and overall the cytotoxicity. The inhibition of primary nucleation by 8 or 9 strongly delayed the fibril formation but increased the amount of oligomers formed as evidenced by the ThT measurements and AFM and TEM results. Moreover, suppression of fibril and seed elongation and disaggregation

porphyrin 8 inside SH-SY5Y cells. The difference in fluorescence between 8 and 9 inside SH-SY5Y cells (Figure 4a,b) may have a direct correlation with the self-association propensity of these porphyrins when dissolved in buffer/ culture medium. Tetrapyrrolic compounds are known to remain aggregated in aqueous solution, thus emitting lesser fluorescence than their monomeric counterparts in organic solvents such as DMSO.35−37 When compared, we observed a higher stacking propensity of 8 than 9 (Figure S2). For the colocalization studies, SH-SY5Y cells incubated for 2 h with 9 were further coincubated with MitoTracker Green FM or LysoTracker Green DND-26, which are specific dyes for the mitochondria and lysosomes, respectively, and then subjected to confocal imaging. The concentration of 9 was kept at 5 μM. It was seen that porphyrin 9 colocalized only in the lysosomes with G


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∼1683 cm−1 (Figure 6a). The same conclusion was also obtained from the second derivative analysis of the FTIR spectra (Figure S5, black trace). The band at 1683 cm−1 was approximately 4-fold weaker than the band at ∼1624 cm−1 (Figure 6a, and Figure S5, black trace, for the second derivative plot) which is suggestive of antiparallel β sheet content.24 On the other hand, oligomers with parallel β structure were formed when 200 μM α-Syn monomers were incubated from the lag phase with 2 μM 8 (Figure 6b) or 9 (Figure 6c) for 28 h. Additionally, the oligomers formed when 200 μM mature fibrils were fragmented by 20 μM porphyrins were also found to be rich in parallel β sheet structures as identified by the presence of a band at 1620−1630 cm−1 and the absence of the band at ∼1683 cm−1 (Figure 6d for 8 or e for 9). ANS binding experiments showed an increase in ANS fluorescence intensity for oligomers generated in the absence of porphyrins (Figure 6f,g) indicating that these oligomers contained large solvent exposed hydrophobic surfaces. In case of porphyrin-stabilized oligomers (prepared by the addition of 8 or 9 either at the lag phase or at the saturation phase of aggregation), the ANS intensity was comparatively lower (Figure 6f: 8 and g: 9).

