Neurotoxic amyloidogenic peptides in the proteome of SARS-COV2: potential implications for neurological symptoms in COVID-19.

AFM and transmission electron microscopy (TEM) imaging of assemblies formed at either 1 or 5 mg mL⁻¹ for 24 h revealed that assemblies from both peptides tend to stack on top of each other forming multi-laminar structures (Fig. 2a–d and Supplementary Figs. 5 and 6). Evidence of lateral assembly of the needles was also observed but this appears to happen more frequently in the ILLIIM assemblies (Fig. 2g) compared to RNYIAQVD. ILLIIM tends to form very large (2–3 μm in width) multi-laminar crystalline assemblies (Fig. 2g), whereas RNYIAQVD predominantly forms long linear needle-like structures. The apparent lower tendency of RNYIAQVD to form large two-dimensional lateral assemblies can be explained by the polymorphism seen in this peptide, which does not promote translational symmetry (i.e., extended crystals). Figure 2i and Supplementary Fig. 4a both clearly show that in addition to the flat needle-like crystals seen elsewhere, RNYIAQVD can also form non-planar partially twisted fibrillar assemblies. This polymorphism observed in RNYIAQVD assemblies may reduce the ability of the crystals to laterally associate and stack into multi-laminar species, simply because of a mismatch in planarity between two adjacent assemblies, the molecular basis for this polymorphism is briefly discussed in the next section of the manuscript. AFM was further used to investigate the height of the individual assemblies of both ILLIIM and RNYIAQVD. Figure 2a, b shows a line section through a multi-laminar RNYIAQVD assembly with two distinct layers with a step height of 5.5 nm between each layer. Similarly for ILLIIM (Fig. 2c and Supplementary Fig. 5), we see multi-laminar stacking with individual step heights that vary between 4 and 12 nm. Turning to RNYIAQVD we see single crystals with step heights varying between 5 and 20 nm (Supplementary Fig. 6). Together this heterogeneous distribution of crystal heights provides further evidence for the polymorphic nature of both the ILLIIM and RNYIAQVD assemblies.

Quantitative analysis of the distribution of assembly heights and contour lengths was performed using the freely available software tool FiberApp³³. The analysis of assembly height distribution was taken from the z-axis (height) of the AFM images, both peptide assemblies show a heterogeneous distribution of fibril heights due to the previously observed tendency of both assemblies to form polymorphic multi-laminar stacks (Fig. 2f, h). Analysis of the distribution of the contour length of the two assemblies showed a biphasic distribution of lengths for both fibrils with two broad sub-populations centred around 1 and 3 µm (Fig. 2j, l). The sub-population at 3 µm was seen to be much larger for the RNYIAQVD peptide (Fig. 2j) compared to the ILLIIM (Fig. 2l). This population of longer fibrils correlates with the observation from Fig. 2 that for RNYIAQVD longer, thinner assemblies are favoured (self-assembly via fibril extension) over the wider shorter assemblies more commonly seen for ILLIIM (assembly via lateral association of protofilaments). Analysis of the persistence length of the fibrils (Supplementary Fig. 7) showed that whilst both peptides formed very straight linear assemblies, the persistence length of RNYIAQVD (λ = 41.92 µm) is greater than that of ILLIIM (λ = 31.96 µm).

To further investigate the polymorphic nature of the assembly of these peptides, we investigated the structures formed from 1:1 mixtures of the two peptides. Interestingly when mixed prior to assembly the peptides form a wide range of polymorphic structures exceeding that of either peptide assembled individually. Supplementary Fig. shows a selection of some of the polymorphs formed, especially interesting are the large flat structures with well-defined edges that seem almost to interlock (Supplementary Fig. ). Such well-ordered 2D crystals were never observed for either peptide individually, and provide clear evidence of co-crystallisation. At this stage we have no evidence for the biological relevance of this co-crystallisation; however, as the ORF proteins from which these peptides are identified are themselves very small proteins (ORF6 is 61 amino acids in length), it is feasible that these small proteins may undergo similar co-crystallisation during their viral replication cycle facilitating an as yet unknown biological function. 8 8c

