Hyperphosphorylated tau aggregation and cytotoxicity modulators screen identified prescription drugs linked to Alzheimer's disease and cognitive functions

The neurodegenerative Alzheimer’s disease (AD) affects more than 30 million people worldwide. There is thus far no cure or prevention for AD. Aggregation of hyperphosphorylated tau in the brain correlates with the cognitive decline of patients of AD and other neurodegenerative tauopathies. Intracerebral injection of tau aggregates isolated from tauopathy brains causes similar pathology in the recipient mice, demonstrating the pathogenic role of abnormally phosphorylated tau. Compounds controlling the aggregation of hyperphosphorylated tau therefore are probable modulators for the disease. Here we report the use of recombinant hyperphosphorylated tau (p-tau) to identify potential tauopathy therapeutics and risk factors. Hyperphosphorylation renders tau prone to aggregate and to impair cell viability. Taking advantage of these two characters of p-tau, we performed a screen of a 1280-compound library, and tested a selective group of prescription drugs in p-tau aggregation and cytotoxicity assays. R-(−)-apomorphine and raloxifene were found to be p-tau aggregation inhibitors that protected p-tau-treated cells. In contrast, a subset of benzodiazepines exacerbated p-tau cytotoxicity apparently via enhancing p-tau aggregation. R-(−)apomorphine and raloxifene have been shown to improve cognition in animals or in humans, whereas benzodiazepines were linked to increased risks of dementia. Our results demonstrate the feasibility and potential of using hyperphosphorylated tau-based assays for AD drug discovery and risk factor identification.

To test the feasibility of applying hyperphosphorylated tau to AD drug discovery, we conducted a proof-ofprinciple pilot screen of a commercial 1280-compound library (95% were approved drugs). Aggregation of 6-µM p-tau under the influence of 30 µM of each of the library compounds or a DMSO vehicle control was monitored in 384-well plates every 10 min for ThS fluorescence changes over 16 h. The full-course aggregation curves of all reactions are shown in Fig. 2a. To assess the quality of the high-throughput p-tau aggregation reactions, we used the T 0 and T 16hr ThS fluorescence units of no-compound control reactions to calculate the Z′-factor and coefficient of variation (CV), two commonly used criteria for robustness of high-throughput screens (HTS) 62 . A Z′-factor greater than 0.5 and CV smaller than 10% indicate robustness 63 . In the case of p-tau aggregation reactions, the Z′-factor and CV were 0.699, and 8.9%, respectively, both exceeding the expectation. To identify compounds with significant p-tau aggregation modulation, the net change of ThS fluorescence was calculated from all reactions (see Supplemental Table 1 for complete listing). The frequency plot (Fig. 2b) summarizes the distribution of these reactions. All compounds deviating 3 standard deviations (SD) or more from the mean, and a small number of selective ones near 2 SD from the mean were subjected to dose-response curve (DRC) tests (1.625-100 µM). Those showing p-tau aggregation modulation activity in DRC were acquired from commercial sources for verification. Nine p-tau aggregation inhibitors (PTAI) and two enhancers (PTAE) were confirmed (see Supplemental Fig. 1 for structures). Table 1 shows these 11 compounds. Quantification of the concentration of a compound needed to reduce aggregation of 6-µM p-tau by 50% (i.e., EC 50 ) revealed a range from 8 to 68 µM among the 9 PTAIs. R-(−)apomorphine and raloxifene are p-tau aggregation inhibitors and protect cells. Of the 9 PTAIs, R-(−)apomorphine and raloxifene were selected for further studies for three reasons. Firstly, we took advantage of the cytotoxicity of p-tau 60 (detailed below) to evaluate whether these compounds modulated p-tau cytotoxicity without the use of the fibril dye, ThS, or any inducer. Three PTAIs [R-(−)apomorphine, idebenone and raloxifene] reduced p-tau cytotoxicity and both PTAEs showed enhancement (Table 1 and Supplemental Fig. 2). PTAIs without an apparent effect on p-tau cytotoxicity were set aside. Secondly, R-(−)apomorphine and raloxifene can pass the blood brain barrier. R-(−)apomorphine is a dopamine receptor agonist used to alleviate symptoms of Parkinson's disease 64 . Raloxifene is a selective estrogen receptor modulator (SERM) that has been shown to penetrate the blood brain barrier 65 . The likelihood of developing effective therapeutics from these two compounds is therefore higher. In contrast, idebenone, being a potent antioxidant and high lipophilicity, has very low bioavailability 66 . Lastly, both R-(−)apomorphine and raloxifene have been linked to the preservation of cognition 67,68 (detailed in "Discussion" below), suggesting that these prior observations might be related to the control of NFT in the brain.
