WO2020232440A1 - Peptide-based inhibitors that block aggregation, seeding and cross-seeding of amyloid beta and tau - Google Patents

Abstract

Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline. Here, we report peptide-based inhibitors that reduce Aβ and tau aggregation and toxicity of already-aggregated species.

Description

PEPTIDE-BASED INHIBITORS THAT BLOCK AGGREGATION, SEEDING AND CROSS-SEEDING OF AMYLOID BETA AND TAU CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application Serial No 62/848,738, filed on May 16, 2019 and entitled“PEPTIDE-BASED INHIBITORS THAT BLOCK AGGREGATION, SEEDING AND CROSS-SEEDING OF AMYLOID BETA AND TAU” which application is incorporated by reference herein. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant Number AG029430, AG054022, NS095661, awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

The invention relates to compositions and methods useful in inhibiting aggregation of Tau and amyloid b proteins. BACKGROUND OF THE INVENTION

Although Alzheimer’s disease (AD) is the most prevalent form of dementia, there are limited treatments to alleviate symptoms and none that halt its progression. Histological features of AD are extracellular senile plaques of amyloid beta (Ab) and intracellular neurofibrillary tangles of tau (1, 2). While Ab aggregation is thought to occur at the early stages of AD, tau aggregation correlates better to disease progression, with characteristic spreading along linked brain areas, and severity of symptoms correlating to the number of observed inclusions (3–8). Structural information about the aggregated forms of Ab and tau is accumulating, but to date this knowledge has not led to successful chemical interventions (9).

A link between the appearance of Ab and tau pathologies has been noted in transgenic mouse models generated by crossing or co-expressing mutant Ab and mutant tau, but the mechanism is not yet understood at the molecular level (10). By injecting Ab seeds derived from synthetic peptide, transgenic mouse or AD patient tissue, tau pathology can be found both at the site of injection, and also in functionally connected brain areas (11–13). Tau aggregation has also been reported to follow Ab seeding in 3D neuronal stem cell cultures that express early onset hereditary mutations to drive overproduction and aggregation of Ab (14). In spite of these observations, the molecular linkage of Ab to tau remains unresolved. Proposed hypotheses include Ab causing downstream cellular changes that trigger tau phosphorylation and eventual aggregation, and/or a direct interaction and seeding of tau by aggregated Ab (15, 16).

Several lines of evidence support the direct interaction model, although questions still remain; for example, how such an interaction could occur since Ab plaques deposit extracellularly, while tau neurofibrillary tangles are intracellular. One possible model for intracellular aggregation could be that Ab is cleaved from APP inside endosomes, and then exported (17). Another model proposes that smaller diffusible Ab oligomers are the toxic species (18–20); indeed oligomers of Ab isolated from AD serum are sufficient to induce tau aggregation (21). Ab has also been found co-localize intra-neuronally with tau as well as at synaptic terminals, with increased interactions correlating with disease progression (5). Furthermore, soluble and insoluble complexes of Ab bound to tau have been detected in AD tissue extracts (5, 22). In vitro, soluble complexes of Ab and tau have been found to promote aggregation of tau (22), while another study found that Ab fibrils can seed tau (23).

Consequently, there is a need for an understanding of the structure of the Ab and tau molecules, particularly information which allows the design of functional inhibitors. Moreover, there is a need for inhibitors having designs that are based upon this functional information, and methods of making and using such inhibitors. SUMMARY OF THE INVENTION

Alzheimer’s disease (AD) pathology is characterized by plaques of amyloid beta (Ab) and neurofibrillary tangles of tau. Ab aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline. Here, we report peptide-based inhibitors that reduce Ab and/or tau aggregation and toxicity of already-aggregated species, and methods of making and using these peptide-based inhibitors.

The invention disclosed herein has a number of embodiments. Embodiments of the invention include compositions of matter comprising a peptide inhibitor of Ab aggregation disclosed herein. Typically the peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10), wherein at least one amino acid is a D-amino acid.

Embodiments of the invention also include a method of inhibiting aggregation of Ab polypeptides by combining Ab polypeptides with one or more peptide inhibitors of Ab aggregation disclosed herein; and allowing the peptide(s) to bind to the Ab polypeptides; so that Ab aggregation is inhibited. In certain embodiments of the invention, a peptide used in the method is selected for an ability to inhibit cross- seeding of tau by Ab polypeptides; and/or inhibit tau homotypic seeding. Typically, the peptide is combined with Ab and/or Tau in vivo. Certain embodiments of the invention include therapeutic methods of inhibiting formation of Ab aggregates in vivo inhibits development or progression of a Ab plaque formation in an individual; and/or inhibiting homotypic seeding of Tau fibrils in vivo inhibits development or progression of a tauopathy in an individual. Yet another embodiment of the invention is a composition of matter comprising a peptide inhibitor of tau aggregation that is disclosed herein. Typically, the peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S- V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q- M-I-N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D- V-W-M-I-N-K-K-R-K (SEQ ID NO: 18). Optionally such compositions further comprise a Tau polypeptide (SEQ ID NO: 1). A related composition of matter comprises a polynucleotide encoding a peptide inhibitor of tau (or Ab) aggregation such as a peptide inhibitor comprising an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I-N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W-M-I-N-K-K-R-K (SEQ ID NO: 18). Typically, the polynucleotide is disposed within a vector selected for its ability to express the peptide inhibitor in a mammalian cell. Optionally, the vector is disposed within a mammalian cell.

Yet another embodiment of the invention is a method of inhibiting formation of tau aggregation, the method comprising combining tau polypeptides with one or more peptide inhibitors of tau aggregation that are disclosed herein; and then allowing the peptide(s) to bind to the tau polypeptides, so that tau aggregation is inhibited. Optionally the peptide(s) used in the method is selected for an ability to inhibit seeding of purified tau fibrils and/or inhibit seeding of tau fibrils present in unpurified or partially purified brain extracts. In certain embodiments of the invention, the peptide is combined with Tau in vivo (e.g. in therapeutic methods designed to inhibit development or progression of a tauopathy in an individual).

In certain compositions of the invention, an inhibitory peptide disclosed herein is coupled to a plurality of heterologous amino acids. For example, in some embodiments of the invention, the peptide is fused, optionally via a linker sequence, to a plurality of heterologous amino acids comprising a cell penetrating peptide (CPP). Optionally, the plurality of heterologous amino acids (e.g. a CPP) is less than 30 amino acids in length. In certain embodiments of the invention, the CPP comprises a plurality of arginine residues, for example 4 to 16 contiguous arginine residues. In some embodiments of the invention, the peptide comprises a non-naturally occurring amino acid such as a D amino acid.

In some embodiments of the invention, the composition comprises a cocktail of different peptide inhibitors such as a combination of the peptide inhibitors of Ab aggregation that are disclosed herein, a combination of the peptide inhibitors of tau aggregation that are disclosed herein, or a combination of the peptide inhibitors of Ab aggregation and the peptide inhibitors of tau aggregation that are disclosed herein. Optionally, relative amounts of a first and a second (and a third etc.) peptide within the composition are selected to effect the ability of the composition to inhibit one or more discreet phenomena observed in pathological protein aggregation (e.g. inhibition of homotypic seeding of protein fibrils).

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C provide disclosure on the microED structure of segment Ab 16-26 D23N from microcrystals. Figure 1A shows an electron micrograph of 3D crystals used for data collection, scale bar is 1mm. Figure 1B provides a schematic showing the crystal structure reveals tightly mated pairs of anti-parallel b-sheets with opposing sheets in grey and cyan. The side-chains interdigitate to form a dry interface. Two neighboring sheets are viewed perpendicular to the b-sheets. Figure 1C provides a view of 6 layers perpendicular to the fibril axis (black line). The b-sheets stack out of register along the fibril axis.

Figures 2A-2E provide disclosure on the development of inhibitors of Ab fibril formation using structure-based design against Ab 16-26 D23N. Figure 2A provides graphed data showing the identification of Ab1-42 inhibitor. 10 mM Ab1-42 was incubated alone or with 100 mM of each candidate peptide inhibitor for 12 hours at 37 ^oC and then diluted 1:10 with pre-plated N2a cells. Cytotoxicity was quantified using MTT dye reduction Bars represent mean with individual technical replicates (n = 3; ns = not significant; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column). Figure 2B and 2C provide schematics of the segment KLVFFAEN (SEQ ID NO: 25), derived from the Ab 16-26 D23N crystal structure, that was used as the design target. Model of peptide inhibitor D1(magenta) bound to the design target, KLVFFAEN (SEQ ID NO: 25) (gray). Smaller hydrophobic residues of D1 mimic interactions with the fibril interface on one side of the peptide (Figure 2B), whereas the other side of the peptide positions large aromatic residues between Ab residues, breaking possible further interactions (Figure 2C). Figure 2D provides an overview of peptide inhibitors in D and L amino acid conformations, as indicated, used in this study and their sequences. Peptide LC is the L-form cognate peptide of D-form peptide D1 and is the negative control for peptide inhibitor D1 and its derivatives D1b and D1d. IC50 values were determined using 4 parameter nonlinear fit. N.D., not determined. Figure 2E provides data showing that peptide inhibitors D1, D1b, and D1d reduce the cytotoxicity of Ab1-42 in a dose dependent manner, whereas control peptide LC does not. 10 mM Ab1-42 was incubated alone or with various concentrations of each peptide inhibitor for 12 hours at 37^oC and then diluted 1:10 with pre-plated N2a cells. Cytotoxicity was quantified using MTT dye reduction. Bars represent mean with individual technical replicates (n = 3-6; ns = not significant; **, p < 0.002; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column).

Figures 3A-3C provide data showing that designed inhibitors reduce aggregation of Ab1-42. Figure 3A provides data showing how peptide inhibitors D1, D1b, and D1d reduce fibril formation of Ab1-42, while negative control peptide LC does not. 10 mM of Ab1-42 was incubated alone or at a 1:10, 1:1, or 10:1 molar ratio to each inhibitor under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Curves show the average of three technical replicates with one standard deviation below. Figure 3B provides data showing a negative-stain TEM analysis that confirms the results of the ThT assays in Figure 3A. Samples were prepared as above and incubated for 72 hours before TEM analysis. Images of Ab1- 42 to D1 (1:10), D1b (1:1) and D1d (1:1) were captured at 3,200x; scale bars are 2mm. All other images were captured at 24,000x; scale bars are 500 nm. Figure 3C provides data showing that peptide inhibitors reduce the formation of Ab1-42 assemblies recognized by conformational monoclonal antibodies, while negative control peptides do not. 10 mM Ab1-42 was incubated alone (left-most column) or with 10-fold molar excess of each peptide-based inhibitor. Aliquots of the reaction were tested for antibody-binding at 6 h, 24 h, and 72 h. Membranes were spliced as indicated for clarity.

Figures 4A-4C provide data showing that inhibitors bind and block toxicity of aggregated Ab1-42. Figure 4A provides data showing that the toxicity of already formed Ab1-42 aggregates is lessened by peptide inhibitors. 10 mM Ab1-42 was incubated alone for 12 hours at 37 °C. Indicated molar ratio of inhibitor was added to the incubated Ab1-42 and then diluted 1:10 with pre-plated N2a cells. Cytotoxicity was quantified using MTT dye reduction. Bars represent mean with individual technical replicates (n = 3-6; ns = not significant; ***, p < 0.0005; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column). Figures 4B and 4C provides data showing that inhibitors bind to Ab1-42 fibrils. Figure 4B provides data showing that peptide inhibitors do not disaggregate Ab. 10 mM Ab1-42 was incubated alone for 72 hours at 37 °. Peptide inhibitors were added at 10-fold molar excess and incubated at RT for 24 hours before TEM analysis. Images were captured at 24,000; scale bars are 500 nm. Figure 4C shows binding isotherm data on inhibitor D1d to fibrillar Ab1-42. The maximal response (RUmax) was derived by fitting sensorgrams obtained over a range of D1d concentrations to the binding model with a Kd of 52 ± 6 mM. . These RUmax values are plotted (mean ± SD, n = 3) as a function of concentration and fitted to a one to one binding model.

Figures 5A-5E provide data showing that tau aggregation is seeded by Ab and reduced by structure-based inhibitors. Figure 5A shows disclosure where 20 mM tau40 was seeded with 10% monomer equivalent of pre-formed fibrils of Ab1-42 or tau-K18m under shaking conditions at 700 RPM at 37 °C in buffer containing 0.5mg/ml heparin. Fibril formation was monitored using ThT fluorescence. Error bars show the standard deviation of the average of three technical replicates. Figure 5B shows disclosure where the number of intracellular aggregates present in tau-K18CY biosensor cells normalized to cell confluence seeded by the addition of 250 nM tau40 or 250 nM Ab1-42 fibrils. Error bars show the standard deviation of the mean of technical replicates (n = 3; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column, and **, p < 0.005 in unpaired t test of Ab vs. vehicle) Figure 5C shows representative images of seeded cells from B at 10x magnification, scale bar 100 mm. Figures 5D and 5E show concentration dependent inhibition of Ab1-42 induced seeding of tau aggregation in tau-K18CY biosensor cells. Figure 5D shows disclosure of an average by Ab as a function of indicated inhibitor concentration. Error bars show the standard deviation of the mean of technical replicates (n = 3; ns = not significant; *, p < 0.02; **, p < 0.005; ***, p < 0.001; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column), and the dotted line shows the mean number of aggregates from untreated Ab1-42 fibrils. Figure 5E shows representative images of tau-K18CY biosensor cells showing the concentration dependent effect of D1b on seeding. Cells are shown at 10X magnification, scale bar 100 mm.

Figures 6A-6D provide disclosure showing that Ab inhibitors also reduce fibril formation and seeding by tau40. Figure 6A shows disclosure on how peptide inhibitors D1, D1b, and D1d reduce fibril formation of tau40. 10 mM tau40 monomer was incubated at a 1:10, 1:1, or 10:1 molar ratio to each inhibitor with 0.5mg/ml heparin under shaking conditions at 700 RPM at 37 ºC. Fibril formation was monitored using ThT fluorescence. Plots show the average of three technical replicates with one standard deviation below. Figure 6B and 6C show disclosure on the effects of the inhibitors on seeding by tau40 fibrils in tau-K18CY biosensor cells. The cells were seeded with 250 nM tau40 fiber (final concentration); in samples with inhibitor, tau40 fibers were incubated with indicated final concentrations of peptide inhibitor for one hour prior to addition to cells. Figure 6B shows disclosure on the average number of aggregates at the indicated inhibitor concentrations, Bars represent mean with individual technical replicates (n = 3; ns = not significant; *, p < 0.02; ***, p < 0.0005; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column). dotted line represents number of aggregates from untreated tau40 fibrils. IC50 value was calculated from the dose–response plot of inhibitor D1b. Figure 6C shows representative images of effect of D1b on seeding. Cells are shown at 10X magnification, scale bar 100 mm. Figure 6D shows disclosure on seeding from tau interface mutation fibrils in tau-K18CY biosensor cells is reduced by D1b. Experiment was performed as above. Average number of normalized aggregates at the indicated inhibitor concentrations, Bars represent mean with individual technical replicates (n = 3; ns = not significant; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column).

Figures 7A and 7B show that peptide inhibitors reduce seeding by crude brain-extract from tauopathy donor tissue. Brain lysate was prepared in TBS buffer from 3 brain regions of one AD patient, and from a one sample of a PSP patient lacking Ab plaques. Cells were seeded with a 1/400 dilution of brain tissue lysate; for samples with inhibitor, lysates were incubated with inhibitor overnight prior to addition to cells. A concentration of 10 mM peptide was used for all of the experiments shown. Figure 7A shows disclosure on the average number of aggregates seeded by lysate from each respective brain region, with or without addition of inhibitors. Bars represent mean with individual technical replicates (n = 3; ns = not significant; *, p < 0.05; ***, p < 0.0005; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column). Figure 7B shows representative images of seeded biosensor cells from Figure 7A shown at 10X magnification, scale bar 100 mm.

Figure 8 provides a schematic showing the crystal packing of the Ab 16-26 D23N atomic structure. View down the‘a’ axis of the unit cell, outlined in red.

Figures 9A-9C show extended toxicity data from peptide inhibitors. Figures 9A and 9B provide data showing that peptide inhibitors are not toxic. 100 mM of each peptide inhibitor was incubated for 12 hours at 37 °C and then diluted 1:10 with pre-plated N2a cells. Cytotoxicity was quantified using MTT dye reduction. Bars represent mean with individual technical replicates. Figure 9C shows disclosure where second generation peptide inhibitors reduce the cytotoxicity of Ab1-42. 10 mM Ab1-42 was incubated alone or with 10 mM and 100 mM of each peptide inhibitor for 12 hours at 37 °C and then diluted 1:10 with pre-plated N2a cells. Cytotoxicity was quantified using MTT dye reduction. (n = 3; ns = not significant; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column).

Figures 10A-10C provide data showing that peptide inhibitors D1a, D1c, D1e, and D1f are less effective ate reducing fibril formation of Ab1-42. Figure 10A shows data from studies where 10 mM of Ab1-42 was incubated alone or at a 1:10, 1:1, or 10:1 molar ratio to each inhibitor under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show the average of three technical replicates with one standard deviation below. Inhibitors D1a and D1c are not shown at (1:10) ratio due to high ThT signal from peptide self-association. Figure 10B shows disclosure where inhibitors D1b and D1d self-associate at high concentrations. Peptide inhibitors were incubated at 10 mM and 100 mM under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show the average of three technical replicates with one standard deviation. Figure 10C shows unspliced dot blots from Figure 4C. Column key displayed at lower right is representative of all displayed blots.

Figure 11 shows a representative sensorgram obtained when D1d solutions at the indicated concentrations were flowed across the Ab1-42 sensor chip.

Figures 12A-12C shows disclosure from tau seeding experiments. Figure 12A shows extended ThT data of Tau seeding experiment. K18 and Ab1-42 seeds have some baseline ThT signal. Figure 12B shows disclosure where 10 mM Ab1-42 was seeded with 10% monomer equivalent of pre-formed seed of Ab1-42 or K18, under quiescent conditions at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show average of three technical replicates. Figure 12C shows disclosure examining the relative seeding efficiency of tau40 and other amyloid fibrils into the HEK293 biosensor that stably expresses K18 yellow fluorescent protein (YFP) fusion. The cells were seeded with 250 nM final concentration of amyloid fibril. (n = 3; ns = not significant; ****, p < 0.0001 using an ordinary one-way ANOVA relative to leftmost column or against all columns excluding leftmost, as indicated ). Amyloid fibrils were confirmed by endpoint ThT or Electron Microscopy.

Figures 13A-13F shows disclosure from tau fibril experiments. Figure 13A shows that a control peptide inhibitor LC does not reduce fibril formation of tau40. 10 mM of tau40 was incubated at a 1:10 molar ratio to LC with 0.5mg/ml heparin under shaking conditions at 700 RPM at 37 °C. Fibril formation was monitored using ThT fluorescence. Lines show the average of three technical replicates with one standard deviation below. esigned inhibitors are not general amyloid inhibitors. Figure 13B shows data where 50 mM of a-synuclein was incubated at a 1:5 molar ratio to peptide inhibitors under shaking conditions at 700 RPM at 37 °C. Lines show the average of three technical replicates. Figure 13C shows data where 10 mM hIAPP was incubated at a 1:5 molar ratio to peptide inhibitors under quiescent conditions at 37 °C. Lines show the average of three technical replicates. Figures 13D-13F show extended data of seeding by tau interface mutation fibrils in tau-K18CY biosensor cells. The cells were seeded with 250 nM tau40 fiber (final concentration); in samples with inhibitor, tau40 fibers were incubated with indicated final concentrations of peptide inhibitor for one hour prior to addition to cells. Average number of aggregates at the indicated inhibitor concentrations, Bars represent mean with individual technical replicates ( n = 3; ns = not significant; *, p < 0.05; ***, p < 0.0005; ****, p < 0.00010001 using an ordinary one-way ANOVA relative to leftmost column in Figure 13D, relative as indicated in Figure 13E, and relative to dotted line representing untreated fibrils in Figure 13E). Figure 13D shows data where interface mutations are less affected by D1b. Figure 13E shows a construct containing all interface mutations is seeding incompetent. Figure 13F shows that seeding interface mutants are not reduced by control inhibitor LC.