of mature fibrils lead to an increase in the amount of oligomers, as has also been reported earlier.38 As a result, the treatment of porphyrins at any stage in the aggregation pathway of α-Syn leads to an overall increase in oligomer content. Since onpathway oligomers are known to generate significant toxicity, while their off-pathway counterparts do not,22−24 determination of the overall cytotoxicity would further validate the onvs off-pathway nature of the oligomers formed by the addition of 8 and 9. Alternatively, these measurements would provide direct information on whether the oligomers formed due to the addition of 8 or 9 at either the lag phase or at the saturation phase were as toxic as the oligomers formed in the absence of the porphyrins. We compared the membrane permeation and cellular toxicity of the oligomers, which formed in the absence and presence of porphyrins. For this purpose, we synthesized calcein-loaded small unilamellar vesicles (SUVs) composed of 3:7 POPC/DOPS and added them to 2 μM oligomeric α-Syn (either treated or untreated with porphyrins) at a protein/lipid ratio of 1:10. Triton X-100 was used to determine 100% calcein release and all results were normalized to this value. Upon addition of oligomers (formed in the absence of porphyrins) to the SUVs, a 62% increase in calcein fluorescence was observed (Figure 5a). In contrast, oligomers formed when 2 μM 8 or 9 were added at the beginning of aggregation kinetics (lag phase) showed a much lesser calcein release of 35% and 41%, respectively. Moreover, the oligomers obtained because of the fragmentation of saturation phase fibrils with 20 μM 8 or 9 also led to a decreased calcein release of 32% and 22%, respectively. Addition of the porphyrins alone as controls (at a concentration of 20 μM) to the SUVs caused a minor calcein release of 300 °C. 1 H NMR (500 MHz, CDCl3 298 K, δ [ppm]): 8.88 (s, 8H, py), 8.25 (m, 8H, Ph−CH), 7.79 (m, 12H, Ph−CH), −2.76 (s, 2H, −NH). UV−vis (CH2Cl2, λ [nm], 298 K): 417, 515, 548, 590, 645. MALDI-TOF MS (m/z): 615.635 (calcd for C44H30N4 exact mass: 614.206). Elemental analysis: Calcd for C44H30N4: C, 85.97; H, 4.92; N, 9.11. Found: C, 86; H, 5.00; N, 9.08. 5,10,15,20-Tetrakis (4-Sulfonatophenyl)porphyrin Tetrasodium Salt (TPPS)27 (2). The mixture of 5,10,15,20-tetraphenylporphyrin 1 (1.0 g, 1.63 mmol) and 100% sulfuric acid (20 mL) was heated at 100 °C for 8 h. The reaction mixture was cooled to room temperature and then poured into ice−water (60 mL). The resulting mixture was neutralized with 10 M NaOH to bring to pH 5, and concentrated to 20 mL. It was then cooled to a temperature below 0 °C and filtered, and the solid was washed with methanol (60 mL). The filtrate was then diluted with methanol (100 mL), and the solid Na2SO4 was removed by filtration. Complete removal of Na2SO4 was performed by repeating the previous operation three times, and the resultant solution was concentrated to give a crude product. This was purified by recrystallizing it with a mixture of methanol and acetone. Yield ∼ 1.21 g (80%). 1 H NMR (500 MHz, DMSO-d6 298 K, δ [ppm]): 8.86 (s, 8H, Py), 8.21 (d, 8H, Ph−CH), 7.6 (d, 8H, Ph−CH), −2.99 (br, 2H, pyrrole NH). UV−vis (H2O, λ [nm], 298 K): 414, 516, 552, 580, 634. HR MS (m/z): 1022.7043 (calcd for C44H30N4O12S4 exact mass: 1022.0026). Elemental analysis: Cacld for C44H26N4Na4O12S4: C, 51.6; H, 2.56; N, 5.48; S, 12.54. Found: C, 51.82; H, 2.27; N, 5.56; S, 12.50. Zn-5,10,15,20-Tetrakis (4-Sulfonatophenyl)porphyrin Tetrasodium (ZnTSPP)27 (3). To (214 mg, 0.21 mmol) 5,10,15,20-tetrakis (4sulfonatophenyl)porphyrin in 20 mL of Milli Q water, 92 mg (0.42 mmol) of zinc acetate dihydrate was added. The solution was stirred under N2 atm. and refluxed for 4 h. The progress of the reaction was monitored by UV−vis spectral observation, and after noticing the absence of free base porphyrin from the UV−vis spectral pattern, the solution was filtered through sintered funnel to get rid of excess metal salt, after which the filtrate was evaporated to dryness under vacuum to yield the pure solid metal complex. Yield ∼ 182 mg (80%). 1 H NMR (500 MHz, DMSO-d6 298 K, δ [ppm]): 8.49 (s, 8H, Py), 8.054 (8H, Ph−CH), 7.86 (8H, Ph−CH). UV−vis (H2O, λ [nm], 298 K): 420, 556, 593. HR MS (m/z): 1083.9302 (calcd for C44H24N4Na4O12S4Zn exact mass: 1083.9161). Elemental analysis: Calcd for C44H24N4Na4O12S4Zn: C, 48.65; H, 2.23; N, 5.16; S, 11.81. Found: C, 48.98; H, 2.34; N, 5.14; S, 11.82. L


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H NMR (500 MHz, DMSO-d6 298 K, δ [ppm]): 8.18 (8H, br, PH−CH), 7.841 (8H, Py H), 7.34 (8H, br, Ph−CH), −2.85 (2H, s, NH). UV−vis (H2O, λ [nm], 298 K): 414, 518, 555, 579, 633. HR MS (m/z): 878.2327 (calcd for C48H26N4Na4O8 exact mass: 878.1347). Elemental analysis: Calcd for C48H26N4Na4O8: C, 65.61; H, 2.98; N, 6.38. Found: C, 65.66; H, 2.99; N, 6.35. Zn-5,10,15,20-Tetrakis (4-Carboxyphenyl)porphyrin Tetrasodium (9). H2TCPP (300 mg, 0.38 mmol) and zinc(II) acetate (166 mg, 0.75 mmol) were dissolved in DMF (80 mL), and the solution was stirred under refluxing conditions for 45 min. The metal complex formation was monitored by UV−vis spectroscopy in DMF. After filtration, the filtrate was dissolved in 30 mL of 0.1 M NaOH. The solution was then evaporated to dryness under vacuum to yield a brown solid. Yield ∼ 322 mg (90%). 1 H NMR (500 MHz, DMSO-d6 298 K, δ [ppm]): 8.39 (s, 8H, Py), 8.18 (d, 8H, Ph−CH), 8.035 (d, 8H, Ph−CH). UV−vis (H2O, λ [nm], 298 K): 423, 559, 597. HR MS (m/z): 940.2432 (calcd for C48H24Na4N4O8Zn exact mass: 940.0482). Elemental analysis: Calcd for C48H24Na4N4O8Zn: C, 61.20; H, 2.57; N, 5.95. Found: C, 61.30; H, 2.66; N, 5.92.