Figure 3b shows the CD spectra of mature assemblies, of both peptides. Assemblies of ILLIIM display a quite simple spectrum indicating the dominance of β-sheets, with a minimum between 225 and 230 nm and a strong maximum at 205 nm²⁹. The CD spectra of RNYIAQVD possess a well-defined minimum at 203 nm and a distinct shoulder at around 215 nm.

To further investigate the predicted secondary structure of both peptide assemblies we employed the secondary structure analysis software BeStSel (Supplementary Table 1)^35,36. As expected from the classic shape of the spectra the predicted secondary structure of ILLIIM at 5 mg mL⁻¹ is exclusively made up of β-sheets (41.8%) and β-turns (58.2%). At these high concentrations, the composition of these β-sheets is shown to be exclusively left twisted, whilst at lower concentrations (1 mg mL⁻¹) a more complex mixture of right, left and non-twisted (relaxed) β-sheets are predicted. At both concentrations, the CD spectra of RNYIAQVD again suggest the secondary structure is dominated by β-sheets; however, now they appear to be exclusively in the form of higher energy right-twisted β-sheets, similar to that observed in the highly strained structure of other amyloidogenic ultra-short peptides³⁰. This additional strain introduced by β-sheets opposing the left-handed chirality seen in natural amino acids may explain the additional polymorphism and twisted microstructures seen in the AFM and TEM images of the RNYIAQVD assemblies (Fig. 2i and Supplementary Fig. 4a)²⁹. The BeStSel fitting algorithm predicted the remainder of the RNYIAQVD secondary structure is composed of α-helical structure and further backbone conformations that could not be assigned (Supplementary Table 1). Part-helical CD spectra do not necessarily imply helical structure, especially considering that a single octapeptide cannot literally be 19% helix (two residues). Backbone conformation as reported by CD correlates through sheet structure to the assembled tertiary structure but no single level of organisation exclusively dictates any other, this is especially true in the case of coupling the twist of a peptide strand to the overall twist of the aggregate, which can relax to meet surface and shape-driven constraints through intersheet and interchain as well as intrachain degrees of freedom²⁹.

To further investigate the conformation of the amyloid assemblies formed we utilised the conformation-specific antibody A11 and the fluorescent probe thioflavin T (ThT). The former binds specifically to non-fibrillar amyloid oligomers and the latter is a commonly used molecular probe that becomes highly fluorescent when binding to amyloid assemblies³⁷. As expected, when ILLIIM and RNYIAQVD assemblies were stained with ThT both demonstrated clearly visible fluorescent emission at 590 nm, providing further evidence of their amyloid nature (Supplementary Fig. 9a, b). Conversely, A11 exhibited no positive binding; specifically, the level of fluorescence observed was similar to the background staining seen in the negative controls, as confirmed by similar fluorescent intensities for both assemblies (Supplementary Fig. 9e, f) and the negative controls (Supplementary Fig. 9d, h). Higher levels of A11 binding were seen for the positive control that consisted of phenylalanine assemblies known to form oligomeric species³⁸ (Supplementary Fig. 9g), with fluorescent intensities for these assemblies over 4 times greater than for ILLIIM or RNYIAQVD. Combined these data confirm the amyloid nature of the two ORF peptide fragments and suggest that non-fibrillar oligomeric amyloid species are absent.

