Ordered and Disordered Segments of Amyloid-Î² Drive Sequential

Subscriber access provided by LUNDS UNIV

Article

Ordered and Disordered Segments of Amyloid# Drive Sequential Steps of the Toxic Pathway Barun Kumar Maity, Anand Kant Das, Simli Dey, Ullhas Kaarthi Moorthi, Amandeep Kaur, Arpan Dey, Dayana Surendran, Rucha Pandit, Mamata Kallianpur, Bappaditya Chandra, Muralidharan Chandrakesan, Senthil Arumugam, and Sudipta Maiti ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00015 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 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

ACS Chemical Neuroscience

Ordered and Disordered Segments of Amyloid-β Drive Sequential Steps of the Toxic Pathway Barun Kumar Maity#1, Anand Kant Das#$1, Simli Dey1, Ullhas Kaarthi Moorthi2, Amandeep Kaur2, Arpan Dey1, Dayana Surendran1, Rucha Pandit$1, Mamata Kallianpur1, Bappaditya Chandra$1, Muralidharan Chandrakesan$1, Senthil Arumugam*2,3and Sudipta Maiti*,1 1Department

of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India. 2EMBL

Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney 2052, Australia 3ARC

Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia

#equal contributions *email: [email protected] or [email protected] ABSTRACT: While the roles of intrinsically disordered protein domains in driving many interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer’s disease (AD) associated amyloid beta (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ~10-40) and a shorter disordered region (residues ~1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aβ fragments and stereo-isomers designed for this purpose. We have measured their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy (FCS). Remarkably, we find that neither part - Aβ10-40 or Aβ1-9, is toxic by itself. The ordered part (Aβ10-40) is the major determinant of how Aβ attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aβ enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) which has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aβ to the toxic 1 ACS Paragon Plus Environment

ACS Chemical Neuroscience 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

pathway. Our findings correlate well with Aβ sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction. KEYWORDS: Intracellular amyloid beta, Amyloid beta fragment, Amyloid beta interactions, Alzheimer’s disease (AD), amyloid-membrane interaction, amyloid enantiomer INTRODUCTION Intrinsically disordered proteins (IDPs) which form amyloid aggregates are associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The disordered regions can potentially provide structural plasticity, conformational adaptability and modulation in their binding to various partners. However, how the disordered domains contribute towards toxicity, if at all, is not properly understood for any of these diseases. Identifying the roles played by the structured and the disordered parts in their interactions with plasma membranes and other cellular organelles, which subsequently leads to pathogenicity, remains a significant challenge. Aβ oligomers, suspected to be the key species responsible for AD1, have been shown to possess distinct ordered and disordered segments2-4, which offers a significant advantage in addressing these questions. The familial AD associated single-point mutations form distinct clusters in both the regions, suggesting that both may be important in determining disease-associated toxicity5. Here we test how each of them contribute to toxicity, whether there are distinct roles for the domains, and how they interact with the cellular membranes and intracellular machinery to bring about the toxic effects. Understanding the ordered and disordered parts of Aβ requires distinguishing its structural features in its distinct aggregated forms. The structural features of the transient oligomers are less well determined than those of the larger, stable, but less toxic, filbrillar aggregates. Mature fibrillar aggregates of Aβ have parallel cross-beta sheet architecture with a turn in the region of residue 256. The N-terminal domain is relatively unstructured7, 8. Solid state NMR (ssNMR) studies show that Aβ1-40 and the N-terminal truncated species, Aβ10-40 form very similar fibril structure9. The structure of the transient oligomeric species is harder to determine, but recent strategies using flash freezing and subsequent ssNMR based examination have yielded some information about the 2 ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33 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


atomic level structure of these toxic species2, 10. Similar regions (spanning residues 10-40) are structured in both the fibrils and the oligomers, but there exists considerable difference at the turn region (residues 22-29) and the N-terminal tail.2 The most striking feature of the oligomer is that the structured region is anti-parallel intra-molecular hydrogen bonded and does not have a cross beta architecture11. Also, a salt bridge between D23 and K28 is present in the fibrils but is absent in the oligomers10. These structural differences between the oligomer and the fibrils in the 10-40 residue region suggest that the specific structure of the ordered region may play a critical role in the enhanced toxicity observed for the oligomers. In comparison to residues 10-40, the N-terminal residues (in some models up to residue 9 or longer12) are less well ordered in the fibrils7, and are even more disordered in the oligomers2, 13. Thus, there is a clear region-specific separation between the ordered and the disordered parts of Aβ. The basic architecture of the structured part is dictated by the 18-35 region, which can fold independently, nucleating the standard architecture of the full-length peptide at least in the fibrillar form14,

15.