5,10,15,20-Tetra(4-pyridyl)porphyrin26 (4). Here, 1.07 g (10 mmol) of 4-pyridinecarboxaldehyde and 50 mL of propionic acid were magnetically stirred together. Freshly distilled pyrrole (0.7 mL, 10 mmol) was then added to the mixture, the temperature was brought to reflux, and the mixture allowed to stir for 2 h at reflux conditions. After allowing the reaction mixture to cool to room temperature, the reaction flask was placed in the freezer overnight to aid precipitation of the porphyrin. The mixture was then vacuumfiltered using a sintered funnel, and a dark purple solid was collected, washed with 5 × 50 mL of DCM and then with methanol, and dried overnight to give 5,10,15,20-tetra(4-pyridyl)porphyrin. Yield 2.472 g (20%). Mp > 300 °C 1 H NMR (500 MHz, CDCl3 298 K, δ [ppm]): 8.94 (d, J = 5 Hz, 8H, pyridyl-H), 8.79 (b, 8H, pyrrole-H), 8.16 (d, J = 5 Hz, 8H, pyridyl-H), −2.99 (b, 2H, pyrrole NH). UV−vis (CH3OH/CH2Cl2, λ [nm], 298 K): 415, 511, 545, 587, 642. MALDI-TOF MS (m/z): 618.629 (calcd for C40H26N8 exact mass: 618.704). Elemental analysis: Calcd for C40H26N8: C, 77.65; H, 4.24; N, 18.11. Found: C, 77.00; H, 4.16; N, 18.07. 5,10,15,20-Tetrakis (4-N-Methylpyridyl)tetraiodide Porphyrin TMPyP28 (5). To 1 g (1.61 mmol) of chromatographically purified 4 in 80 mL of DMF, 6 mL (96.37 mmol) of methyl iodide was added, and the mixture was refluxed for 1 h under N2 atm. The solution was then cooled and diluted with 100 mL of acetone. The resulting precipitate was filtered, washed with acetone, and dried. Yield 1.36 g (75%). 1 H NMR (500 MHz, DMSO-d6, 298 K, δ [ppm]): −3.11 (s, 2H, NH), 4.72 (s, 12H, N−CH3), 9.27 (d, 8H, Ph-o CH), 9.13 (s, 8H, pyrrole β-H), 8.94 (d, 8H, Ph-m CH). UV−vis (DMF, λ [nm], 298 K): 421, 518, 553, 584, 642. MALDI-TOF MS (m/z) 1187.319 (calcd for C44H38I4N8 exact mass: 1185.939). Elemental analysis: Calcd for C44H38I4N8: C, 44.54; H, 3.23; N, 9.44. Found: C, 44.59; H, 3.26; N, 9.40. Zn-5,10,15,20-Tetrakis (4-N-Methylpyridyl)tetraiodide Porphyrin (TMPyPZn)28 (6). To 249 mg (0.21 mmol) of tetra(N-methyl-4pyridyl)porphyrin tetraiodide in 20 mL of Milli-Q water, 92 mg (0.42 mmol) of zinc acetate dihydrate was added and refluxed under N2 atmosphere. After the conversion to Zn (II)-porphyrin was complete, as indicated by the absence of free base porphyrin absorption bands in visual spectroscopic measurements, the solution was evaporated to dryness under vacuum to yield a purple-brown solid. Yield ∼ 220 mg (84%). 1 H NMR (500 MHz, DMSO-d6 298 K, δ [ppm]): 9.207 (d, 8H, Ph−CH), 9.03 (s, 8H, pyrrole-H), 8.86 (d, 8H, Ph−CH). UV−vis (H2O, λ [nm], 298 K): 435, 560, 606. MALDI-TOF MS (m/z): 1248.1101 (calcd for C44H36I4N8Zn exact mass: 1247.8533). Elemental analysis: Calcd for C44H36I4N8Zn: C, 42.28; H, 2.90; N, 8.97. Found: C, 44.40; H, 2.96; N, 8.94. 5,10,15,20-Tetrakis (4-Carboxyphenyl)porphyrin65,66 (7). Here, 1.5 g (10 mmol) of 4-formylbenzoic acid and 50 mL of propionic acid were stirred at ambient temperature. To increase the solubility of 4formylbenzoic acid, the reaction mixture was heated to 80 °C, at which point the aldehyde was fully dissolved. Freshly distilled pyrrole (0.7 mL; 10 mmol) was then added to the mixture, and the mixture was stirred for 2 h under reflux. The reaction mixture was cooled to room temperature and then placed in the freezer overnight to aid precipitation of the porphyrin. The mixture was then vacuum-filtered using a sintered funnel and a dark purple solid was collected, washed with 5 × 50 mL of DCM, then washed with methanol and dried overnight to give 1.1 g of 5,10,15,20-tetrakis(4-carboxyphenyl) porphyrin (∼4.3 g, 55% yield). Mp > 300 °C. 1 H NMR (500 MHz, DMSO-d6 298 K, δ [ppm]): 13.14 (4H, br, −COOH), 8.86 (8H, s, Py β-H), 8.38 (16H, dd, phenyl H), −2.93 (2H, s, NH). UV−vis (CH3OH, λ [nm], 298 K): 413, 513, 545, 589, 646. 5,10,15,20-Tetrakis (4-Carboxyphenyl)porphyrin Tetrasodium)29 (8). H2TCPP (100 mg, 0.126 mmol) was dissolved in 10 mL of 0.1 M NaOH. The solution was evaporated to dryness under vacuum to yield a brown solid. Yield ∼ 107 mg (98%).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00436.