Figures 14A-14C show schematics of peptide inhibitor interactions. The spines of Ab 16-26 D23N and tau are structurally similar. Figures 14A-14B show Ab 16-26 D23N overlay with tau 274-283 in parallel and antiparallel orientations. Figure 14A shows 32 backbone atoms differ from each other by 0.53 Å RMSD. RMSD values were calculated using LSQ in Coot. Figure 14B shows the C_a atoms differ from each other by 0.56 Å RMSD. Backbone and side chain rotomers were optimized with Foldit over 2000 iterations to minimize energy to -603 REU. Figure 14C shows Ab 16-22 overlay with tau 304-310 in parallel, backbone atoms differ from each other by 0.54 Å RMSD. RMSD values were calculated using LSQ in Coot. Figures 15A-15B show schematics of tau. The VQIVYK (SEQ ID NO: 27) segment of tau harbors two aggregation-prone surfaces. Figure 15A shows (Top) Schematic of full-length Tau showing the positions of the VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) aggregation segments in the microtubule binding domain which contains four repeats (R1-4), together termed K18. Also shown are the domain boundaries of the cryoEM AD fibril core (13), and a modified K18 construct used in this work called K18+, which includes an 8 additional amino acids at the C- terminus to match the AD fibril core. (Bottom) ZipperDB (17) plot showing the aggregation propensity for each of the given hexapeptides shown in the blowup of the tau microtubule binding repeat. The vertical axis plots the energy of a steric zipper formed by a hexapeptide beginning with the residue above, and extending 5 additional amino acids to the right (N-to-C). Residues exceeding a predetermined threshold of - 23 kcal/mol are predicted to form steric zippers. Figure 15B shows the crystal structure of the SVQIVY (SEQ ID NO: 29) steric zipper segment determined in this work. The structure contains two different steric zippers formed on opposite faces of the peptide, a Class 1 interface that is created by the top two strands colored in tan and orange, and a Class 3 interface that is formed by the bottom two strands colored grey and orange. For both the Class 1 and 3 interfaces, the buried surface area (Ab) and shape complementarity (Sc) are shown in inset boxes.

Figures 16A-16D show disclosure that provides evidence that the Class 3 steric zipper interface is involved in the formation of disease-relevant tau fibrils. The Class 3 steric zipper interface maps to a region of unmodeled density in the tau PHF and SF. Overlay of the SVQIVY (SEQ ID NO: 29) crystal structure with the tau Figure 16(A) straight filament, SF, (PDB 5O3T) and Figure 16(B) paired helical filament, PHF, (PDB 5O3L). Residues VQIVY from a single protomer chain of the steric zipper were aligned to the same sequence of the respective cryoEM structures, and show that the mated strand that forms the Class 3 interface lays in a patch of unmodeled density present in both the SF and PHF cryoEM maps. Numbering along the backbone corresponds to residue positions that were modified in the panel of capping inhibitors of Table 1. Figure 16C shows seeding inhibition by VQIVYK (SEQ ID NO: 27) and VQIINK (SEQ ID NO: 28) capping inhibitors. Unlabeled fibrils of Tau40 were transfected into HEK293 biosensor cells that stably express P301S tau-K18 YFP. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor concentration, and IC50s were calculated by fitting dose-response curves. Figure 16D shows seeding following transient transfection of a PiggyBac vector encoding the WIW capping inhibitor peptide, or a scrambled peptide as a control, into tau biosensor cells.

Figures 17A-17E show disclosure from inhibition of seeding by AD- derived tau fibrils using the VQIVYK (SEQ ID NO: 27) and VQIINK (SEQ ID NO: 28) panel of capping inhibitors. Seeding in tau biosensor cells was induced by (Figure 17A and Figure 17B) crude brain extract or (Figure 17C) fibrils purified by size exclusion chromatography from donors with AD. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor. Figure 17D shows negative-stain electron micrograph of fibrils used for seeding in 17C. Figure 17E shows representative images from seeding inhibition experiments from 17B (seeded with crude brain extract), and 17C (seeded with purified AD fibrils). Red arrows indicate representative cells containing seeded tau aggregates, and white arrows point to representative cells lacking aggregated tau.

Figures 18A-18F show disclosure from inhibitor profiling studies. Figure 18(A) shows CTE-derived tau seeds from the temporal cortex, Figure 18(B) shows recombinant tau K18+ that was seeded with CTE-derived tau seeds, and Figure 18(C) shows recombinant tau K18+ fibrils aggregated with heparin. Seeding inhibition measurements for the seeded recombinant fibrils from Figure 18B were carried out in tau biosensor cells after 3 sequential rounds of in vitro seeding. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor. In Figures 18A-C, red arrows were used to mark capping inhibitors that were effective at blocking seeding by CTE-derived tau from crude brain extracts, blue arrows mark inhibitors effective at blocking seeding by recombinant tau fibrils, and a purple arrow marking IN-W3 in Figure 18B emphasizes that it is the only of the different inhibitors that blocks seeding by both CTE-derived tau and recombinant tau fibrils. Figures 18D-18F show representative images from Figures 18A, 18B and 18C, respectively showing seeding and inhibition in tau biosensor cells. Red arrows indicate representative cells containing seeded tau aggregates, and white arrows point to representative cells lacking aggregated tau.

Figures 19A-19K show disclosure from inhibitor profiling studies. Inhibitor profiling in biosensor cells seeded by brain extract from 4 different PSP donors. Figure 19A shows tissue sections from donors 1 and 2 were harvested from the midbrain, and from the locus coeruleus for donor 3. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor. VQIINK (SEQ ID NO: 28) inhibitors showing greater than 70% inhibition are highlighted on bar graphs with a red outline. Figure 19B shows seeding by extracts from PSP donors 1, 2 and 3 after treatment with the capping peptide W4. Figure 19C shows representative images from a showing seeding and inhibition in tau biosensor cells. Red arrows indicate representative cells containing seeded tau aggregates, and white arrows point to representative cells lacking aggregated tau. Figure 19D and 19E show that, as in Figure 19A, except tissue sections came for a two different brain regions, the cerebellum (Figure 19D) or frontal cortex (Figure 19E), of a 4^th PSP donor. Figures 19F-19H show representative images from Figure 19D. Figures 19I- 19K show representative images from 19E.

Figure 20 provides a schematic summary of sensitivities to panel of capping peptides measured by seeding inhibition using extracts from donors with different tauopathies. The top panel of cartoons shows locations of segments in tau targeted by different inhibitors of the panel, and crystal structures of corresponding interfaces. The Table shows efficacies of respective inhibitors for each donor tested in this study. For this analysis, inhibitors were scored as effective (filled box) if seeding was inhibited by 50% or more. Otherwise inhibitors were scored ineffective (open box).

Figures 21A-21C provide photos and schematics of tau. Figure 21A: Left panel– microcrystals of SVQIVY (SEQ ID NO: 29) in hanging drop crystallization screens. Middle panel– Electron micrograph of SVQIVY (SEQ ID NO: 29) microcrystals (Scale bar 1 micron). Right panel– representative electron diffraction image from SVQIVY (SEQ ID NO: 29) microcrystals. Figure 21B: Refined atomic model for SVQIVY (SEQ ID NO: 29) shown with 2Fo-Fc map (grey) and Fo-Fc map (green and red). Figure 21C: Alignment of VQIVYK (SEQ ID NO: 27) (2ON9) and Class 1 interface of SVQIVY (SEQ ID NO: 29).

Figures 22A-22D provide schematics of tau. Class 1 steric zipper structures formed by the VQIVYK (SEQ ID NO: 27) segment from (Figure 22A) peptide crystal structure SVQIVY (SEQ ID NO: 29), and (Figure 22C - Figure 22D) cryoEM fibril structures from PiD (6GX5) and AD (5O3L and 5O3T). The VQIVYK (SEQ ID NO: 27) strand is colored yellow, and the mated sheet, which varies in sequence in the different structures, is colored magenta.

Figure 23 shows seeding inhibition in tau biosensor cells by titration of the D-peptide TLKIVW (SEQ ID NO: 30) into recombinant fibrils of tau40 prepared using heparin. No inhibition of seeding was observed up to a concentration of 50 mM peptide.

Figure 24 provides data from seeding inhibition studies with crude brain extract from two different donors (left and right panels) with AD. Seeding was measured by counting the number of fluorescent puncta in tau biosensor cells after transfecting with crude brain lysate, and seeding inhibition was determined by plotting the number of counted aggregates when crude brain lysate was pre-treated with the indicated capping inhibitor at a final concentration of 10 mM. Figure 25 shows an inhibitor profile of tau biosensor cells seeded by crude brain extract from a donor with CBD. Seeding inhibition was measured by counting the number of fluorescent puncta as a function of inhibitor.

Figures 26A-26F provide data showing that seeding by soluble oligomers of tau are inhibited by SVQIVY (SEQ ID NO: 29)- and VQIINK (SEQ ID NO: 28)- based capping inhibitors. Figure 26A shows shaking recombinant tau-K18 with ionic liquid 15 (Hampton Research) allows for slower aggregation compared to heparin. Figure 26B and Figure 26C show endpoints of solutions aggregated with IL15 produce fibrils, as shown for the IL15 endpoint of (Figure 26B) Tau40 and (Figure 26C) tau-K18. Figure 26D shows data from studies of aliquots removed between 16-20 hours for tau-K18 with IL15 were subjected to size exclusion chromatography on a superdex 200 column and result in 2 peaks. Figure 26E shows the early eluting species has immunoreactivity to the oligomer antibody A11, whereas the late eluting species corresponding to the tau monomer lacks A11 immunoreactivity. Oligomers purified by size exclusion chromatography of (Figure 26E) tau-K18 and (Figure 26F) tau-K18+ show similar sensitivity to the panel of SVQIVY (SEQ ID NO: 29) and VQIINK (SEQ ID NO: 28) based capping, suggesting that IL15 induces on-pathway oligomers, and that these soluble species share characteristics that are similar to the fibril. DETAILED DESCRIPTION OF THE INVENTION

In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

Alzheimer’s disease (AD) pathology is characterized by plaques of amyloid beta (Ab) and neurofibrillary tangles of tau. Ab aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline. Here, we report peptide-based inhibitors that reduce Ab and/or tau aggregation and toxicity of already-aggregated species, and methods of using these peptide-based inhibitors.

The invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, a composition of matter comprising at least one peptide inhibitor of Ab aggregation wherein the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10); and the at least one peptide inhibitor comprises at least one D-amino acid. Optionally the composition comprises a plurality of peptide inhibitors of Ab aggregation. In some embodiments of the compositions of the invention, the peptide inhibitor sequence comprises less than 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids. In certain embodiments of the invention, the peptide inhibitor sequence is coupled to a plurality of heterologous amino acids, for example a linker amino acid sequence and/or a cell penetrating peptide (CPP) amino acid sequence. Optionally the composition further comprises an Ab polypeptide (SEQ ID NO: 2).

Another embodiment of the invention is a composition of matter comprising a polynucleotide encoding at least one peptide inhibitor of Ab aggregation wherein the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10). Typically, these polynucleotides are disposed within a vector selected for its ability to express the peptide inhibitor in a mammalian cell. In certain embodiments of the invention, the composition comprises a vector disposed within a mammalian cell.

Another embodiment of the invention is a method of inhibiting aggregation of Ab polypeptides, the method comprising combining Ab polypeptides with at least one peptide inhibitor of Ab aggregation that is disclosed herein; and then allowing the at least one peptide inhibitor of Ab aggregation to bind to the Ab polypeptides, such that that Ab aggregation is inhibited. In certain embodiments of these methods, the at least one peptide inhibitor of Ab aggregation used in the method is selected for an ability to inhibit cross-seeding of tau by Ab polypeptides; and/or to inhibit tau homotypic seeding. In certain embodiments, the method uses a plurality of peptide inhibitors of Ab aggregation. Optionally the at least one peptide inhibitor of Ab aggregation is combined with Ab and/or Tau in vivo. In certain such embodiments, inhibiting formation of Ab aggregates in vivo inhibits development or progression of a Ab plaque formation in an individual; and/or inhibiting homotypic seeding of Tau fibrils in vivo inhibits development or progression of a tauopathy in an individual.

Yet another embodiment of the invention is a composition of matter comprising at least one peptide inhibitor of tau aggregation wherein the at least one peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W- I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I- N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W- M-I-N-K-K-R-K (SEQ ID NO: 18). Typically, the composition comprises a plurality of peptide inhibitors of tau aggregation; and/or the composition comprises a pharmaceutical excipient selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar or a pH adjusting agent. In some embodiments of the compositions of the invention, the peptide inhibitor sequence comprises less than 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids. In certain embodiments of the invention, the peptide inhibitor sequence is coupled to a plurality of heterologous amino acids, for example a linker and/or a cell penetrating peptide (CPP). Optionally the composition further comprises a tau polypeptide (SEQ ID NO: 1).

Another embodiment of the invention is a composition of matter comprising a polynucleotide encoding a peptide inhibitor of tau aggregation wherein the peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V- Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I-N-K-K-L- K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W-M-I-N-K- K-R-K (SEQ ID NO: 18). In typical embodiments, the polynucleotide is disposed within a vector selected for its ability to express the peptide inhibitor in a mammalian cell. Optionally the composition comprises the vector is disposed within a mammalian cell.

Yet another embodiment of the invention is a method of inhibiting aggregation of tau polypeptides comprising combining tau polypeptides with a composition comprising at least one peptide inhibitor of tau aggregation that is disclosed herein; and then allowing the at least one peptide inhibitor of tau aggregation to bind to the tau polypeptides, such that tau aggregation is inhibited. In certain embodiments of these methods, the at least one peptide inhibitor of tau aggregation used in the method is selected for an ability to inhibit seeding of purified tau fibrils and/or to inhibit seeding of tau fibrils present in unpurified brain extracts. In some embodiments of the invention, the at least one peptide inhibitor of tau aggregation is combined with Tau in vivo, for example in methods designed to inhibit seeding of Tau fibrils in vivo so as to inhibit development or progression of a tauopathy in an individual. In certain embodiments of the invention, the method uses a plurality of peptide inhibitors of tau aggregation. Optionally, the at least one peptide inhibitor of tau aggregation is coupled to a plurality of heterologous amino acids.

As noted above, in certain embodiments of the invention, a tau and/or Ab inhibitory peptide disclosed herein is coupled to heterologous amino acids such as a cell penetrating peptide (CPP) amino acid sequence, typically one less than 30 amino acids in length. Optionally the peptide is coupled to the heterologous amino acids by a peptide linker comprising 1-7 amino acids. In some embodiments of the invention, the heterologous amino acids forms a polycationic structure. In other embodiments of the invention, the heterologous amino acids forms an amphipathic structure. Embodiments of the invention include peptides wherein the peptide comprises at least one D amino acid (e.g. a peptide comprising all D-amino acids). Embodiments of the invention can further compare metabolic stability and efficacy of L- and D form peptide inhibitors.

Embodiments of the invention include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein by chemical or biological (genetic) means to proteins or peptides to carry the peptide inhibitor across the blood-brain-barrier (BBB), as a therapy for neurodegeneration or movement disorder. For example, embodiments of the invention include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to a cell penetrating peptide and then coupling the resulting peptide by chemical or biological (genetic) means to proteins or peptides to carry the inhibitor across the blood-brain-barrier (BBB), as a therapy for neurodegeneration or movement disorder. Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to small molecules that aid in the ability of the peptide inhibitors to cross the BBB and/or cell membranes. Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to enzymes or small molecules (e.g. fluorophores) to aid in the diagnosis of pathology (ante and/or postmortum). Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein to small molecules that aid in the ability of the peptide inhibitors to degrade amyloid aggregates.

Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein by chemical means to a nano-particle capable of crossing the BBB and possibly also entering into cells, for example for use as a therapy for neurodegenation or movement disorder. Embodiments of the invention also include coupling of any of a tau and/or Ab inhibitory peptides disclosed herein by chemical means to a metal-containing nano-particle capable of crossing the BBB to create a diagnostic/biomarker for MRI or PET diagnosis of neurodegenerative disease or movement disorders (e.g. Parkinson's disease).

Embodiments of the invention also include insertion of any of a tau and/or Ab inhibitory peptides disclosed herein into the CDRs of an antibody to produce inhibitors of greater potency and/or specificity. The antibody can be a full antibody, and Fab domain, or a single-chain antibody. Coupling of the resulting antibody to a nano-particle provides embodiments of the invention useful for therapy or diagnosis as noted above.

An inhibitory peptide or CPP inhibitor of the invention can be synthesized (e.g., chemically or by recombinant expression in a suitable host cell) by any of a variety of art-recognized methods. In order to generate sufficient quantities of an inhibitory peptide for use in a method of the invention, a practitioner can, for example, using conventional techniques, generate nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of an expression control sequence such as a promoter or an enhancer, which can then direct the synthesis of the peptide. For example, one can (a) synthesize the DNA de novo, with suitable linkers at the ends to clone it into the vector; (b) clone the entire DNA sequence into the vector; or (c) starting with overlapping oligonucleotides, join them by conventional PCR-based gene synthesis methods and insert the resulting DNA into the vector. Suitable expression vectors (e.g., plasmid vectors, viral, including phage, vectors, artificial vectors, yeast vectors, eukaryotic vectors, etc.) will be evident to skilled workers, as will methods for making the vectors, inserting sequences of interest, expressing the proteins encoded by the nucleic acid, and isolating or purifying the expressed proteins. In illustrative embodiments of the invention, capping inhibitor peptides are loaded into adeno-associated virus (AAV) capsids and hydrogel-based polymers to mediate delivery across the blood brain barrier

One aspect of the invention is a method for reducing or inhibiting Tau aggregation, comprising contacting Tau amyloid protofilaments with an effective amount of one or more of the inhibitory peptides or CPP inhibitors of the invention. Such a method can be carried out in vitro (in solution) or in vivo (e.g. cells in culture or in a subject). Another aspect of the invention is a method for restoring the conformation of a Tau protein molecule having an aberrant conformation. An “aberrant conformation,” as used herein, refers to a conformation which is different from the wild type conformation, and which results in a loss of function of the molecule. Such aberrant conformation is sometimes referred to herein as pathological conformation. The aberrant conformation can take the form of amyloid aggregates or fibers (fibrils) of Tau molecules with other Tau molecules or with other proteins. Alternatively, the aberrant conformation can take the form of misfolding (e.g., partial or complete unfolding) of the Tau protein due to mutations or other factors. In this method for restoring the conformation of a Tau protein having an aberrant conformation, the Tau molecule having the aberrant conformation is contacted with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention. The contacted Tau molecule has a restored conformation, and exhibits a restored or reactivated biological or biochemical activity. Another aspect of the invention is a method for reducing or inhibiting Ab aggregation, comprising contacting Ab proteins with an effective amount of one or more of the inhibitory peptides or CPP inhibitors of the invention. Such a method can be carried out in vitro (in solution) or in vivo (e.g. cells in culture or in a subject). Another aspect of the invention is a method for restoring the conformation of a Ab protein molecule having an aberrant conformation. An“aberrant conformation,” as used herein, refers to a conformation which is different from the wild type conformation, and which results in a loss of function of the molecule. Such aberrant conformation is sometimes referred to herein as pathological conformation. The aberrant conformation can take the form of amyloid aggregates or fibers (fibrils) of Ab molecules with other Ab molecules or with other proteins. Alternatively, the aberrant conformation can take the form of misfolding (e.g., partial or complete unfolding) of the Ab protein due to mutations or other factors. In this method for restoring the conformation of a Ab protein having an aberrant conformation, the Ab molecule having the aberrant conformation is contacted with an effective amount of an inhibitory peptide or a CPP inhibitor of the invention. The contacted Ab molecule has a restored conformation, and exhibits a restored or reactivated biological or biochemical activity.

Another aspect of the invention is a method for reactivating or restoring a biological or biochemical activity (function) of Tau and/or Ab which results from aberrant conformation of the Tau and/or Ab proteins. The method comprises contacting the Tau and/or Ab protein molecule having an aberrant conformation with an effective amount of an inhibitor peptide or CPP inhibitor of the invention. As a result of contacting the Tau and/or Ab protein having the aberrant conformation, the lost biological or biochemical activity of the Tau and/or Ab molecule is reactivated or restored.

Another aspect of the invention is a method for inhibiting or preventing a loss of a biological or biochemical activity (function), of a Tau and/or Ab protein which results from aberrant conformation of the Tau and/or Ab protein. The method comprises contacting the Tau and/or Ab protein molecule having an aberrant conformation with an effective amount of an inhibitor peptide or CPP inhibitor of the invention. As a result of contacting the Tau and/or Ab protein having the aberrant conformation, the loss of activity of the Tau and/or Ab molecule is inhibited or prevented.