The N-terminal disordered part thus does not have substantial influence on the

architecture of the ordered 10-40 part. However, 6 of the known 13 familial AD mutations cluster in this disordered region5, and the N-terminal has become the focus of recent debates about Aβ toxicity16. Naturally occurring fragments of the precursor APP protein which contain a significant part of the N-terminus (e.g. residues 1-16, together with an extension of the N-terminus) tend to display toxicity17. N-terminus directed antibodies reduce plaque burden and restore cognitive deficits in mice model of AD18. There is a structural change at 1-16 region of Aβ when zinc binds to it, and Zn binding is known to modulate Aβ toxicity19. Aβ peptide starting with pyroglutamate at position 3 is a component of the amyloid deposits in the AD brain20 which shows prion like behavior and tau dependent toxicity21. Thus, the N-terminal also plays an important role in toxicity, despite being disordered. Here, we determine the roles played by the ordered and disordered regions at different steps of cellular interaction leading to toxicity. In physiological scenarios, Aβ aggregates are primarily found in the extracellular space of the brain of AD patients22. The initial step of toxicity of an extracellular peptide would likely involve its interaction with the cell membrane. This interaction by itself may cause toxicity, or it may facilitate the entry of Aβ for further localization in the intracellular space. One of the predominant hypotheses related to Aβ mediated toxicity is the disruption of the lipid bilayer membrane23. Aβ 3 ACS Paragon Plus Environment


Page 4 of 33

assemblies cause either generic24 or specific perturbation, leading to the formation of unregulated ion-channel like structures (‘pores’) on the membrane25-30. Electrostatic interactions between the cell membrane and Aβ oligomers are thought to drive pore formation27, which can lead to calcium dis-homeostasis28, synapse alteration, and uncontrolled nitric oxide release.29,

25, 30

Another

hypothesis states that major damage is caused by Aβ localized in critical cellular organelles31-34. There is some evidence that the major location of intracellular Aβ is the endosome-lysosomal system35, 36. There is also some evidence that Aβ binds directly to the DNA and may modulate gene expression37, 38. All these points towards a complex multi-modal action of Aβ that needs further detailed investigations in neuronal systems. In this article, using the N-terminal deleted fragment of the peptide (Aβ10-40, called ‘CTF’), the disordered fragment (Aβ1-9, called ‘NTF’), the full length peptide (Aβ1-40) and a concatenated peptide (Aβ

(1-9)-(18-35)),

we investigate the distinct contributions to toxicity by the ordered and

disordered domains of Aβ. We also use a full-length peptide with alternative D-isomers in the disordered part (AβD/L) to probe the role of any residual or emergent structure in this part. Using this experimental system, we ask which part of Aβ is responsible for its cell membrane interaction, for cell entry, for its localization in the specific organelles, and whether particular intracellular localizations are required for its toxicity. Our experiments suggest a toxicity model based on separable roles of the ordered and disordered segments of Aβ1-40. The N-terminal holds the key to the final toxic step, which likely takes place at the late endosomes, and requires a stereo-specific interaction of the disordered domain with an unknown partner.

RESULTS AND DISCUSSIONS A. Role of the ordered and disordered regions in Aβ1-40 toxicity: To test whether the ordered or the disordered region is sufficient for the toxic effect of the full-length peptide, we have performed cell viability studies on rat primary cortical neuronal cultures. The cells were incubated with the peptide for 60 hrs in cell culture conditions. The ratio of the number of cells marked with propidium iodide (PI) to the cells marked with Hoechst 33342 provides a measurement for the fraction of dead cells. The data is expressed as viability relative to the vehicle treated control cells 4 ACS Paragon Plus Environment

Page 5 of 33 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


(Fig. 1), where the viability of control cells is assumed to be 100 % (actual viability in control cells: 68 ± 3 %). We find that oligomers of the CTF are almost non-toxic to the cells (viability: 93 ± 3 %), at a concentration where Aβ1-40 exposed cells for same duration is rather toxic (39 ± 5 %) (Fig. 1). The dose response curve for these peptides also confirms that even at higher concentrations, CTF is non-toxic, while the full-length peptide exerts its toxicity in a dose dependent manner (Fig. S1). This study indicates that though the ordered region is structurally similar to the full length, it is still non-toxic in nature. Also, NTF is itself non-toxic (cell viability: 97 ± 3 %). The peptide, Aβ(1-9)-(18-35) which has the N-terminal attached to the core structured region of the full length peptide, is also non-toxic. Further, we ask if the ability of the disordered residues 1-9 to adopt specific conformations, perhaps in association with other Aβ monomers or with a different intracellular component, is important for triggering downstream toxic events. To probe this, we prepare Aβ with alternate D/L amino acids at positions 1-9 (i.e. D amino acids at positions 2, 4, 6, and 8), such that any stereospecific interaction with the disordered part is disrupted. We find that AβD/L is much less toxic (cell viability: 74 ± 3 %) compared to full-length Aβ1-40 (cell viability: 38 ± 4 %) (Fig. 1). This suggests that the 1-9 part is involved in some intermolecular interactions, which has at least some degree of stereo-specificity. This is in partial agreement with earlier reports that full-D enantiomers of Aβ can be toxic.39 It is important to consider whether the N-terminal can be altered independently, or do such perturbations couple to the structure or aggregation properties of the ordered part of Aβ. We know that the structures of the Aβ10-40 and Aβ18-35 fibrils are similar to the same region of the full length peptide9, 14. We also know that residues 10-40 of the oligomers of the full length peptide are structured while residues 1-9 are unstructured2. So it is reasonable to assume that perturbations in the unstructured region, either when it is a part of the full length peptide (in the case of AβD/L), or when it is concatenated to the core region (Aβ(1-9)-(18-35)) would not majorly disturb the structure of the structured region. We test the aggregation properties by measuring the hydrodynamic radius of the small oligomers, using fluorescence correlation spectroscopy. The results (Fig. S2) show that the hydrodynamic radii of the oligomers are nearly identical for the wild type and all the Nterminal altered peptides. Therefore, alterations of the N-terminal do not substantially affect the structure or aggregation properties of the ordered part. We note that this inference is based mostly on results obtained in vitro, and the properties of the peptide may be different in special intracellular environments. 5 ACS Paragon Plus Environment


Our results, taken together, show that the N-terminal (residues 1-9) is essential for the full-length peptide to be toxic, but it cannot execute some of the essential steps of the toxic pathway without the help of the ordered segment. Also, the final toxic step is at least partially dependent on some conformational features of the disordered segment. We now proceed to dissect the sequential step involved in toxicity.

