
==== Front
eLife
Elife
eLife
eLife
2050-084X
eLife Sciences Publications, Ltd

39109999
83320
10.7554/eLife.83320
version of record
Research Article
Biochemistry and Chemical Biology
Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates
Sangar Daljit 1
Hill Elizabeth 1
Jack Kezia 1
Batchelor Mark https://orcid.org/0000-0001-6847-5131
1
Mistry Beenaben 1
Ribes Juan M 1
Jackson Graham S 1
Mead Simon 1
Bieschke Jan https://orcid.org/0000-0002-3485-9767
j.bieschke@ucl.ac.uk
1
1 https://ror.org/043j90n04 MRC Prion Unit at UCL, Institute of Prion Diseases London United Kingdom
Mapa Koyeli Reviewing Editor https://ror.org/05aqahr97 Department of Life Sciences, School of Natural Sciences, Shiv Nadar University India

Ron David Senior Editor https://ror.org/013meh722 University of Cambridge United Kingdom

07 8 2024
2024
13 e8332007 9 2022
20 7 2024
This manuscript was published as a preprint at bioRxiv.25 8 2022

© 2024, Sangar et al
2024
Sangar et al
https://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt–Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an ‘anti-chaperone’, which promotes toxic aggregation intermediates by inhibiting fibril formation.

prion
syntaxin
aggregation
chaperone
toxicity
amyloid
Research organism

E. coli
Mouse
http://dx.doi.org/10.13039/100000065 National Institute of Neurological Disorders and Stroke 1R21NS101588-01A1 Bieschke Jan http://dx.doi.org/10.13039/501100000265 Medical Research Council MC_UU_00024/6 Sangar Daljit Batchelor Mark Jackson Graham S Bieschke Jan http://dx.doi.org/10.13039/501100000265 Medical Research Council MRC Prion Unit Graduate Programme Hill Elizabeth Jack Kezia Mistry Beenaben The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Author impact statementA new native prion protein aggregation assay shows that syntaxin-6, a risk factor for sporadic Creutzfeldt–Jakob disease, delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates.
==== Body
pmcIntroduction

Prion replication and prion-like mechanisms are believed to drive dozens of human neurodegenerative and systemic diseases, as well as scrapie, bovine spongiform encephalopathy, and chronic wasting disease in animals (Prusiner, 1998; Brundin et al., 2010; Frost and Diamond, 2010). All of these disorders are caused by protein misfolding, but the details of the self-replication mechanism, the cellular factors involved in prion replication, and the molecular basis for neurotoxicity are far from clear despite decades of research (Collinge and Clarke, 2007). Prions are fibrillar assemblies of misfolded cellular prion protein (PrPC), which form highly ordered parallel in register intermolecular β-sheet amyloid structures (prion rods, Manka et al., 2022; Kraus et al., 2021). While purified prion rods are not directly neurotoxic, prion-associated toxicity can be blocked by anti-PrP antibodies (Benilova et al., 2020; Reilly et al., 2022) suggesting that non-fibrillar PrP assemblies, which form after prion titers plateau (Sandberg et al., 2011; Sandberg et al., 2014; Collinge, 2016), could be the toxic agents, as is the case for other amyloid-forming proteins (Haass and Selkoe, 2007; Corbett et al., 2020).

Prions can be highly infectious. However, accidental exposure only accounts for a tiny percentage of Creutzfeldt–Jakob disease (CJD) cases. Similarly, mutations in the PRNP gene, which codes for the prion protein PrP, cause only 10–15% of CJD cases (Mead et al., 2019). The majority of prion disease cases are sporadic in nature, where PrP is thought to spontaneously misfold. It is likely that hidden genetic risk factors modulate either the susceptibility to prion replication, prion-associated neurotoxicity, or the initial prion formation. A recent genome-wide association study to discover non-PRNP risk factors of sporadic CJD (sCJD) identified two highly significant risk loci including one in and near to STX6 (Jones et al., 2020). Elevated STX6 mRNA in the caudate and putamen nuclei, two regions implicated in CJD pathology (Meissner et al., 2009), correlated with CJD risk.

STX6 encodes syntaxin-6, an 8-exon, 255 amino-acid protein that predominantly localizes to the trans-Golgi network (TGN), and is mainly involved in recycling of cargo between the TGN and early endosomes (Jung et al., 2012). Syntaxin-6 is thought to form part of the t-SNARE complex involved in the decision of a target membrane to accept the fusion of a vesicle (Wendler and Tooze, 2001). Misfolded prion protein in infected cells localizes at the plasma membrane, the likely site of prion conversion (Goold et al., 2011), as well as in perinuclear compartments. Early and late endosomes, the endocytic recycling compartment and multivesicular bodies have also been implicated as prion replication sites (Yamasaki et al., 2018; Yim et al., 2015). These observations raise two alternative mechanistic hypotheses for the role of syntaxin-6 in prion disease: (1) a direct interaction with PrP in the course of misfolding and prion replication, or (2) an indirect effect through the cellular processing of PrPC or prions.

STX6 is also a risk gene for progressive supranuclear palsy (Höglinger et al., 2011), where the protein interacts directly with tau through its transmembrane domain (Lee et al., 2021). STX6 has been identified in a proteome-wide association study in Alzheimer’s disease (Wingo et al., 2021). Downregulation of syntaxin-6 expression in a murine neuroblastoma scrapie cell model (PK1) did not alter PrPC expression and had no obvious effect on PrPC trafficking (Jones et al., 2020), suggesting that it may directly alter prion replication by a yet undiscovered chaperone-like activity.

An array of chaperone proteins aid and tightly control protein folding and protein homeostasis in the cell (Hartl, 2011; Balch et al., 2008). Chaperones, such as the heat shock protein HSP70, inhibit protein misfolding and can delay amyloid formation in vitro (Glover and Lindquist, 1998) and in model organisms (Thackray et al., 2022). It seems likely that more proteins with chaperone-like functions are yet to be discovered.