Another aspect of the invention is a method for treating a subject having a disease or condition which is mediated by loss of function of Tau and/or Ab, such as a pathological syndrome in which Tau and/or Ab has an abnormal conformation (e.g. is aggregated or misfolded). That is, the pathological syndrome is associated with Tau and/or Ab having an aberrant conformation. The method comprises administering to the subject an effective amount of one or more peptides of the invention. In some embodiments, a cocktail of two of more of the peptides or CPP inhibitor peptides is used.

Yet another embodiment of the invention is a method of observing the presence or absence of Tau amyloid fibrils and/or Ab in a biological sample comprising combining a biological sample with a peptide disclosed herein that binds to Tau or Ab, allowing the peptide to bind to Tau amyloid fibrils and/or Ab that may be present in the biological sample, and then monitoring this combination for the presence of complexes formed between Tau amyloid fibrils and/or Ab and the peptide; wherein the presence of said complexes show the presence of Tau amyloid fibrils and/or Ab in the biological sample. Optionally in this method, the presence of complexes formed between Tau amyloid fibrils and/or Ab and the peptide is monitored using a detectable label that is coupled to the peptide (e.g. a heterologous peptide tag). Typically, the method is performed on a biological sample obtained from an individual suspected of suffering from a tauopathy. Such embodiments of the invention can be used, for example, in diagnostic methods designed to observe the presence or status of Alzheimer’s disease, for example to detect disease beginnings before clinical symptoms, and to follow the effectiveness (or lack of effectiveness), of a therapeutic treatment.

Peptide inhibitors of the invention bind specifically (selectively, preferentially) to Tau and/or Ab rather than to unintended proteins. The protein to which the peptide inhibitor binds may be, e.g., a monomer, small aggregate, oligomer, or fibril. For example, the binding can be 2 times, 5 times, 10 times, 100 times or 200 times stronger, or no binding at all can be detected to an unintended target. Conventional methods can be used to determine the specificity of binding, such as e.g. competitive binding assays or other suitable analytic methods.

Active variants of the inhibitory peptides described above are also included. An“active variant” is a variant which retains at least one of the properties of the inhibitory peptides described herein (e.g., the ability to bind to Tau and/or Ab and/or to block, inhibit or prevent Ab or Tau fibrillation (aggregation) and/or Tau cytotoxicity). Fibrilization, as used herein, refers to the formation of fiber or fibrils, such as amyloid fibrils.

Suitable active variants include peptidomimetic compounds (any compound containing non-peptidic structural elements that is capable of mimicking the biochemical and/or biological action(s) of a natural mimicked peptide), including, for example, those designed to mimic the structure and/or binding activity (such as, for example, hydrogen bonds and hydrophobic packing interactions) of the peptides according to the methods disclosed herein). Inhibitory peptides of the invention, including active variants thereof, are sometimes referred to herein as“peptidic compounds” or“compounds.”

In one embodiment, active variants of the inhibitory peptides are shortened by 1-3 (e.g., 1, 2 or 3) amino acids at either the N-terminus, the C-terminus, or both of the starting inhibitory peptide. In another embodiment, the active variants are lengthened (extended) by 1, 2, 3 or 4 amino acids at the C-terminal end of the starting inhibitory peptide, e.g. with amino acid residues at the position in which they occur in Tau and/or Ab.

A variety of other types of active variants are included in embodiments of the invention. In some embodiments, amino acids other than the ones noted above are substituted. These amino acids can help protect the peptide inhibitors against proteolysis or otherwise stabilize the peptides, and/or contribute to desirable pharmacodynamic properties in other ways. In some embodiments, the non-natural amino acids allow an inhibitor to bind more tightly to the target because the side chains optimize hydrogen bonding and/or apolar interactions with it. In addition, non- natural amino acids offer the opportunity of introducing detectable markers, such as strongly fluorescent markers which can be used, e.g., to measure values such as inhibition constants. Also included are peptide mimetics, such as, e.g., peptoids, beta amino acids, N-ethylated amino acids, and small molecule mimetics.

In one embodiment, non-natural amino acids are substituted for amino acids in the sequence. More than 100 non-natural amino acids are commercially available. These include, for example,

Non-natural amino acids which can substitute for LEU:

L - cyclohexylglycine 161321 - 36 - 4

L - phenylglycine 102410 - 65 - 1

4 - hydroxy - D - phenylglycine 178119-93-2

L - Į - t - butylglycine 132684 - 60 - 7

cyclopentyl - Gly - OH 220497 - 61– 0\

Non-natural amino acids which can substitute for THR:

Thr(tBu)-OH 71989-35-0

(RS) - 2 - amino - 3 - hydroxy - 3 - 105504 - 72 - 1

methylbutanoic acid

Non-natural amino acids which can substitute for ILE: allo-Ile-OH 251316-98-0

N-Me-allo-Ile-OH 136092-80-3

Homoleu-OH 180414-94-2

Non-natural amino acids which can substitute for ARG:

N^ - nitro - L - arginine 58111-94-7

L - citrulline 133174 - 15 - 9

Non-natural amino acids which can substitute for TYR:

3 - amino - L - tyrosine 726181-70-0

3 - nitro - L - tyrosine 136590 - 09 - 5

3 _{- methoxy - L - tyrosine}

3 - iodo - L - tyrosine 134486 - 00 - 3

3-chloro-L-tyrosine 478183-58-3

3,5-dibrimo- L- tyrosine 201484-26-6

Non-natural amino acids which can substitute for LYS:

Lys(retro-Abz-)-OH 159322-59-5

Lys(Mca)-OH 386213-32-7

(Nį - 4 - methyltrityl) - L - 343770-23-0

ornithine

N - Į - - N - İ - (d - Biotin) - L - 146987 - 10 - 2

lysine In another embodiment, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of the L-amino acids are substituted with a D amino acid. In another embodiment, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) N-methylated residues are included in the peptide. An inhibitory peptide of the invention can comprise, e.g., L-amino acids, D- amino acids, other non-natural amino acids, or combinations thereof.

Active variants include molecules comprising various tags at the N-terminus or the C-terminus of the peptide (e.g. tags comprising a stretch of heterologous amino acids). For example, an inhibitory peptide of the invention can comprise as tags at its N-terminus and/or at its C-terminus: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Lysine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Arginine residues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Glutamate residues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Aspartate residues; combinations of these amino acid residues; or other polar tags that will be evident to a skilled worker. Other active variants include mutations of the Tau and/or Ab sequence which increase affinity of the inhibitory peptides for Tau and/or Ab.

In one embodiment of the invention, an inhibitory peptide of the invention is isolated or purified, using conventional techniques such as the methods described herein. By“isolated” is meant separated from components with which it is normally associated, e.g., components present after the peptide is synthesized. An isolated peptide can be a cleavage product of a protein which contains the peptide sequence. A “purified” inhibitory peptide can be, e.g., greater than 90%, 95%, 98% or 99% pure.

In one embodiment, to enhance the cell permeability of an inhibitory peptide of the invention, the peptide is fused to any of a variety of cell penetrating peptides (CPPs). CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non- polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPP’s are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake. Some typical CPP’s that can be fused to an inhibitory peptide of the invention are provided in Table Alpha below. Table Alpha

Name Sequence

polyARG nR where 4<n<17 (e.g., n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) (SEQ ID NO:19) polyLYS nK where 4<K<17 (e.g., K=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) D-polyARG nR where 4<n<17 (e.g., n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) D-polyLYS nK where 4<K<17 (e.g., K=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) SynB1 RGGRLSYSRRRFSTSTGR (SEQ ID NO:20)

SynB3 RRLSYSRRRF (SEQ ID NO:21)

Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO:22)

PenArg RQIRIWFQNRRMRWRR (SEQ ID NO:23)

PenLys KQIKIWFQNKKMKWKK (SEQ ID NO:24)

TatP59W GRKKRRQRRRPWQ (SEQ ID NO:25)

Tat (48-60) GRKKRRQRRRPPQ (SEQ ID NO:26)

R9-Tat GRRRRRRRRRPPQ (SEQ ID NO:27)

Tat YGRKKRRQRRR (SEQ ID NO:28)

D-Tat GRKKRRQRRRPPQ (SEQ ID NO:23).

BMVGag(7-25) KMTRAQRRAAARRNRWTAR (SEQ ID NO:24) Other represntative CPPs useful in embodiments of the invention are found, for example in WO 2018/005867, the contents of which are incorporated herein by refernce.

In typical embodiments of the invention, the CPP comprises a plurality of arginine residues (e.g. R1-16). In general, it is advisable that the length of the CPP is rather short, e.g. less than about 30 amino acids, in order to improve stability and pharmacodynamic properties once the molecule enters a cell. In some embodiments, the CPP is directly attached (fused) to a peptide of the invention. In other embodiments, it is desirable to separate the highly charged CPP from the inhibitor peptide with a linker, to allow the inhibitor to retain its activity. Any of a variety of linkers can be used. The size of the linker can range, e.g., from 1-7 or even more amino acids (e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids).

In embodiments of the invention, the inhibitory peptide can be detectably labeled. Labeled peptides can be used, e.g., to better understand the mechanism of action and/or the cellular location of the inhibitory peptide. Suitable labels which enable detection (e.g., provide a detectable signal, or can be detected) are conventional and well-known to those of skill in the art. Suitable detectable labels include, e.g., radioactive active agents, fluorescent labels, and the like. Methods for attaching such labels to a protein, or assays for detecting their presence and/or amount, are conventional and well-known.

An inhibitory peptide of the invention can be synthesized (e.g., chemically or by recombinant expression in a suitable host cell) by any of a variety of art- recognized methods. In order to generate sufficient quantities of an inhibitory peptide for use in a method of the invention, a practitioner can, for example, using conventional techniques, generate nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of an expression control sequence such as a promoter or an enhancer, which can then direct the synthesis of the peptide. For example, one can (a) synthesize the DNA de novo, with suitable linkers at the ends to clone it into the vector; (b) clone the entire DNA sequence into the vector; or (c) starting with overlapping oligonucleotides, join them by conventional PCR-based gene synthesis methods and insert the resulting DNA into the vector. Suitable expression vectors (e.g., plasmid vectors, viral, including phage, vectors, artificial vectors, yeast vectors, eukaryotic vectors, etc.) will be evident to skilled workers, as will methods for making the vectors, inserting sequences of interest, expressing the proteins encoded by the nucleic acid, and isolating or purifying the expressed proteins.

Another aspect of the invention is a pharmaceutical composition comprising one or more of the inhibitory peptides and a pharmaceutically acceptable carrier. Optionally, the components of the pharmaceutical composition can be detectably labeled, e.g. with a radioactive or fluorescent label, or with a label, for example one that is suitable for detection by positron emission spectroscopy (PET) or magnetic resonance imaging (MRI). For example, peptides of the invention can be coupled to a detectable label selected from the group consisting of a radioactive label, a radio- opaque label, a fluorescent dye, a fluorescent protein, a colorimetric label, and the like. In some embodiments, the inhibitory peptide is present in an effective amount for the desired purpose. The compositions may contain preservatives and/or antimicrobial agents as well as pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.

"Pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use. For example,“pharmaceutically acceptable salts" of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound.

Another aspect of the invention is a polynucleotide encoding an inhibitory peptide of the invention. In embodiments of the invention, the polynucleotide is operably linked to a regulatory control sequence (e.g., a promoter or an enhancer) to facilitate production of the encoded protein following introduction (e.g. by transfection) into a suitable cell. Other embodiments include a cell comprising the expression vector; and a method of making an inhibitory peptide of the invention comprising cultivating the cell and harvesting the peptide thus generated.

Another aspect of the invention is a kit for carrying out any of the methods described herein. The kit may comprise a suitable amount of an inhibitory peptide of the invention; reagents for generating the peptide; reagents for assays to measure their functions or activities; or the like. Kits of the invention may comprise instructions for performing a method. Other optional elements of a kit of the invention include suitable buffers, media components, or the like; a computer or computer-readable medium providing the structural representation of a crystal structure described herein; containers; or packaging materials. Reagents for performing suitable controls may also be included. The reagents of the kit can be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in single reaction form for administering to a subject.

Characterization of candidate inhibitory peptides of the invention can be carried out by any of a variety of conventional methods. For example, the peptides can be assayed for the ability to reduce or inhibit Tau and/or Ab aggregation or cytotoxicity or cell-to-cell spread. The assays can be carried out in vitro or in vivo. Suitable assays will be evident to a skilled worker; some suitable assays are described herein.

One aspect of the invention is a method for reducing or inhibiting Tau and/or Ab aggregation, comprising contacting Tau and/or Ab proteins with an effective amount of one or more of the inhibitory peptides of the invention. Such a method can be carried out in solution or in a cell (e.g. cells in culture or in a subject).

Another aspect of the invention is a method for treating a subject having a disease or condition which is mediated by the presence of fibrillated Tau (sometimes referred to herein as a Tauopathy or a Tau-mediated disease or condition), comprising administering to the subject an effective amount of an inhibitory peptide or pharmaceutical composition of the invention. Among such diseases or conditions are, e.g., Alzheimer’s disease. Another aspect of the invention is a method to prevent the onset of such diseases or conditions (e.g., Alzheimer’s disease), or to treat a subject in the early stages of such diseases or conditions, or that is developing such a disease or condition, in order to prevent or inhibit development of the condition or disease.

An inhibitory peptide or pharmaceutical composition of the invention is sometimes referred to herein as an“inhibitor.” An“effective amount” of an inhibitor of the invention is an amount that can elicit a measurable amount of a desired outcome, e.g. inhibition of Tau and/or Ab aggregation or cytotoxicity; for a diagnostic assay, an amount that can detect a target of interest, such as an Tau and/or Ab aggregate; or in a method of treatment, an amount that can reduce or ameliorate, by a measurable amount, a symptom of the disease or condition that is being treated.

A“subject” can be any subject (patient) having aggregated (fibrillated) Tau and/or Ab molecules associated with a condition or disease which can be treated by a method of the present invention. In one embodiment of the invention, the subject has Alzheimer’s disease. Typical subjects include vertebrates, such as mammals, including laboratory animals, dogs, cats, non-human primates and humans.

The inhibitors of the invention can be formulated as pharmaceutical compositions in a variety of forms adapted to the chosen route of administration, for example, orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into tissue.

Suitable oral forms for administering the inhibitors include lozenges, troches, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.

The inhibitors of the invention can be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or insufflation. They can be enclosed in coated or uncoated hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the compounds can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For compositions suitable for administration to humans, the term "excipient" is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (hereinafter Remington's). The inhibitors can be combined with a fine inert powdered carrier and inhaled by the subject or insufflated. Such compositions and preparations should contain at least 0.1% compounds. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of a given unit dosage form.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.

Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac or sugar and the like. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.

In addition, the inhibitors can be incorporated into sustained-release preparations and devices. For example, the inhibitors can be incorporated into time release capsules, time release tablets, and time release pills. In some embodiments, the composition is administered using a dosage form selected from the group consisting of effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.

The inhibitors may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the inhibitors can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the compounds which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.

Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Other solid carriers include conventional nontoxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols or water/alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Useful dosages of the peptides or pharmaceutical compositions of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. For example, the concentration of the compounds in a liquid composition, such as a lotion, can be from about 0.1-25% by weight, or from about 0.5-10% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder can be about 0.1-5% by weight, or about 0.5- 2.5% by weight.

Effective dosages and routes of administration of agents of the invention are conventional. The exact amount (effective dose) of the agent will vary from subject to subject, depending on, for example, the species, age, weight and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like. A therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an, effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutic dose can also be selected by analogy to dosages for comparable therapeutic agents.

The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.

In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg of body weight per day, such as above about 0.1 mg per kilogram, or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day. For example, a suitable dose can be about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 30 mg/kg of body weight per day.

The inhibitors are conveniently administered in unit dosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form. In some embodiments, the dosage unit contains about 0.1 mg, about 0.5 mg, about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg, of active ingredient. EXAMPLES

The Examples below provide illustrative methods and materials that can be used in the practice the various embodiments of the invention disclosed herein. EXAMPLE 1: STRUCTURE BASED INHIBITORS OF AMYLOID BETA CORE SUGGEST A COMMON INTERFACE WITH TAU

Alzheimer’s disease (AD) pathology is characterized by plaques of amyloid beta (Ab) and neurofibrillary tangles of tau. Ab aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline. Here, we report the crystal structure of an Ab core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Ab aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Ab-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Ab and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.

Although Alzheimer’s disease (AD) is the most prevalent form of dementia, there are limited treatments to alleviate symptoms and none that halt its progression. Histological features of AD are extracellular senile plaques of amyloid beta (Ab) and intracellular neurofibrillary tangles of tau (1, 2). While Ab aggregation is thought to occur at the early stages of AD, tau aggregation correlates better to disease progression, with characteristic spreading along linked brain areas, and severity of symptoms correlating to the number of observed inclusions (3–8) . Structural information about the aggregated forms of Ab and tau is accumulating, but to date this knowledge has not led to successful chemical interventions (9)

A link between the appearance of Ab and tau pathologies has been noted in transgenic mouse models generated by crossing or co-expressing mutant Ab and mutant tau, but the mechanism is not yet understood at the molecular level (10). By injecting Ab seeds derived from synthetic peptide, transgenic mouse or AD patient tissue, tau pathology can be found both at the site of injection, and also in functionally connected brain areas (11–13). Tau aggregation has also been reported to follow Ab seeding in 3D neuronal stem cell cultures that express early onset hereditary mutations to drive overproduction and aggregation of Ab (14). In spite of these observations, the molecular linkage of Ab to tau remains unresolved. Proposed hypotheses include Ab causing downstream cellular changes that trigger tau phosphorylation and eventual aggregation, and/or a direct interaction and seeding of tau by aggregated Ab (15, 16).

Several lines of evidence support the direct interaction model, although questions still remain; for example, how such an interaction could occur since Aȕ plaques deposit extracellularly, while tau neurofibrillary tangles are intracellular. One possible model for intracellular aggregation could be that Ab is cleaved from APP inside endosomes, and then exported (17). Another model proposes that smaller diffusible Ab oligomers are the toxic species (18–20); indeed oligomers of Ab isolated from AD serum are sufficient to induce tau aggregation (21). Ab has also been found co-localize intra-neuronally with tau as well as at synaptic terminals, with increased interactions correlating with disease progression (5). Furthermore, soluble and insoluble complexes of Ab bound to tau have been detected in AD tissue extracts (5, 22). In vitro, soluble complexes of Ab and tau have been found to promote aggregation of tau(22), while another study found that Ab fibrils can seed tau (23). Taking the evidence together, we hypothesize that cross-seeding of tau by Ab promotes tangle formation in AD, which could be prevented not only by inhibiting Ab aggregation, but also by disrupting the binding site of Ab with tau.

A number of interaction sites have been proposed on both proteins. In Ab, both the amyloid core KLVFFA (SEQ ID NO: 31), along with region spanning the carboxy terminal residues were found to bind tau (22). Conversely peptides from regions of tau in exons 7 and 9, well as aggregation prone sequences VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) located at the beginning of repeat 2 (R2) and repeat 3 (R3) of the microtubule domain (K18), respectively, were found to bind Ab (22). A computational seeding model predicts that the amyloid core of Ab can form intermolecular b-sheet interactions with VQIINK (SEQ ID NO: 28) or VQIVYK (SEQ ID NO: 27) (24).

On this basis, we hypothesized that an inhibitor capable of targeting the amyloid core, which itself is an important sequence for Ab aggregation (25–27), might block both Ab aggregation and tau seeding by Ab. However, this segment has been observed in multiple conformations in steric zipper structures (28) and fiber models (29–33), impeding structure-based inhibitor design. In an effort to characterize a toxic conformation of this sequence, we focused our efforts on determining the structure of the segment 16-26, containing the Iowa early onset hereditary mutation, D23N (34). Based on this structure, we designed several inhibitors and found that they indeed blocked aggregation of Ab, prevented cross- seeding of tau by Ab, and surprisingly, also blocked tau homotypic seeding. We suggest that the efficacy of these structure-based inhibitors against both proteins, but not other amyloid fibrils, implies there is a similar binding interface displayed on both Ab and tau aggregates, supporting the cross-amyloid cascade hypothesis in AD.