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Binding studies of 8 and 9 with α-Syn, self-association propensity analyses of 8 and 9, flow cytometry analyses data for apoptosis, mitochondrial membrane depolarization, second derivative of FTIR spectra, ESI-TOF and MALDI-TOF mass spectra, NMR spectra (1H NMR, 13 C NMR, 1H−1H COSY), and UV−vis spectral information on the porphyrin macrocycles studied (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +913324995843. *E-mail: [email protected]. Tel: +913324734971. ORCID

Harapriya Rath: 0000-0002-5507-5275 Krishnananda Chattopadhyay: 0000-0002-1449-8909 Author Contributions

S.S. and N.H. synthesized and characterized the macrocycles reported in the paper. R.C. designed and performed the biological studies. K.C., H.R., and R.C. wrote the manuscript. Funding

R.C. and S.S. thank UGC and CSIR for their research fellowship. N.H. thanks IACS for SRF position. The study has been funded by Department of Biotechnology Grant (MED/ 2017/50) to K.C. The work at IACS was supported by DSTSERB (EMR/2016/004705), New Delhi, India. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS α-Syn, alpha synuclein; TCPP, tetrakis (4-carboxyphenyl)porphyrin tetrasodium; ZnTCPP, zinc-tetrakis (4carboxyphenyl)porphyrin tetrasodium; NAC, nonamyloid component; ThT, Thioflavin T; MPP+, N-methyl-4-phenylpyridinium Iodide; EC50, half maximal effective concentration; AFM, atomic force microscopy; TEM, transmission electron M