Assessing the role of proteins with chaperone-like activity in PrP misfolding, fibril formation, and toxicity requires assays under native or near-native conditions. The established fibril formation assays for PrP in vitro require non-physiological conditions, such as high temperatures (Atarashi et al., 2011), denaturants (Legname et al., 2004), and detergents or low pH (Post et al., 1998). These conditions facilitate the unfolding and conversion of PrPC, but preclude the analysis of other folded proteins in the context of PrP fibril formation. Here, we have developed a fibril formation assay for recombinant murine and human PrPC (23-231) under near-native conditions to study the effect of syntaxin-6 and other cellular factors, which may be risk factors or potential therapeutic targets in prion disease.

Results

Full-length prion protein forms amyloid fibrils under near-native conditions

In order to study PrP fibril formation under near-native conditions in vitro, full-length murine PrPC 23-231 (mPrP23) and human PrPC 23-231 (hPrP23) were expressed recombinantly (Figure 1—figure supplement 1A), purified via Ni-NTA chromatography, and refolded into their native conformation (Figure 1—figure supplement 1E and F). Proteins were incubated in a non-binding 96-well plate under agitation, and amyloid formation was monitored via thioflavin T (ThT) fluorescence (Figure 1A, Figure 1—figure supplement 2). We tested different temperatures pH values and agitation conditions to assess whether PrP could form amyloid fibrils in vitro under near-native conditions (Figure 1—figure supplement 2A–C). While PrP was slow to form aggregates de novo without the addition of seeds, elevated temperatures accelerated fibril formation (Figure 1—figure supplement 2A) as had been previously observed (Ziaunys et al., 2021). Aggregation at 42°C, pH 6.8 yielded highly reproducible conditions for the formation of fibrils (Figure 1A–C, Figure 1—figure supplement 2B and C) when agitated by glass or silanized zirconia beads. Fibrils had diameters of 8–12 nm and morphologies typical of amyloid fibrils (Figure 1C, Figure 1—figure supplement 2G). Fibrils formed in native aggregation assay (NAA) were resistant to digestion by proteinase K to the same degree as authentic prion rods prepared from RML-infected mice, producing a main PK-resistant fragment of ~12 kD (Figure 1D, Figure 1—figure supplement 2H). The addition of preformed fibrillar aggregates as seeds accelerated fibril formation in a concentration-dependent manner by decreasing lag-times (t50, Figure 1A and B, Figure 1—figure supplements 2D and 3) but had no significant influence on rates of fibril elongation (Figure 1—figure supplement 2E). Lag phases and elongation rates of mPrP23 in vitro were indistinguishable when using synthetic seed formed from recombinant PrPC or using authentic prion rods, isolated from mice infected with scrapie strains RML or ME7 in (Figure 1—figure supplements 2D, E and 3). Interestingly, fibrils formed from synthetic seeds displayed increased ThT fluorescence when compared to RML and ME7 seeded fibrils, hinting at a difference in fibril structure imprinted by the different seed templates (Figure 1H, Figure 1—figure supplement 2F). For safety reasons, seeding of hPrP23 was restricted to synthetic PrP fibrils.

Figure 1. Native aggregation assay (NAA) of human PrP (23-231).

(A) Seed titration experiment using seed generated from hPrP23 fibrils formed de novo in NAA, seed fraction (w/w), 2.5 µM hPrP23, pH 6.8, 42°C. (B) Plot of lag phase vs. fraction of seed, mean ± SD, n = 3. (C) TEM image of hPrP23 fibrils formed after 166 hr incubation. (D) Western blot (ICSM35) of ex vivo RML prions and NAA aggregation endpoint samples after digestion with proteinase K (50 µg/ml, 30 min) and NaPTA precipitation. (E–H) Titration of PrP concentration (0.3–20 µM) in ThT aggregation assays containing 0.01% seed, pH 6.8, 42°C. (E) Plot of ThT fluorescence vs. time. (F) Plot of lag time t50 vs. PrP concentration. (G) Plot of elongation rate constant k vs. PrP concentration. (H) Plot of fluorescence amplitude A vs. PrP concentration. Source data is available at https://doi.org/10.17632/yggpkrgnx8.1.

Figure 1—figure supplement 1. Protein characterization.

(A) Coomassie/silver stained gels of purified hPrP23, mPrP23, and candidate proteins. (B) Circular dichroism spectrum of purified syntaxin-6 (STX6) protein showing a mostly alpha helical shape with minima at 208 nm and 223 nm. (C–F) Circular dichroism spectra of Hsp70 (HSPA1A), stathmin 1 (STMN1), human PrP 23-231,129M (hPrP23), and mouse PrP 23-231 (mPrP23). Source data is available at https://doi.org/10.17632/yggpkrgnx8.1.

Figure 1—figure supplement 2. Optimization of native aggregation assay (NAA).

(A) Plot of mPrP23 ThT fluorescence over time at 37, 42, and 50°C (pH 7.4). (B) mPrP23 NAA aggregation kinetics at pH 6.6, 6.8, 7.0, and 7.2 at 42°C. (C) NAA aggregation kinetics of hPrP23 (2.5 µM) with Zr vs. glass beads, pH 6.8 at 42°C. (D–F) mPrP23 seed titration experiments comparing seeding from synthetic PrP fibrils (Sp) and purified prion rods from mouse prion strains ME7 and RML. (D) Plot of time to half-maximal fluorescence (t50) (E) elongation rate constant (k), and (F) ThT amplitudes as functions of seed concentration; mean ± SD, n = 5. (G) TEM image of mPrP23 fibrils formed after 47 hr incubation at pH 6.8, 42°C; scale bar 100 nm. (H) Silver stain gel of hPrP23 monomer and NAA product after 30 min PK digestion (50 µg/ml). Source data is available at https://doi.org/10.17632/yggpkrgnx8.1.

Figure 1—figure supplement 3. mPrP23 seeding kinetics.

Graphs of native aggregation assay (NAA) on mPrP23 (10 µM) seed titration experiments demonstrate seed concentration-dependent acceleration of fibril formation. (A) First-generation (spontaneous) seed. (B) Seed generated from RML mouse strain. (C) Seed generated from ME7 mouse strain. Solid lines indicate sigmoidal fits of the data, n = 3. Post aggregation decay of ThT fluorescence, which was likely due to adsorption and sedimentation, was not included in the fits.