RESULTS

Atomic structure of Aȕ16-26 D23N determined using MicroED

With crystals only a few hundred nanometers thick, we used micro-electron diffraction (MicroED) to determine the structure of Ab residues 16-26 containing the hereditary mutation D23N, (Figure 1A), KLVFFAENVGS (SEQ ID NO: 26). The structure revealed pairs of anti-parallel b-sheets each composed of ~4000 strands, stacked into a fibril that spans the entire length of the crystal. Neighboring sheets are oriented face to back (Figure 1B, Table 1) defining a Class 7 steric zipper motif. In addition, the three C-terminal residues adopt an extended, non-b conformation which stabilizes the packing between steric zippers (Figure 8). The sheet-sheet interface is strengthened by interdigitating side chains, Lys 16, Val18, Phe20, Glu22 from the face of one strand, and Leu17, Phe19, and the N-terminus from the back of the other. The zipper has an extensive interface with a high shape complementarity of 0.76 and a total buried solvent accessible surface area of 258 Å2.

This structure is partly identical to that of a shorter peptide segment, Ab16-21, KLVFFA (SEQ ID NO: 31) (crystal form-I) (28), which we used successfully as a search model for molecular replacement. Both the longer and shorter segments have class 7 symmetry. However, the two segments differ in registry. The shorter segment maintains an in-register hydrogen bonding pattern while the longer segment is out-of- register. That is, the strands of Ab16-26 are tilted away from perpendicular to the fibril axis—a departure from canonical cross-b architecture. The antiparallel architecture and lack of registration of Ab16-26 suggest this “fibrillar” assembly has some characteristics of an amyloid oligomer. Structural studies of amyloid oligomers most frequently reveal anti-parallel b sheet architecture (35–37), whereas most fibril structures have revealed parallel (29, 30, 33, 38), with the exception of some short segments of Ab (39) and the early onset hereditary mutation D23N which leads to anti-parallel fiber deposition in plaques (31, 40). The out-of-register stacking of anti-parallel b strands has been proposed to be the defining trait of toxic oligomers (36, 41). The segment Ab16-22 has been proposed to be able to form such oligomers in silica (42). The structures of Ab16-21 and Ab16-26 may offer clues to designing inhibitors that impede both fibrillar and oligomeric assemblies. Efficacy of inhibitors of Aȕ aggregation designed against Aȕ 16-26 D23N

As the zipper motif observed in the atomic structure of Ab_16-26 D23N may be relevant to a variety of amyloid beta assemblies, we sought to use it to develop structure-based peptide inhibitors of Ab1-42. Our laboratory has developed a Rosetta based design strategy using steric zipper structures to design capping peptide inhibitors for a number of amyloid proteins implicated in disease (43–47). We chose to truncate our structure to residues 16-22 for the search model, omitting the residues not in the b strand. We threaded amino acids onto a capping b strand and minimized energies of sidechains. From our first round of design we chose 6 distinct inhibitor candidates; those that were identified as good candidates but containing strong amino acid similarities to other top inhibitors were discarded. Our initial pool of inhibitors contained four L-form peptides, 2 each of 6 and 8 amino acids length, termed L1-L4, and two D-peptides 6 amino acids long, termed D1 and D2.

We assessed the efficacy of the inhibitors at a 10 molar excess by testing if they prevented Ab_1-42 toxicity on Neuro-2a (N2a) cells, a mouse neuroblastoma cell line, (48). We measured cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye reduction (49, 50). Our toxicity assay revealed one inhibitor, D1 with the sequence (D)-LYIWVQ (SEQ ID NO: 3) , that was able to eliminate the toxic effect of Ab1-42 (Figure 2A, Table S1); none of the inhibitors were toxic to N2a cells alone (Figure 9). In our molecular model of the inhibitor, smaller hydrophobic residues of D1 mimic interactions with the fibril interface on one side of the peptide, which promotes recognition, (Figure 2B), while the other side of the peptide positions large aromatic residues between Ab residues, blocking possible further interactions (Figure 2C).

We focused on these key features of the inhibitor sequence for our second round of design and aimed to improve efficacy. We lengthened our peptides to extend over more of our available structure towards the carboxy-terminus and made conservative residue changes to the face containing smaller hydrophobic residues. We selected and tested 6 new designs. Of the six, four were eight amino acids long such that the inhibitor would extend over more of our crystal structure, which we called D1a-D1d. The additional two, termed D1e and D1f, were six amino acids long featuring slight sequence perturbations from D1 (Table S1). We identified 2 of the 8 amino acid long inhibitors, D1b and D1d, that were also effective at reducing AB1-42 toxicity at both a tenfold excess and at an equimolar ratio (Figure 9). We then tested these two inhibitors, as well as D1, across a range of concentrations with final concentrations ranging from 100 nM to 10 mM (Figure 2D, E). We found that all inhibitors elicited a dose dependent response, with all having an IC50 of less than 1uM. The 6 residue long inhibitors, D1e and D1g, also had a similar effect on toxicity reduction as D1, however they did not perform as well as D1 in additional characterization and were not explored further (Figure 10). The cognate negative peptide control, LC, the L-form peptide of inhibitor D1, did not reduce toxicity (Figure 2E). Reduction of toxicity by designed inhibitors is explained by a reduction of Aȕ_1-42 aggregation

We next sought to understand the mechanism by which our peptide inhibitors reduce the toxic effect of Ab1-42. We therefore assayed fibril formation to discern if this reduction of toxicity could be explained by reduced aggregation. We incubated Ab1-42 with our inhibitors at 10:1, 1:1, and 1:10 molar ratios and monitored fibril formation by thioflavin-T (ThT) fluorescence at 37 °C under quiescent conditions. We observe that all of our inhibitors reduce fibril formation in a dose dependent manner, while the negative control peptide, LC, does not (Figure 3A). The longer inhibitors, D1b and D1d, appear effective at an equimolar ratio. However, when assayed at higher concentrations, the inhibitors appear to self-assemble, but remain effective at reducing Ab_1-42 toxicity (Figure 10). After 72 hours, samples were taken for negative-stain TEM analysis, which confirmed the reduced abundance of Ab1-42 fibrils. D1b and D1d were more effective at reducing fibril formation than D1, although all three inhibitors showed near equal efficiency in reducing toxicity. Fibrils were observed in the equimolar ratio sample of Ab1-42 with D1, whereas the comparable samples with D1b and D1d did not contain fibrils. Inhibitors that were not efficient at preventing toxicity, such as D1a and D1c, were also less effective at blocking fiber formation (Figure 9,Figure 10).

Since oligomers, and not fibrils, are considered to be the more toxic species of Ab (18–21), we then investigated if our inhibitors affect the formation of oligomers or other cytotoxic Ab_1-42 species. We used conformational antibodies to probe samples of Ab_1-42 incubated with a 10-molar excess of inhibitor overnight at 37°C. Binding by oligomer specific conformational antibody A11-O9, a monoclonal variant of A11, was reduced by all of our inhibitors (Figure 3C, Figure 10). Additionally, the inhibitors reduced the abundance of Ab conformations recognized by antibodies mOC24, mOC64, mOC104, and mOC116. These antibodies bind fibrillar plaques from patient derived AD tissue and/or 3xTg-AD mouse tissue (51). Overall, these results indicate that our inhibitors may reduce disease relevant, and oligomeric conformations.

Inhibitors bind and reduce toxicity of Aȕ aggregates

As AD is only diagnosable long after Ab aggregation has initiated, we wondered if these inhibitors would not only prevent amyloid aggregates from forming, but also if they can reduce the toxic effect of already formed aggregates. First, we incubated 10 mM Ab at 37°C for 12hours to form oligomers, and then added inhibitors at various concentrations just prior to addition to N2a cells and assayed toxicity by MTT dye reduction. We found that adding the inhibitor to monomeric Ab1-42 prior to incubation had a marked difference from adding inhibitor to pre-formed Ab_1-42 oligomers. When co-incubated with monomeric Ab, the shorter D1 inhibitor was as effective as D1b and D1d at reducing toxicity; however, when added to pre-formed Ab assemblies, only the longer inhibitors D1b and D1d were effective at reducing toxicity. (Figure 4A). Both of the longer inhibitors could fully ameliorate toxicity of aggregates at 10 mM, but D1d is more potent, with effective reduction of toxicity to 1 mM. D1b differs from D1d only at amino acid positions 6 and 7. We suspect the difference in efficacy is conferred from residue 6, because both inhibitors contain positively charged residues at position 7, but at position 6 D1b contains a Gln while D1d has a much bulkier Trp. Our results indicate that while peptide inhibitors can both prevent aggregation initiation and block toxicity of aggregated assemblies, the latter appears to be more sensitive to slight perturbations in inhibitor composition.

We next performed TEM to determine if our inhibitors could disaggregate fibers, or if the fibers are being capped, as our inhibitor design would predict. We aggregated 10 mM Ab_1-42 for 72 hours at 37°C under shaking conditions, then added inhibitors at 100 mM and incubated overnight. As the fibers are still present, we presume that our inhibitors are indeed capping or coating the fibers at toxicity inducing interfaces, thus preventing further seeding or toxic effects (Figure 4B). We performed SPR to verify that our inhibitors bind to fibers. We find that the most potent inhibitor of aggregated assemblies, D1d, binds to Ab_1-42 fibrils with an apparent K_d of 46 mM (Figure 4C, Figure 11). We used a one-inhibitor-to-one-protein substrate model to fit the data; however the true Kd may be lower due to the complication of D1d self-interaction and polymorphic Ab fibrils. Thus, we have shown that inhibitors D1b and D1d not only prevent aggregation of monomeric Ab, but also bind aggregated states.

Inhibitors reduce seeding of tau by aggregated Aȕ1-42

Having demonstrated that our inhibitors block a toxic interface on Ab, we next questioned if this interface could also be involved in cross seeding tau. First, we sought to validate the direct seeding mechanism that has been reported by others (22, 24, 52). We tested seeding of full length tau (tau40) in a ThT assay at 37 °C under shaking conditions and found that fibrils of Ab_1-42 seeded aggregation as efficiently as fibrils of the microtubule binding domain of tau (K18) (Figure 5A, Figure 12). Conversely, K18 was unable to seed Ab (Figure 12).

Next, we tested seeding in a well-established HEK293 biosensor cell line, tau- K18 (P301S) EYFP, which stably expresses the microtubule binding domain of tau P301S mutant. This cell line, referred to hereafter as tau-K18 biosensor cells, has been used to demonstrate prion like seeding from transfected tau fibrils to cells and has been used as a model system to test tau inhibitors (44, 53). We transfected biosensor cells with tau40 or Ab fibrils to a final concentration of 250 nM. We found that Ab was able to produce intracellular aggregates significantly greater than the vehicle alone, but only at around 2.5% efficiency of tau40. It is not altogether surprising that Ab has such a low efficiency of cross-seeding; this mirrors a previous result in a similar system (23). It is possible that tau fibrils contain multiple polymorphs and interfaces capable of homotypic seeding, whereas Ab may have a more limited number of tau seeding-competent conformations. Additionally, in vitro Ab aggregation may create disproportionate ratios of assemblies compared to those present in AD. Regardless, it remains that some Ab species is tau-seeding competent. The finding that Ab is indeed able to seed aggregation in tau-K18 expressing cell lines suggests that the cross-interacting region of tau is located on this microtubule binding domain.

We found other amyloid protein fibrils are not seeding-competent in this system (Figure 12), indicating the biosensor cell assay can faithfully differentiate between amyloid proteins that form morphologically similar fibers, but that differ in their underlying structures and sequences.

If our inhibitors block the interface responsible for seeding, we would expect Ab treated with inhibitors to no longer to be seeds for tau. To test this hypothesis, we treated 250 nM Ab fibers with indicated concentrations of inhibitor for 1 hour and transfected these into the biosensor cell line. All of our inhibitors were able to reduce seeding at 20 mM final concentration, while D1b showed a reduction in seeding at a concentration as low as1 mM (Figure 5DE, Figure 12). While both D1b and D1d reduced Ab aggregate toxicity on N2a cells, D1d was the more effective inhibitor of Ab toxicity, whereas D1b is the more effective inhibitor at reducing tau seeding. Inhibitors reduce tau aggregation and seeding

Our data support previous studies that suggest the tau binding surface on Ab is localized to the segment whose structure we determined and targeted for design of inhibitors against Ab aggregation and Ab-mediated seeding of tau (22, 24). We hypothesized that the tau fibril could contain a similar self-complementary surface and would also be susceptible to treatment with our inhibitors. We first asked if the Ab inhibitors, D1, D1b, and D1d could prevent monomeric tau from aggregating. We performed a ThT assay on 10 mM tau40, at 37 °C with shaking and 0.5 mg/mL heparin and found that all inhibitors function in a dose dependent manner similar to our results with Ab monomer, while the control inhibitor LC does not reduce tau aggregation (Figure 6A, Figure 13). The peptide inhibitors are not able to block aggregation of the amyloid forming proteins hIAPP or alpha synuclein, indicating that these inhibitors are specific for Abeta and tau, and are not general amyloid inhibitors (Figure 13).

Because we had observed differences in inhibitor efficacy on monomer versus aggregated species of Ab, we next tested if the inhibitor was effective against the seeding ability of tau40 fibrils. We formed tau40 fibrils, treated them with indicated inhibitor concentration and transfected into tau-K18 biosensor cells to measure seeding inhibition. We found that similar to our Ab-mediated tau biosensor seeding experiment, D1b was the best inhibitor, with an IC50 of 4.5 mM. D1 was slightly effective, while D1d showed seeding reduction only when increased to 75 mM (Figure 6B, C). It could be that D1b plays a dual role to inhibit both Ab and tau, and this combined effect could explain the drastically reduced seeding from Ab fibrils in our prior experiment (Figure 5D).

Next, we sought to determine potential binding sites on tau for D1b. We postulated that regions know to be drivers of tau aggregation could share structural features with the Ab core, and thus be inhibited by D1b. We designed mutants of tau40 that disrupt key interactions in steric zipper interfaces determined from crystal structures of VQIINK (SEQ ID NO: 28)(44) and VQIVYK (SEQ ID NO: 27) (54), and cryoEM models of AD tau fibrils (55). In total we tested 6 different constructs, each designed to block all but one aggregation interface of tau. The first 3 mutants were engineered to block the VQIVYK (SEQ ID NO: 27) aggregation interfaces in addition to all but 1 of the 3 different known VQIINK (SEQ ID NO: 28) interfaces. Mutant 1 (Q276W, L282R, I308P) leaves only interface A of VQIINK (SEQ ID NO: 28) available for aggregation, mutant 2 (Q276W, I277M, I308P) leaves only interface B for aggregation, and mutant 3 (I277M, L282R, I308P) leaves only interface C accessible for aggregation. Constructs 4 and 5 were designed to test the effect of blocking VQIINK (SEQ ID NO: 28) and all but 1 of the VQIVYK (SEQ ID NO: 27) surfaces. Mutant 4 (Q276W, I277M, L282R, Q307W, V309W) leaves only the dry interface of VQIVYK (SEQ ID NO: 27) available for aggregation and mutant 5 (Q276W, I277M, L282R, I308W) leaves only the solvent accessible surface for aggregation. In addition, we tested the effect of D1b on blocking seeding by 3R tau, which lacks the VQIINK (SEQ ID NO: 28) aggregation segment and leaves the VQIVYK (SEQ ID NO: 27) interface intact (Figure 6D, Figure 13).

To test if specific interfaces are inhibited by D1b, fibrils were formed from all of the different mutants, and then each was incubated with the indicated concentration of D1b and used to seed wild type tau-K18 biosensor cells, as described previously with wild type tau fibrils. We found that D1b was most effective at inhibiting seeding by fibrils of mutants that left intact: interface A of VQIINK (SEQ ID NO: 28) which is thought to involve aggregation at site I277 of tau, the solvent accessible interface of VQIVYK (SEQ ID NO: 27) as well as 3R (Figure 6D). D1b also showed moderate inhibition of several other tau mutants, but required high concentrations to inhibit seeding (Figure 13). As a control, we tested seeding by a mutant of tau40 that combined all of the different mutations, and found this mutant did not induce any seeding in tau-K18 biosensor cells (Figure 13), indicating that at least one of the known interfaces is needed for seeding. Control inhibitor LC has little to no effect on seeding from any construct (Figure 13). Taken together, these data show that both the VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) aggregation segments of tau are inhibited by D1b, and suggest that each may share common structural features with the Ab core that could allow for cross-seeding of tau by Ab. Designed inhibitor D1b targets disease relevant conformations

Amyloid polymorphs may differ depending on whether they were aggregated in vitro or extracted from human brain tissue (56). We sought to determine if our inhibitors are capable of blocking pathological forms of either tau, or Ab. As suggested previously in our conformational antibody assay and structural alignment (Figure 3C), we hypothesized that our inhibitors would block disease-relevant amyloid polymorphs. Since we also found that our inhibitors blocked both homotypic and heterotypic tau seeding by aggregated tau and Ab, we tested our inhibitor series on crude lysate from AD donor patient brain tissue.

We homogenized tissue from three different brain regions of a single AD patient brain, corresponding to early (hippocampal), middle (frontal cortex), and late (occipital lobe) stage affected areas. We also prepared samples from patient tissue with progressive supranuclear palsy (PSP), which is a tau aggregation disease that displayed no Ab aggregation by immunostaining. We transfected brain lysates into the biosensor cells; samples with inhibitor were treated with 10 mM D1, D1b, or D1d.

We found that treating the brain-derived lysates with D1b significantly reduced seeding by all tested brain tissue samples (Fig. 7). D1b treatment reduced seeding in all stages of AD brain, with more pronounced effects in areas affected later in the disease. Although our inhibitor D1b showed reduction of seeding in the hippocampal sample, the fibril load of this region due to the late stage of the patient may have been too great to have been efficiently halted by the dose used. Interestingly, the PSP tauopathy tissue was also responsive to treatment with each of the inhibitors, with D1b displaying the most pronounced inhibition. We surmise that D1b recognizes a common toxic epitope found in both Ab, and in a variety of tau polymorphs. Discussion

The search for druggable targets in AD is muddied by the numerous proteins involved and incomplete understanding of whether or not the two histological protein hallmarks, Ab and tau, interact directly with each other. On top of this, Ab, the apparent initiator of the disease, aggregates into a wide variety of species, from soluble oligomers ranging from dimers to those that contain dozens of copies, to polymorphic fibril deposits. While there may be numerous toxic assemblies, targeting a specific sequence or structure of a toxic motif that is present in a variety of these assemblies could be an effective strategy for designing pharmaceuticals. We targeted the amyloid core segment of Ab due to its defined amyloidogenicity, and putative interaction with the late-stage aggregating protein, tau. We focused our efforts on the Ab16-26 segment with a hereditary mutation D23N, whose structure we determined by MicroED. Although the structure of this segment is fibrillar, and resembles a previously observed zipper interface, the out-of-register interface of the b-strands suggests that portions of this conformation may be present in a number of toxic oligomeric intermediates as well as in fibrils. We successfully used this structure to design a series of related inhibitors that reduce toxicity of Ab in model N2a cells.

Our biochemical and toxicity studies indicate that these inhibitors function in two ways. The first is by preventing monomeric Ab from aggregating. The second is by reducing toxicity of pre-formed oligomeric Ab, possibly by binding to and blocking a surface that is responsible for conferring toxicity or seeding. While all of our designed inhibitors prevent monomeric Ab from aggregating, only the longer D1b and D1d versions are effective at reducing toxicity of preformed assemblies. These two peptides were designed by extending the C-terminus. D1b and D1d could conceivably act by obscuring resides important for conferring toxicity, as supported by early onset hereditary mutations clustering at residues 21-23 (57–59).

Our data implicate an extended Ab core in the spread of the disease, because targeting inhibitors to this region appears to block the templating interface needed to cross-seed tau. Ab fibers treated with D1b showed a dramatic reduction of cross seeding in tau-K18 biosensor cells. Tau fibers treated with D1b showed similarly inhibited seeding in biosensor cells. The dual efficacy of the inhibitor D1b designed against the 16-23 region of Ab suggests that these two pathological aggregates, Ab and tau, share a common structural motif and aggregation pathway in AD. By using mutant constructs of tau with only one available amyloid interface, we were able to determine two interfaces on tau where seeding was highly reduced by D1b. Both the R2 and R3 amyloid-prone regions of tau contain a D1b sensitive interface. Of note, the surface of R2 blocked by D1b contains residue I277, which previously has been shown to be critical for tau aggregation (60). We find that overlaying our Ab segment crystal structure with structures of tau R2 and R3 reveals a high degree of structural similarity both in the backbone, and also apparently in the complementarity of sidechains from each to intrinsically interdigitate (Figure 13). These models support the hypothesis that Ab and tau may interact with favorable energy to form a stable heterozipper. Interestingly, Ab overlays well with these regions of tau in both parallel and anti-parallel orientations, suggesting that either fiber or smaller oligomers could be capable of cross seeding. On this basis, we suggest that the amyloid core of Ab and the regions VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) form similar structures in AD that are biochemically capable of cross-seeding.