B. Role of ordered and disordered regions of Aβ1-40 in membrane affinity: In order to be toxic, an extracellular protein/peptide has to first interact with the cell membrane. To understand the membrane interaction of the NTF and the CTF oligomers, we labeled both Aβ1-40 and CTF with Rhodamine-B at the N-terminus and quantified their affinity for small unilamellar vesicles (SUVs with diameter of 25-40 nm), which serve as well-characterized mimics for the cell membrane. We use a quantitative assay developed in-house, based on Fluorescence Correlation Spectroscopy (FCS), which has been described elsewhere40. Briefly, FCS monitors the timescale of diffusion through a small region41, and if the fluorescently-labeled Aβ (or fragment) oligomers attach to the much larger sized SUV’s, their diffusion slows down. A comparison of the slow and fast diffusing species can therefore yield the degree of SUV binding by Aβ. The SUVs, composed of POPC, POPG and cholesterol in 1:1:1 molar ratio, are incubated with 100 nM Rhodamine-B labeled Aβ1-40 (RAβ1-40) and CTF (RAβ10-40) oligomers for 30 min. Then, these solutions are subjected to FCS measurements. Our results show that both have similar affinity to SUVs (fraction bound for Aβ140:

37 ± 6 %, and CTF: 47 ± 5 %) (Fig.-2). We note that the property of Rhodamine-B labeled

Aβ1-40 is mainly dictated by the peptide, not by the labeling dye, since Rhodamine-B labeled Aβ140

monomers do not attach to cell membranes while the oligomers do so strongly42. Also,

single/double mutations at F19 and L34 positions of Aβ1-40 abolish membrane affinity, as estimated using Rhodamine-B labeled peptides43. This shows that the structure of the ordered part of the peptide, and not the labeling dye, plays the defining role in membrane interaction. Hence, our result suggests that the disordered region does not play a crucial role for attachment to the lipidic component of cell membranes. We then test their membrane affinity with primary cortical neuron cells using confocal imaging. In this case, though exact quantitative comparison is not possible due to unwanted 6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33 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


contribution from cellular auto-fluorescence, we can measure the relative extent of attachment from the change in brightness of the cell images. Rat primary cortical neurons are incubated with modified Thomson’s buffer (TB) containing 250 nM oligomeric solution of Rhodamine-B labeled peptide at room temperature for 30 min. The degree of binding of the peptide to the cell membrane is assessed by analyzing the change in the brightness of the membrane during the incubation period as measured in a confocal microscope. Fig. 3 shows the membrane regions of the cells, both at the initial time and after 30 min of incubation with the oligomers. Fig. 3A and 3E show a vehicle treated control set of cells at 0 and 30 min respectively, where the mean intensity changes by a factor of 1.0 ± 0.1. We find that Aβ1-40 (Fig. 3B, 0 min; Fig. 3F, 30 min) binds to a somewhat higher extent than CTF (Fig. 3C, 0 min; Fig. 3G, 30 min), as the mean fluorescence intensity increases by a factor of 3.5 ± 0.7 for Aβ1-40, and by 2.8 ± 0.2 for CTF. When the cells are incubated with the oligomers for 24 hrs, we find that the brightness within the cells incubated with Aβ1-40 is ~1.7 fold higher than those incubated with CTF (Fig. S3, 4). However, NTF does not bind to the cell membrane (Fig. 3D, 0 min; Fig. 3H, 30 min). It suggests that the residues 1-9 can modulate cell membrane binding and cellular entry, but the dominant role is played by the ordered CTF. Also, these results suggest that the lack of toxicity of NTF may stem from its inability to interact with and penetrate the cell membrane, and it needs the ordered part at least as a carrier for its cellular interactions. C. Role of ordered and disordered regions in intracellular localization: While membrane interaction may itself lead to toxicity by causing disruptions or by disturbing the ionic homeostasis there26, Aβ is also known to enter the cell and this may be important in causing toxicity. We therefore examine the cellular entry and localization of Aβ post membrane attachment. Though the ordered CTF region is mainly responsible for membrane affinity, it is possible that without the NTF, it may get trafficked to a different cellular location. Here, we test the degree of colocalization of the full length Aβ and the CTF using two color confocal imaging of primarycultured neurons from the rat hippocampus. The NTF is not probed in these experiments, as it does not even enter the cells. The cells are incubated with 100 nM Rhodamine-110 labeled Aβ1-40 (Rh110-Aβ1-40) and Rhodamine-B labeled Aβ10-40 (RAβ10-40) for 24 hrs in culture media at 37 ⁰C. After washing with TB, the cells are subjected to confocal imaging. The images obtained from two channels (Fig. 4B, Rh110-Aβ1-40; Fig. 4C: RAβ10-40) clearly shows that both Aβ1-40 and CTF enter into cells and localize in similar punctate organelles. We have quantified the extent of co7 ACS Paragon Plus Environment