Research Article


that alpha-synuclein oligomers are toxic. Proc. Natl. Acad. Sci. U. S. A. 108, 4194−9. (17) Lashuel, H. A., Petre, B. M., Wall, J., Simon, M., Nowak, R. J., Walz, T., and Lansbury, P. T., Jr. (2002) Alpha-synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322, 1089−102. (18) Gaspar, R., Meisl, G., Buell, A. K., Young, L., Kaminski, C. F., Knowles, T. P. J., Sparr, E., and Linse, S. (2017) Secondary nucleation of monomers on fibril surface dominates alpha-synuclein aggregation and provides autocatalytic amyloid amplification. Q. Rev. Biophys. 50, e6. (19) Knowles, T. P. J., Waudby, C. A., Devlin, G. L., Cohen, S. I. A., Aguzzi, A., Vendruscolo, M., Terentjev, E. M., Welland, M. E., and Dobson, C. M. (2009) An Analytical Solution to the Kinetics of Breakable Filament Assembly. Science 326, 1533−1537. (20) Linse, S. (2017) Monomer-dependent secondary nucleation in amyloid formation. Biophys. Rev. 9, 329−338. (21) Ingelsson, M. (2016) Alpha-Synuclein Oligomers-Neurotoxic Molecules in Parkinson’s Disease and Other Lewy Body Disorders. Front. Neurosci. 10, 408. (22) Paslawski, W., Mysling, S., Thomsen, K., Jørgensen, T. J., and Otzen, D. E. (2014) Co-existence of two different α-synuclein oligomers with different core structures determined by hydrogen/ deuterium exchange mass spectrometry. Angew. Chem., Int. Ed. 53, 7560−7563. (23) Pieri, L., Madiona, K., and Melki, R. (2016) Structural and functional properties of prefibrillar α-synuclein oligomers. Sci. Rep. 6, 24526. (24) Gonzalez-Lizarraga, F., Socias, S. B., Avila, C. L., TorresBugeau, C. M., Barbosa, L. R., Binolfi, A., Sepulveda-Diaz, J. E., DelBel, E., Fernandez, C. O., Papy-Garcia, D., Itri, R., Raisman-Vozari, R., and Chehin, R. N. (2017) Repurposing doxycycline for synucleinopathies: remodelling of alpha-synuclein oligomers towards non-toxic parallel beta-sheet structured species. Sci. Rep. 7, 41755. (25) Dehay, B., Bourdenx, M., Gorry, P., Przedborski, S., Vila, M., Hunot, S., Singleton, A., Olanow, C. W., Merchant, K. M., Bezard, E., Petsko, G. A., and Meissner, W. G. (2015) Targeting α-synuclein for treatment of Parkinson’s disease: mechanistic and therapeutic considerations. Lancet Neurol. 14, 855−866. (26) Adler, A. D., Sklar, L., Longo, F. R., Finarelli, J. D., and Finarelli, M. G. (1968) A mechanistic study of the synthesis of mesotetraphenylporphin. J. Heterocycl. Chem. 5, 669−678. (27) Beyene, B. B., Mane, S. B., and Hung, C.-H. (2015) Highly efficient electrocatalytic hydrogen evolution from neutral aqueous solution by a water-soluble anionic cobalt(ii) porphyrin. Chem. Commun. 51, 15067−15070. (28) Kanetada, N., Matsumura, C., Yamazaki, S., and Adachi, K. (2013) Mechanism of Peripheral Substituent Effects on Adsorption− Aggregation Behaviors of Cationic Porphyrin Dyes on Tungsten(VI) Oxide Nanocolloid Particles. ACS Appl. Mater. Interfaces 5, 12991− 12999. (29) Shahroosvand, H., Zakavi, S., Sousaraei, A., and Eskandari, M. (2015) Saddle-shaped porphyrins for dye-sensitized solar cells: new insight into the relationship between nonplanarity and photovoltaic properties. Phys. Chem. Chem. Phys. 17, 6347−6358. (30) Joshi, N., Basak, S., Kundu, S., De, G., Mukhopadhyay, A., and Chattopadhyay, K. (2015) Attenuation of the Early Events of αSynuclein Aggregation: A Fluorescence Correlation Spectroscopy and Laser Scanning Microscopy Study in the Presence of Surface-Coated Fe3O4 Nanoparticles. Langmuir 31, 1469−1478. (31) Ghosh, S., Kundu, A., and Chattopadhyay, K. (2018) Small Molecules Attenuate the Interplay between Conformational Fluctuations, Early Oligomerization and Amyloidosis of Alpha Synuclein. Sci. Rep. 8, 5481. (32) LeVine, H., 3rd (1999) Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 309, 274−84. (33) Kalivendi, S. V., Cunningham, S., Kotamraju, S., Joseph, J., Hillard, C. J., and Kalyanaraman, B. (2004) α-Synuclein Upregulation and Aggregation during MPP+-induced Apoptosis in