Consistent with our finding that Ab and tau share structural similarities, we found that the Ab inhibitor D1b is able to reduce seeding from brain homogenates, indicating that the inhibitor is recognizing a disease-related structural motif, while D1 and D1d are much less effective. It is curious that seeding by both AD and PSP is greatly reduced by the inhibitor D1b, as PSP pathology does not include Ab aggregates. It is thought that different disease phenotypes, which display distinct fiber morphologies commonly referred to as strains, are determined by the formation of different steric zipper cores (61). Thus, PSP fibers may contain a different core than our in vitro aggregated tau or AD derived tau. However, our tau mutagenesis results suggest that inhibitor D1b can recognize at least 2 unique core interfaces, and thus could be able to act on multiple strains of tau fibers.

Similar to other peptide-based amyloid inhibitors, the effective dose to reduce toxicity of aggregated species is higher than to delay aggregation of monomeric species. This is emphasized by the differing efficacies of our related inhibitor series, where some inhibitors were able to prevent initial aggregation, but not toxicity or seeding from various assemblies. It appears that inhibitors to prevent an aggregation nucleus are much more promiscuous than those that ameliorate toxicity by binding to a distinct structure. This trend was observed in both Ab and tau, suggesting a common inhibitory mechanism for both proteins, and highlights the need for multiple experimental measures to validate inhibitor efficacy.

In summary, our results suggest that a direct interaction between the Ab core and the amyloid-prone regions of tau facilitates cross seeding. Our inhibitors designed for the Ab core segment prevent cross seeding of tau, as well as tau homotypic seeding. The entwined nature of these two proteins in AD suggests it is necessary to control aggregation of both in order to treat the disease. Early detection is still crucial, but these data provide a platform on which further inhibitors can be designed for optimized inhibition of amyloid seeding in Alzheimer’s disease. Experimental Procedures

Recombinant Amyloid Beta Peptide purification- Ab was purified as described in Krotee et. Al (47) After purification, the protein was lyophilized. Dried peptide powders were stored in desiccant jars at -20 ºC.

Peptide Preparation- Candidate inhibitors were custom made and purchased from Genscript (Piscataway, NJ). Lyophilized candidate inhibitors were dissolved at 10mM in 100% DMSO. 10mM stocks were diluted as necessary. All stocks were stored frozen at -20°C.

Amyloid Beta was prepared by dissolving lyophilized peptide in 100% DMSO or 100mM NaOH. Next, the sample was spin-filtered and the concentration was assessed by BCA assay (Thermo Scientific, Grand Island, NY). The DMSO or NaOH peptide stocks were diluted 100-fold in filter-sterilized Dulbecco’s PBS (Cat. # 14200-075, Life Technologies, Carlsbad, CA).

Crystallization- 16-Ac-KLVFFAENVGS-NH₃-26 (SEQ ID NO: 26) (Ab 16- 26 D23N) was dissolved at 4.5 mg/ml in 20% DMSO. Micro crystals were grown in batch in 0.2M magnesium formate, 0.1M Tris base pH 8.0, and 15% isopropanol at room temperature under quiescent conditions. Crystals grew within 4 days to a maximum of 2 weeks.

MicroED data collection- The procedures for MicroED data collection and processing largely follow published procedures (62, 63). Briefly, a 2-3 ml drop of crystals in suspension was deposited onto a Quantifoil holey-carbon EM grid then blotted and vitrified by plunging into liquid ethane using a Vitrobot Mark IV (FEI, Hillsboro, OR). Blotting times and forces were optimized to keep a desired concentration of crystals on the grid and to avoid damaging the crystals. Frozen grids were then either immediately transferred to liquid nitrogen for storage or placed into a Gatan 626 cryo-holder for imaging. Images and diffraction patterns were collected from crystals using FEI Tecnai 20 TEM with field emission gun (FEG) operating at 200 kV and fitted with a bottom mount TVIPS TemCam-F416 CMOS-based camera. Diffraction patterns were recorded by operating the detector in a video mode using electronic rolling shutter with 2 x 2 pixel binning (64). Exposure times for these images were either 2 or 3 seconds per frame. During each exposure, crystals were continuously unidirectionally rotated within the electron beam at a fixed rate of 0.3 degrees per second, corresponding to a fixed angular wedge of 0.6 or 0.9 degrees per frame.

Crystals that appeared visually undistorted produced the best diffraction. Datasets from individual crystals were merged to improve completeness and redundancy. Each crystal dataset spanned a wedge of reciprocal space ranging from 40-80°. We used a selected area aperture with an illuminating spot size of approximately 1 mm. The geometry detailed above equates to an electron dose rate of less than 0.01 e-/Ų per second being deposited onto our crystals.

Measured diffraction images were converted from TIFF format into SMV crystallographic format, using publicly available software (available for download at https://cryoem.janelia.org/downloads). We used XDS to index the diffraction images and XSCALE (65) for merging and scaling together datasets originating from thirteen different crystals.

Structure determination- We determined the structure of Ab 16-26 D23N using molecular replacement. KLVFFA (SEQ ID NO: 31) (pdb 2Y2A) led us to our atomic model. The solution was identified using Phaser (66). Subsequent rounds of model building and refinement were carried out using COOT and Phenix, respectively (67, 68). Electron scattering factors were used for refinement. Some reflections extended to 1.40 Å resolution. Calculations of the area buried and Sc were performed with AREAIMOL (69, 70)) and Sc (71–73), respectively.

Computational structure-based design- Computational designs were carried out using the RosettaDesign software as described previously (74). The atomic structure of the 16-KLVFFAENVGS-26 (SEQ ID NO: 26) Ab segment was used as a starting template for computational design. An extended L-peptide (or D-peptide, six to eight residues) was first placed at the end of the starting template of atomic structure. The design procedure then built side-chain rotamers of all residues onto the nine-residue peptide backbone placed at growing end of fibril. The optimal set of rotamers was identified as those that minimize an energy function containing a Lennard-Jones potential, an orientation-dependent hydrogen bond potential, a solvation term, amino acid-dependent reference energies, and a statistical torsional potential that depends on the backbone and side-chain dihedral angles. Area buried and shape complementarity calculations were performed with areaimol and Sc, respectively, from the CCP4 suite of crystallographic programs (69). The solubility of each peptide was evaluated by hydropathy index (75). The designed peptides were selected based on calculated binding energy of top or bottom binding mode, shape complementarity and peptide solubility. Each structural model of selected peptides went through human inspection using Pymol, where those peptides with sequence redundancy and less binding interactions were omitted. Finally, select peptides were synthesized and tested experimentally. Sample preparation for electron microscopy- Ab1-42 was dissolved and diluted as previously described. Inhibitor stocks were prepared in 100% DMSO and were added such that the sample contained 10 mM monomeric Ab1-42 the indicated ratio of inhibitor with final concentration of 1% DMSO. Samples were incubated for 72 hours at 37°C under quiescent conditions. Ab1-42 fibrils were formed as described, and then treated with indicated ration of inhibitor for 24 hours at 37°C under quiescent conditions. Fibril abundance was checked using electron microscopy. Transmission electron microscopy-Samples were spotted onto non-holey grids and left for 160 to 180 seconds. Remaining liquid was wicked off and then left to dry before analyzing. Samples for negative-stain TEM were treated with 2% uranyl acetate after sample was wicked off the grid. After 1 minute, the uranyl acetate was wicked off. The grids were analyzed using a T12 Electron Microscope (FEI, Hillsboro, OR). Images were collected at 3,200 or 24,000x magnification and recorded using a Gatan 2kX2k CCD camera.

Thioflavin-T (ThT) kinetic assays- Thioflavin-T (ThT) assays were performed in black 96-well plates (Nunc, Rochester, NY) sealed with UV optical tape. The total reaction volume was 150 mL per well. Ab1-42 was prepared as described. Inhibitors were added at indicted concentrations, with a final concentration of 1%DMSO. ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a Varioskan Flash (Thermo Fisher Scientific, Grand Island, NY). Experiments were performed at 37°C without shaking in triplicate and readings were recorded every 5 minutes. ThT assays with tau40 were prepared as above with the following exceptions. 0.5mg/mL heparin (Sigma cat. no. H3393) was added to the reaction mixture and experiments were performed at 37°C with double orbital shaking at 700 rpm. Seeding assay included 10% monomer equivalent of preformed fibrils, sonicated for 10 minutes prior to addition.

Cell culture- Neuro2a (N2a) cells were a gift from the Pop Wongpalee in the laboratory of Douglas Black at UCLA. Cells were cultured in MEM media (Cat. # 11095-080, Life Technologies) plus 10% fetal bovine serum and 1% pen-strep (Life Technologies). Cells were cultured at 37°C in 5% CO2 incubator.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay for cell viability- N2a cells were plated at 5,000 cells per well in 90 mL of culture media, in clear 96-well plates (Cat. # 3596, Costar, Tewksbury, MA). Cells were allowed to adhere to the plate for 20-24 hours. Ab1-42 samples were incubated at 10mM with or without inhibitors at varying ratios for 12 hours at 37°C and then applied to N2a cells. 10 mL of sample was added to cells. By doing this, samples were diluted 1/10 from in vitro stocks. Experiments were done in triplicate.

After a 24-hour incubation, 20 mL of Thiazolyl Blue Tetrazolium Bromide MTT dye (Sigma, St. Louis, MO) was added to each well and incubated for 3.5 hours at 37°C under sterile conditions. The MTT dye stock is 5 mg/mL in Dulbecco’s PBS. Next, the plate was removed from the incubator and the MTT assay was stopped by carefully aspirating off the culture media and adding 100 mL of 100% DMSO to each well. Absorbance was measured at 570 nm using a SpectraMax M5. A background reading was recorded at 700 nm and subsequently subtracted from the 570 nm value. Cells treated with vehicle alone (PBS+0.1%DMSO) were designated at 100% viable and cells treated with 100% DMSO designated as 0% viable, and cell viability of all other treatments was calculated accordingly. We employed one-way ANOVA as our statistical test for significance.

Dot Blot Assay- Ab1-42 samples were incubated at 10 mM with or without inhibitors for 6, 24, and 72 hours at 37°C, and spotted onto a nitrocellulose membrane (Cat. # 162–0146, BioRad, Hercules, CA). 20mL was loaded for each condition; 2mL was spotted at a time and allowed to dry between application. The membranes were blotted as previously described (76), with the exception of the primary antibodies used. The antibodies used in the assay were previously generated and characterized (51). Surface Plasmon Resonance (SPR)- SPR experiments were performed using BiacoreT200 instrument (GE Healthcare). Ab42 fibrils/tau K18 fibrils were immobilized on a CM5 sensor chip. The fibrils of Ab42 were prepared by placing a sample of 50 mM Ab42 in PBS pH 7.4 in two wells of a Nunc 96-well optical bottom plate (Thermo Scientific), 150 ml/well and incubating the plate in a microplate reader (FLUOstar Omega, BMG Labtech) at 37 °C with double orbital shaking at 600 rpm overnight. Sample from the two wells were pooled together and Ab42 fibrils were isolated from the incubation mixture by centrifuging it at 13,000 xg, 4°C for 45 minutes. The supernatant was removed and the pellet was re-dissolved in an equal volume of PBS as that of supernatant. The isolated fibrils were sonicated using a probe sonicator for 1-2 minutes at 18% amplitude with 2 sec on, 5 sec off pulses. The sonicated fibrils were filtered through a 0.22 m filter to remove large aggregates. The sonicated and filtered fibrils were diluted to 60 mg/ml in 10 mM NaAc, pH 3 and then, immobilized immediately on a CM5 sensor chip using standard amine coupling chemistry. Briefly, the carboxyl groups on the sensor surface were activated by injecting 100 ul of 0.2 M EDC and 0.05 M NHS mixture over flow cells 1–2. The fibrils were then injected at a flow rate of 5 ml/min over flow cell 2 of the activated sensor surface for 900 seconds. The remaining activated groups in both the flow cells were blocked by injecting 120 ml of 1 M ethanolamine-HCl pH 8. 5.. For the binding assay each peptide inhibitor was dissolved in 100 % DMSO at a concentration of 1 mM and diluted in PBS pH 7.4+1.2% DMSO to concentrations ranging from 5 mM to 260 mM. Each peptide was injected at a flow rate of 30 ml/min over both flow cells (1 and 2) at increasing concentrations (in running buffer, PBS, pH 7.4+1.2% DMSO) at 25°C. For each sample the contact time and dissociation time were 120 seconds and 160 seconds, respectively. 3 M NaCl was used as regeneration buffer. The data were processed and analyzed using Biacore T200 evaluation software 3.1. The data of flow cell 1 (blank control) was subtracted from the data of flow cell 2 (with immobilized fibrils/monomers). The equilibrium dissociation constant (Kd) was calculated by fitting the plot of steady-state peptide binding levels (Req) against peptide concentration (C) with 1:1 binding model (Eq 1).

Eq 1. Req = CRmax + RI

Kd + C

Rmax = Analyte binding capacity of the surface

RI = Bulk refractive index contribution in the sample

Recombinant Tau purification-Human Tau40 (residues 1-441) WT, 3R and mutants: : interface A (Q276W, L282R, I308P), interface B (Q276W, I277M, I308P), interface C (I277M, L282R, I308P)interface 1 (Q276W, I277M, L282R, Q307W, V309W), interface 2 (Q276W, I277M, L282R, I308W), were expressed in pET28b with a C-terminal His-tag in BL21-Gold E. coli cells grown in TB to an OD600 = 0.8. Cells were induced with 0.5 mM IPTG for 3 hours at 37°C and lysed by sonication in 50 mM Tris (pH 8.0) with 500 mM NaCl, 20 mM imidazole, 1 mM beta- mercaptoethanol, and HALT protease inhibitor. Cells were lysed by sonication, clarified by centrifugation at 15,000 rpm for 15 minutes, and passed over a 5 ml HisTrap affinity column. The column was washed with lysis buffer and eluted over a gradient of imidazole from 20 to 300 mM. Fractions containing purified Tau40 were dialyzed into 50 mM MES buffer (pH 6.0) with 50 mM NaCl and 1 mM beta- mercaptoethanol and purified by cation exchange as described for K18. Peak fractions were polished on a HiLoad 16/600 Superdex 200 pg in 1X PBS (pH 7.4), and concentrated to ~20-60 mg/ml by ultrafiltration using a 10 kDa cutoff.

Fibril incubation with inhibitors for tau biosensor cell-seeding assays. Ab fibrils were prepared at 200 mM at 37°C for 72 hours before diluting to 50 mM in PBS buffer (pH 7.4) for seeding experiments. Tau40 WT and interface mutation fibrils were prepared by shaking 50 mM tau40 in PBS buffer (pH 7.4) with 0.5 mg/ml heparin (Sigma cat. no. H3393) and 1 mM dithiothreitol (DTT) for 3–6 days. Fibrillization was confirmed with an endpoint ThT reading, and fibrils were then diluted 20-fold to 1.25 mM in OptiMEM (Life Technologies, cat. no. 31985070). Inhibitors dissolved in DMSO were added to 20 ml of diluted fibrils at a concentration 20-fold greater than the final desired concentration. Fibrils were incubated for ~16 h with the inhibitor, and subsequently were sonicated in a Cup Horn water bath for 3 min before seeding the cells. The resulting‘pre-capped fibrils’ were mixed with one volume of Lipofectamine 2000 (Life Technologies, cat. no. 11668027) prepared by diluting 1 ml of Lipofectamine in 19 ml of OptiMEM. After 20 min, 10 ml of fibrils were added to 90 ml of the tau-K18CY biosensor cells to achieve the final indicated ligand concentration. Quantification of seeding was determined by imaging the entire well of a 96-well plate seeded in triplicate and imaged using a Celigo Image Cytometer (Nexcelom) in the YFP channel. Aggregates were counted using ImageJ (77) by subtracting the background fluorescence from unseeded cells and then counting the number of peaks with fluorescence above background using the built-in Particle Analyzer. Dose-response curves were constructed for inhibitor peptides exhibiting concentration dependence by fitting to a nonlinear regression model in Graphpad Prism. High resolution images were acquired using a ZEISS Axio Observer D1 fluorescence microscope.

Preparation of Brain lysate. Human brain tissue was obtained from the Neuropathology Laboratory at UCLA Medical Center. AD and PSP cases were confirmed by the Neuropathology Laboratory by immunostaining autopsied brain tissue sections, and the PSP donor was confirmed to be free of amyloid immunoreactivity. Tissue sections from the indicated brain regions were manually homogenized using a disposable ultra-tissue grinder (Thermo Fisher) in TBS (pH 7.4) supplemented with 1X HALT protease inhibitor. Homoginzed tissue was aliquoted to several PCR tubes and prepared for seeding in biosensor cells by sonication as described by Kaufman et al. (78), except tissue sections were sonicated twice as long, for a total of 2 h, in an ice cooled circulating water bath with individual sample tubes stirring to ensure each tube received the same sonication energy. Subsequently, seeding was measured by transfection into biosensor cells and quantified as described above.

Table 1. Statistics of MicroED data collection and atomic refinement.

EXPERIMENTAL PROCEDURES

Aggregation Inhibition Assay with a-synuclein- a-synuclein was expressed and purified as described previously in Rodriguez, et al. with the following exceptions to the expression protocol. An overnight starter culture was grown in 15 mL instead of 100 mL, 7 mL of which was used to inoculate 1 L. After induction, cells were allowed to grown for 3-4 hours at 34 °C (instead of 4-6 hours at 30 °C). Cells were then harvested by centrifuging at 5,000 x g. ThT assays with a-synuclein were performed in black 96-well plates (Nunc, Rochester, NY) sealed with UV optical tape. The total reaction volume was 180 mL per well. ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a Varioskan Flash (Thermo Fisher Scientific, Grand Island, NY). Experiments were performed at 37°C, shaking at 600 rpm with a teflon bead, in triplicate and readings were recorded every 15 minutes. Alpha synuclein at 105 mM in PBS was diluted to a final concentration of 50 mM in 25 mM Thioflavin-T and PBS. Inhibitors were added at the specified concentration by diluting 10 mM stocks in 100% DMSO 1 to 40 in the same manner. Thus, inhibitors were tested at 5:1 molar excess of a-synuclein.

Aggregation Inhibition Assay with IAPP- Human IAPP1-37NH₂ (hIAPP) was purchased for Innopep (San Diego, CA). Peptides were prepared by dissolving lyophilized peptide in 100% 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) at 250mM for 2 hours. Next, the sample was spin-filtered and then HFIP was removed with a CentriVap Concentrator (Labconco, Kansas City, MO). After removal of the HFIP, the peptide was dissolved at 1mM or 10mM in 100% DMSO (IAPP alone) or 100% DMSO solutions containing 1mM or 10mM inhibitor. The DMSO peptide stocks were diluted 100-fold in filter-sterilized Dulbecco’s PBS (Cat. # 14200-075, Life Technologies, Carlsbad, CA). Thioflavin-T (ThT) assays with hIAPP were performed in black 384-well plates (Nunc, Rochester, NY) sealed with UV optical tape. hIAPP1- 37NH2 and mIAPP1-37NH2 were prepared as described. The total reaction volume was 150 mL per well. ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a Varioskan Flash (Thermo Fisher Scientific, Grand Island, NY). Experiments were performed at 25°C without shaking in triplicate and readings were recorded every 5 minutes.