The amyloid nature of the two assemblies is further confirmed by the wide-angle X-ray scattering (WAXS) spectra (Fig. 3d) of the peptide assemblies which possessed a number of strongly diffracting Bragg peaks. Both peptides have a clear peak at 1.38 Å⁻¹ corresponding to a d-spacing of 4.6 Å which is indicative of an amyloid assembly composed of extended β-sheets³⁹. It is worth noting that the apparent intrastrand spacing of ILLIIM assemblies is very slightly lower than the typically reported values (4.7–4.8 Å). This may be explained by the BeStCell analysis of the ILLIIM assemblies at 1 mg mL⁻¹, which suggests that the β-sheets in these assemblies are composed of a complex mixture of left-handed, strained right-handed and relaxed β-sheets (Supplementary Table 1); therefore, it is perhaps not surprising that the observed average intersheet spacing very slightly differs from that which is commonly reported. Furthermore, Lomont et al.⁴⁰ report that the observed intrastrand spacing from a range of amyloid crystal structures can vary by as much as 0.45 Å. ILLIIM also has a very strong Bragg reflection at 0.58 Å⁻¹ (11 Å) corresponding to a typical intersheet spacing given moderately bulky hydrophobic sidechains forming a steric zipper. RNYIAQVD has a number of well-defined Bragg peaks between 0.3 and 0.75 Å⁻¹ that are consistent with a mixture of first and second-order reflections corresponding to an amyloid-like 3D symmetry. Typical reflections arising from the combinations of the longer two axes of the unit cell of short peptide amyloid crystals arise in the 0.3–0.75 Å⁻¹ region with a qualitatively similar appearance to the pattern from RNYIAQVD, although in this case the peaks could not be individually assigned.

The ThT stain, which becomes highly fluorescent upon binding to β-sheet rich amyloid assemblies was used to assess the assembly kinetics of both ILLIIM and RNYIAQVD (Fig. 3c). Both peptides show rapid kinetics with significant assembly occurring almost instantaneously and reaching a plateau after 30–60 min. Longer amyloidogenic polypeptides typically show a distinct lag phase in their assembly kinetics; however, this was not observed in these sequences. This apparent lack of a lag phase in the assembly kinetics behaviour is typical of amyloidogenic short peptides, which have been previously seen to assemble very rapidly^29,41. This is highly likely to be due to a lack of additional non-amyloidogenic amino acid sequences acting as a kinetic barrier to amyloid formation. The ThT signal for ILLIIM at 5 mg mL⁻¹ plateaus at about 300 a.u., this is slightly stronger than the maximum signal generated from mature fibrils of the somewhat homologous peptide ILQINS, which was around 250 a.u^29–31,42, suggesting that the amyloidogenicity of the two peptides is comparable. RNYIAQVD, whilst showing similar ThT values at low concentrations (1 mg mL⁻¹), generated a ThT signal nearly 3 times as large at 5 mg mL⁻¹ suggesting that the assembly of this peptide is highly concentration dependent. For reasons yet unknown, it seems that RNYIAQVD appears to reach a maximum ThT value and then begin to drop, this can be seen at both concentrations but is most obvious at the higher concentration. This could be due to a reversible self-assembly as seen in other functional amyloids^39,41,43,44, or to self-quenching of the amyloid bound aromatic ThT molecules, or simply to a reduction of exposed ThT-binding sites as larger aggregates with a lower surface area to volume ratio come to dominate the solution.

Given the physical evidence and the discussions referred to in the introduction of various means by which SARS-CoV-2 and other viral infections could enhance their fitness (to the detriment of the host) by the production of amyloidogenic peptides, we hypothesised that the SARS-CoV-2 viral transcript fragments ILLIIM and RNYIAQVD are toxic to human neurons. This is in particular supported by the previously reported neuroinvasive capabilities of SARS-CoV-2^7,8, the noted similarities of the symptoms to a (hopefully transient form of) AD⁵ and the previous detection of amyloid assemblies driven by other viruses²⁰. To investigate this further we performed a number of cytotoxicity assays of the two peptide sequences against a human-derived neuroblastoma cell line (SH-SY5Y) often used as a model cell line for studying Parkinson's and other neurodegenerative diseases⁴⁶. Using an MTT assay we found that cells grown in the presence of both peptide assemblies possessed much lower viability after 48 h incubation. Concentrations as low as 0.04 and 0.03 mM (for RNYIAQVD and ILLIIM, respectively) were seen to reduce the viability of cultured cells after 48 h to <50% (IC₅₀) compared to the cells cultured without the peptides (Fig. 5a, b). This toxicity in relation to concentration is similar to that reported for Aβ42⁴⁷ although expression levels and time-scales (sudden for COVID versus chronic for AD) are likely to be very different.