Figure 1—figure supplement 4. Amylofit analysis of concentration-dependent native aggregation assay (NAA) data.

Aggregation data from experiment shown in Figure 1E were smoothed by moving average and normalized (A) and then analyzed in the Amylofit framework to generate t50 values (B; n = 3). (C–F) Global fitting of data using different models for aggregation kinetics as indicated. Only the model including saturated elongation adequately fitted the data.

The lag phase of hPrP23 aggregation was largely independent of PrP monomer concentration (Figure 1E and F), while elongation rates even showed a slight inverse reaction to monomer concentration (Figure 1G). Therefore, PrP refolding rather than elongation may be rate-limiting under the assay conditions. ThT amplitude at the end of growth was proportional to monomer concentration (Figure 1H). However, the lag phase increased and ThT amplitude dropped at monomer concentrations above 16 µM, suggesting the formation of off-pathway PrP aggregates at high monomer concentrations, which do not contribute to fibril formation (Powers and Powers, 2008).

Analysis of aggregation kinetics using the Amylofit framework confirmed this interpretation (Figure 1—figure supplement 4). The concentration dependence of fibril formation could be described adequately only under the assumption that kinetics were dominated by saturated elongation. It should be noted that kinetic curves in the NAA have higher fluctuations in the ThT signal than found under denaturing conditions (see Sun et al., 2023). This is most likely due to the formation of large PrP aggregates in the NAA. These fluctuations had no influence on the accuracy of kinetic fitting when comparing lag times derived from raw data (Figure 1E and F) with those from smoothed aggregation curves (Figure 1—figure supplement 4A and B).

Syntaxin-6 inhibits PrP fibril formation

The STX6 gene is a proposed risk factor for sCJD, suggesting that it may alter PrP fibril formation. We therefore tested what effect the syntaxin-6 protein had on PrP fibril formation under native conditions and compared it to two reference proteins: a well-characterized amyloid inhibitor, the heat shock protein HSPA1A, which is the human, inducible form of HSP70, and the α-helical microtubule-associated protein stathmin 1 (STMN1) as positive and negative controls, respectively (Figure 1—figure supplement 1A). Circular dichroism confirmed that all proteins retained their native, mostly α-helical folds under assay conditions (Figure 1—figure supplement 1B–F).

To our surprise, syntaxin-6 delayed the lag phase of hPrP23 fibril formation even at highly sub-stoichiometric molar ratios of 1:100 and prevented fibril formation entirely at equimolar ratio (STX6, Figure 2A and B). At the same time, it only slightly lowered fibril elongation rates (Figure 2C). Syntaxin-6 promoted the formation of aggregate clusters (Figure 2D and E) rather than distinct amyloid fibrils (Figure 1C). Immuno-gold staining revealed that syntaxin-6 was incorporated into these aggregates (Figure 2E). Similarly, HSPA1A prevented fibril formation in favor of amorphous PrP aggregates (Figure 2—figure supplement 1A), while hPrP23 formed fibrillar aggregates in the presence of control protein STMN1 (Figure 2—figure supplement 1B). However, sedimentation after 0–90 hr incubation revealed that syntaxin-6 delayed but did not prevent the formation of hPrP23 aggregates. In contrast, a substantial fraction of hPrP23 remained soluble when incubated with HSPA1A at a 1:10 molar ratio (Figure 2—figure supplement 2).

Figure 2. Syntaxin-6 (STX6) delays hPrP23 fibril formation at molar ratios of 1:1–1:100; 2.5 µM hPrP23, pH 6.8, 42°C, 0.01% seed.

(A) ThT fluorescence vs. time. (B) Plot of lag phase t50 vs. molar ratio of STX6/PrP; mean ± SD, n = 3. (C) Plot of elongation rate constant k vs. molar ratio of syntaxin-6/PrP. (D) TEM image of hPrP23 co-aggregated with syntaxin-6 at 1:10 (STX6/PrP) molar ratio under standard native aggregation assay (NAA) conditions for 116 hr. (E) Immuno-TEM image of hPrP23 – syntaxin-6 co-aggregate cluster after 100 hr aggregation. Syntaxin-6 is labeled with anti-syntaxin-6 Ab/10 nm anti-rabbit immunogold beads; scale bars 200 nm.

Figure 2—figure supplement 1. TEM images of native aggregation assay (NAA) aggregation endpoints of hPrP23 co-aggregated with HSP70 (HSPA1A) or stathmin 1 (STMN1).

(A) PrP forms amorphous aggregates in the presence of HSPA1A at 1:10 molar ratio. (B) PrP forms fibrillar aggregates in the presence of STMN1 at 1:10 molar ratio; scale bar 100 nm.

Figure 2—figure supplement 2. Sedimentation assay of hPrP23.

hPrP23 was incubated for 0–90 hr alone or co-aggregated with syntaxin-6 (1:10 molar ratio) or HSPA1A (1:10 molar ratio). Protein samples were centrifuged; supernatants were collected, and analyzed by silver-stained SDS-PAGE. Source data is available at https://doi.org/10.17632/yggpkrgnx8.1.

Figure 2—figure supplement 3. Seeding capacity of aggregation intermediates.

Aggregates of hPrP23 (black) or hPrP23 co-aggregated with syntaxin-6 (STX6, 1:10 molar ratio; orange) collected at 20 hr (A), 40 hr (B), 60 hr (C), and 90 hr (D) were used to seed a second aggregation assay using hPrP23 as substrate; seed concentration 0.1% (w/w). Aggregation time points correspond to those used in toxicity assays. Graphs represent averaged data of three replicate experiments.

Figure 2—figure supplement 4. Secondary seeding seed dilution experiment of hPrP23 aggregates.

(A) Native aggregation assay (NAA) experiment of hPrP23 (2.5 µM) in the absence (orange) or presence of stx6 (0.25 µM, gray). (B) Endpoint aggregates (t = 70 hr) were separated into total, soluble (supernatant), and insoluble (pellet) fractions, which were resuspended to the original volume and sonicated to generate secondary seeds. (C–E) Box plots of times to half maximal fluorescence (t50) in secondary hPrP23 seeding assays seeded at 10–3 to 10–8 seed to monomer ratio using total, supernatant, and pellet seed fractions; n = 3, data points at the bottom of graphs indicate samples that failed to aggregate.