Atomic structure overlay- A structural superposition of Ab 16-26 and tau (5V5B, 6HRE) was performed using LSQ from coot (Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–32). We calculated root mean square deviation (RMSD) of main chains for parallel orientations fitting 6-8 resisdues. Anti-paprallel LSQ computation of Ab 16-22 and tau 275-281 (5V5B) of C_a atoms was calculated, and side chain rotomers optimized with foldit (Kleffner, R., Cooper, S., Khatib, F., Flatten, J., Leaver-Fay, A., Baker, D., and Siegel, J. B. (2017) Foldit Standalone: a video game-derived protein structure manipulation interface using Rosetta. Bioinformatics. 33, 2765–2767) over 2000 iterations to minimize energy to -603 REU. Supplemental Table 1 (Table S1). Computed binding properties of designed inhibitors to amyloid-beta ₁₆KLVFFAEN₂₃ (SEQ ID NO: 25)

REFERENCES CITED IN TEXT ABOVE

1. Glenner, G. G., Wong, C. W., Quaranta, V., and Eanes, E. D. (1984) The amyloid deposits in Alzheimer’s disease: their nature and pathogenesis. Appl. Pathol. 2, 357–369

2. Goedert, M., Eisenberg, D. S., and Crowther, R. A. (2017) Propagation of Tau Aggregates and Neurodegeneration. Annu. Rev. Neurosci.40, 189–210

3. Tanzi, R. E. (2012) The genetics of Alzheimer disease (7). Cold Spring Harb. Perspect. Med.2, a006296- 4. Hardy, J., and Selkoe, D. J. (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science.297, 353–356 5. Manczak, M., and Reddy, P. H. (2014) Abnormal interaction of oligomeric amyloid-b with phosphorylated tau: implications to synaptic dysfunction and neuronal damage. J Alzheimers Dis.36, 285–295

6. Seward, M. E., Swanson, E., Norambuena, A., Reimann, A., Cochran, J. N., Li, R., Roberson, E. D., and Bloom, G. S. (2013) Amyloid- signals through tau to drive ectopic neuronal cell cycle re-entry in Alzheimer’s disease. J. Cell Sci.126, 1278– 1286

7. Brier, M. R., Gordon, B., Morris, J. H., Benzinger, T. L. S., and Ances, B. M. (2016) Tau and Aß imaging, CSF measures, and cognition in Alzheimer’ s disease. Sci. Transl. Med.8, 1–10

8. Schwarz, A. J., Yu, P., Miller, B. B., Shcherbinin, S., Dickson, J., Navitsky, M., Joshi, A. D., Devous, M. D., and Mintun, M. S. (2016) Regional profiles of the candidate tau PET ligand18F-AV-1451 recapitulate key features of Braak

histopathological stages. Brain.139, 1539–1550

9. Chen, G., Xu, T., Yan, Y., Zhou, Y., Jiang, Y., Melcher, K., and Xu, H. E. (2017) Amyloid betaௗ: structure , biology and structure-based therapeutic development. Nat. Publ. Gr.38, 1205–1235

10. Oddo, S., Caccamo, A., Shepherd, J. D., Murphy, M. P., Golde, T. E., Kayed, R., Metherate, R., Mattson, M. P., Akbari, Y., and LaFerla, F. M. (2003) Triple- transgenic model of Alzheimer’s Disease with plaques and tangles: Intracellular Ab and synaptic dysfunction. Neuron.39, 409–421

11. Bolmont, T., Clavaguera, F., Meyer-Luehmann, M., Herzig, M. C., Radde, R., Staufenbiel, M., Lewis, J., Hutton, M., Tolnay, M., and Jucker, M. (2007) Induction of tau pathology by intracerebral infusion of amyloid-b-containing brain extract and by amyloid-b deposition in APP x tau transgenic mice. Am. J. Pathol.171, 2012– 2020

12. Gotz, J. (2001) Formation of Neurofibrillary Tangles in P301L Tau

Transgenic Mice Induced by Abeta 42 Fibrils. Science (80-. ).293, 1491–1495 13. Morales, R., Bravo-Alegria, J., Duran-Aniotz, C., and Soto, C. (2015)

Titration of biologically active amyloid-b seeds in a transgenic mouse model of Alzheimer’s disease. Sci. Rep.5, 9349

14. Choi, S. H., Kim, Y. H., Hebisch, M., Sliwinski, C., Lee, S., D’Avanzo, C., Chen, H., Hooli, B., Asselin, C., Muffat, J., Klee, J. B., Zhang, C., Wainger, B. J., Peitz, M., Kovacs, D. M., Woolf, C. J., Wagner, S. L., Tanzi, R. E., and Kim, D. Y. (2014) A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature.515, 274–278

15. Ittner, L. M., and Götz, J. (2011) Amyloid-b and tau - A toxic pas de deux in Alzheimer’s disease. Nat. Rev. Neurosci.12, 67–72

16. Stancu, I. C., Vasconcelos, B., Terwel, D., and Dewachter, I. (2014) Models of b-amyloid induced Tau-pathology: the long and“folded” road to understand the mechanism. Mol. Neurodegener.9, 51

17. Rajendran, L., Honsho, M., Zahn, T. R., Keller, P., Geiger, K. D., Verkade, P., and Simons, K. (2006) Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci.103, 11172–11177

18. Lesné, S., Ming, T. K., Kotilinek, L., Kayed, R., Glabe, C. G., Yang, A., Gallagher, M., and Ashe, K. H. (2006) A specific amyloid-b protein assembly in the brain impairs memory. Nature.440, 352–357

19. Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed, R., Liosatos, M., Morgan, T. E., Rozovsky, I., Trommer, B., Viola, K. L., Wals, P., Zhang, C., Finch, C. E., Krafft, G. A., and Klein, W. L. (1998) Diffusible, nonfibrillar ligands derived from Ab 1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci.95, 6448–6453

20. Benilova, I., Karran, E., and De Strooper, B. (2012) The toxic Ab oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat. Neurosci.

10.1038/nn.3028

21. Jin, M., Shepardson, N., Yang, T., Chen, G., Walsh, D., and Selkoe, D. J. (2011) Soluble amyloid -protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc. Natl. Acad. Sci. 108, 5819–5824 22. Guo, J.-P., Arai, T., Miklossy, J., and McGeer, P. L. (2006) Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A.103, 1953–8 23. Vasconcelos, B., Cosmin, I., Arjan, S., Matthew, B., Wang, P., Vanoosthuyse, A., Kolen, K. Van, Verheyen, A., Kienlen, P., Jean, C., Octave, N., Baatsen, P., and Moechars, D. (2016) Heterotypic seeding of Tau fibrillization by pre ^ aggregated Abeta provides potent seeds for prion ^ like seeding and propagation of Tau ^ pathology in vivo. Acta Neuropathol.131, 549–569

24. Miller, Y., Ma, B., and Nussinov, R. (2011) Synergistic interactions between repeats in tau protein and Ab amyloids may be responsible for accelerated

aggregation via polymorphic States. Biochemistry.50, 5172–5181

25. Tjernberg, L. O., Callaway, D. J., Tjernberg, a, Hahne, S., Lilliehöök, C., Terenius, L., Thyberg, J., and Nordstedt, C. (1999) A molecular model of Alzheimer amyloid beta-peptide fibril formation. J. Biol. Chem.274, 12619–12625

26. Bernstein, S. L., Wyttenbach, T., Baumketner, A., Shea, J. E., Bitan, G., Teplow, D. B., and Bowers, M. T. (2005) Amyloid b-protein: Monomer structure and early aggregation states of Ab42 and its Pro19alloform. J. Am. Chem. Soc.127, 2075–2084

27. Marshall, K. E., Vadukul, D. M., Dahal, L., Theisen, A., Fowler, M. W., Al- Hilaly, Y., Ford, L., Kemenes, G., Day, I. J., Staras, K., and Serpell, L. C. (2016) A critical role for the self-assembly of Amyloid-b1-42 in neurodegeneration. Sci. Rep. 6, 1–13

28. Colletier, J.-P., Laganowsky, A., Landau, M., Zhao, M., Soriaga, A. B., Goldschmidt, L., Flot, D., Cascio, D., Sawaya, M. R., and Eisenberg, D. (2011) Molecular basis for amyloid- polymorphism. Proc. Natl. Acad. Sci.108, 16938– 16943 29. Lührs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Döbeli, H., Schubert, D., and Riek, R. (2005) 3D structure of Alzheimer’s amyloid-beta(1-42) fibrils. Proc. Natl. Acad. Sci. U. S. A.102, 17342–17347

30. Colvin, M. T., Silvers, R., Ni, Q. Z., Can, T. V., Sergeyev, I., Rosay, M., Donovan, K. J., Michael, B., Wall, J., Linse, S., and Griffin, R. G. (2016) Atomic Resolution Structure of Monomorphic Ab 42 Amyloid Fibrils. J. Am. Chem. Soc.138, 9663–9674

31. Qiang, W., Yau, W.-M., Luo, Y., Mattson, M. P., and Tycko, R. (2012) Antiparallel -sheet architecture in Iowa-mutant -amyloid fibrils. Proc. Natl. Acad. Sci.109, 4443–4448

32. Huber, M., Ovchinnikova, O. Y. u, Schütz, A. K., Glockshuber, R., Meier, B. H., and Böckmann, A. (2015) Solid-state NMR sequential assignment of Osaka- mutant amyloid-beta (Ab1-40 E22ǻ) fibrils. Biomol. NMR Assign.9, 7–14

33. Wälti, M. A., Ravotti, F., Arai, H., Glabe, C. G., Wall, J. S., Böckmann, A., Güntert, P., Meier, B. H., and Riek, R. (2016) Atomic-resolution structure of a disease-relevant Ab(1-42) amyloid fibril. Proc. Natl. Acad. Sci. U. S. A.113, E4976- 84

34. Van Nostrand, W. E., Melchor, J. P., Cho, H. S., Greenberg, S. M., and Rebeck, G. W. (2001) Pathogenic Effects of D23N Iowa Mutant Amyloid b-Protein. J. Biol. Chem.276, 32860–32866

35. Tay, W. M., Huang, D., Rosenberry, T. L., and Paravastu, A. K. (2013) The Alzheimer’s amyloid-b(1-42) peptide forms off-pathway oligomers and fibrils that are distinguished structurally by intermolecular organization. J. Mol. Biol.425, 2494– 2508

36. Laganowsky, A., Liu, C., Sawaya, M. R., Whitelegge, J. P., Park, J., Zhao, M., Pensalfini, A., Soriaga, A. B., Landau, M., Teng, P. K., Cascio, D., Glabe, C., and Eisenberg, D. (2012) Atomic view of a toxic amyloid small oligomer. Science.335, 1228–31 37. 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-b Oligomers and Less-Toxic Fibrils in Regions Known To Harbor Familial Alzheimer’s Mutations. Angew. Chem. Int. Ed.

10.1002/anie.201402636

38. Krotee, P., Griner, S. L., Sawaya, M. R., Cascio, D., Rodriguez, J. A., Shi, D., Philipp, S., Murray, K., Saelices, L., Lee, J., Seidler, P., Glabe, C. G., Jiang, L., Gonen, T., and Eisenberg, D. S. (2018) Common fibrillar spines of amyloid-b and human islet amyloid polypeptide revealed by microelectron diffraction and structure- based inhibitors. J. Biol. Chem.10.1074/jbc.M117.806109

39. Colletier, J.-P., Laganowsky, A., Landau, M., Zhao, M., Soriaga, A. B., Goldschmidt, L., Flot, D., Cascio, D., Sawaya, M. R., and Eisenberg, D. (2011) Molecular basis for amyloid-beta polymorphism. Proc. Natl. Acad. Sci. U. S. A.108, 16938–43

40. Tycko, R., Sciarretta, K. L., Orgel, J. P. R. O., and Meredith, S. C. (2009) Evidence for novel b-sheet structures in Iowa mutant b-amyloid fibrils. Biochemistry. 48, 6072–6084

41. Liu, C., Zhao, M., Jiang, L., Cheng, P.-N., Park, J., Sawaya, M. R., Pensalfini, A., Gou, D., Berk, A. J., Glabe, C. G., Nowick, J., and Eisenberg, D. (2012) Out-of- register b-sheets suggest a pathway to toxic amyloid aggregates. Proc. Natl. Acad. Sci. U. S. A.109, 20913–8

42. Sun, Y., Ge, X., Xing, Y., Wang, B., and Ding, F. (2018) b-barrel Oligomers as Common Intermediates of Peptides Self-Assembling into Cross-b Aggregates. Sci. Rep.8, 10353

43. Sievers, S. A., Karanicolas, J., Chang, H. W., Zhao, A., Jiang, L., Zirafi, O., Stevens, J. T., Münch, J., Baker, D., and Eisenberg, D. (2011) Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature.475, 96–100 44. Seidler, P. M., Boyer, D. R., Rodriguez, J. A., Sawaya, M. R., Cascio, D., Murray, K., Gonen, T., and Eisenberg, D. S. (2018) Structure-based inhibitors of tau aggregation. Nat. Chem.10, 170–176

45. Saelices, L., Johnson, L. M., Liang, W. Y., Sawaya, M. R., Cascio, D., Ruchala, P., Whitelegge, J., Jiang, L., Riek, R., and Eisenberg, D. S. (2015)

Uncovering the mechanism of aggregation of human transthyretin. J. Biol. Chem.290, 28932–28943

46. Soragni, A., Janzen, D. M., Johnson, L. M., Lindgren, A. G., Thai-Quynh Nguyen, A., Tiourin, E., Soriaga, A. B., Lu, J., Jiang, L., Faull, K. F., Pellegrini, M., Memarzadeh, S., and Eisenberg, D. S. (2016) A Designed Inhibitor of p53

Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas. Cancer Cell. 29, 90–103

47. Krotee, P., Griner, S. L., Sawaya, M. R., Cascio, D., Rodriguez, J. A., Shi, D., Philipp, S., Murray, K., Saelices, L., Lee, J., Seidler, P., Glabe, C. G., Jiang, L., Gonen, T., and Eisenberg, D. S. (2018) Common fibrillar spines of amyloid-b and human islet amyloid polypeptide revealed by microelectron diffraction and structure- based inhibitors. J. Biol. Chem.293, 2888–2902

48. Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F., and Rosenbaum, J. (1970) Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proc. Natl. Acad. Sci. U. S. A.65, 129–136

49. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods.65, 55–63

50. Liu, Y., Peterson, D. A., Kimura, H., and Schubert, D. (1997) Mechanism of cellular 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) reduction. J. Neurochem.6, 581–593

51. Hatami, A., Albay, R., Monjazeb, S., Milton, S., and Glabe, C. (2014) Monoclonal antibodies against A??42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain. J. Biol. Chem.289, 32131– 32143

52. Manuscript, A., Iii, G. E., Salvadores, N., Shahnawaz, M., Soto, C., May, A., Responsible, B., Tatarnikova, O. G., Orlov, M. A., Bobkova, N. V, Chen, G., Xu, T., Yan, Y., Zhou, Y., Jiang, Y., Melcher, K., Xu, H. E., Baughman, H. E. R., Clouser, A. F., Klevit, R. E., Nath, A., Vasconcelos, B., Cosmin, I., Arjan, S., Matthew, B., Wang, P., Vanoosthuyse, A., Kolen, K. Van, Verheyen, A., Kienlen, P., Jean, C., Octave, N., Baatsen, P., and Moechars, D. (2017) Heterotypic seeding of Tau fibrillization by pre ^ aggregated Abeta provides potent seeds for prion ^ like seeding and propagation of Tau ^ pathology in vivo. Acta Neuropathol.131, 1327–1334

53. Kfoury, N., Holmes, B. B., Jiang, H., Holtzman, D. M., and Diamond, M. I. (2012) Trans-cellular propagation of Tau aggregation by fibrillar species. J. Biol. Chem.287, 19440–19451

54. Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I., Thompson, M. J., Balbirnie, M., Wiltzius, J. J. W., McFarlane, H. T., Madsen, A. Ø., Riekel, C., and Eisenberg, D. (2007) Atomic structures of amyloid cross-b spines reveal varied steric zippers. Nature.447, 453

55. Fitzpatrick, A. W. P., Falcon, B., He, S., Murzin, A. G., Murshudov, G., Garringer, H. J., Crowther, R. A., Ghetti, B., Goedert, M., and Scheres, S. H. W. (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature.547, 185

56. Falcon, B., Zhang, W., Schweighauser, M., Murzin, A. G., Vidal, R.,

Garringer, H. J., Ghetti, B., Scheres, S. H. W., and Goedert, M. (2018) Tau filaments from multiple cases of sporadic and inherited Alzheimer’ s disease adopt a common fold. Acta Neuropathol.136, 699–708

57. Lazo, N. D., Grant, M. A., Condron, M. C., Rigby, A. C., and Teplow, D. B. (2009) On the nucleation of amyloid b-protein monomer folding. Protein Sci.14, 1581–1596 58. Krone, M. G., Baumketner, A., Bernstein, S. L., Wyttenbach, T., Lazo, N. D., Teplow, D. B., Bowers, M. T., and Shea, J. (2008) Effects of Familial Alzheimer’s Disease Mutations on the Folding Nucleation of the Amyloid b-Protein. J. Mol. Biol. 381, 221–228

59. Chong, S.-H., Yim, J., and Ham, S. (2013) Structural heterogeneity in familial Alzheimer’s disease mutants of amyloid-beta peptides. Mol. BioSyst. Mol. BioSyst. 9, 997–1003

60. Kirschner, D. A., Abraham, C., and Selkoe, D. J. (1986) X-ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross-beta conformation. Proc. Natl. Acad. Sci.83, 503– 507

61. Sanders, D. W., Kaufman, S. K., DeVos, S. L., Sharma, A. M., Mirbaha, H., Li, A., Barker, S. J., Foley, A. C., Thorpe, J. R., Serpell, L. C., Miller, T. M.,

Grinberg, L. T., Seeley, W. W., and Diamond, M. I. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron.82, 1271–1288 62. Shi, D., Nannenga, B. L., de la Cruz, M. J., Liu, J., Sawtelle, S., Calero, G., Reyes, F. E., Hattne, J., and Gonen, T. (2016) The collection of MicroED data for macromolecular crystallography. Nat. Protoc.11, 895–904

63. Hattne, J., Reyes, F. E., Nannenga, B. L., Shi, D., de la Cruz, M. J., Leslie, A. G. W., and Gonen, T. (2015) MicroED data collection and processing. Acta

Crystallogr. Sect. A Found. Adv.71, 353–360

64. Nannenga, B. L., Shi, D., Leslie, A. G. W., and Gonen, T. (2014) High- resolution structure determination by continuous-rotation data collection in MicroED. Nat. Methods.11, 1–5

65. Kabsch, W. (2010) XDS. Acta Crystallogr. D. Biol. Crystallogr.66, 125–32 66. McCoy, A. J. (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D. Biol. Crystallogr.63, 32–41 67. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr.60, 2126–32

68. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and Read, R. J. (2005) Likelihood-enhanced fast translation functions. Acta Crystallogr. D. Biol. Crystallogr. 61, 458–64

69. Collaborative Computational Project Number 4 (1994) The CCP4 suite:

programs for protein crystallography. Acta Crystallogr. D. Biol. Crystallogr.50, 760– 763

70. Lee, B., and Richards, F. M. (1971) The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol.10.1016/0022-2836(71)90324-X 71. Connolly, M. L. (1983) Solvent-accessible surfaces of proteins and nucleic acids. Science.221, 709–13

72. Lawrence, M. C., and Colman, P. M. (1993) Shape complementarity at protein-protein interfaces. J. Mol. Biol.93, 946–950

73. Richards, F. M. (1977) Areas, volumes, packing, and protein structure. Annu. Rev. Biophys. Bioeng.5, 151–176

74. Sievers, S. A., Karanicolas, J., Chang, H. W., Zhao, A., Jiang, L., Zirafi, O., Stevens, J. T., Münch, J., Baker, D., and Eisenberg, D. (2011) Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature.475, 96–100 75. Kyte, J., and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol.157, 105–132

76. Krotee, P., Rodriguez, J. A., Sawaya, M. R., Cascio, D., Reyes, F. E., Shi, D., Hattne, J., Nannenga, B. L., Oskarsson, M. E., Philipp, S., Griner, S., Jiang, L., Glabe, C. G., Westermark, G. T., Gonen, T., and Eisenberg, D. S. (2017) Atomic structures of fibrillar segments of hIAPP suggest tightly mated b-sheets are important for cytotoxicity. Elife.10.7554/eLife.19273 77. Eliceiri, K., Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJௗ: 25 years of image analysis HISTORICAL commentary NIH Image to ImageJௗ: 25 years of image analysis. Nat. Methods.9, 671–675 EXAMPLE 2: A PANEL OF DESIGNED INHIBITORS HALTS SEEDING BY TARGETING AGGREGATED POLYMORPHS FROM DIFFERENT TAUOPATHIES

Aggregated tau is a histological hallmark of Alzheimer’s disease (AD) and a group of neurological disorders called tauopathies. Tau pathology accompanies progressive neurodegeneration, and aggregated tau is thought to spread to adjacent neurons and anatomically connected brain regions by prion-like seeding. Hence, inhibitors of seeding offer one possible route to therapy. Here, we report the MicroED structure of an aggregation-prone segment of tau with sequence SVQIVY (SEQ ID NO: 29), which is present in the cores of patient-derived fibrils from AD and Pick’s disease. Our structure illuminates a previously unappreciated surface of SVQIVY (SEQ ID NO: 29) that contributes to proteopathic seeding by patient-derived fibrils. Using this structure as a template, we designed peptide-based capping inhibitors that add to a panel of inhibitors that target different segments and polymorphs of aggregated tau. We show that inhibitors from the panel block seeding by extracts from donors with different tauopathies, and the sensitivity to given inhibitors of the panel varied for different tauopathies, consistent with the hypothesis that different polymorphs are linked to different tauopathies. Furthermore, some donors with the same tauopathy exhibited different profiles of inhibitor sensitivity, suggesting that aggregated tau polymorphs can vary among patients with the same diagnosed tauopathy, and even within the brains of individuals. Nevertheless, certain capping inhibitors of the panel blocked seeding by extracts from all of the tauopathies tested, and moreover as we show, could be transiently expressed in HEK293 tau biosensor cells, suggesting that nucleic acid based vectors can be used as an alternative form of inhibitor delivery. Tau pathology is a marker of neurodegeneration in AD and related tauopathies (1, 2). The spreading of minimal units of tau pathology– misfolded aggregates of tau, termed seeds–converts inert, soluble tau monomers in recipient cells into pathological aggregates through a process called prion-like seeding, in principle connecting seeding to the progressive cascade of degeneration that is characteristic of tauopathies, and other amyloid diseases (2-5). The amyloid core of tau, like other amyloidogenic proteins, is formed by steric zippers with rich b character, tightly interdigitated sidechains and strong shape complementarity (6, 7). Amyloid structures are particularly stable relative to other protein folds owing to steric zipper interactions, and an extensive network of hydrogen bonds that form along the fibril axis (8, 9), potentially explaining their ability to spread transcellularly to distant, anatomically connected brain regions. CryoEM models recapitulate all of the structural features, and additionally reveal novel protein folds and polymorphs that are unique to amyloids and contribute to their unusual stability and diversity.