To gain a better understanding of the potential of R-(−)apomorphine and raloxifene for AD drug development, we first compared the kinetics of p-tau aggregation inhibition by these two chemicals. Figure 3 shows different patterns. The action of R-(−)apomorphine consisted of multiple phases: a lag phase in which ThS fluorescence increased at the "normal" rate briefly, followed by stabilization or a slow reduction, then a sharp decline. Eventually, the highest dose of R-(−)-apomorphine caused the ThS fluorescence to drop well below the initial signal. At the same dosages, on the other hand, raloxifene elicited a typical inhibitory pattern by preventing the elevation of ThS fluorescence. P-tau aggregation appeared to be stalled from the beginning by 50 and 100 µM of the inhibitor. The distinctions in kinetics suggested that R-(−)-apomorphine and raloxifene targeted different aspects of p-tau fibrillogenesis.
Many "hits" from prior tau-based HTS were non-selective redox modulators 49 , due most likely to the susceptibility of unmodified tau fibrils to the oxidation of two cysteine residues of tau, C291 and C322. Because p-tau aggregation was not affected by changing the two cysteine residues to alanine or serine 60 , we suspected that R-(−)-apomorphine and raloxifene would act independently of these two cysteine. Figure 3c and Supplemental Fig. 3a show that the aggregation of the C/A or C/S mutant p-tau was effectively inhibited by either compound.  Table 1. Summary of p-tau aggregation inhibitors and enhancers identified in the library screen. EC 50 was defined as the concentration of each PTAI that reduced the aggregation of 6-µM p-tau to 50% of the maximal. NA, not applicable. *Hexachlorophene consistently showed PTAI activity but was inconsistent in protecting cells from p-tau toxicity. www.nature.com/scientificreports/ By itself, the C/S p-tau gained 130 units of ThS fluorescence over the course of the aggregation reaction (blue column). With increasing doses of either compound, the net gain of ThS fluorescence decreased progressively. The extent of reduction mimicked that from the use of the wildtype p-tau ( Fig. 3a,b) in that higher doses of R-(−)-apomorphine caused the ThS signals to drop below the initial level. Raloxifene, on the other hand, showed milder but still easily recognizable inhibition. It was therefore concluded that R-(−)-apomorphine and raloxifene antagonized p-tau aggregation in a mechanism(s) not related to the two cysteine residues of p-tau. Another unique character of p-tau that is relevant to AD pathology is that p-tau causes cell death at submicromolar concentrations 60 (see below). If this cytotoxicity is part of the underlying reasons for neurodegeneration, PTAIs that quenched p-tau cytotoxicity would be better candidates to be funneled through further development. To examine whether R-(−)-apomorphine or raloxifene could protect cells from p-tau, the neuroblastoma SH-SY5Y cells were treated with different doses of p-tau for 16-20 h before propidium iodide (PI) and fluorescein diacetate (FDA) staining and microscopic inspection. FDA is converted by cellular esterases into a fluorogenic fluorescein, therefore revealing live cells. PI is excluded by living cells, but diffuses into dead cells where it binds nucleic acid and emits fluorescence. FDA/PI differential staining thus provides a quantitative assessment of cellular viability. The blue curves in Fig. 4a,b depict