Figure 2—figure supplement 5. Secondary seeding seed dilution kinetics.

Kinetic traces of hPrP23 native aggregation assay (NAA) secondary seeding assays at 10–3 to 10–8 seed to monomer ratio described in Figure 2—figure supplement 4: (A) total, (B) pellet, and (C) supernatant seed fractions and unseeded kinetics; n = 3, only dilutions 10–3 to 10–5 are plotted for supernatant seeds.

Figure 2—figure supplement 6. Salt-dependent inhibition of fibril formation.

Syntaxin-6, STMN1, HSPA1A 1:10 molar ratio were incubated with hPrP23. (A) Plot of relative ThT fluorescence profiles vs. time in NAA (150 mM NaCl, 42 °C, 0.01% seed,). (B) Plot of lag phase t50 vs. NaCl concentration for hPrP23 alone vs. co-aggregated with syntaxin-6 (STX6) at 1:10 molar ratio. (C) Plot of elongation rate constant k vs. NaCl concentration. (D) Plot of lag phase t50 vs. NaCl concentration for hPrP23 alone vs. co-aggregated with HSPA1A at 1:10 molar ratio. (E) Plot of lag phase t50 vs. NaCl concentration for hPrP 23–231 alone vs. co-aggregated with STMN1 at 1:10 molar ratio; mean ± SD, n = 3.

To probe the nature of PrP aggregates formed in the presence of syntaxin-6, we performed two secondary seeding assays. In the first assay, we tested the seeding capacity of PrP samples harvested at different incubation times (20, 40, 60, 90 hr) in the presence and absence of syntaxin-6 in a NAA seeded with 0.1% fibrillar hPrP23 (Figure 2—figure supplement 3). Samples harvested during the lag phase of either reactions were not seeding competent, but both aggregates formed in the presence and absence of syntaxin-6 (1:10) were seeding competent after the formation of ThT-positive aggregates, indicating that, at sub-stoichiometric ratios, syntaxin-6 delays, but does not prevent the formation of seeding competent PrP fibrils. We then generated seeds in a second experiment, in which the primary seed was added at a lower concentration (0.01%), which prolonged the delay in fibril formation by syntaxin-6 (Figure 2—figure supplement 4A). Seed preparations harvested after 70 hr incubation were then separated into total, soluble, and insoluble fractions and added to secondary seeding assays at 10–3 to 10–8 molar ratio monomer equivalents (Figure 2—figure supplement 4B–E). Both in syntaxin-6 and in untreated samples, seeding activity was only found in the total and insoluble fractions. The presence of syntaxin-6 lowered the amount of seeding competent aggregates by at least three orders of magnitude (Figure 2—figure supplement 4C–E and 5).

To further probe the mechanism of syntaxin-6, we tested the influence of NaCl concentration on fibril formation in the presence and absence of syntaxin-6 (1:10 molar ratio) in the NAA (Figure 2—figure supplement 6, Figure 3—figure supplements 2 and 3). We compared kinetics in the presence of syntaxin-6 to those in the presence of HSPA1A and STMN1, respectively, at the same molar ratio (Figure 2—figure supplement 6A). HSPA1A delayed fibril formation to a similar degree as syntaxin-6, whilst, as expected, STMN1 had no significant effect on fibril formation kinetics. In the absence of syntaxin-6, the lag phase of PrP fibril formation decreased only weakly with NaCl concentration while the elongation rate increased (Figure 2—figure supplement 6B and C). Syntaxin-6 prolonged the lag phase at low and physiological salt concentrations, but lag phases decreased with increasing NaCl concentration, suggesting that ionic interactions may mediate the binding of syntaxin-6 to PrP. A similar salt dependence was observed for HSPA1A (Figure 2—figure supplement 6D), while STMN1 did not inhibit fibril formation at any salt concentration (Figure 2—figure supplement 6E). Syntaxin-6 did not significantly reduce fibril elongation compared with untreated PrP independently of salt concentration (Figure 2—figure supplement 6C).

To visualize the interaction of syntaxin-6 with PrP aggregates and fibrils, we imaged NAA endpoints by dSTORM super-resolution microscopy (Figure 3—figure supplement 1) revealing that hPrP23 and syntaxin-6 co-aggregated into large aggregate clusters. Super-resolution microscopy indicated a fibrillar substructure of these aggregates (Figure 3—figure supplement 1A and B) similar to that observed in TEM (Figure 2D and E). When, conversely, syntaxin-6 labeled with AlexaFluor647 was added to pre-formed fibrillar hPrP23 formed under NAA conditions, it preferentially bound to specific ‘hotspots’ on the PrP fibril (Figure 3A and B, indicated by arrows). In this experiment, hPrP23 fibrils were unlabeled and were visualized by transient amyloid binding (TAB) imaging using the amyloidophilic dye Nile red (Spehar et al., 2018). We repeated the experiment with syntaxin-6-AlexaFluor488 (Figure 3E) and with unlabeled proteins using immuno-TEM (Figure 3F) to exclude that the interaction was influenced by the choice of fluorescent dye. Both assays confirmed the presence of interaction hotspots. Interestingly, interaction sites were often located at fibril ends or kinks in the fibril. Since syntaxin-6 only weakly affected apparent fibril elongation rates in bulk assays, this may suggest a role of syntaxin-6 in fibril breakage or secondary nucleation. PrP formed longer, unbranched fibrils with increasing salt concentration. As had been seen in TEM, syntaxin-6 induced the formation of aggregate clusters when visualized by TAB imaging (Figure 3—figure supplements 2 and 3) when compared with untreated fibrils, supporting the hypothesis that the protein may alter secondary nucleation/branching of fibrils.