In some cases, crystal structures of steric zippers determined from short, aggregation-prone peptide segments reveal the exact molecular interfaces that are seen in cryoEM structures of fibrils containing the same aggregation-prone segments. For a-synuclein, cryoEM structures of two different fibril polymorphs revealed homomeric interfaces, each formed by different homo-steric zippers (10). Both were identical to steric zipper interfaces determined from crystal structures of the same respective peptide segments (11), demonstrating that peptide structures recapitulate subsets of the interactions that are also seen in the fibril core.

The aggregation of amyloid proteins can involve different surfaces of peptides with the same sequence, leading to different structural polymorphs that are associated with different neurodegenerative diseases. For tau, this is exemplified by 4 cryoEM structures of patient-derived fibrils from AD (12, 13) and Pick’s disease (14): two polymorphs from AD (referred to as PHFs and SFs, which are ultrastructural polymorphs of each other) and two from Pick’s disease (NPFs and WPFs). Given these observations, which are supported by biological studies (3, 5), it has been suggested that tauopathies possess different disease-specific polymorphs of aggregated tau.

The apparent contribution of seeding to the progressive spread of tau pathology, combined with emerging evidence of disease-specific tau polymorphs, underscores the need for a panel of inhibitors, with each inhibitor of the panel targeting different polymorphs of aggregated tau, in order to classify and inhibit the various disease-specific polymorphs. The rational design of inhibitors of seeding has so far been unsuccessful using small molecules and antibodies. Instead, we previously developed peptide-based inhibitors of seeded aggregation that work by“capping” the ends of growing fibrils (15), thereby halting fibril growth. Using this approach, we developed capping inhibitors from crystal structures of the steric zipper segments of tau with sequences ₂₇₅VQIINK₂₈₀ (SEQ ID NO: 28) and ₃₀₆VQIVYK₃₁₁ (SEQ ID NO: 27) (15, 16). We found that a capping inhibitor TLKIVW (SEQ ID NO: 30), designed to target one polymorph of VQIVYK (SEQ ID NO: 27), was a poor inhibitor of seeding in spite of being a strong inhibitor of primary aggregation of 3R tau, whereas capping inhibitors that targeted 3 different steric zipper polymorphs of VQIINK (SEQ ID NO: 28) strongly inhibited seeding by recombinant tau fibrils.

In this Example, we design a panel of capping inhibitors, each inhibitor of the panel targeting 1 of 5 different aggregation-prone interfaces of tau. Three inhibitors of the panel target different steric zipper polymorphs formed by VQIINK (SEQ ID NO: 28). Using the crystal structure of ₃₀₅SVQIVY₃₁₀, (SEQ ID NO: 29) a polymorph of ₃₀₆VQIVYK₃₁₁ (SEQ ID NO: 27) that we determined as part of this work, we designed additional capping inhibitors to target 2 steric zipper polymorphs formed by VQIVYK (SEQ ID NO: 27). We benchmark our panel of capping peptides by testing inhibition of seeding by pathological fibrils from ten different donors with four different tauopathies, and show that sensitivity to inhibition by different inhibitors of the panel is largely specific to the given tauopathy from which the pathological extracts derived. Results

To aid the design of our panel of inhibitors that targets different segments and polymorphs of aggregated tau, we sought to determine the structure of an alternative polymorph of VQIVYK (SEQ ID NO: 27). We scanned the tau sequence using ZipperDB (17), and found that a peptide with sequence 305SVQIVY310 (SEQ ID NO: 29) had a slightly greater predicted propensity to form a steric zipper compared to the canonical aggregation-prone segment with sequence ₃₀₆VQIVYK₃₁₁ (SEQ ID NO: 27) (Fig. 15A). To determine whether SVQIVY (SEQ ID NO: 29), shifted by -1 residue compared to VQIVYK (SEQ ID NO: 27), forms a different steric zipper polymorph, we grew nanocrystals of the SVQIVY (SEQ ID NO: 29) peptide and determined the MicroED structure by direct methods at a resolution of 1.0 Å (Fig.21 and Table B).

As shown in Fig. 15B, the SVQIVY (SEQ ID NO: 29) structure contains two different steric zipper interfaces that belong to two different classes. A Class 1 steric zipper is formed by two copies of the peptide that are related by crystallographic symmetry (Fig. 15B), and is nearly identical to the Class 1 steric zippers formed by the VQIVYK (SEQ ID NO: 27) segment determined previously (7). The opposite face of SVQIVY (SEQ ID NO: 29) forms a second steric zipper that is a Class 3 interface created by two molecules in the asymmetric unit. The Class 3 interface is more interdigitated than the Class 1, having a shape complementarity (S_c) of 0.87 and solvent-accessible surface area buried (A_b) of 110.9 Ų (compared to 0.79 and 106.1 Ų, respectively, for the Class 1 zipper). A Class 3 steric zipper interface was also observed previously in the cryoEM structure of an 11 residue segment from TDP43 (18), but has not been observed, or at least recognized, in any longer fibril structures determined by cryoEM to date. A comparison of SVQIVY (SEQ ID NO: 29) to previously determined cryoEM structures of tau from patient-derived fibrils suggests the SVQIVY (SEQ ID NO: 29) structure offers a high resolution template for the design of inhibitors to halt seeding by these fibrils. The Class 3 interface, formed by Ser305, Gln307, and Val309 of the SVQIVY (SEQ ID NO: 29) crystal structure maps to a solvent exposed surface of patient-derived fibrils from AD (Fig. 16A and B), and Pick’s disease. Also contained in the SVQIVY (SEQ ID NO: 29) structure are residues Val 306 and Ile308, which form a Class 1 steric zipper interface that is buried in the cores of the same patient-derived fibrils. The Class 1 steric zipper from the peptide crystal structure is formed by a homomeric steric zipper with a mated sheet of identical sequence, while the Class 1 interface from patient-derived fibrils forms through heteromeric steric zipper interactions. Nevertheless the features of the interface from the peptide crystal structure, mainly the zipper class and solvent-accessible surface area buried, is reminiscent of the cryoEM fibril structures (Fig. 22). Similarly, we suggest the Class 3 interface of SVQIVY (SEQ ID NO: 29) highlights a bona fide aggregation surface that could contribute to fibril stability and/or seeding, even though the sequences of the mated strands in the fibrillar and crystalline states are likely to differ.

Importantly, the Class 1 and 3 steric zipper interfaces are not mutually exclusive as they are both observed simultaneously in the SVQIVY (SEQ ID NO: 29) crystal structure. In fact, as shown in Fig. 16A and B, aligning the SVQIVY (SEQ ID NO: 29) crystal structure with AD-derived filaments reveals that unmodeled electron density overlaps with the same interface that forms the Class 3 steric zipper in both the PHF and SF, suggesting that similar interactions could form on the surfaces of these patient-derived fibrils. In contrast to the PHF, the surface that forms the Class 3 interface is accessible to only one of the two protomers in the SF, as the other is occluded by a key intermolecular contact that forms with the mated protomer. In summary, we find that a second steric zipper interface can be formed by the aggregation-prone SVQIVY (SEQ ID NO: 29) segment, and the same surface of the PHF and SF harbors unmodeled, contiguous electron density. These observations suggest that a Class 3 interface resembling the one that we observe in the SVQIVY (SEQ ID NO: 29) structure could also form in pathogenic fibrils of tau.

To test the hypothesis that the Class 3 interface reflects adhesive interactions that are important for fibril pathology, we used our atomic resolution structures to design a panel of capping inhibitors, peptides that incorporate into growing fibrils and block further extension, by introducing steric clashes into the space that is occupied by mated promoters. Using the SVQIVY (SEQ ID NO: 29) structure, we designed two types of capping inhibitors; inhibitors of the Class 1 and the Class 3 interfaces. To achieve this, we modeled single tryptophan substitutions at each position of the SVQIVY (SEQ ID NO: 29) peptide to determine which sites would most potently inhibit seeding. Modeling suggested that substitutions at positions 3 and 5 (Fig. 16A and B) would disrupt the Class 3 interface, while substitution at position 4 disrupts the Class 1 interface.

To test inhibition by the different SVQIVY (SEQ ID NO: 29) capping inhibitors, we measured seeding by recombinant fibrils of full length tau (tau40) with each of the capping inhibitors listed in Table A. As shown in Fig. 16C, Class 3 interface inhibitors WIV and QIW, which have tryptophan substitutions at positions 3 and 5, both modestly inhibit seeding with IC50s of 11.9 and 17.4 mM, respectively. In stark contrast, the Class 1 capping inhibitor W4, which contains a tryptophan substitution at position 4, has no measurable inhibition of seeding by recombinant tau fibrils, even at 50 mM. Consistent with this finding, a D-peptide inhibitor TLKIVW (SEQ ID NO: 30), which was previously designed using the Class 1 VQIVYK (SEQ ID NO: 27) structure and potently inhibited the primary aggregation of the 3R tau (15), likewise had no measurable inhibition of seeding by full length tau fibrils at concentrations up to 50 mM (Fig.23).

Table A Amino acid sequences of Tau VQIINK (SEQ ID NO: 28) and VQIVYK (SEQ ID NO: 27) capping inhibitors. The top row contains the residue position number for the referenced inhibitor peptide. The second row is the native sequence from wild-type human Tau40 from which the respective capping inhibitor peptides were derived, and subsequent rows are inhibitor peptide sequences tested in this paper. Residues modified from the wild- type sequence for each given capping inhibitor are highlighted in yellow and are listed in red font. Because both Class 3 inhibitors WIV and QIW exhibited modest inhibition of seeding, we combined the two tryptophan substitutions at positions 3 and 5 from WIV and QIW into a single capping inhibitor peptide referred to as WIW. The IC50 of WIW determined using recombinant fibrils of tau40 improved to 4 mM (Fig. 16C). In comparison, the series of VQIINK (SEQ ID NO: 28) capping inhibitors: WMINK (SEQ ID NO: 32), W3, M4 and R9 have similar IC50’s of about 1 mM. Since capping inhibitors are composed of L-peptides, we wondered whether they could be delivered to cells by transfecting DNA that encoded the inhibitor sequence. To test this, we transfected the WIW Class 3 capping inhibitor peptide into tau biosensor cells, and 24 hours after transfection, biosensor cells were seeded with recombinant fibrils of tau K18+ (residues Q244-E380), which contains the entire core observed in the AD PHF and SF cryoEM fibril structures. Seeding inhibition was measured for cells that were transfected with inhibitor and a scrambled peptide as negative control peptide. As shown in Fig. 16D, cells transfected with WIW resisted seeding, whereas cells seeded with vehicle only or a scrambled peptide were robustly seeded. These data demonstrate that capping peptides that are encoded by DNA can be expressed in mammalian cells while retaining the ability to suppress aggregation that is seeded by exogenous fibrils.

Next, we aimed to test our panel inhibitors on postmortem brain tissue extracts from donors with AD. As shown in Fig. 17A, seeding by crude AD brain extract in tau biosensor cells was inhibited by several peptides from our panel. Of the inhibitors targeting VQIVYK (SEQ ID NO: 27), only WIW blocked seeding. A VQIINK (SEQ ID NO: 28) inhibitor, WMINK (SEQ ID NO: 32), which targets 3 different aggregation interfaces with engineered steric clashes at positions 3, 4 and 9 of the inhibitor sequence, failed to block seeding, in spite of being one of the most potent inhibitors of recombinant tau fibrils that we discovered (16). On the other hand the 3 VQIINK (SEQ ID NO: 28) inhibitors IN-W3, IN-M4 and IN-R9, which each target a single interface of the three that are targeted by WMINK (SEQ ID NO: 32), all blocked seeding. Seeding inhibition measured from crude brain extracts from 3 additional AD donors likewise produced similar profiles of inhibitor sensitivity (Fig. 17B and Fig. 24). These data establish that both VQIINK (SEQ ID NO: 28)- and VQIVYK (SEQ ID NO: 27)-based capping inhibitors block seeding by extracts from AD brain, although clearly the profiles of inhibition differed from recombinant fibrils since WMINK (SEQ ID NO: 32), a strong inhibitor of recombinant fibrils, poorly inhibited seeding by AD brain extract. These data are consistent with structural studies that show heparin-induced recombinant tau fibrils form mixtures of polymorphs that differ from pathological fibrils (19).

As a control to ensure that our panel of capping inhibitors targets fibrillar tau and not some other hypothetical seeding-competent species or cofactor in crude brain extract, we purified PHFs and SFs from brain tissue of a fourth donor with AD, and compared seeding inhibition of purified fibrils and the crude brain extract. As shown in Fig. 17B and C, inhibition of the purified AD fibrils and crude brain extracts by our panel of capping inhibitors essentially mirrored one another, with W4 and TLKIVW (SEQ ID NO: 30) showing no inhibition, WIV and QIW showing mediocre inhibition, and WIW, W3, M4 and R9 strongly inhibiting seeding. Surprisingly even though seeding by purified fibrils in biosensor cells was an order of magnitude greater, capping inhibitors blocked seeding more effectively compared to the crude brain extract. We suspect the disparity is due to increased degradation of capping peptides in the crude extracts. Nevertheless, these data show that seeding and inhibitor measurements can be made from crude brain extract, and that by this approach, the efficacy of seeding inhibition by our panel of capping inhibitors can be ascertained.

Since different fibril polymorphs are thought to be associated with different tauopathies, we asked whether donors with other tauopathies would exhibit different patterns of sensitivity to our panel of inhibitors. To test this, we assayed seeding inhibition of crude brain extract from a donor with CTE. Like AD-derived extracts, CTE extracts were well inhibited by the VQIINK (SEQ ID NO: 28) inhibitors IN-W3 and IN-M4 (Fig. 18A). By contrast, CTE brain extract was not inhibited by IN-R9 or WIW. Instead, seeding was inhibited also by WIV and W4, two VQIVYK (SEQ ID NO: 27) inhibitors that did not inhibit seeding by extracts from the AD donors.

We sought to amplify the CTE polymorph of tau by in vitro seeding, since CTE is a relatively rare tauopathy. We carried out three sequential rounds of in vitro seeding using purified tau-K18+, which comprises residues 244-380, and contains the entire microtubule binding domain plus additional C-terminal residues that were found in the cores of pathological fibrils from AD and Pick’s disease. In vitro seeding was performed using the sarkosyl insoluble fraction from CTE brain extract, and we compared the seeding inhibition profiles of the resulting daughter fibrils to the CTE- derived parent fibrils. Daughter fibrils exhibited a wide range of inhibitor sensitivities similar to the parent fibrils (Fig. 18B). However unlike parent fibrils, the daughter fibrils also exhibited an unexplained sensitivity to WIW. We found that heparin induced fibrils of recombinant K18+ likewise exhibit sensitivity to WIW (Fig. 18C), suggesting that daughter fibrils inherited the inhibitor sensitivities of both the parent and recombinant fibrils. Of note, heparin was not used for in vitro seeding to amplify the CTE-derived polymorph. These data suggest that in vitro seeding produces a heterogeneous mixture of fibril polymorphs that is indeed characteristic of the parent fibril, but also bears hallmarks of the polymorphs that are characteristic of the recombinant protein.

We next expanded our experiments to test inhibitor efficacies on extracts from 4 PSP donors and 1 CBD donor. Contrary to the seeding inhibition profile measured for CTE-derived tau, which was inhibited by nearly half of the capping peptides in our panel, inhibition of seeding by extract from the CBD donor was poor for all of the inhibitors except M4 (Fig. 25). On the other hand, the inhibitor profiles observed for the different PSP donors more closely mirrored the 4 AD donors, with TLKIVW (SEQ ID NO: 30) and WMINK (SEQ ID NO: 32) showing no inhibition and W4, in some cases, stimulating seeding (Fig. 19). The VQIVYK (SEQ ID NO: 27) inhibitors WIV, QIW and WIW, produced variable effects generally ranging from no inhibition, to mild inhibition (Fig. 19A), whereas VQIINK (SEQ ID NO: 28) inhibitors IN-W3, IN-M4 and IN-R9 were stronger inhibitors, and interestingly inhibited seeding with different efficacies for the different PSP donors tested. In particular, donors 1 and 3 shared similar profiles of inhibition in spite of deriving from two different brain regions, and were particularly sensitive to inhibition by IN-M4 and WIW, whereas little-to-no response was seen for IN-W3 and the other two VQIVYK (SEQ ID NO: 27) inhibitors, WIV and QIW. By contrast, seeding by extract from donor 2 was potently inhibited by all of the VQIINK (SEQ ID NO: 28)-based inhibitors: IN-W3, IN-M4 and IN-R9, but vulnerability to the VQIVYK (SEQ ID NO: 27)-based inhibitor WIW was less pronounced.

Because PSP donors 1-3 appeared to show variable profiles of inhibitor sensitivities, we tested the susceptibility of a fourth PSP donor using tissue sections from two different brain regions, the frontal cortex and cerebellum using a subset of the inhibitors including IN-W3, IN-M4 and IN-R9, which proved to be the greatest discriminators observed for donors 1-3. The inhibitor profile from the cerebellum of PSP donor 4 closely matched the profiles we observed for PSP donors 1 and 3, with strong inhibitor sensitivity limited mainly to the VQIINK (SEQ ID NO: 28)-based inhibitor IN-M4 (Fig. 19D and F-H). On the other hand, the inhibitor profile we observed from a tissue section from the frontal cortex of the same donor more closely matched the profile that we observed from PSP donor 2, with the VQIINK (SEQ ID NO: 28)-based inhibitors IN-W3, IN-M4 and IN-R9 all showing strong inhibition (Fig. 19E and I-K). Unlike donor 2 however, extract from the frontal cortex of donor 4 additionally exhibited a strong sensitivity to WIV. Collectively, these data suggest that the patterns of sensitivity to our panel of inhibitors may be indicative of different fibril polymorphs, or possibly mixtures of polymorphs, and that the sensitivity profiles differed among tauopathy donors. Moreover these data show that different patterns of inhibitor sensitivity can be found not only among donors with the same tauopathy, but in the case of PSP donor 4, even in different brain regions of the same donor. Discussion

It is not known what surfaces of amyloid fibrils are most amenable to being targeted by inhibitors. Here, we use high resolution crystal structures and moderate resolution cryoEM structures of patient fibrils to design inhibitors for different segments and surfaces of aggregated tau. Inhibitors from our panel target either of two aggregation-prone segments, VQIINK (SEQ ID NO: 28) or VQIVYK (SEQ ID NO: 27). The VQIVYK (SEQ ID NO: 27) inhibitors WIV, QIW and WIW target a surface that is solvent exposed in cryoEM structures of AD fibrils, whereas the inhibitors TLKIVW (SEQ ID NO: 30) and W4 target a surface that is buried in the cores of the same fibrils (13). We find that inhibitors targeting the solvent exposed surface blocked seeding, whereas inhibitors targeting the buried surface poorly inhibited seeding by AD fibrils and crude brain extracts. These data demonstrate that inhibitors of seeding can be designed by targeting solvent exposed surfaces, and suggest that these surfaces contribute to seeding by pathological fibrils, perhaps by acting as scaffolds to allow for nucleation and growth of new fibrils by offering an adhesive interface on which tau monomers can accumulate. We speculate that these interactions are transient and dynamic, and that nucleated fibrils eventually break off to become independent fibrils. In support of this, we note that structures of patient- derived fibrils from AD harbor unmodeled electron density that overlaps with the position of the Class 3 interface observed in our SVQIVY (SEQ ID NO: 29) crystal structure. Alternatively, the Class 3 interface that is targeted by our inhibitors might contribute to fibril stability through intra- or interprotomer contacts that are poorly resolved in the cryoEM maps due to heterogeneity and/or partial occupancy.