To gain further insight into the mechanism of cell death occurring in the peptide exposed cells, we performed a detailed flow cytometry analysis using the apoptotic stain Annexin V and the viability dye 7-AAD. Figure 5c shows representative flow cytometry plots; cells can be identified as viable (bottom left quadrant), viable but undergoing early apoptosis (bottom right), non-viable and necrotic (top left) or non-viable due to late-stage apoptosis (top right). The percentages of cells in these quadrants are roughly equal for all conditions tested except in the case of late-stage apoptosis where we see a large increase in the cells exposed to the peptide assemblies (a 6.25-fold increase for RNYIAQVD at 2.5 mg mL⁻¹). Quantification over a range of concentrations showed that on average cells exposed to both ILLIIM and RNYIAQVD had a 3–5-fold increase in late-stage apoptosis compared to SH-SY5Y cells cultured in the absence of peptide assemblies (Fig. 5d, e). No evidence of increasing necrosis was seen in any of the samples, suggesting that the amyloid assemblies are triggering programmed cell death via an apoptotic pathway. This triggering of late-stage apoptosis in the cells was more pronounced for ILLIIM than for RNYIAQVD, showing statistically significant increases in apoptotic cells at concentrations as low as 0.04 mg mL⁻¹ for ILLIIM compared to 0.15 mg mL⁻¹ for RNYIAQVD. This increase in apoptosis down to low concentration provides convincing evidence, especially for ILLIIM, that the amyloid aggregates are responsible for this toxicity, as at these low concentrations we would expect very little un-assembled peptide to exist. The mechanisms of cell death in neurodegenerative diseases are complex and can vary between different diseases⁴⁸, and here we provide evidence that induction of apoptosis may be an important mechanism of neuronal death in COVID-19. Intriguingly, the conserved protein ORF6 from SARS-CoV-1 (not SARS-CoV-2) has previously been shown to induce apoptosis⁴⁹. Furthermore, we performed a series of cell counting experiments and demonstrated that after 48 h incubation we saw statistically significant decreases in cell number for both peptides at concentrations as low as 0.04 mg mL⁻¹ for ILLIIM and 0.32 mg mL⁻¹ for RNYIAQVD. These results confirm that in addition to the cytotoxic nature of the peptide assemblies, they significantly reduce cell number especially in the case of ILLIIM. The significant increase in apoptosis and reduction in cell number seen for ILLIIM correlates with the work of Lee et al. who have previously shown that the ORF6 protein (that contains the ILLIIM sequence) is the most cytotoxic protein in the proteome of SARS-CoV-2¹³. Combined with our data, this suggests that this toxicity might be due to the amyloidogenic nature of this short protein.