localization by spectrally de-convoluting the images and then calculating the overlap coefficient between the two images using a program written in Matlab. We find that the overlap coefficient is 0.72 ± 0.03. We also observed similar results for the cell line of raphe origin (RN46A)44 (Overlap coefficient: 0.61 ± 0.05, Fig.-S5). Our results suggest that the deletion of the disordered N-terminal does not substantially affect the ability of the ordered region to localize in specific cellular organelles. To further test whether the labeling dyes may have an influence over the intra-cellular localization of these peptides, we have followed the co-localization of Aβ1-40 labeled with Rhodamine-110 and Rhodamine-B respectively, in RN46A cell line. We find that they have similar localization inside cells (Fig.-S6) indicating that the dye does not affect localization. D. Probing the identity of the Aβ-associated cellular organelles and their dynamics using lattice light sheet microscopy: Previous studies have indicated that cellular uptake of Aβ1-40 and CTF occurs via clathrin and dynamin independent endocytosis45. Typically, endocytosis results in membranous vesicles that mature through various compartments. Consistent with this, Aβ has been shown to accumulate within lysosomes, subsequently promoting neuronal death through lysosomal destabilization34, 46, 47. We therefore, hypothesize that the punctate structures observed in our experiments are Aβ1-40 and CTF localized in endosomes. Another hypothesis pertaining to lysosomes and aggregates is that lysosomal activation results in enlargement of aggregate containing lysosomes to clear them in quiescent neural stem cells48. To test these hypotheses, we followed the colocalization of Aβ1-40 and CTF with Rab7, a late endosomal marker. To further examine if Aβ1-40 or CTF result in dysfunction of lysosomes leading to pathogenicity, we measured the motilities of endosomes bearing Aβ1-40 and CTF using lattice light-sheet microscopy (LLSM)49. LLSM allowed precise quantification of motilities in the 3-dimensional volume of the cell as well as the absolute number of vesicles in the entire volume of the cell as a function of time (see Movie 1, SI). Directly correlating trajectories, independently acquired for Rab7 and Aβ1-40 or CTF using multi-channel imaging, allowed unbiased quantification of dynamic co-localization as well as motility characteristics in Rab7 endosomes bearing either Aβ1-40 or CTF. We found that the normalized fraction of Aβ1-40 or CTF positive structures that co-tracked with Rab7 were larger for CTF (0.68 ± 0.09, Mean ± SD, n = 15 cells, > 1500 tracks) compared to Aβ1-40 (0.54 ± 0.10, Mean ± SD, n = 15 cells, > 1500 tracks, difference significant at p 2 = 4𝐷𝑡α +2σ2 ……………….. (1) where D is the diffusion coefficient, t is the lag-time and α, the scaling exponent, σ is the measurement error measured on the LLSM using beads adsorbed onto glass, fixed as 35 nm for the fits. The trajectories were characterized according to the scaling exponent α, retrieved from the fits. Fluorescence lifetime imaging. The RN46A cells were incubated with RAβ1-40 and RAβ10-40 oligomers for 3 hrs in the culture media at 37°C. After washing with TB, the cells were subjected to FLIM experiments. The experiment was performed in a commercial confocal microscope based set up (LSM-710, Carl Zeiss, Germany). Briefly, 530 ± 22 nm laser light obtained from OYSL supercontinuum white light laser source after filtering with a band pass filter (Semrock 530/43 band pass filter) was used as the excitation source. The repetition rate was 10 MHz. The beam was routed through the IR laser path in the scan box. An 80/20 beam-splitter, which allows only 20% of the excitation light to follow the excitation path and a mirror right below the objective were used to guide the light along the excitation path. It is then focused into the sample volume by a 40X water immersion objective of NA = 1.2. The fluorescence was collected by using the same objective lens and routed outside the scan box, focused by a 5 cm convex lens to an optical fiber of core diameter 50 µm after filtering the excitation light using a suitable filter. The other side of the fiber was coupled to a detector (SPAD, Micro Photon Devices, Italy), which sent the signal to SPC150 TCSPC card. For fluorescence lifetime imaging (FLIM), the fluorescence was collected by scanning a sample in XY plane, which was achieved by pixel-wise raster scanning of the galvanic mirrors of the confocal microscope. Time correlated single photon counting (TCSPC) data is collected from each individual pixel, and analyzed for the fluorescence lifetime to produce the lifetime image. Therefore, it required synchronization between the TCSPC acquisition and the pixel clocks of the scanning mirror, which was achieved through a cable connecting the laser scanning microscope (LSM710) with SPC150 TCSPC data acquisition card. SPCImage software was used for data processing. Only the pixels occupying the cellular regions were accounted to obtain the cumulative distribution of fluorescence lifetimes. Now, to account the changes in quantum efficiency, we adjusted the fluorescence intensity by the fluorescence lifetime at that pixel to acquire the knowledge about the relative of population at different locations of the cells.