the cellular viability after treatment with 0-0.8 µM of p-tau. The LD 50 of p-tau (concentration of p-tau causing 50% cell death) was found to be around 0.5 µM for this cell line (red horizontal lines). Co-incubating p-tau with increasing amounts of R-(−)-apomorphine or raloxifene caused the LD 50 values of p-tau to increase progressively, demonstrating p-tau cytotoxicity attenuation. The more potent cytoprotection by R-(−)-apomorphine was consistent with its stronger power in inhibiting p-tau aggregation (Fig. 3). To obtain additional quantitative assessment of the observed cytoprotection, SH-SY5Y cells were again treated with a fixed dose of p-tau (0.8 µM) along with different amounts of R-(−)-apomorphine. The resultant cellular viability was plotted as a function of R-(−)-apomorphine dosages to determine EC 50 , the concentration of a compound to reduce p-tau cytotoxicity by 50%. In the two independent assays, the EC 50 of R-(−)-apomorphine against 0.8 µM of p-tau was found to be 2.9 and 3.4 µM ( Fig. 4c and data not shown). Under the same condition, the EC 50 of raloxifene was 4.8 µM (data not shown), again manifesting that both PTAIs antagonized the cytotoxicity of p-tau, with the former being more potent. In addition, the C/A and C/S mutants that could not go through Cys-Cys disulfide bond formation were also tested for their effects on cell survival in the absence or 3b,c demonstrated the reduction of p-tau C/A and C/S cytotoxicity by these two compounds. Again, R-(−)-apomorphine exhibited more potent protective power than did raloxifene. One likely mechanism for p-tau-inflicted cell death was apoptosis 60 , evidenced by cellular staining of an apoptosis marker, annexin V (Fig. 4d). Cells treated with p-tau showed a significant increase staining of annexin V and PI. Microscopic quantification showed that without p-tau, 90% of cells were viable and were refractory to annexin V staining (first column, Fig. 4e). Treatment with 0.6 µM of p-tau caused 47% of cells stained positive with both annexin V and PI (orange sector, second column). A small number of cells were annexin V-positive but PI-negative, suggesting early apoptosis. In the presence of 10 µM of R-(−)-apomorphine, the live and nonapoptotic cells increased from 47 to 77% (P < 0.001). The reduced apoptotic population was consistent with the overall improved cell viability shown in Fig. 4a,c.

EC50 (µM) p-tau cytotox Medical uses
We next examined whether R-(−)-apomorphine could benefit cells after p-tau assault had begun. Figure 5a shows the assay workflow. Fifteen micromolar of R-(−)-apomorphine or DMSO was added to cells that had been exposed to 0.5 or 1 µm of p-tau for 24 h. Cells were harvested for ThS/PI staining after additional 24 or 48 h. ThS staining helped visualize intracellular fibrils 45 . In this experiment, we used HEK293T cells to avoid potential interference from the endogenous tau. A quick microscopic survey revealed that p-tau treatment caused many cells to be stained by both PI and ThS (DMSO panel, Fig. 5b), indicating cell death with cytoplasmic fibrils buildup. Adding R-(−)-apomorphine 24 h after the initial p-tau attack resulted in a visible increase of viable cells (i.e., free of PI staining); some of these cells remained to be stainable by ThS (white arrows, Fig. 5b). To gain a quantitative assessment, the numbers of the four possible combinations of ThS/PI staining were tallied. Figure 5c shows that most