Figure 3. Imaging of hPrP23 fibrils incubated with syntaxin-6.

hPrP23 (2.5 µM) was pre-aggregated in native aggregation assay (NAA) for 115 hr and incubated with syntaxin-6 (250 nM) for 1 hr. hPrP was visualized by TAB imaging using 10 nM Nile red dye; syntaxin-6 was labeled with AlexaFluor488 (A–D) or AlexaFluor647 (E) and imaged by dSTORM. (A) SR image overlay shows syntaxin-6 binding at hotspots and PrP fibril ends. (B) Magnified area from (A). (C) Widefield image taken with green laser (561 nm) illumination. (D) Widefield image taken under blue laser (473 nm) illumination. (E) SR image overlay of PrP and syntaxin-6-AF647 images; scale bars 2 µm. (F) TEM Immuno-gold staining of hPrP23 fibrils incubated with syntaxin-6; scale bar 100 nm.

Figure 3—figure supplement 1. Overlay of dSTORM SR images taken at 473 nm and 638 nm excitation.

Co-aggregates of hPrP23 and syntaxin-6 formed in native aggregation assay (NAA) under conditions of Figure 3. (A) Co-aggregation of hPrP23 labeled with AlexaFluor 647-NHS (2.5 µM, 3% labeled, 95% unlabeled) with 10 nM syntaxin-6 labeled with AlexaFluor488-NHS. (B) Co-aggregation of PrP labeled with AlexaFluor 488-NHS (2.5 µM, 5% labeled, 95% unlabeled) with syntaxin-6-AlexaFluor647 at 1:10 molar ratio (syntaxin-6, 250 nM, 4% labeled, 96% unlabeled). Insets: widefield images with 473 nm and 638 nm excitation and composite image of both channels.

Figure 3—figure supplement 2. TAB SR microscopy images of aggregation endpoints of hPrP23.

hPrP23 was aggregated in NAA with increasing salt concentrations from 0 mM to 600 mM NaCl for 140 hr; scale bars 2 µm.

Figure 3—figure supplement 3. TAB SR microscopy images of aggregation endpoints of hPrP23 co-aggregated with syntaxin-6.

Native aggregation assay (NAA) was performed for hPrP23/syntaxin-6 co-aggregation (1:0.1 molar ratio) at increasing salt concentrations from 0 mM to 600 mM NaCl for 140 hr; scale bars 2 µm.

Syntaxin-6 interacts with PrP in cell models of prion disease

Syntaxin-6 and PrP need to interact in order to directly affect prion replication and/or prion-associated toxicity in vivo. We therefore probed the interaction of syntaxin-6 with PrP via Förster resonance energy transfer (FRET) imaging in the PK1 cell model (Klöhn et al., 2003), which can be persistently infected with RML prions. Proteins were stained with anti-syntaxin-6 antibody (C34B2)/anti-rabbit-Alx647 and three monoclonal anti-PrP antibodies (5B2, 6D11, 8H4)/anti-mouse-RhX, which bound different regions of PrP (Figure 4A, Figure 4—figure supplements 1 and 2). PixFRET analysis indicated a perinuclear interaction between both proteins, both in non-infected (PK1) and infected (iS7) cells (Figure 4A, wide arrows). An additional FRET signal on the plasma membrane of infected cells suggests that syntaxin-6 may be recruited into misfolded PrP assemblies (Figure 4A, narrow arrows). This interaction resulted in FRET signals with all three anti-PrP antibodies, whereas only the two antibodies directed to the unfolded N-terminal domain of PrP (5B2 and 6D11) displayed FRET in membrane-associated compartments (Figure 4—figure supplement 1). This may suggest a different binding mode of syntaxin-6 to PrP species in both compartments.

Figure 4. Interaction of PrP and syntaxin-6 in vivo.

(A) Non-infected PK1 cells and persistently infected PK1 cells (iS7) were immuno-stained with anti-PrP antibodies 6D11 and 5B2 (green), with anti-syntaxin-6 antibody (red) and with DAPI (blue). Förster resonance energy transfer (FRET) analysis reveals interaction in perinuclear compartments (wide arrows) and at membranes in infected cells (narrow arrows). Panels show zoomed regions of images in Figure 4—figure supplement 1. (B, C) In vitro prion replication by protein misfolding cyclic amplification (PMCA) using Stx6+/+ and Stx6-/- mouse brains as substrate. PMCA reactions were seeded with RML prions from terminally ill mice and subjected to PMCA for 96 cycles over 48 hr. (B) Representative western blot (ICSM35) after PK digestion. Molecular weight markers are indicated on the left. (C) The PrPSc signal was quantified using densitometry and normalized to the control unamplified reaction. Bar graphs each represent mean ± SD of biological replicates from three separate mice, each blotted as two technical replicates. Source data is available at https://doi.org/10.17632/yggpkrgnx8.1.

Figure 4—figure supplement 1. hPrP/syntaxin-6 Förster resonance energy transfer (FRET) analysis.

(A, B) PixFRET analysis of non-infected PK1 cells and persistently infected PK1 cells (iS7). Cells were immuno-stained with anti-PrP antibodies 6D11, 8H4, or 5B2 (green), with anti-syntaxin-6 antibody (STX6, red) and with DAPI. (C) Schematic of binding locations of antibodies used in FRET analysis (5B2, 6D11, 8H4). Numbers represent putative epitopes. (C) was adapted from Figure 2B from Castle and Gill, 2017.

Figure 4—figure supplement 2. hPrP/syntaxin-6 Förster resonance energy transfer (FRET) analysis controls.

PixFRET analysis of control cells. iS7 cells were immuno-stained with anti-PrP antibodies 6D11, 8H4, or 5B2 (green) only, with anti-syntaxin-6 antibody (STX6, red) only, or with secondary antibodies only and with DAPI.

Syntaxin-6 knockout does not affect the replication of prions in vitro

Infectious prions can be replicated in cell-free conditions using protein misfolding cyclic amplification (PMCA), whereby the conversion of PrPC into PK-resistant PrPSc is enhanced by cyclic bursts of sonication in a brain homogenate substrate (Bieschke et al., 2004). Here, we employed PMCA to explore whether the presence or absence of syntaxin-6 in the substrate affected prion conversion. To this end, we seeded PMCA substrates derived from Stx6+/+ and Stx6-/- mice, respectively, (Hill, 2023) with RML prions. Both substrates generated comparable amounts of PK-resistant PrP (Figure 4B), indicating that syntaxin-6 does not directly alter prion replication in vitro under PMCA conditions, which are dominated by fibril fragmentation/elongation.