There is a wealth of literature suggesting that in neurodegenerative diseases like Alzheimer's and Parkinson's amyloid oligomers are the main toxic culprits and mature amyloid fibrils are a more inert assembly end-point. This is seemingly at odds with our data; however, there have also been multiple studies that show mature assemblies can also display significant toxicity^37,53,54. Alternatively, it may be the nature of the amyloids species seen here that differs from amyloids in neurodegenerative diseases; the amyloids seen here appear to be largely crystalline (especially in the case of ILLIIM). Previous work has shown that amyloid crystals are deeper in the free energy landscape compared to twisted protofilaments and amyloid ribbons^30,55, representing a global energy minima. AFM and TEM data have shown that these stable amyloid crystals are the dominant polymorph for ILLIIM (Fig. 2c, g, k) and that RNYIAQVD shows examples of higher energy (partially) twisted fibrils (Fig. 2i and Supplementary Figs. 4a and 6). Therefore, we hypothesise that the low energy ILLIIM crystalline assemblies are more slowly metabolised and cells are exposed for longer timeframes to the cytotoxic effect compared to RNYIAQVD assemblies. To date, there have been few investigations into the toxicity of amyloid crystals compared to other more commonly reported amyloid species. For the reasons above, the toxic nature of these amyloid assemblies warrants further investigations into the potential presence of amyloid aggregates from SARS-CoV-2 in the CNS of COVID-19 patients, and the potential role of amyloids in the neurological symptoms observed.

In conclusion, using a bioinformatics approach we identified two strongly amyloidogenic sub-sequences from the ORF6 and ORF10 sections of the SARS-COV-2 proteome. Nanoscale imaging, X-ray scattering, molecular modelling, spectroscopy and kinetic assays revealed that these self-assembled structures are amyloid in nature, and screening against neuronal cells revealed that they are highly toxic (approximately as toxic as the toxic amyloid assemblies in AD) to a cell line frequently used as a neurodegenerative diseases model. The neuroinvasive nature of SARS-COV-2 has been established previously^7,8; therefore, it is entirely plausible that amyloid assemblies either from these ORF proteins or other viral proteins could be present in the CNS of COVID-19 patients. The cytotoxicity and protease-resistant structure of these assemblies may result in their persistent presence in the CNS of patients post-infection that could partially explain the lasting neurological symptoms of COVID-19, especially those that are novel in relation to other post-viral syndromes such as that following the original SARS-CoV-1. The outlook in relation to triggering of progressive neurodegenerative disease remains uncertain. Given the typically slow progress of neurodegenerative disease if such a phenomenon exists, it will most probably take some time to become evident epidemiologically.

The online amyloid prediction algorithms TANGO and ZIPPER were used to predict peptide sequences with a tendency to form β-rich amyloid assemblies. TANGO is an algorithm that predicts aggregation nucleating regions in unfolded polypeptide chains⁵⁶. It works on the assumption that the aggregating regions are buried in the hydrophobic core of the natively folded protein. ZIPPER is an algorithm that predicts hexapeptides within larger polypeptide sequences that have a strong energetic drive to form the two complementary β-sheets (known as a steric zipper) that give rise to the spine of an amyloid fibril⁵⁷. Both methods are physically motivated but rely on statistically determined potentials.

NH2-ILLIIM-CO2H and Ac-RNYIAQVD-NH2 (>95% pure) were purchased from GL Biochem Ltd (Shanghai, China). Ideally, it would have been preferred to have both peptides capped (N-terminus: Acetyl and C-terminus: Amide), as they would better represent small fragments of a larger peptide sequence. Due to the fact ILLIIM contains no charged sidechains, synthesising capped sequences to high purity would have been very challenging, therefore only the RNYIAQVD sequence remained capped and the ILLIIM sequence had regular carboxyl and amino termini. To ensure that all peptide seeds were fully dissolved before self-assembly was initiated the peptides were solubilised in warmed PBS (90 °C) at either 1 or 5 mg mL⁻¹ The warmed peptide solutions were vortexed vigorously and held at 90 °C for 3 h to ensure maximum dissolution. After the second round of vortexing, the peptide suspensions were cooled slowly. This protocol has been previously used to maximise a homogenous starting population of monomeric peptide³². Alternatively, self-assembly was carried out at a constant temperature of 37 °C without pre-solubilising the peptides in hot PBS.