p-tau-treated cells were dead with cytoplasmic ThS staining (red columns; 85% in 24-h, 94% Selective benzodiazepine drugs enhance p-tau aggregation and cytotoxicity. Given the tremendous socioeconomical burdens of AD, measures that prevent or delay the onset of the devastating neurodegeneration of AD will be highly impactful. If hyperphosphorylated tau-inflicted neuronal damages are an underpinning for AD and other tauopathies, enhancers of p-tau aggregation (PTAEs) may be risk factors for neurodegeneration. The library screen mentioned above identified prednicarbate and carmofur as PTAEs (see Supplemental Fig. 1 for structures). Prednicarbate is a synthetic corticosteroid for dermatological use, and carmofur (1-hexylcarbamoyl-5-fluorouracil) is an anti-neoplastic pyrimidine analogue. While both compounds appeared to exacerbate p-tau cytotoxicity (Supplemental Fig. 2b), these drugs are unlikely to pose a significant neurodegeneration threat to the general population due to their primary medical use. On the other hand, epidemiological studies reported increased risks of dementia from long-term use of benzodiazepine drugs (BZDs) 14,69 . BZDs are among the most widely prescribed medication, in particular to the elderly, for depression and insomnia 70,71 . We hypothesized that certain BZDs acted as a PTAE to potentiate p-tau aggregation and cytotoxicity. The compound library used in the aforementioned screen did not include BZD family drugs. We therefore randomly picked nine prescription BZDs for p-tau aggregation assessment ( Fig. 6; see Supplemental Fig. 5 for chemical structures). To 6-µM p-tau aggregation reactions, increasing doses of each of these BZDs were added. Comparison of the 16-h net changes of ThS fluorescence showed that prazepam, flurazepam, temazepam, diazepam, and devazepide enhanced p-tau aggregation in a dose-dependent manner, whereas nitrazepam, nimetazepam, oxazepam, and nordiazepam did not (Fig. 6a). None of these compounds raised ThS fluorescence by themselves (Fig. 6b). It was therefore concluded that selective BZDs were able to stimulate p-tau aggregation in vitro. www.nature.com/scientificreports/ To see whether the PTAE activity of BZDs would lead to enhancement of p-tau-inflicted cell death, SH-SY5Y cells were treated with p-tau pre-incubated with different concentrations of nitrazepam (a non-PTAE), prazepam or devazepide. P-tau caused cell death (blue squares, Fig. 7) with an LD 50 value of approximately 0.54 µM. Without p-tau, none of the three BZDs affected cellular viability (i.e., 0-µM p-tau). The curve of cell death shifted progressively to the left (i.e., decreasing LD 50 ) when increasing concentrations of prazepam and devazepide were present. Between these two drugs, devazepide consistently showed stronger PTAE activity (data not shown), which was in excellent agreement with its stronger p-tau cytotoxicity enhancement (note the use of lower concentrations of devazepide). In contrast, nitrazepam, which had no PTAE activity, did not alter the cytotoxicity of p-tau at all. The five cell death curves with 0 to 15 µM of nitrazepam were indistinguishable from each other. Together, biochemical and cell-based experiments in Figs. 6, 7 and Supplemental Fig. 2b demonstrated that compounds that enhanced the in vitro aggregation of p-tau might also exacerbate the cytotoxicity associated with this protein, suggesting a possible explanation for increased AD risks by BZD drugs.