Syntaxin-6 prolongs the presence of toxic PrP aggregation intermediates

The previous data would suggest a protective rather than deleterious effect of syntaxin-6 in prion disease. However, the oligomer toxicity hypothesis posits that mature fibrils may not be the toxic species in prion nor in other amyloid diseases (Collinge, 2016; Haass and Selkoe, 2007). Correspondingly, purified, highly infectious prion rods were not toxic to primary neurons (Benilova et al., 2020). To test whether the same logic applied to the effect of syntaxin-6 on PrP aggregation, we assessed the toxicity of PrP to mouse primary neurons at different stages of the aggregation kinetics by neurite length (Figure 5) and by counting the number of viable neurons (Figure 5—figure supplement 1). PrP aggregates were highly toxic to primary neurons during lag and early growth phases (20 hr and 40 hr incubation), but neurotoxicity was diminished at the plateau phase of fibril formation, so that endpoint aggregates (90 hr) were no more toxic than PrP monomers or buffer controls (Figure 5A and C, Figure 5—figure supplement 1). Notably, PrP toxicity preceded the formation of seeding-competent assemblies (Figure 2—figure supplement 3), supporting the hypothesis that the toxic PrP species is a pre-fibrillar assembly.

Figure 5. Toxicity in mouse primary neurons incubated with hPrP23/syntaxin-6 at different aggregation time points.

Native aggregation assay (NAA) was performed as in Figure 2 and samples were diluted 1:10 into cell culture media at the indicated time points (0 hr, 20 hr, 40 hr, 64 hr, 90 hr). (A) Plot of effect of PrP (250 nM) on neurite length compared to effect of media or NAA assay buffer at the same dilution from four independent wells. (B) Plot of effect of hPrP23 co-aggregated with syntaxin-6 (STX6, 1:10 molar ratio). (C) Box plot of average relative neurite lengths between 80–100 hr incubation of PrP in the cell culture media. p-Values were derived from ANOVA testing between PrP vs. PrP/syntaxin-6 samples at each time point.

Figure 5—figure supplement 1. Survival of primary neurons.

Numbers of live primary neurons after incubation with hPrP23 or hPrP23 co-aggregated with syntaxin-6 (STX6, 1:10 molar ratio) as in Figure 5. Relative numbers of neurons after 4 hr incubation in four fields of view per sample were normalized to the same FOV at t = 0 hr; *p<0.05, **p<0.001, ***p<0.0001, n.s. not significant from ANOVA statistical analysis.

In contrast, PrP aggregated in the presence of syntaxin-6 (1:0.1 molar ratio) retained its neurotoxicity significantly longer than PrP incubated in the absence of syntaxin-6 (Figure 5B and C, Figure 5—figure supplement 1). While PrP species formed early in the lag phase of aggregation at 20 hr were neurotoxic both in the presence and absence of syntaxin-6 when compared to t = 0 hr, PrP was significantly more toxic in the presence of syntaxin-6 at later time points when PrP would form fibrils in the absence of syntaxin-6 (Figure 5C, Figure 5—figure supplement 1). Thus, in delaying or preventing PrP fibril formation, syntaxin-6 prolonged the presence of toxic aggregation intermediates and exacerbated neurotoxicity.

Discussion

Development of a novel native aggregation assay

We developed a fibril formation assay for full-length mouse and human PrPC to be able to study the mechanistic effect of cellular proteins such as the proposed sCJD risk factor syntaxin-6 on PrP misfolding and self-assembly under near-native conditions. A multitude of in vitro fibril formation assays have been developed previously (Schmidt et al., 2015). While the generation of authentic prion infectivity was reported for several assays (Legname et al., 2004; Schmidt et al., 2015), fibrils generally form under highly non-physiological conditions in the presence of high concentrations of guanidine or urea (Legname et al., 2004), at low pH (Swietnicki et al., 2000), in the presence of SDS (Stöhr et al., 2008), or at very high temperatures (Atarashi et al., 2007). None of these condition provide a native environment for protein–protein interaction, and many cellular proteins will be unfolded under these assay conditions.

Full-length PrP formed fibrils with morphology and β-sheet secondary structure characteristic of amyloid under conditions of the NAA (Willbold et al., 2021). Fibril formation could be tracked by binding of the amyloidophilic dye ThT and displayed sigmoid aggregation kinetics typical of a nucleated polymerization mechanism (Cohen et al., 2013). As had been observed for other amyloidogenic proteins, solution conditions such as pH and temperature affected aggregation kinetics (Buell et al., 2014; Milto et al., 2014). Aggregation proceeded faster at neutral pH, while monomeric protein was stable at pH of 6.5 and below.

Seeding by preformed fibrils reduced the lag phase of aggregation (Jarrett and Lansbury, 1993; Cohen et al., 2006). The limiting dilution for seeding was ~10–10 (w/w), which corresponds to approximately 2000 PrP molecules per well or 5 prion rods of a typical length of 200 nm (Terry et al., 2016), similar to other seeding assays (Stöhr et al., 2008; Atarashi et al., 2007; Du et al., 2011). In NAA, both lag phase t50 and elongation rate constant k displayed little dependence on monomer concentration, which suggests that monomer refolding was rate-limiting for fibril elongation. While in general aggregation kinetics scale with monomer concentration, elongation saturates at high monomer concentration as conformational change of PrP becomes rate-limiting (Jain and Udgaonkar, 2008; Honda and Kuwata, 2017). ThT fluorescence amplitudes scaled with PrP concentration between 2 and 16 µM, then dropped and lag phase increased. A competing non-fibrillar aggregation pathway at high protein concentration could account for this observation (Powers and Powers, 2008; Bieschke et al., 2005). High salt concentration shields charges, which promotes hydrophobic interactions and lowers the critical concentration of nucleated polymerization, often leading to the formation of amyloid fibril clusters (Klement et al., 2007). Notably, while PrP aggregated faster in the NAA at high salt concentration, it tended to form longer, more isolated fibrils.