microscopy; KSV, Stern−Volmer quenching constant; Kd, dissociation constant

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REFERENCES

(1) Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) Alpha-synuclein in Lewy bodies. Nature 388, 839−40. (2) Tong, J., Wong, H., Guttman, M., Ang, L. C., Forno, L. S., Shimadzu, M., Rajput, A. H., Muenter, M. D., Kish, S. J., Hornykiewicz, O., and Furukawa, Y. (2010) Brain alpha-synuclein accumulation in multiple system atrophy, Parkinson’s disease and progressive supranuclear palsy: a comparative investigation. Brain 133, 172−88. (3) Jankovic, J. (2008) Parkinson’s disease: clinical features and diagnosis. J. Neurol., Neurosurg. Psychiatry 79, 368−376. (4) Moore, D. J., West, A. B., Dawson, V. L., and Dawson, T. M. (2005) Molecular pathophysiology of Parkinson’s disease. Annu. Rev. Neurosci. 28, 57−87. (5) El-Agnaf, O. M. A., Jakes, R., Curran, M. D., Middleton, D., Ingenito, R., Bianchi, E., Pessi, A., Neill, D., and Wallace, A. (1998) Aggregates from mutant and wild-type α-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β-sheet and amyloid-like filaments. FEBS Lett. 440, 71− 75. (6) Uéda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A., Kondo, J., Ihara, Y., and Saitoh, T. (1993) Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 90, 11282−11286. (7) Dedmon, M. M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M., and Dobson, C. M. (2005) Mapping Long-Range Interactions in α-Synuclein using Spin-Label NMR and Ensemble Molecular Dynamics Simulations. J. Am. Chem. Soc. 127, 476−477. (8) Bertoncini, C. W., Jung, Y.-S., Fernandez, C. O., Hoyer, W., Griesinger, C., Jovin, T. M., and Zweckstetter, M. (2005) Release of long-range tertiary interactions potentiates aggregation of natively unstructured α-synuclein. Proc. Natl. Acad. Sci. U. S. A. 102, 1430− 1435. (9) Cohen, F. E., and Kelly, J. W. (2003) Therapeutic approaches to protein-misfolding diseases. Nature 426, 905. (10) Bulawa, C. E., Connelly, S., DeVit, M., Wang, L., Weigel, C., Fleming, J. A., Packman, J., Powers, E. T., Wiseman, R. L., Foss, T. R., et al. (2012) Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade. Proc. Natl. Acad. Sci. U. S. A. 109, 9629−9634. (11) Basak, S., Prasad, G. V. R. K., Varkey, J., and Chattopadhyay, K. (2015) Early Sodium Dodecyl Sulfate Induced Collapse of αSynuclein Correlates with Its Amyloid Formation. ACS Chem. Neurosci. 6, 239−246. (12) Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R., Engemann, S., Pastore, A., and Wanker, E. E. (2008) EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 15, 558−66. (13) Garcia, G. A., Cohen, S. I. A., Dobson, C. M., and Knowles, T. P. J. (2014) Nucleation-conversion-polymerization reactions of biological macromolecules with prenucleation clusters. Phys. Rev. E 89, 032712. (14) Iljina, M., Garcia, G. A., Horrocks, M. H., Tosatto, L., Choi, M. L., Ganzinger, K. A., Abramov, A. Y., Gandhi, S., Wood, N. W., Cremades, N., Dobson, C. M., Knowles, T. P. J., and Klenerman, D. (2016) Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading. Proc. Natl. Acad. Sci. U. S. A. 113, E1206−E1215. (15) Uversky, V. N. (2007) Neuropathology, biochemistry, and biophysics of alpha-synuclein aggregation. J. Neurochem. 103, 17−37. (16) Winner, B., Jappelli, R., Maji, S. K., Desplats, P. A., Boyer, L., Aigner, S., Hetzer, C., Loher, T., Vilar, M., Campioni, S., Tzitzilonis, C., Soragni, A., Jessberger, S., Mira, H., Consiglio, A., Pham, E., Masliah, E., Gage, F. H., and Riek, R. (2011) In vivo demonstration N


