Detection of α-syn pores.

<p><b>A</b>) Pore formation results in increased current-flows over the membrane (black trace) compared to intact bilayers (grey trace) when a voltage ramp is applied. <b>B</b>) Pore detection rate for oligomers obtained with 2.1 µM α-syn with 1% DMSO and 20 µM Fe³⁺ at RT. 4 and 24 h of incubation did not lead to any pore detections (N = 8, each). In 33% of all 48 h-samples (N = 9) pore formations could be detected, increasing to a maximum at 72 h (N = 10). Further incubation results in a decrease of pore detection (N = 10). Non-incubated α-syn monomers did not lead to pore detections (N = 4), as well as DMSO and Fe³⁺ (“buffer”) after all tested incubation times (N = 4, each). α-hemolysin was used as a positive control (α-HL; N = 4). <b>C</b>) In a further set of experiments, the effects of different α-syn concentrations and the effect of the aggregation inhibitor baicalein were investigated after incubation of α-syn for 72 h with DMSO and Fe³⁺. Shown is the probability of pore detection following sequential application of aliquots of the respective samples to the bilayer. α-syn was used at 7.0 µM (N = 69), 2.1 µM (N = 35) and 0.7 µM (N = 8) with up to 9 aliquots applied per sample. With decreasing α-syn concentration, cumulative pore detection rate decreases significantly (p<0.001). Co-incubation of 2.1 µM of α-syn with 50 µM of baicalein (N = 8) significantly decreases pore detection compared to 2.1 µM control condition (p<0.005). <b>D</b>) When different voltages were clamped to membranes with inserted pores, step-like changes in conductivity were consistently observed during the duration of the voltage-pulse.</p

Schematic illustration of different models for increased membrane permeability caused by α-syn oligomers.

<p>In principle, α-syn oligomers could cause increased conductance of lipid membranes by different modes of action. <b>A</b>) A diffuse damage of the bilayer could lead to an unspecific increase in transmembrane current flow. <b>B</b>) Distinct pores could be formed in the bilayer that switch between two or more different conformational states, resulting in corresponding changes in conductivity. <b>C</b>) Different numbers of uniform pores could spontaneously insert and de-insert into the membrane leading to step-like changes in conductivity. <b>D</b>) The number of “open” pores could fluctuate due to open and closure events of permanently inserted pore complexes.</p

Development and Implementation of a High-Throughput Compound Screening Assay for Targeting Disrupted ER Calcium Homeostasis in Alzheimer's Disease

<div><p>Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.</p></div

Active structures identified from primary HTS screen.

<p>Shown are 53 small molecules identified from the primary screen with their chemical structure and the corresponding normalized mean ER calcium response ± standard deviation values generated at 10 µM, as a measure for their activity.</p

Validation of primary hits, preliminary structure-activity-relationship (SAR) analysis and their <i>in vitro</i> cytotoxicity.

<p>(<b>a</b>) The activity of all 53 primary hits was validated in PS1-M146L/YC3.6 line. All the hits were capable of reducing the peak response of CCh-induced calcium release to <90% of DMSO-treated controls. (<b>b</b>) The structure-activity-relationship (SAR) map of the screened compounds. The symbols represent compound clusters generated by Benchware DataMiner software. The distance between the clusters correlates with the similarity between their chemical structures. The number of compounds within a cluster is illustrated by the size of the symbol. A cluster with more than 50% active compounds is represented by a star, and marked in blue if the actual number of active compounds is greater than four. The highlighted identified lead structures belong to compound classes Thiazolidine (blue), Phenothiazine (green), Imidazole (turquoise) and Benzhydrilpiperidinamine (brown). Primary hits were also active in HEK293 cells expressing (<b>e</b>) PS1-DeltaE9/YC3.6, (<b>f</b>) PS1-C92S/YC3.6, and (<b>g</b>) PS1-D385N/YC3.6, by attenuating the mutant PS1-induced amplified calcium release. (<b>c</b>) and (<b>d</b>) The hits from the primary screen were classified into 8 categories based on their efficacy in lowering the CCh-evoked calcium release. These categories are separated according to the value of normalized ER calcium response. The noted numbers in each category indicates the number of compounds belonging to that category. (<b>h</b>) Viability of HEK293 cells treated with the primary screen hits was assessed by means of MTT assay after 24 h compound treatment. Values are presented as percentage of viable cells. In (a), (e), (f) and (g), the peak response of DMSO-treated control is set to one. Each color denotes a different lead structure in (a), (b), (e), (f), (g) and (h). The data for analogous molecules belonging to the same lead structure are marked with the same color.</p

Effect of on Bepridil on APP processing.

<p>(<b>a</b>) Reduced production of Aβ38, Aβ40 and Aβ42 after 16 h Bepridil (30 µM) treatment in HEK293 cells coexpressing APPsw and PS1-M146L. Sulindac sulfide (50 µM) was used as a γ-secretase modulator control. (<b>b</b>) Increased levels of sAPPα and decreased sAPPβ secreted fragments after 16 h treatment with Bepridil in HEK293 cells coexpressing APPsw and PS1-M146L. (n.s.: non-significant; * P<0.05, ** P<0.01 and *** P<0.001).</p

CCh–induced calcium release in HEK293 carrying PS1 mutations.