Discussion
According to the World Health Organization, 50 million people in the world are living with dementia, of which 2/3 are Alzheimer's disease. Finding an effective therapeutic or prophylactic regimen for AD will have tremendous health and socioeconomical impacts. This work presents proof-of-principle evidence for a novel, hyperphosphorylated tau-based platform supporting AD drug discovery and risk factor initial assessments. The center of this platform is p-tau produced by the PIMAX approach. We have recently reported that p-tau produced by PIMAX possesses phosphorylation marks intimately associated with cognitive impairments in AD patients 60 . Hyperphosphorylation apparently renders tau prone to form cytotoxic aggregates that trigger apoptosis and cell death in a manner that does not require an inducer, such as heparin. The character of heparin-independent aggregation and cytotoxicity is critically advantageous over the unmodified tau that has been used in multiple futile drug screens [45][46][47][48][49] , for the pathophysiological relevance of heparin in AD remains elusive. Compounds identified in a tau-based aggregation primary screen may contain a large number of molecules that act on the interface between tau and heparin, not tau aggregates per se. In addition, changing the two cysteine residues to either alanine or to serine does not impact the inducer-independent aggregation of PIMAX p-tau 60 , suggesting that non-specific redox modulators are unlikely to cause false positive results in high-throughput screens, as www.nature.com/scientificreports/ they did for unmodified tau-based drug screens 49 . Using a combination of inducer-free aggregation and cell assays, we identified modulators for both p-tau aggregation and cytotoxicity. Both p-tau aggregation inhibitors and enhancers were identified, and that, importantly, these compounds have previously linked to dementia and Alzheimer's disease. The assay platform described in this work therefore holds a promising prospect for exploration of AD disease-modifying compounds. The current AD drug discovery field has been dominated by Aβ-centric strategies and trials with limited successes 2 . The trial failures and the fact that NFTs, but not Aβ, predict cognitive decline, led to heightened interests in NFT-targeting drugs. Unfortunately, such endeavors also encountered setbacks 49,72 . One possibility is that the subject proteins used for tau aggregation inhibitor (TAI) screens lack the hyperphosphorylation mark that is likely key to converting a normal tau to one that not only forms fibrils but also propagates pathology between cells 73 . The inducer-free aggregation and substantial impacts on cell viability of hyperphosphorylated tau produced by PIMAX suggest that TAIs, identified from heparin-induced tau aggregation assays, may not have the same inhibitory effect on p-tau aggregation. This appears to be the case. For example, two TAIs found in a high-throughput screen using heparin-induced K18 P301L aggregation as the primary assay 49 were subjected to heparin-free p-tau aggregation reactions (Supplemental Fig. 4). At 50 or 200 µM, compound 1 had no effect on p-tau aggregation (Supplemental Fig. 4a), whereas compound 2 showed moderate inhibition. However, even when the doses were raised to 400 µM or 1 mM, p-tau aggregation was reduced to only 57% and 46%, respectively. In comparison, 50 µM of raloxifene completely blocked p-tau aggregation (Fig. 4b). These results demonstrated the unique nature of tau aggregation resulting from hyperphosphorylation, and that using p-tau aggregation as the primary assay for drug screen may uncover compounds that might escape unmodified tau-based screens.
For high-throughput drug screening, p-tau cytotoxicity appears to be an effective orthogonal assay following the aggregation modulator screen. Firstly, cell-based assays were done without heparin or ThS. Molecules that affect heparin or ThS action but not p-tau aggregation per se will likely be ruled out by the lack of influence on p-tau cytotoxicity. Secondly, using the ability of p-tau to damage tissue culture cells as a model for neurodegeneration, compounds that alter p-tau cytotoxicity are therefore better connected to AD onset and progression. From 1280 compounds in the chemical library, 9 PTAIs and 2 PTAEs were found in the initial screen and the subsequent secondary DRC tests (Table 1). When they were tested in cell viability assays, only R-(−)-apomorphine, raloxifene, and idebenone showed consistent cytoprotection (Supplemental Fig. 2a). Hexachlorophene showed inconsistent results in cell protection assays, due likely to its high propensity to oxidation (data not shown). Idebenone is a coenzyme Q analogue with limited bioavailability 66 . Prednicarbate, a PTAE, appeared to enhance the cell killing by p-tau, while carmofur exhibited borderline effects (Supplemental Fig. 2b). Prednicarbate is a synthetic corticosteroid for dermatological use 74 . Carmofur (1-hexylcarbamoyl-5-fluorouracil) is an anti-neoplastic pyrimidine analogue that inhibits acid ceramidase upregulated in many tumors 75 . These four compounds, while having limited implications in AD, may provide a useful tool to study the mechanisms by which p-tau aggregation and cytotoxicity might be controlled.