Research Article


pathogenesis and treatment of Parkinson’s disease. Biochemistry 40, 7812−9. (50) Zakharov, S. D., Hulleman, J. D., Dutseva, E. A., Antonenko, Y. N., Rochet, J. C., and Cramer, W. A. (2007) Helical alpha-synuclein forms highly conductive ion channels. Biochemistry 46, 14369−79. (51) Kostka, M., Hogen, T., Danzer, K. M., Levin, J., Habeck, M., Wirth, A., Wagner, R., Glabe, C. G., Finger, S., Heinzelmann, U., Garidel, P., Duan, W., Ross, C. A., Kretzschmar, H., and Giese, A. (2008) Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers. J. Biol. Chem. 283, 10992−1003. (52) Celej, M. S., Sarroukh, R., Goormaghtigh, E., Fidelio, G. D., Ruysschaert, J. M., and Raussens, V. (2012) Toxic prefibrillar alphasynuclein amyloid oligomers adopt a distinctive antiparallel beta-sheet structure. Biochem. J. 443, 719−26. (53) Lashuel, H. A., Hartley, D., Petre, B. M., Walz, T., and Lansbury, P. T., Jr (2002) Amyloid pores from pathogenic mutations. Nature 418, 291. (54) Bolognesi, B., Kumita, J. R., Barros, T. P., Esbjorner, E. K., Luheshi, L. M., Crowther, D. C., Wilson, M. R., Dobson, C. M., Favrin, G., and Yerbury, J. J. (2010) ANS binding reveals common features of cytotoxic amyloid species. ACS Chem. Biol. 5, 735−40. (55) Zampagni, M., Cascella, R., Casamenti, F., Grossi, C., Evangelisti, E., Wright, D., Becatti, M., Liguri, G., Mannini, B., Campioni, S., Chiti, F., and Cecchi, C. (2011) A comparison of the biochemical modifications caused by toxic and non-toxic protein oligomers in cells. J. Cell. Mol. Med. 15, 2106−16. (56) Campioni, S., Mannini, B., Zampagni, M., Pensalfini, A., Parrini, C., Evangelisti, E., Relini, A., Stefani, M., Dobson, C. M., Cecchi, C., and Chiti, F. (2010) A causative link between the structure of aberrant protein oligomers and their toxicity. Nat. Chem. Biol. 6, 140− 7. (57) Lamberto, G. R., Binolfi, A., Orcellet, M. L., Bertoncini, C. W., Zweckstetter, M., Griesinger, C., and Fernandez, C. O. (2009) Structural and mechanistic basis behind the inhibitory interaction of PcTS on alpha-synuclein amyloid fibril formation. Proc. Natl. Acad. Sci. U. S. A. 106, 21057−62. (58) Lamberto, G. R., Torres-Monserrat, V., Bertoncini, C. W., Salvatella, X., Zweckstetter, M., Griesinger, C., and Fernández, C. O. (2011) Toward the Discovery of Effective Polycyclic Inhibitors of αSynuclein Amyloid Assembly. J. Biol. Chem. 286, 32036−32044. (59) Ardah, M. T., Paleologou, K. E., Lv, G., Abul Khair, S. B., Kazim, A. S., Minhas, S. T., Al-Tel, T. H., Al-Hayani, A. A., Haque, M. E., Eliezer, D., and El-Agnaf, O. M. (2014) Structure activity relationship of phenolic acid inhibitors of alpha-synuclein fibril formation and toxicity. Front. Aging Neurosci. 6, 197. (60) Oertel, W. H. (2017) Recent advances in treating Parkinson’s disease. F1000Research 6, 260. (61) Lakowicz, J. R. Principles of Fluorescence Spectroscopy., Second ed.; Kluwer Academic/Plenum: New York, 1999. (62) van Rooijen, B. D., Claessens, M. M., and Subramaniam, V. (2009) Lipid bilayer disruption by oligomeric alpha-synuclein depends on bilayer charge and accessibility of the hydrophobic core. Biochim. Biophys. Acta, Biomembr. 1788, 1271−8. (63) van Engeland, M., Nieland, L. J. W., Ramaekers, F. C. S., Schutte, B., and Reutelingsperger, C. P. M. (1998) Annexin V-Affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1−9. (64) Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G. D., and Chen, L. B. (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a Jaggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. U. S. A. 88, 3671−3675. (65) Adler, A. D., Longo, F. R., Finarelli, J. D., Goldmacher, J., Assour, J., and Korsakoff, L. (1967) A simplified synthesis for mesotetraphenylporphine. J. Org. Chem. 32, 476−476. (66) Adler, A. D., Longo, F. R., and Shergalis, W. (1964) Mechanistic Investigations of Porphyrin Syntheses. I. Preliminary Studies on ms-Tetraphenylporphin. J. Am. Chem. Soc. 86, 3145−3149.