Inhibition of fibril formation by syntaxin-6

Syntaxin-6 delayed the formation of PrP fibrils at highly sub-stoichiometric ratios and co-aggregated with PrP into aggregate clusters. It delayed, but did not prevent, the formation of insoluble seeding-competent PrP assemblies. On the face, its effect was similar to that of the heat shock protein family A (HSP70) member 1A (Figure 2, Figure 2—figure supplement 3), which would suggest a role of syntaxin-6 as a potent aggregation inhibitor with chaperone-like activity. Chaperones assist folding of newly synthesized proteins, sequester soluble misfolded polypeptides, and target them for degradation, which makes them promising therapeutic targets in protein misfolding diseases (Balch et al., 2008; Hartl, 1996; Muchowski and Wacker, 2005).

HSP70 has been described as a ‘holdase’, which inhibits amyloid formation of various proteins at sub-stoichiometric concentrations even in the absence of ATP by binding to early-stage aggregation intermediates (Evans et al., 2006; Wacker et al., 2004). Overexpression or exogenous addition of HSP70 rescued cytotoxicity in cell cultures and model organisms (Rosas et al., 2016; Bongiovanni et al., 2018; Fernandez-Funez et al., 2016). Sequestering aggregation intermediates into inert off-pathway aggregates can be a powerful strategy against amyloid toxicity (Bieschke et al., 2010; Ehrnhoefer et al., 2008). However, our data indicate that, far from detoxifying PrP aggregation intermediates, the delay of PrP fibril formation by syntaxin-6 exacerbated PrP toxicity by allowing toxic aggregation intermediates to persist for longer (Figure 5).

Conversely, natural and chemical chaperones can detoxify aggregation intermediates by promoting fibril formation and stabilizing mature amyloid fibrils (Cohen et al., 2006; Bieschke et al., 2012; Lam et al., 2016). Syntaxin-6 seems to present the flip side of this benign activity in delaying PrP fibril formation and thus prolonging the presence of toxic aggregation intermediates.

The nature of the toxic species in prion disease is under active investigation. Brain homogenates from scrapie-infected mice have a specific toxic component, which can be blocked by the addition of anti-PrP antibody (Benilova et al., 2020). However, highly purified infectious prion rods were shown not to be directly neurotoxic (Benilova et al., 2020), suggesting that toxicity may be caused by non-prion assemblies, which accumulate after the infectious titer has peaked and which, unlike prions, are sensitive to sarkosyl (Reilly et al., 2022; Sandberg et al., 2011; Sandberg et al., 2014) or by transient aggregation intermediates, whose formation may be catalyzed by prions or other fibrillar PrP assemblies (Collinge and Clarke, 2007).

The chaperone Brichos breaks generation of toxic Aβ oligomers, which are formed through surface-catalyzed secondary nucleation (Cohen et al., 2015). Conceivably, syntaxin-6 could have the opposite effect on PrP fibril formation, in which toxic oligomer formation is promoted, by delaying competing fibril formation pathways. PrP itself binds to and inhibits the elongation of Aβ amyloid fibrils (Amin and Harris, 2021; Bove-Fenderson et al., 2017) oligomers and nanotubes. The presence of PrP during aggregation led to the formation of larger numbers of shorter fibrils and increased Aβ neurotoxicity in a dose-dependent manner (Amin and Harris, 2021). This suggests that PrP not only acts as a receptor for toxic amyloid oligomers (Freir et al., 2011; Nicoll et al., 2013), but may exacerbate neurotoxicity in a mechanism similar to syntaxin-6. Notably, syntaxin-6, when added to preformed fibrils, preferentially bound to fibril ends and other hotspots (Figure 3). Previous studies found only weak binding of syntaxin-6 to monomeric PrPC (Jones et al., 2020), which, together with the inhibition at sub-stoichiometric concentrations, strongly suggests an interaction with early aggregation intermediates and with fibrillary assemblies, possibly promoting secondary nucleation.

While variants at the STX6 locus are known risk factors for sCJD (Jones et al., 2020), its deletion only modestly delayed the incubation period in RML prion infected mice, an observation that is open to different interpretations (Hill, 2023). Rather than directly altering prion replication kinetics, the protein may confer risk of disease by either facilitating the initial generation of prions in sporadic disease or exacerbating prion-associated toxicity. Our results lend support to the second hypothesis as syntaxin-6 interacted with early aggregation intermediates of PrP in vitro and exacerbated their toxicity (Figure 5) but did not accelerate the formation of seeding-competent PrP species (Figure 2—figure supplement 3) nor directly affected the replication of pre-existing prion seeds in PMCA reactions (Figure 4B). This finding argues against the hypothesis that syntaxin-6 binding to mature PrP fibrils induces fibril breakage. Correspondingly, an increase in fibrils branching/secondary nucleation as observed in the presence of syntaxin-6 (Figure 3—figure supplements 1 and 3) would have little effect on replication kinetics dominated by fragmentation/elongation.

Localization of syntaxin-6 and PrP

A direct effect of syntaxin-6 on PrP assembly and/or prion-associated toxicity in vivo obviously requires the two proteins to be present in the same sub-cellular compartment. PrPC is present mainly in the TGN and on the outer plasma membrane, while mature PrPC is tethered to the membrane by a C-terminal glycophosphadityl-inositol anchor. At the plasma membrane, PrPC is incorporated into lipid rafts and caveolae, which are membrane microdomains enriched in cholesterol and sphingolipids. However, it has also been detected in multi-vesicular bodies and, to a small degree, in the cytosol (Grassmann et al., 2013). Both lipid rafts and the endocytic pathway are implicated as sites of prion replication (Grassmann et al., 2013).