18 ACS Paragon Plus Environment

Page 18 of 33

Page 19 of 33 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


This parameter from the pixels occupying the cellular regions was employed to derive the population distribution as a function of fluorescence lifetimes. Fluorescence quenching studies of RAβ1-40 and RAβ10-40 oligomers by tryptophan in solution. The stock solutions of both RAβ1-40 and RAβ10-40 of 100 µM at pH 11 were diluted by 1000 folds with TB to prepare the oligomer solutions at pH 7.4. Required amount of tryptophan solution from stock solution of 54 mM was added to it to maintain its different concentrations, and used for fluorescence lifetime measurements. These experiments were performed in the same instrumental set up in point mode, where the instrument response function (IRF) is ~160 ps. The lifetime traces were fitted with two components decay using FluoFit software. The fitted parameters were accounted to obtain the average lifetimes at different concentrations of tryptophan. The dynamic quenching constant was derived from these average lifetimes using Stern-Volmer relationship (Eqn. 2). 𝜏0 𝜏

= 1 + 𝐾𝑞 𝜏0 [𝑄]

……………………. (2)

Where 𝜏0, 𝜏 is the fluorescence lifetime in absence and presence of the quencher, tryptophan respectively, Kq is dynamic quenching constant and [Q] is the concentration of the quencher. ASSOCIATED CONTENT Supporting information Dose dependent toxicity studies in presence of Aβ1-40 and its fragments; Size of oligomers of different peptides; Cellular peptide uptake by RN46A cells; Spatial localization of Aβ1-40 and CTF in RN46A cells; Spatial localization of Aβ1-40, labeled with Rhodamine-110 and Rhodamine-B respectively, in RN46A cells; Quantitation of run lengths of the Aβ1-40 or CTF bearing vesicles positive for Rab7; Description of the movie showing the tracks of Aβ1-40 obtained from LLSM experiments; Analysis of FLIM results from oligomers in RN46A cells. AUTHOR INFORMATIONS Corresponding author *E-mail: [email protected], [email protected] $ Current

address 19 ACS Paragon Plus Environment


AKD: Vienna University of Technology, Institute for Applied Physics - Biophysics group, Getreidemarkt 9, 1060 Vienna, Austria RP: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St Lucia QLD 4072, Australia BC: St. Jude Children’s Research Hospital, Memphis, TN 38105, USA MC: USV private Ltd, Mumbai 400008, India Author contributions BKM performed vesicle binding, co-localization and quenching experiments; AKD performed toxicity and cell attachment experiments; BKM, MK and AD performed fluorescence life-time imaging (FLIM) experiments; SD helped for FLIM experiment; UK and AM did lattice light sheet imaging (LLSI) experiments; BC, MC and RP synthesized the peptides; SA conceptualized the LLSI experiments; SM conceptualized the other experiments; BKM, AKD, SA and SM co-wrote the manuscript. Notes The authors declare no competing financial interest.

Abbreviations AD, Alzheimer’s disease; Aβ, amyloid β; FCS, fluorescence correlation spectroscopy; RH, hydrodynamic radius; PBS, phosphate buffered saline; TB, Thomson’s buffer; CTF, C-terminal fragment (Aβ10-40); NTF, N-terminal fragment (Aβ1-9); RAβ1-40, Aβ1-40 labelled with RhodamineB at the N-terminal; RAβ10-40 , Aβ10-40 labelled with Rhodamine-B at the N-terminal; RAβ(1-9)-(1835),

Aβ(1-9)-(18-35) labelled with Rhodamine-B at the N-terminal; CRAβ1-40, Aβ1-40 labelled with

Rhodamine-B at the C-terminal; PI, propidium iodide. Acknowledgement We acknowledge the help of Prof. Trevor Smith and Dr. Hamid Soleimaninejad for collecting preliminary lifetime imaging data. REFERENCES