Neuroblastoma Cells: Intermediacy Of Transferrin Receptor Iron And Hydrogen Peroxide. J. Biol. Chem. 279, 15240−15247. (34) Vila, M., Vukosavic, S., Jackson-Lewis, V., Neystat, M., Jakowec, M., and Przedborski, S. (2000) α-Synuclein Up-Regulation in Substantia Nigra Dopaminergic Neurons Following Administration of the Parkinsonian Toxin MPTP. J. Neurochem. 74, 721−729. (35) Caughey, W. S., Priola, S. A., Kocisko, D. A., Raymond, L. D., Ward, A., and Caughey, B. (2007) Cyclic tetrapyrrole sulfonation, metals, and oligomerization in antiprion activity. Antimicrob. Agents Chemother. 51, 3887−94. (36) Laia, C. A., and Costa, S. M. (2008) Interaction of zinc tetrasulfonated phthalocyanine with cytochrome C in water and Triton-X 100 micelles. J. Phys. Chem. B 112, 4276−82. (37) Woodburn, K. W., Vardaxis, N. J., Hill, J. S., Kaye, A. H., and Phillips, D. R. (1991) Subcellular localization of porphyrins using confocal laser scanning microscopy. Photochem. Photobiol. 54, 725− 32. (38) Arosio, P., Vendruscolo, M., Dobson, C. M., and Knowles, T. P. (2014) Chemical kinetics for drug discovery to combat protein aggregation diseases. Trends Pharmacol. Sci. 35, 127−35. (39) Banerjee, K., Sinha, M., Pham, C. L. L., Jana, S., Chanda, D., Cappai, R., and Chakrabarti, S. (2010) α-Synuclein induced membrane depolarization and loss of phosphorylation capacity of isolated rat brain mitochondria: Implications in Parkinson’s disease. FEBS Lett. 584, 1571−1576. (40) Arrondo, J. L., Muga, A., Castresana, J., and Goni, F. M. (1993) Quantitative studies of the structure of proteins in solution by Fourier-transform infrared spectroscopy. Prog. Biophys. Mol. Biol. 59, 23−56. (41) Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson, R. E., and Lansbury, P. T., Jr. (2000) Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. U. S. A. 97, 571−6. (42) Giehm, L., Svergun, D. I., Otzen, D. E., and Vestergaard, B. (2011) Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation. Proc. Natl. Acad. Sci. U. S. A. 108, 3246−3251. (43) Chen, S. W., Drakulic, S., Deas, E., Ouberai, M., Aprile, F. A., Arranz, R., Ness, S., Roodveldt, C., Guilliams, T., De-Genst, E. J., Klenerman, D., Wood, N. W., Knowles, T. P. J., Alfonso, C., Rivas, G., Abramov, A. Y., Valpuesta, J. M., Dobson, C. M., and Cremades, N. (2015) Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation. Proc. Natl. Acad. Sci. U. S. A. 112, E1994−E2003. (44) Danzer, K. M., Haasen, D., Karow, A. R., Moussaud, S., Habeck, M., Giese, A., Kretzschmar, H., Hengerer, B., and Kostka, M. (2007) Different species of alpha-synuclein oligomers induce calcium influx and seeding. J. Neurosci. 27, 9220−32. (45) Bertoncini, C. W., Fernandez, C. O., Griesinger, C., Jovin, T. M., and Zweckstetter, M. (2005) Familial mutants of α-synuclein with increased neurotoxicity have a destabilized conformation. J. Biol. Chem. 280, 30649−30652. (46) Buell, A. K., Galvagnion, C., Gaspar, R., Sparr, E., Vendruscolo, M., Knowles, T. P., Linse, S., and Dobson, C. M. (2014) Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation. Proc. Natl. Acad. Sci. U. S. A. 111, 7671−6. (47) Bieschke, J., Russ, J., Friedrich, R. P., Ehrnhoefer, D. E., Wobst, H., Neugebauer, K., and Wanker, E. E. (2010) EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity. Proc. Natl. Acad. Sci. U. S. A. 107, 7710−5. (48) Danzer, K. M., Krebs, S. K., Wolff, M., Birk, G., and Hengerer, B. (2009) Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha-synuclein pathology. J. Neurochem. 111, 192−203. (49) Volles, M. J., Lee, S. J., Rochet, J. C., Shtilerman, M. D., Ding, T. T., Kessler, J. C., and Lansbury, P. T., Jr. (2001) Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the O


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