AFM imaging was performed on a Bruker Multimode 8 AFM and a Nanoscope V controller. Tapping mode imaging was used throughout, with antimony (n)-doped silicon cantilevers having approximate resonant frequencies of 525 or 150 kHz and spring constants of either 200 or 5 Nm⁻¹ (RTESPA-525, Bruker or RTESPA-150, Bruker). No significant differences were observed between cantilevers. 50 µL aliquots of the peptide (either at 1 or 5 mg mL⁻¹) were drop cast onto freshly cleaved muscovite mica disks (10 mm diameters) and incubated for 20 min before gently rinsing in MQ water and drying under a nitrogen stream. All images were flattened using the first order flattening algorithm in the nanoscope analysis software and no other image processing occurred. Statistical analysis of the AFM images was performed using the open-source software FiberApp³³ from datasets of no less than 900 fibres.

Copper TEM grids with a formvar-carbon support film (GSCU300CC-50, ProSciTech, Qld, Australia) were glow discharged for 60 s in an Emitech k950x with k350 attachment. Then, 5 µL drops of sample suspension were pipetted onto each grid, allowed to adsorb for at least 30 s and blotted with filter paper. Two drops of 2% uranyl acetate were used to negatively stain the particles with excess negative stain removed by blotting with filter paper after 10 s each. Grids were then allowed to dry before imaging. Grids were imaged using a Joel JEM-2100 (JEOL (Australasia) Pty Ltd) transmission electron microscope equipped with a Gatan Orius SC 200 CCD camera (Scitek Australia).

SAXS/WAXS experiments were performed at room temperature on the SAXS/WAXS beamline at the Australian Synchrotron. Peptide assemblies in PBS prepared at both 1 and 5 mg mL⁻¹ were loaded into a 96-well plate held on a robotically controlled x-y stage and transferred to the beamline via a quartz capillary connected to a syringe pump. Data from the 5 mg mL⁻¹ assemblies were discarded due to sedimentation of the assemblies preventing reliable sample transfer into the capillaries. The experiments used a beam wavelength of λ = 1.03320 Å⁻¹ (12.0 keV) with dimensions of 300 µm × 200 µm and a typical flux of 1.2 × 10¹³ photons per second. 2D diffraction images were collected on a Pilatus 1M detector. SAXS experiments were performed at q ranges between 0.002 and 0.25 Å⁻¹ and WAXS experiments were performed at a q range between 0.1 and 2 Å⁻¹. These overlapping spectra provide a total q range of 0.002–2.2 Å⁻¹. Spectra were recorded under flow (0.15 mL min⁻¹) to prevent X-ray damage from the beam. Multiples of approximately 15 spectra were recorded for each time point (exposure time = 1 s) and averaged spectra are shown after background subtraction against PBS in the same capillary.

CD spectroscopy was performed using an AVIV 410-SF CD spectrometer. Spectra were collected between 190 and 260 nm in PBS using 1 mm quartz cuvettes with a step size of 0.5 nm and 2 s averaging time. Data were analysed using the BeStSel (Beta Structure Selection) method of secondary structure determination³⁵.

Atomistic models were constructed using the Nucleic Acid Builder⁵⁸. Simulations were run in explicit water (TIP3P⁵⁹) using the ff15ipq forcefield⁶⁰ and the pmemd time integrator⁶¹. In order to hold the unit cell geometry to values consistent with the observed scattering, the alpha carbon of the central residue of each chain was subjected to a restraining force with spring constant 2 kcal mol⁻¹ Å⁻². Periodic boundaries were applied to the system such that it formed a truncated octahedron, which was relaxed during equilibration to a volume of 10,310 nm³, giving a density of 0.973 reference with 323,535 water molecules. The system state after 10 ns of equilibration was stripped of water molecules more than 10 Å from any non-hydrogen solute atom, and passed to CRYSOL3 for calculation of orientationally averaged scattering profile given the example state (including the ordered waters from the explicit solvent shell, and also including an approximate treatment of ordered water beyond this shell)^42,62.