<p>(<b>a</b>) The average peak amplitude of CCh-induced calcium release is significantly potentiated in FAD and inactive PS1 mutants compared to wild type PS1 cells (*** P<0.001). (<b>b</b>) The average number of responsive cells to CCh is remarkably increased in cells expressing FAD and inactive PS1 mutants compared to wild type PS1 cells (*** P<0.001).</p

Workflow of the high-throughput FRET calcium imaging based compound screening assay and data analysis.

<p>(<b>a</b>) Structure of the calcium sensor YC3.6 that is a fusion protein composed of CFP and YFP attached via calmodulin (CaM) and a CaM-binding peptide (M13). Calcium binding brings CFP and YFP together, shifting the emission of 480 nm to 535 nm upon excitation at 440 nm. (<b>b</b>) CCh application initiates a pathway, which results in calcium release from ER. CCh exposure leads to G-coupled activation of PLC catalyzing the hydrolysis of the membrane-associated PIP₂ molecule to IP₃. Binding of IP₃ molecule to IP₃ receptor channels (IP₃R) on the ER membrane in turn leads to opening of IP₃R channels and calcium release from ER to cytosol. (<b>c</b>) Representative calcium transients of CCh-evoked calcium release in cells expressing FAD-linked PS mutant versus wild type PS. FAD-PS expressing cells exhibit an exaggerated calcium release upon CCh exposure. The arrow shows the time point at which CCh is applied. The HTS screening rationale was to identify drugs that can restore the FAD-PS-associated potentiation of CCh-evoked calcium release to the level of wild type PS. (<b>d</b>) HEK293 cells stably expressing PS1-M146L and YC3.6 calcium indicator are seeded in 384-well format plates. 6–8 h post seeding, using a pipetting robot, library compounds are added to separate wells. After 24 h, to stain nuclei, DRAQ5 is added to each well using the pipetting robot. After 2 h, plates are confocally imaged by “Opera” system, which is equipped with a fast dispensing unit applying CCh to each well during time-lapse imaging. An image analysis tool within the “Acapella” software is developed to automatically analyze single cell calcium transients. Using DRAQ5 nuclear segmentation, image analysis tool detects the boundaries of individual cells in the first time point and measures then the intensities of in FRET–acceptor and –donor over the course of imaging. The FRET efficiency of individual cells are then calculated and normalized. For each cell, the signal maximum (peak) is determined. The compounds which attenuated the peak amplitude of CCh-induced calcium release to <90% of the DMSO controls were regarded as hit. Finally, by data mining and determining the structure-activity-relationships (SAR) of the entire library consisting of over 20,000 compounds, active lead structures were identified. (<b>e</b>) Z′-factor as a measure for the robustness of the screening assay is evaluated for ten randomly selected imaged plates. The average Z′-factor for the screened plates exceeded 0.8.</p

Schematic illustrating the influence of mimicking Ser129 phosphorylation on asyn membrane interactions.

<p>A. For stressed gel-state membranes (DPPC-SUV), mimicking phosphorylation at Ser129 mildly reduces asyn binding affinity. B. Both pseudophosphorylated and unphosphorylated asyn monomers show no affinity to membranes in the liquid-crystalline state (POPC). C. Fe³⁺ induced asyn oligomers show a high membrane affinity and potentially act as membrane pores. Pseudophosphorylation at Ser129 shows a differential influence on asyn aggregation and binding behaviour. While increasing oligomer formation in presence of trivalent metal-ions, Ser129 pseudophosphorylation inhibits membrane binding and may thus allow oligomer sequestration into larger aggregates such as fibrils.</p

Effect of Ser129 pseudophosphorylation on asyn monomer binding to lipid vesicles.

<p>aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ were coincubated with POPC- and DPPC-SUV⁴⁸⁸. Schematic A. demonstrates the appearance of vesicles, protein monomers and oligomers in 2D FIDA histograms (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098906#pone.0098906-Hogen1" target="_blank">[7]</a>). B. aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ monomers show no interactions with POPC-SUV. Conversely, extensive monomer binding to DPPC-SUV is seen irrespective of pseudophosphorylation status. C. Quantitative FIDA analysis shows a mild tendency towards reduced vesicle binding of asyn129E⁶⁴⁷. D. Quantitative 2D-FIDA analysis of monomers bound to DPPC-SUV demonstrates an overall higher amount of bound monomers (total brightness), determined through brightness and concentration of bicolored particles. Furthermore, the higher fluorescence intensity of the bicolored particles (single vesicle brightness) indicates that more monomers are bound per vesicle. E. In a control experiment, asyn⁶⁴⁷ and asyn129E⁶⁴⁷ particle brightness (Q) as determined by 1D FIDA analysis (one component fit) is unchanged in presence of POPC-SUV as compared to control measurements in the absence of vesicles, confirming that no membrane binding takes place. However, Q increases in presence of DPPC-SUV. Dissolution of DPPC-SUV by SDS yields an asyn⁶⁴⁷ particle brightness similar to monomeric asyn⁶⁴⁷, indicating that oligomer formation is not induced by binding to DPPC-SUV. Levels of significance are depicted as * = p<0.05; n = 3.</p