Page 20 of 33

Page 21 of 33 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


[1] Benilova, I., Karran, E., and De Strooper, B. (2012) The toxic Abeta oligomer and Alzheimer's disease: an emperor in need of clothes, Nat Neurosci 15, 349-357. [2] Sarkar, B., Mithu, V. S., Chandra, B., Mandal, A., Chandrakesan, M., Bhowmik, D., Madhu, P. K., and Maiti, S. (2014) Significant Structural Differences between Transient Amyloid-beta Oligomers and Less-Toxic Fibrils in Regions Known To Harbor Familial Alzheimer's Mutations, Angew Chem Int Ed Engl 53, 6888-6892. [3] Fawzi, N. L., Ying, J. F., Ghirlando, R., Torchia, D. A., and Clore, G. M. (2011) Atomic-resolution dynamics on the surface of amyloid-beta protofibrils probed by solution NMR, Nature 480, 268-U161. [4] Fawzi, N. L., Ying, J. F., Torchia, D. A., and Clore, G. M. (2010) Kinetics of Amyloid beta Monomer-toOligomer Exchange by NMR Relaxation, Journal of the American Chemical Society 132, 9948-9951. [5] Hatami, A., Monjazeb, S., Milton, S., and Glabe, C. G. (2017) Familial Alzheimer's Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloidbeta Peptide, J Biol Chem 292, 3172-3185. [6] Paravastu, A. K., Qahwash, I., Leapman, R. D., Meredith, S. C., and Tycko, R. (2009) Seeded growth of beta-amyloid fibrils from Alzheimer's brain-derived fibrils produces a distinct fibril structure, Proc Natl Acad Sci U S A 106, 7443-7448. [7] Petkova, A. T., Ishii, Y., Balbach, J. J., Antzutkin, O. N., Leapman, R. D., Delaglio, F., and Tycko, R. (2002) A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR, Proc Natl Acad Sci U S A 99, 16742-16747. [8] Bertini, I., Gonnelli, L., Luchinat, C., Mao, J., and Nesi, A. (2011) A new structural model of Abeta40 fibrils, J Am Chem Soc 133, 16013-16022. [9] Paravastu, A. K., Petkova, A. T., and Tycko, R. (2006) Polymorphic fibril formation by residues 10-40 of the Alzheimer's beta-amyloid peptide, Biophys J 90, 4618-4629. [10] Chandra, B., Bhowmik, D., Maity, B. K., Mote, K. R., Dhara, D., Venkatramani, R., Maiti, S., and Madhu, P. K. (2017) Major Reaction Coordinates Linking Transient Amyloid-beta Oligomers to Fibrils Measured at Atomic Level, Biophys J 113, 805-816. [11] Bhowmik, D., Mote, K. R., MacLaughlin, C. M., Biswas, N., Chandra, B., Basu, J. K., Walker, G. C., Madhu, P. K., and Maiti, S. (2015) Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation, Acs Nano 9, 9070-9077. [12] Tompa, P. (2009) Structural disorder in amyloid fibrils: its implication in dynamic interactions of proteins, FEBS J 276, 5406-5415. [13] Tarus, B., Tran, T. T., Nasica-Labouze, J., Sterpone, F., Nguyen, P. H., and Derreumaux, P. (2015) Structures of the Alzheimer's Wild-Type Abeta1-40 Dimer from Atomistic Simulations, J Phys Chem B 119, 10478-10487. [14] Chandrakesan, M., Sarkar, B., Mithu, V. S., Abhyankar, R., Bhowmik, D., Nag, S., Sahoo, B., Shah, R., Gurav, S., Banerjee, R., Dandekar, S., Jose, J. C., Sengupta, N., Madhu, P. K., and Maiti, S. (2013) The basic structural motif and major biophysical properties of Amyloid-β are encoded in the fragment 18-35, Chemical Physics 422, 80-87. [15] Spencer, R. K., Li, H., and Nowick, J. S. (2014) X-ray crystallographic structures of trimers and higherorder oligomeric assemblies of a peptide derived from Abeta(17-36), J Am Chem Soc 136, 55955598. [16] Murray, B., Sharma, B., and Belfort, G. (2017) N-Terminal Hypothesis for Alzheimer's Disease, ACS Chem Neurosci 8, 432-434. [17] Willem, M., Tahirovic, S., Busche, M. A., Ovsepian, S. V., Chafai, M., Kootar, S., Hornburg, D., Evans, L. D., Moore, S., Daria, A., Hampel, H., Muller, V., Giudici, C., Nuscher, B., Wenninger-Weinzierl, A., Kremmer, E., Heneka, M. T., Thal, D. R., Giedraitis, V., Lannfelt, L., Muller, U., Livesey, F. J., 21 ACS Paragon Plus Environment


Meissner, F., Herms, J., Konnerth, A., Marie, H., and Haass, C. (2015) eta-Secretase processing of APP inhibits neuronal activity in the hippocampus, Nature 526, 443-447. [18] McLaurin, J., Cecal, R., Kierstead, M. E., Tian, X., Phinney, A. L., Manea, M., French, J. E., Lambermon, M. H., Darabie, A. A., Brown, M. E., Janus, C., Chishti, M. A., Horne, P., Westaway, D., Fraser, P. E., Mount, H. T., Przybylski, M., and St George-Hyslop, P. (2002) Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis, Nat Med 8, 1263-1269. [19] Zirah, S., Kozin, S. A., Mazur, A. K., Blond, A., Cheminant, M., Segalas-Milazzo, I., Debey, P., and Rebuffat, S. (2006) Structural changes of region 1-16 of the Alzheimer disease amyloid betapeptide upon zinc binding and in vitro aging, J Biol Chem 281, 2151-2161. [20] Harigaya, Y., Saido, T. C., Eckman, C. B., Prada, C. M., Shoji, M., and Younkin, S. G. (2000) Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain, Biochemical and biophysical research communications 276, 422-427. [21] Nussbaum, J. M., Schilling, S., Cynis, H., Silva, A., Swanson, E., Wangsanut, T., Tayler, K., Wiltgen, B., Hatami, A., Ronicke, R., Reymann, K., Hutter-Paier, B., Alexandru, A., Jagla, W., Graubner, S., Glabe, C. G., Demuth, H. U., and Bloom, G. S. (2012) Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-beta, Nature 485, 651-655. [22] Hardy, J., and Allsop, D. (1991) Amyloid deposition as the central event in the aetiology of Alzheimer's disease, Trends Pharmacol Sci 12, 383-388. [23] Kotler, S. A., Walsh, P., Brender, J. R., and Ramamoorthy, A. (2014) Differences between amyloid-beta aggregation in solution and on the membrane: insights into elucidation of the mechanistic details of Alzheimer's disease, Chem Soc Rev 43, 6692-6700. [24] Yip, C. M., and McLaurin, J. (2001) Amyloid-beta peptide assembly: a critical step in fibrillogenesis and membrane disruption, Biophys J 80, 1359-1371. [25] Quist, A., Doudevski, I., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J., and Lal, R. (2005) Amyloid ion channels: a common structural link for protein-misfolding disease, Proc Natl Acad Sci U S A 102, 10427-10432. [26] Lal, R., Lin, H., and Quist, A. P. (2007) Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm, Biochim Biophys Acta 1768, 1966-1975. [27] Del Mar Martinez-Senac, M., Villalain, J., and Gomez-Fernandez, J. C. (1999) Structure of the Alzheimer beta-amyloid peptide (25-35) and its interaction with negatively charged phospholipid vesicles, Eur J Biochem 265, 744-753. [28] Kawahara, M., and Kuroda, Y. (2000) Molecular mechanism of neurodegeneration induced by Alzheimer's beta-amyloid protein: channel formation and disruption of calcium homeostasis, Brain Res Bull 53, 389-397. [29] Muehlhauser, F., Liebl, U., Kuehl, S., Walter, S., Bertsch, T., and Fassbender, K. (2001) AggregationDependent interaction of the Alzheimer's beta-amyloid and microglia, Clin Chem Lab Med 39, 313316. [30] Jang, H., Arce, F. T., Ramachandran, S., Capone, R., Lal, R., and Nussinov, R. (2010) beta-Barrel topology of Alzheimer's beta-amyloid ion channels, J Mol Biol 404, 917-934. [31] Wang, X., Su, B., Perry, G., Smith, M. A., and Zhu, X. (2007) Insights into amyloid-beta-induced mitochondrial dysfunction in Alzheimer disease, Free Radic Biol Med 43, 1569-1573. [32] Unterberger, U., Hoftberger, R., Gelpi, E., Flicker, H., Budka, H., and Voigtlander, T. (2006) Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo, J Neuropathol Exp Neurol 65, 348-357. [33] Joshi, G., Bekier, M. E., 2nd, and Wang, Y. (2015) Golgi fragmentation in Alzheimer's disease, Front Neurosci 9, 340. 22 ACS Paragon Plus Environment