As summarized in Fig. 20, extracts from all of the donors we tested were inhibited by VQIINK (SEQ ID NO: 28)-based inhibitors. Yet, structures of the AD PHF and SF show that VQIINK (SEQ ID NO: 28) localizes to the solvent exposed fuzzy coat. Collectively, given this observation, and that the best VQIVYK (SEQ ID NO: 27)-based inhibitor, WIW, also targets a solvent exposed surface, we speculate that adhesive segments on VQIINK (SEQ ID NO: 28) that reside in the fuzzy coat likewise play an important role in seeding. Along these lines, immune-EM of patient- derived tau fibrils shows that epitopes in the fibril core are inaccessible to antibody labeling (12-14). In the same respect, we propose that aggregation-prone segments in the fibril core are likely inaccessible to tau monomers in the cellular milieu, and instead it is solvent exposed aggregation-prone segments that drive seeded aggregation.

The observed inhibitor sensitivity profiles varied between of donors with different tauopathies, despite a shared sensitivity to IN-M4. These data suggest that the profiles of sensitivity to our panel of inhibitors could be diagnostic of different tauopathies. Although seeding inhibition profiles were measured from a relatively small number of donors and brain regions, our analysis of 4 AD donors revealed similar inhibitor sensitivity profiles, consistent with structural studies that suggest that the fibrillar polymorphs in AD could be limited to the PHF and SF. On the other hand, we measured at least two different inhibitor sensitivity profiles from four different PSP donors, and in one case we found distinct profiles from a single donor when two different brain regions were sampled. These data suggest that polymorphisms could occur among tauopathy donors, and even within the brain of a given donor, although it is not clear how widespread this phenomenon is, and whether it is limited to donors with specific tauopathies. To answer these questions, additional studies will focus on sampling larger numbers of patients and tauopathies, and evaluating inhibitor sensitivity profiles from multiple brain regions of individual donors.

It is our hypothesis that the prion-like spread of aggregated tau drives progressive neurodegeneration, and inhibitors of seeding could thus slow or even halt disease progression. The delivery of peptides as therapeutics to the brain is liable to be challenging, and the half-lives of peptides could be short. As such, we wondered whether capping inhibitor peptides could be functionally synthesized by ribosomes following delivery to cells in a DNA vector. We found that, at least for the WIW capping inhibitor peptide, that transfection of plasmid DNA encoding the WIW sequence into tau biosensor cells allowed cells to resist seeding with recombinant fibrils. These data suggest that capping inhibitors could, at least in theory, be delivered to cells as nucleic acids.

The prion-like nature of amyloid aggregates is thought to perpetuate specific disease-specific polymorphs, and thus, it is thought that seeding produces daughter fibrils that are identical to parent fibrils. Contrary to this, we found that daughter fibrils seeded using the sarkosyl insoluble fraction from a donor with CTE possessed a different profile of inhibitor sensitivity compared to the parent fibril polymorph. Parent fibrils exhibited a broad response to our panel of inhibitors, whereas daughter fibrils exhibited characteristics of both the parent and recombinant fibrils. These data suggest that while in vitro seeding faithfully propagates features that are characteristic to the parent polymorph, imprints of the recombinant protein remain, at least in our hands, in this case, and the resulting daughter fibrils are not perfect replicates of the parent. It is possible that some polymorphs require mixtures of different tau isoforms or the addition of specific co-factors to faithfully propagate, even in the presence of the authentic parent seeds. Nevertheless, these data exemplify the diagnostic power of the inhibitor profiling approach by showing that the profile of inhibition of CTE- derived daughter fibrils is the product of its derivatives: recombinant protein and the CTE parent fibril.

In summary, we show that the aggregation-prone segment SVQIVY (SEQ ID NO: 29) forms two simultaneous steric zipper interfaces of different classes. Inhibitors targeting the solvent exposed surface of SVQIIVY (SEQ ID NO: 40) and VQIINK (SEQ ID NO: 28) blocked seeding by pathological tau with sensitivities that differed for extracts deriving from different tauopathies. In some cases, variations in inhibitor sensitivity were observed for donors with the same diagnosed tauopathy, and even in different brain regions of individual donors, suggesting that fibril polymorphisms may occur in a certain subsets of tauopathies. Further studies with larger sample sizes that encompass multiple brain regions will be needed to clarify how widespread the phenomenon of fibril polymorphism is among the different tauopathies. We suggest that the response of patient-derived seeds to our panel of inhibitors provides a“fingerprint” that is diagnostic of the fibrillar polymorphs it contains, and that the profile of inhibitor sensitivity might be useful for distinguishing tauopathies, and detecting unique polymorphs that are associated with abnormal clinical characteristics. Methods:

Crystallization

305SVQIVY310 (SEQ ID NO: 29) synthetic peptide was purchased from GenScript and microcrystals were grown in batch at 3.3 mg/mL in 0.667 M DL-Malic acid pH 7.0, 8% w/v PEG 3350 at 18C. Micro-ED Data Collection and Processing

Crystal solution was applied to a glow discharged Quantifoil R1/4 cryo-EM grid, and plunge frozen using a Vitrobot Mark 4. Micro-ED data was collected in a manner similar to previous studies(19). Briefly, plunge-frozen grids were transferred to an FEI Technai F20 electron microscope operating at 200 kV and diffraction data were collected using a TVIPS F416 CMOS camera with a sensor size of 4,096 x 4,096 pixels, each 15.6 x 15.6 mm. Diffraction data was indexed using XDS, and XSCALE was used for merging and scaling together data sets from different crystals(20). XXX diffraction movies were merged using XSCALE to produce the final data set. Structure Determination

The SHELX macromolecular structure determination suite was used for phasing the measured intensities(21). A combination of REFMAC, Phenix, and Buster refinement programs were used with electron scattering factors to refine the atomic coordinates determined by the direct-methods protocol. Recombinant protein expression and purification

Human Tau tau K18+ (residues Q244-E380) was expressed in a pNG2 vector in BL21-Gold E. coli cells grown in LB to an OD600 = 0.8. Cells were induced with 0.5 mM IPTG for 3 hours at 37 ºC and lysed by sonication in 20 mM MES buffer (pH 6.8) with 1 mM EDTA, 1 mM MgCl2, 1 mM DTT and HALT protease inhibitor before addition of NaCl 500 mM final concentration. Lysate was boiled for 20 minutes and the clarified by centrifugation at 15,000 rpm for 15 minutes and dialyzed to 20 mM MES buffer (pH 6.8) with 50 mM NaCl and 5 mM DTT. Dialyzed lysate was purified on a 5 ml HighTrap SP ion exchange column and eluted over a gradient of NaCl from 50 to 550 mM. Protein was polished on a HiLoad 16/600 Superdex 75 pg in 10 mM Tris (pH 7.6) with 100 mM NaCl and 1 mM DTT, and concentrated to ~20-60 mg/ml by ultrafiltration using a 3 kDa cutoff.

Human Tau40 (residues 1-441) was expressed in pET28b with a C-terminal His-tag in BL21-Gold E. coli cells grown in TB to an OD600 = 0.8. Cells were induced with 0.5 mM IPTG for 3 hours at 37 ºC and lysed by sonication in 50 mM Tris (pH 8.0) with 500 mM NaCl, 20 mM imidazole, 1 mM beta-mercaptoethanol, and HALT protease inhibitor. Cells were lysed by sonication, clarified by centrifugation at 15,000 rpm for 15 minutes, and passed over a 5 ml HisTrap affinity column. The column was washed with lysis buffer and eluted over a gradient of imidazole from 20 to 300 mM. Fractions containing purified Tau40 were dialyzed into 50 mM MES buffer (pH 6.0) with 50 mM NaCl and 1 mM beta-mercaptoethanol and purified by cation exchange as described for K18. Peak fractions were polished on a HiLoad 16/600 Superdex 200 pg in 1X PBS (pH 7.4), and concentrated to ~20- 60 mg/ml by ultrafiltration using a 10 kDa cutoff. Preparation of crude and purified brain-derived tau seeds Tissue was received for neuropathlogically confirmed tauopathy cases from brain regions. Tissue was cut into a 0.2-0.3 g section on a block of dry ice, and then manually homogenized in a 15 ml disposable tube in 1 ml of 50mM Tris, pH 7.4 with 150mM NaCl containing 1X HALT protease. Samples were then aliquoted to PCR tubes and sonicated in a cuphorn bath for 120 min under 30% power at 4 °C in a recirculating ice water bath. For purification of PHFs and SFs from AD brain tissue, extractions were performed according to the previously published protocol (Fitzpatrick AWP, et al. (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547(7662):185–190) without any modifications. Inhibitor peptides

Inhibitor peptides were designed using the native crystal structure as a starting point. Bulky sidechains were modeled at sites in the VQIINK (SEQ ID NO: 28) structure that were in close contact with residues in the mated sheet of the steric zipper interface. Capping residues were chosen by modelling all possible rotamers to find sidechains without any compatible conformer with the steric zipper interface (that is, sidechains that clashed with the mated beta sheet at every rotamer conformer were selected). All of the inhibitor peptides shown in Table A were synthesized by Genscript with minimum purities of 90% and dissolved in deionized water or DMSO to a working concentration of 1.4 mM. Tau biosensor cell line maintenance and seeding

HEK293 cell lines stably expressing tau-K18 P301S-eYFP , referred to as“tau biosensor cells” were engineered by Marc Diamond’s lab at UTSW (5) and used without further characterization or authentication. Cells were maintained in DMEM (Life Technologies, cat. 11965092) supplemented with 10% (vol/vol) FBS (Life Technologies, cat. A3160401), 1% penicillin/streptomycin (Life Technologies, cat. 15140122), and 1% Glutamax (Life Technologies, cat. 35050061) at 37 °C, 5% CO2 in a humidified incubator. Fibrils and patient-derived seeds were incubated for 16 hours with inhibitor to yield a final inhibitor concentration of 10 mM (on the biosensor cells), except for IC50 determinations, which instead used adjustments to achieve the final indicated inhibitor concentration. For seeding, inhibitor-treated seeds were sonicated in a cuphorn water bath for 3 minutes, and then mixed with 1 volume of Lipofectamine 3000 (Life Technologies, cat. 11668027) prepared by diluting 1 ml of Lipofectamine in 19 ml of OptiMEM. After twenty minutes, 10 ml of fibrils were added to 90 ml of tau biosensor cells. The number of seeded aggregates was determined by imaging the entire well of a 96-well plate in triplicate using a Celigo Image Cytometer (Nexcelom) in the YFP channel. Aggregates were counted using ImageJ by subtracting the background fluorescence from unseeded cells and then counting the number of peaks with fluorescence above background using the built-in Particle Analyzer. The number of aggregates was normalized to the confluence of each well, and dose-response plots were generated by calculating the average and standard deviations from triplicate measurements. For IC50 calculations, does- response curves were fit by nonlinear regression in Graphpad Prism. For high quality images, cells were photographed on a ZEISS Axio Observer D1 fluorescence microscope using the YFP fluorescence channel. Data Availability

Atomic coordinates and structure factors have been deposited in the Protein Data Bank.

Table B Crystallographic Data Collection and Refinement Statistics. Values in parentheses represent highest resolution shell. EXAMPLE 2 REFERENCES

1. Goedert M, Eisenberg DS, Crowther RA (2017) Propagation of Tau Aggregates and Neurodegeneration. Annu Rev Neurosci 40(1):189–210. 2. Mudher A, et al. (2017) What is the evidence that tau pathology spreads through prion-like propagation? Acta Neuropathol Commun 5. doi:10.1186/s40478- 017-0488-7.

3. Harper JD, Lansbury PT (1997) Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time- dependent solubility of amyloid proteins. Annu Rev Biochem 66:385–407.

4. Sievers SA, et al. (2011) Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature 475(7354):96–100.

5. Seidler PM, et al. (2018) Structure-based inhibitors of tau aggregation. Nat Chem 10(2):170–176.

6. Falcon B, et al. (2018) Structures of filaments from Pick’s disease reveal a novel tau protein fold. doi:10.1101/302216.

7. Mirbaha H, et al. (2018) Inert and seed-competent tau monomers suggest structural origins of aggregation. eLife 7. doi:10.7554/eLife.36584.

8. Jackson SJ, et al. (2016) Short Fibrils Constitute the Major Species of Seed- Competent Tau in the Brains of Mice Transgenic for Human P301S Tau. J Neurosci 36(3):762–772.

9. Fitzpatrick AWP, et al. (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547(7662):185–190.

10. Gibbons GS, et al. (2018) Detection of Alzheimer Disease (AD)-Specific Tau Pathology in AD and NonAD Tauopathies by Immunohistochemistry With Novel Conformation-Selective Tau Antibodies. J Neuropathol Exp Neurol 77(3):216–228. 11. Peng C, et al. (2018) Cellular milieu imparts distinct pathological a-synuclein strains in a-synucleinopathies. Nature 557(7706):558.

12. Laferrière F, et al. (2019) TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates. Nat Neurosci 22(1):65. 13. von Bergen M, et al. (2000) Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence segment ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci U S A 97(10):5129–5134.

14. Falcon B, et al. (2015) Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates. J Biol Chem 290(2):1049–1065.

15. Sawaya MR, et al. (2007) Atomic structures of amyloid cross-b spines reveal varied steric zippers. Nature 447(7143):453–457.

16. Goldschmidt L, Teng PK, Riek R, Eisenberg D (2010) Identifying the amylome, proteins capable of forming amyloid-like fibrils. Proc Natl Acad Sci 107(8):3487–3492.

17. Wiltzius JJW, et al. (2009) Molecular mechanisms for protein-encoded inheritance. Nat Struct Mol Biol 16(9):973–978.

18. Törnquist M, et al. (2018) Secondary nucleation in amyloid formation. Chem Commun. doi:10.1039/C8CC02204F.

19. Rodriguez JA, et al. (2015) Structure of the toxic core of a-synuclein from invisible crystals. Nature 525(7570):486–490.

20. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66(Pt 2):125–132. 21. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A 64(Pt 1):112–122. Tau 40 (SEQ ID NO: 1) and Amyloid Beta (SEQ ID NO: 2) Polypeptide Sequence NCBI Reference Sequence: NP_005901.2

UniProtKB/Swiss-Prot Reference Sequence: P05067.3

REFERENCES

Note: This application references a number of different publications as indicated throughout the specification by reference numbers. A list of these different publications ordered according to these reference numbers can be found above.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. CONCLUSION

This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS: 1. A composition of matter comprising:

at least one peptide inhibitor of Ab aggregation wherein:

the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10); and

the at least one peptide inhibitor comprises at least one D-amino acid.

2. The composition of claim 1, wherein the composition comprises a plurality of peptide inhibitors of Ab aggregation.

3. The composition of claim 1, wherein the peptide inhibitor comprises less than 10 amino acids.

4. The composition of claim 1-3, wherein the peptide is coupled to a plurality of heterologous amino acids.

5. The composition of claim 4, wherein the plurality of heterologous amino acids comprises a linker and/or a cell penetrating peptide (CPP).

6. The composition of claim 1, further comprising an Ab polypeptide (SEQ ID NO: 2).

7. A composition of matter comprising a polynucleotide encoding at least one peptide inhibitor of Ab aggregation wherein:

the at least one peptide inhibitor comprises an amino acid sequence LYIWVQ (SEQ ID NO: 3), LYIWIQMQ (SEQ ID NO: 4), LYIWIWRT (SEQ ID NO: 5), LYIWIWFS (SEQ ID NO: 6), LYIWIQKT (SEQ ID NO: 7), MYIWVQ, MYIWRQ (SEQ ID NO: 9) or MLIVRN (SEQ ID NO: 10).

8. The composition of claim 7, wherein the polynucleotide is disposed within a vector selected for its ability to express the peptide inhibitor in a mammalian cell.

9. The composition of claim 8, wherein the vector is disposed within a mammalian cell.

10. A method of inhibiting aggregation of Ab polypeptides comprising:

combining Ab polypeptides with at least one peptide inhibitor of Ab aggregation of claims 1-4; and

allowing the at least one peptide inhibitor of Ab aggregation to bind to the Ab polypeptides;

such that that Ab aggregation is inhibited.

11. The method of claim 10, wherein the at least one peptide inhibitor of Ab aggregation used in the method is selected for an ability to:

inhibit cross-seeding of tau by Ab polypeptides; and/or

inhibit tau homotypic seeding.

12. The method of claim 10, wherein the at least one peptide inhibitor of Ab aggregation is combined with Ab and/or Tau in vivo.

13. The method of claim 12, wherein:

inhibiting formation of Ab aggregates in vivo inhibits development or progression of a Ab plaque formation in an individual; and/or

inhibiting homotypic seeding of Tau fibrils in vivo inhibits development or progression of a tauopathy in an individual.

14. The method of claim 10, wherein the at least one peptide inhibitor of Ab aggregation is combined with Ab and/or Tau in vitro.

15. The method of claim 10, wherein the method uses a plurality of peptide inhibitors of Ab aggregation.

16. A composition of matter comprising:

at least one peptide inhibitor of tau aggregation wherein:

the at least one peptide inhibitor comprises an amino acid sequence: S-V- W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W- Y-E (SEQ ID NO: 13), S-V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L- K (SEQ ID NO: 15), D-V-Q-M-I-N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K- K-R-K (SEQ ID NO: 17) or D-V-W-M-I-N-K-K-R-K (SEQ ID NO: 18).

17. The composition of claim 16, wherein:

the composition comprises a plurality of peptide inhibitors of tau aggregation; and/or

the composition further comprise a pharmaceutical excipient selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar or a pH adjusting agent.

18. The composition of claim 16, wherein the at least one peptide inhibitor of tau aggregation comprises less than 10 amino acids.

19. The composition of claim 16-18, wherein the peptide is coupled to a plurality of heterologous amino acids.

20. The composition of claim 19, wherein the plurality of heterologous amino acids comprises a linker and/or a cell penetrating peptide (CPP).

21. The composition of claim 16, further comprising a Tau polypeptide (SEQ ID NO: 1).

22. A composition of matter comprising a polynucleotide encoding a peptide inhibitor of tau aggregation wherein:

the peptide inhibitor comprises an amino acid sequence: S-V-W-I-V-Y-E (SEQ ID NO: 11), S-V-Q-W-V-Y-E (SEQ ID NO: 12), S-V-Q-I-W-Y-E (SEQ ID NO: 13), S-V-W-I-W-Y-E (SEQ ID NO: 14), D-V-W-I-I-N-K-K-L-K (SEQ ID NO: 15), D-V-Q-M-I-N-K-K-L-K (SEQ ID NO: 16), D-V-Q-I-I-N-K-K-R-K (SEQ ID NO: 17) or D-V-W-M-I-N-K-K-R-K (SEQ ID NO: 18).

23. The composition of claim 22, wherein the polynucleotide is disposed within a vector selected for its ability to express the peptide inhibitor in a mammalian cell.

24. The composition of claim 23, wherein the vector is disposed within a mammalian cell.

25. A method of inhibiting aggregation of tau polypeptides comprising:

combining tau polypeptides with a composition comprising at least one peptide inhibitor of tau aggregation of claims 16-20; and

allowing the at least one peptide inhibitor of tau aggregation to bind to the tau polypeptides;

such that tau aggregation is inhibited.

26. The method of claim 25, wherein the at least one peptide inhibitor of tau aggregation used in the method is selected for an ability to:

inhibit seeding of purified tau fibrils and/or

inhibit seeding of tau fibrils present in unpurified brain extracts.

27. The method of claim 25, wherein at least one peptide inhibitor of tau aggregation is combined with Tau in vivo.

28. The method of claim 27, wherein inhibiting seeding of Tau fibrils in vivo inhibits development or progression of a tauopathy in an individual.

29. The method of claims 16, wherein the method uses a plurality of peptide inhibitors of tau aggregation.

30. The method of claims 16, wherein the at least one peptide inhibitor of tau aggregation is coupled to a plurality of heterologous amino acids.