Peptide assemblies were made up to concentrations of 1 or 5 mg mL⁻¹ suspensions containing 25 µM ThT in PBS. The first fluorescence measurement (t = 0) was recorded immediately after sample preparation. All the samples were then stored at room temperature and fluorescence intensity was recorded at different time points. Measurements were performed in triplicate using a ClarioStar fluorimeter equipped with a 96-well plate reader (excitation wavelength: 440 nm, emission wavelength: 482 nm).

Human-derived neuroblastoma cells (SH-SY5Y, ATCC Product Number: CRL-2266) were cultured in DMEM-F12 (Invitrogen) medium supplemented with 10% (v/v) foetal calf serum (FCS), 100 UmL⁻¹ penicillin and 100 µgmL⁻¹ streptomycin (Invitrogen, Carlsbad, CA). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂.

ThT staining was performed by incubating the amyloid assemblies with a 25 µM solution of ThT in PBS for 15 min, in an 8-well Labtek II chamber (Nunc). For the antibody stain the same assemblies were incubated in a 1:200 dilution of the A11 polyclonal antibody raised in rabbit (Invitrogen, REF: AHB0052, LOT:VF299837) in 2% BSA in PBS for 1 h. Following this the wells were carefully washed in PBS and a solution of the 2° antibody (Goat-Anti Rabbit IgG-Alexa Fluor 647, Product A21244, Lot #: 1871168, Molecular Probes) at 1:1000 dilution in PBS was incubated with the peptides for 1 h. Finally the assemblies were once again washed in PBS before being imaged via laser scanning confocal microscopy using a FV3000 microscope (Olympus) and 60× objective lens (1.35 NA Oil Plan Apochromat) using the following settings: ThT channel λex = 450 nm, λem = 490 nm, Alexa Fluora 647 channel λex = 650 nm, λem = 665 nm. The same imaging settings were used for all samples and the negative controls (peptide assemblies + 2° antibody only) were used to determine the level of background fluorescence. For positive controls, amyloid assemblies of phenylalanine were used under concentrations known to readily form oligomeric amyloid assemblies³⁸, which were shown here to bind strongly to the A11 antibody (Supplementary Fig. 9e, f).

Cells were seeded into 96-well plates at 1 × 10⁵ cells per mL and incubated for 24 h to ensure good attachment to the surface. A stock solution of peptide assemblies (10 mg mL⁻¹) was serially diluted into DMEM-F12 media 2.5–0.02 mg mL⁻¹ or 3.3–0.027 mM for ILLIIM and 2.45–0.02 mM for RNYIAQVD) and seeded onto the SH-SY5Y cells and incubated for 48 h, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as described previously⁶³. Equivalent MTT assays were performed on cells cultured in the same ratios of PBS to media, but in the absence of peptide, assemblies to confirm that the culture conditions were non-toxic (Supplementary Fig. 10). Absorbance readings of untreated control wells in 100% cell culture media were designated as 100% cell viability. Statistical analysis was performed by one-way ANOVA tests with Tukey comparison in the software GraphPad (Prism) ***p < 0.001. Flow cytometry assays to determine cell viability were performed in a similar manner to the MTT assays. Briefly, to determine the effect of the peptides on cellular viability SH-SY5Y cells were cultured in the presence of the peptides for 48 h, harvested and stained with the apoptosis stain Annexin V for 10 min on ice (Cat No. 550474, BD Biosciences, 5 µL in 100 µL of 2% FCS in PBS). Samples were diluted with 300 µL of 2% FCS in PBS and stained with the viability dye 7-AAD (559925 BD Biosciences, 5 µL per sample) and analysed using flow cytometry (FACS Aria III; BD Biosciences). Cell counts were performed manually using a hemocytometer, with tryphan blue to differentiate non-viable cells.

Further information on research design is available in the linked to this article. Nature Research Reporting Summary

Supplementary Information Peer Review File Reporting Summary