Page 22 of 33

Page 23 of 33 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


[34] Ditaranto, K., Tekirian, T. L., and Yang, A. J. (2001) Lysosomal membrane damage in soluble Abetamediated cell death in Alzheimer's disease, Neurobiol Dis 8, 19-31. [35] Li, J., Kanekiyo, T., Shinohara, M., Zhang, Y., LaDu, M. J., Xu, H., and Bu, G. (2012) Differential regulation of amyloid-beta endocytic trafficking and lysosomal degradation by apolipoprotein E isoforms, J Biol Chem 287, 44593-44601. [36] Cataldo, A. M., Petanceska, S., Terio, N. B., Peterhoff, C. M., Durham, R., Mercken, M., Mehta, P. D., Buxbaum, J., Haroutunian, V., and Nixon, R. A. (2004) Abeta localization in abnormal endosomes: association with earliest Abeta elevations in AD and Down syndrome, Neurobiol Aging 25, 12631272. [37] Maloney, B., and Lahiri, D. K. (2011) The Alzheimer's amyloid beta-peptide (A beta) binds a specific DNA A beta-interacting domain (A beta ID) in the APP, BACE1, and APOE promoters in a sequencespecific manner: Characterizing a new regulatory motif, Gene 488, 1-12. [38] Gezen-Ak, D., Atasoy, I. L., Candas, E., Alaylioglu, M., and Dursun, E. (2018) The Transcriptional Regulatory Properties of Amyloid Beta 1-42 may Include Regulation of Genes Related to Neurodegeneration, Neuromol Med 20, 363-375. [39] Cribbs, D. H., Pike, C. J., Weinstein, S. L., Velazquez, P., and Cotman, C. W. (1997) All-D-enantiomers of beta-amyloid exhibit similar biological properties to all-L-beta-amyloids, J Biol Chem 272, 74317436. [40] Bhowmik, D., Das, A. K., and Maiti, S. (2014) Rapid, Cell-Free Assay for Membrane-Active Forms of Amyloid-beta, Langmuir. [41] Maiti, S., Haupts, U., and Webb, W. W. (1997) Fluorescence correlation spectroscopy: diagnostics for sparse molecules, Proc Natl Acad Sci U S A 94, 11753-11757. [42] Sarkar, B., Das, A. K., and Maiti, S. (2013) Thermodynamically stable amyloid-beta monomers have much lower membrane affinity than the small oligomers, Front Physiol 4, 84. [43] Das, A. K., Rawat, A., Bhowmik, D., Pandit, R., Huster, D., and Maiti, S. (2015) An early folding contact between Phe19 and Leu34 is critical for amyloid-beta oligomer toxicity, ACS Chem Neurosci 6, 1290-1295. [44] White, L. A., Eaton, M. J., Castro, M. C., Klose, K. J., Globus, M. Y., Shaw, G., and Whittemore, S. R. (1994) Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons, J Neurosci 14, 6744-6753. [45] Wesen, E., Jeffries, G. D. M., Matson Dzebo, M., and Esbjorner, E. K. (2017) Endocytic uptake of monomeric amyloid-beta peptides is clathrin- and dynamin-independent and results in selective accumulation of Abeta(1-42) compared to Abeta(1-40), Sci Rep 7, 2021. [46] Nixon, R. A., Cataldo, A. M., and Mathews, P. M. (2000) The endosomal-lysosomal system of neurons in Alzheimer's disease pathogenesis: a review, Neurochem Res 25, 1161-1172. [47] Yang, A. J., Chandswangbhuvana, D., Margol, L., and Glabe, C. G. (1998) Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis, J Neurosci Res 52, 691-698. [48] Leeman, D. S., Hebestreit, K., Ruetz, T., Webb, A. E., McKay, A., Pollina, E. A., Dulken, B. W., Zhao, X., Yeo, R. W., Ho, T. T., Mahmoudi, S., Devarajan, K., Passegue, E., Rando, T. A., Frydman, J., and Brunet, A. (2018) Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging, Science 359, 1277-1283. [49] Chen, B. C., Legant, W. R., Wang, K., Shao, L., Milkie, D. E., Davidson, M. W., Janetopoulos, C., Wu, X. S., Hammer, J. A., 3rd, Liu, Z., English, B. P., Mimori-Kiyosue, Y., Romero, D. P., Ritter, A. T., Lippincott-Schwartz, J., Fritz-Laylin, L., Mullins, R. D., Mitchell, D. M., Bembenek, J. N., Reymann, A. C., Bohme, R., Grill, S. W., Wang, J. T., Seydoux, G., Tulu, U. S., Kiehart, D. P., and Betzig, E. (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution, Science 346, 1257998. 23 ACS Paragon Plus Environment