R-(−)-apomorphine and raloxifene hold a promise for further AD therapeutics development due to their brain permeability and association with cognitive preservation. R-(−)-apomorphine is a dopamine receptor agonist, whereas raloxifene is a selective estrogen receptor modulator (SERM). Dopamine and several other SERMs including tamoxifen and diethylstilbestrol were present in the pilot screen library analyzed above, but none of them affected p-tau aggregation (see Supplemental Table 1), suggesting additional structural features unique www.nature.com/scientificreports/ to these two compounds for the observed PTAI activity. In a 3xTg mice (APP Swedish, MAPT P301L, PSEN1 M146V) study 67 , R-(−)-apomorphine elevated levels of the insulin-degrading enzyme and reduced Aβ, resulting in memory improvement. Given the link between R-(−)-apomorphine and p-tau, it will be very interesting to see whether this drug also antagonizes tau aggregation in 3xTg mice. Clinically, R-(−)-apomorphine has a potent emetic property and is administered subcutaneously as acute, intermittent treatment of the "off " episodes associated with advanced Parkinson's disease. This drug is therefore not ideal for AD treatment. On the other hand, it has been reported that a spike of tau hyperphosphorylation happens after brain injury 76 and general anesthesia 77 , due likely to acutely diminished phosphatase activities 78,79 . Whether this transient tau hyperphosphorylation is related to the positive association between dementia and traumatic brain injury 13 or exposure to general anesthesia 80 remains an open question. Animal studies may provide a clue as to whether R-(−)-apomorphine can be used to neutralize hyperphosphorylated tau following brain injury or general anesthesia exposure, thus reducing the likelihood of AD and ADRD after recovery. Raloxifene is used to treat osteoporosis of postmenopausal women 81 . A randomized, placebo-controlled, 3-year trial on 5386 participants (mean age 66.3 years, range 35.7-80.9) showed reduced risks of mild cognitive impairment (MCI) among women who received a daily dose of 120 mg of raloxifene 68 . However, another 12-month trial involving 42 women (mean age 76 years, range 68-84) with late-onset AD of mild to moderate severity showed no significant benefits of raloxifene for AD treatment 82 . Some of the key differences between these two studies, such as the age (66.3 vs. 76 years) and the initial cognitive status (mostly cognitively normal vs. mild-to-moderate severity of AD) of the participants, may underlie the deviating outcomes, and suggest that raloxifene may only be effective when given at an early stage of cognitive decline.
It remains a formidable challenge to identify early AD patients for trials or treatment. Preventative measures against modifiable risk factors appears to be a more manageable approach at the present time. Several benzodiazepines were found to be enhancers for p-tau aggregation. The biochemical PTAE activity of BZDs, or the lack thereof, correlated with changes in p-tau cytotoxicity (Figs. 6, 7). Interestingly, the cytotoxicity enhancement activity of these BZDs required pre-incubation with p-tau (24 or 48 h; data not shown), suggesting that the observed exacerbation of cell death was through the production of a more potent p-tau toxin, not via the known targets of BZDs, which include the GABA A receptor 83 and the mitochondrial outer membrane translocator protein TSPO 84 .
Besides BZDs, certain anticholinergic drugs 15,16 and DMARD (disease modifying anti-rheumatic drugs) 17 were also linked to increased risks of dementia. Some of these drugs were included in the library used for the pilot screen above, but had insignificant effects on the aggregation of the 1N4R p-tau used in this study (Supplemental Table 1). While these drugs may promote dementia through a molecular target unrelated to tau, it remains formally possible that other isoforms of tau (e.g., 4R vs. 3R) phosphorylated by another kinase (e.g., CDK5) may be more responsive to some of these drugs. A panel of different p-tau species will therefore provide a valuable tool for more comprehensive studies.