[50] Zajac, A. L., Goldman, Y. E., Holzbaur, E. L., and Ostap, E. M. (2013) Local cytoskeletal and organelle interactions impact molecular-motor- driven early endosomal trafficking, Curr Biol 23, 1173-1180. [51] Mithu, V. S., Sarkar, B., Bhowmik, D., Chandrakesan, M., Maiti, S., and Madhu, P. K. (2011) Zn(++) binding disrupts the Asp(23)-Lys(28) salt bridge without altering the hairpin-shaped cross-beta Structure of Abeta(42) amyloid aggregates, Biophys J 101, 2825-2832. [52] Sengupta, P., Balaji, J., and Maiti, S. (2002) Measuring diffusion in cell membranes by fluorescence correlation spectroscopy, Methods 27, 374-387. [53] Culbertson, C. T., Jacobson, S. C., and Michael Ramsey, J. (2002) Diffusion coefficient measurements in microfluidic devices, Talanta 56, 365-373.

Figures


Page 24 of 33

Page 25 of 33 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


Fig.-1: Cell viability assay in presence of Aβ and its variants. The bar graphs denote the effect on viability when rat primary cortical neurons are exposed to 100 μM of Aβ1-40, CTF, NTF, Aβ(19)-(18-35), AβD/L for 60 hours. The cell viability was assessed by 0.01 mg/ml Hoechst 33342 and 0.01mg/ml PI staining. Cell viability is expressed as percentage relative to control assuming 100 % cell viability for control cells. The values are mean ± SEM. Single asterisk: p < 0.01, double: p< 0.001. The comparisons are with respect to the control.



Fig.-2: Vesicle binding measurement of RAβ40 and CTF using FCS. Normalized autocorrelation function as a function of delay time for 100 nM RAβ40 (black), CTF (green) oligomers in PBS; 100 nM RAβ40 (pink) and CTF (blue) oligomers after incubation with SUVs for 30 min. The triangular and encircled traces are experimentally determined, while the bold traces are fitted. Lower panel denotes the residual of fitting of above mentioned traces. Color remains consistent with the samples. (Inset) The bar graphs represent binding percentage of RAβ40 and CTF with SUVs. Values represent mean ± SEM. The difference is not statistically significant (p = 0.24).

Fig.-3: Binding studies of RAβ1-40, RAβ10-40 and RAβ1-9 oligomers with primary cortical neurons. (A)-(D): Confocal sections of 4 set of cells (excitation 543 nm, emission 550-700nm). 26 ACS Paragon Plus Environment

Page 26 of 33

Page 27 of 33 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


(E)-(H): Corresponding sets of cells after 30 min of incubation with TB (E), 250 nM oligomeric RAβ1-40 (F), 250 nM oligomeric RAβ10-40(G), 250 nM oligomeric RAβ1-9(H).The intensity is false coded. Scale bar: 10 μm.



Fig.-4: Co-localization studies in primary neuronal cultures incubated with 100 nM Rh110Aβ1-40 and 100 nM RAβ10-40/100 nM RAβ(1-9)-(18-35) for 24hrs. (A), (E): the transmission images of two set of cells, (B), (F): confocal images for Rh110-Aβ1-40 (excitation 488 nm, emission 500535 nm) corresponding to the transmission images (A) and (E) respectively, (C): for RAβ10-40 (excitation 543 nm, emission 550-680 nm) corresponding to the transmission images (A); (G): for RAβ(1-9)-(18-35) (excitation 543 nm, emission 550-680 nm) corresponding to the transmission images (E), (D): co-localized image of (B) and (C); (H): co-localized image of (F) and (G). Scale bar: 10 µm.


Page 28 of 33

Page 29 of 33 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


Fig.-5: Measuring the dynamics of Aβ1-40 and CTF (Aβ10-40) using 2- color Lattice Light Sheet Microscopy. (A) Schematic and analytical workflow. Rapid 2-colour volumetric movies were obtained and the endosomes were tracked using Imaris. The trajectories were subjected to categorization by their co-tracking with Rab7 or independent of Rab7. These trajectories were then analyzed using MSD analysis fit to further categorize distinct mobilities defined by the scaling exponent in the fit. (B) Fraction of all Aβ tracks co-travelling with Rab7. Using ANOVA, the fratio value is 14.99. The p-value is .000592. The result is significant at p < .01. (C) MSD based mobility analysis of Aβ1-40 and CTF that co-travel with Rab7 as well as independent of Rab7. Asterisks represents significance (*, p

Ordered and Disordered Segments of Amyloid-Î² Drive Sequential

Recommend Documents