In conclusion, this work presents proof-of-principle evidence for a novel, hyperphosphorylated tau-based AD drug and risk factor screening platform. This platform can be scaled up for high-throughput screens of a much larger number of compounds, and can also be applied to more targeted categories of molecules to enable drug repurposing and risk factor assessment, thereby facilitating the development of therapeutic and preventive regimens for Alzheimer's disease.

Materials and methods
Materials. Prestwick FDA Approved Chemical Library was provided by MSU Assay Development and Drug Repurposing Core (ADDRC). R-(−)-apomorphine hydrochloride, raloxifene hydrochloride, fosinopril, nifedipine, nisoldipine, idebenone, itraconazole, prednicarbate, carmofur, diazepam, flurazepam, temazepam, prazepam, nitrazepam, nimetazepam, nordiazepam and oxazepam were from Cayman Chemical (Ann Arbor, MI). Devazepide was purchased from Santa Cruz Biotechnology (Dallas, Texas). Fluorescein diacetate, propidium iodide, clofazimine, hexachlorophene, hydralazine hydrochloride, thioflavin S and Amicon Centrifugal Filter Unit were purchased from Sigma Aldrich (St. Louis, MO). Gibco Dulbecco's Modified Eagle Medium (DMEM), HyClone™ Fetal Bovine Serum, Optical Adhesive Film and FITC-Annexin V were purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals for common buffers and solutions were from Sigma Aldrich. plasmids and recombinant genes. Plasmid and expression procedures for the 1N4R tau and p-tau were previously described 59 . All constructs were verified by DNA sequencing. The final tau and p-tau expressed from the PIMAX system contained a 7-residue remnant from cloning: GSSPEQP at the N' end.
Hyperphosphorylated tau expression, purification, and aggregation assays. Procedures for the preparation of PIMAX p-tau, as well as the conditions for p-tau aggregation assays can be found in reference 60 . The aggregation assays over the course of this project have been done with multi-plate readers from different sources. The absolute fluorescence units of thioflavin S recorded by each instrument might vary, but the trend of relative changes remained the same.
High-throughput screening. A common mix of 6 µM p-tau, 20 mM Tris pH 7.4, 1 mM DTT, and 20 µM ThS was assembled and dispensed to 384-well low-volume plates ( www.nature.com/scientificreports/ by Biotek Synergy Neo Plate Reader. Four plates were used to cover the 1280-compound library. The real-time kinetics of ThS signals were collected using Gen5 software. The net change of ThS of each reaction was calculated and was used to assess the standard deviation. The Z′-value of p-tau aggregation reactions was derived by first calculating the ThS net change over 16 h of no-protein, ThS-only reactions as the negative control (− c), and p-tau-containing reactions (with DMSO vehicle) as the positive control (+ c). The Z′-value was calculated by the equation: where σ +c , σ -c , µ +c , and µ -c are the standard deviations (s) and the averages (µ) of the positive and negative controls 62 . All compounds showing greater than 3 standard deviations were picked for dose response curves (DRC). Compounds showing greater than 2 standard deviations and used for treating CNS conditions were selected for DRC as well. Those that showed significant deviation from no-compound controls were purchased from commercial venders and repeated for DRC. cytotoxicity of p-tau. Cell viability assays were tested according to reference 60 with modifications. PTAIs were typically added to cells along with (Fig. 4) or after p-tau was added to cells (Fig. 5). For PTAEs, 10 × concentration of p-tau and the test compounds were assembled for pre-aggregation (without heparin or ThS) for 48 h before adding 10 µl to 100 µl of cells in 96-well plates. Twenty-four or 48 h after the addition of protein/ compound, cells were trypsinized and transferred to microcentrifuge tubes, and pelleted at 1000 × g for 5 min at room temperature for FDA, PI, annexin V, and ThS staining 60 . For PI/ThS double staining, cell pellets were resuspended in 20 mM Tris, pH7.4 buffer and incubated with 100 µM ThS for 5 min at room temperature. 5-µg/ ml propidium iodide was then added to cell suspension for observation under a microscope. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.