Interestingly, syntaxin-6 has role in caveolin membrane recycling (Choudhury et al., 2006). As a SNARE protein, syntaxin-6 sits on the outside of vesicles/intracellular compartments and on the inner plasma membrane, so that a direct interaction with PrP would require mislocation of either protein. Both PrP and syntaxin-6 contain a trans-membrane domain and PrP can populate transmembrane forms in neurons (Hegde et al., 1998; Stewart and Harris, 2005). It is therefore plausible that, similar to the tau protein, PrP and prions bound to the membrane bilayer could interact with the transmembrane domain of syntaxin-6. Alternatively, mislocation of misfolded PrP assemblies could facilitate interaction with syntaxin-6. Amyloid assemblies can disrupt membrane integrity through mechanical rupture, pore formation, or altering membrane curvature (Shi et al., 2015; Lashuel et al., 2002). Disruption of membranes on the cell surface and in vesicles of the endocytic pathway is a central step in the internalization of other misfolded polypeptides, such as Aβ (Jin et al., 2016; Friedrich et al., 2010). Similarly, it has been hypothesized to be necessary for cellular prion propagation (Yim et al., 2015). These processes could bring misfolded PrP into direct contact with syntaxin-6. FRET imaging suggests a direct contact between PrP and syntaxin-6 in a membrane-associated compartment in prion-infected cells (Figure 4A, Figure 4—figure supplement 1B), supporting this hypothesis.

Conclusions

We developed an aggregation assay of murine and human prion protein under near-native conditions. PrP forms amyloid fibrils under the assay conditions, which bind ThT and can seed further fibril formation. Unlike natively unfolded proteins, however, PrP fibril formation does not scale strongly with monomer and seed concentrations, suggesting that structural conversion of native PrP into the amyloid fold is rate-limiting. This is consistent with the observation that the time constant of exponential prion replication in vivo is independent of PrP expression (Sandberg et al., 2011).

In the NAA, both the PrP and potential protein modulators of aggregation are in their native conformation, which means that the mechanisms by which protein and small modulators alter amyloid formation can be assessed. We analyzed the effect of syntaxin-6, a recently proposed risk factor for sCJD, on PrP self-assembly. To our surprise, we discovered that the protein acts as an ‘anti-chaperone’, which, by delaying PrP fibril formation, prolonged the persistence of toxic aggregation intermediates in vitro. Genetic variants in or near to STX6 that enhance brain expression of the protein might therefore modify the risk of CJD by direct interaction with PrP and changing aggregation pathways, including the possibility of favoring more toxic aggregation intermediates.

While at this point we cannot assess whether the toxicity of PrP aggregation intermediates formed in vitro recapitulates the authentic PrP species responsible for neurotoxicity in prion disease in vivo, our data highlight a new mechanism by which protein modulators of amyloid formation can have counter-intuitive deleterious effects.

Materials and methods

Protein expression and purification

Human PrP (23-231), mouse PrP (23-231)

The open-reading frame of the human PrP gene (PRNP) (residues 23-231), containing methionine at residue 129 and the mouse PrP gene (Prnp) (residues 23-231, including S231), was synthesized de novo by Eurofins MWG Operon, with a thrombin-cleavable His-Tag added to the PrP N-terminus. The ligated pTrcHisB/PRNP and pTrcHisB/Prnp constructs were used to transform the Escherichia coli host strain BL21(DE3) (Novagen), genotype F′ ompT hsdSB (rB- mB-) gal dcm (DE3), which was then plated onto Luria-Bertoni (LB) agar plates containing 100 μg/ml carbenicillin. Cultures were grown for purification using a modification of protocols previously described (Hosszu et al., 2005). Briefly, following harvesting, cells were sonicated and their inclusion bodies containing PrP resolubilized in 6 M guanidine hydrochloride (GuHCl), 50 mM Tris-HCl, 0.8% β-mercaptoethanol, pH 8.0. These were loaded onto a Ni-NTA column equilibrated in 6 M GuHCl, 10 mM Tris-HCl, 100 mM Na2PO4, 10 mM glutathione pH 8.0, and eluted from the column using 10 mM Tris-HCl, 100 mM Na2PO4, 2 M imidazole pH 5.8. Residual GuHCl was removed through dialysis against 20 mM Bis-Tris.HCl pH 6.5, CaCl2 added to a final concentration of 2.5 mM, and the N-terminal His-tag cleaved by thrombin for 16 hr at room temperature (RT) (0.1 U thrombin [Novogene]/1 mg of PrP added). The cleaved protein was loaded onto a second Ni-NTA column equilibrated with 20 mM Bis-Tris pH 6.5, 25 mM imidazole pH 6.5, and the eluted PrP peak was collected and dialyzed against 10 mM Bis-Tris pH 6.5, and aliquoted and stored at –80°C. Protein concentrations were determined by UV absorption at 280 nm using a calculated molar extinction of 56,667 M–1 cm–1 and 62,268 M–1 cm–1 for human and mouse PrP, respectively (https://web.expasy.org/protparam).

SEC of syntaxin-6/hPrP23/mPrP23

2 ml pre-concentrated, pre-cleared protein was loaded via injection valve onto a Sephacryl S100HR column (GE Healthcare) (26/60: 320 ml bed volume) that had been pre-equilibrated with 20 mM Tris-HCl, 0.2 M NaCl, pH 8. A flow rate of 2 ml/min was used; all eluted peaks were checked on a silver-stained NuPage Bis-Tris 12% gel, using the protocol according to the silver staining kit (SilverQuest Silver Staining Kit-1, LC6070, Life Technologies Ltd) and the purified protein was aliquoted and stored at −80°C until use.

Stathmin 1 (STMN1)/heat-shock protein 70 (HSPA1A)

Glycerol stocks of E. coli BL21 strains expressing His-tagged STMN1 and HSPA1A were a gift by E. Wanker, MDC-Berlin, Germany. Small-scale cultures of STMN1 and HSPA1A were cultured in LB/Amp (100 µg/ml) by inoculating with stabs from glycerol stocks. Cells were pelleted and then inclusion bodies lysed in 1 ml lysis buffer (containing 50 mM Tris, 0.2 M NaCl, pH 8 containing 0.1% Tween20, 0.5% NP40, 50 U/ml benzonase, 10 µg/ml lysozyme, and 1 mM PMSF) placed on ice for 1 hr with gentle vortexing every 15 min. (All centrifugations were done at 4°C to further prevent non-specific cleavage by proteases and samples were kept on ice.) The lysate was cleared by centrifugation (1 hr, 16,100 rpm) and then loaded onto 2 ml NiNTA resin bed packed in mini spin columns, pre-equilibrated with buffer containing 50 mM NaH2PO4, 0.3 M NaCl, 20 mM imidazole at pH 8. The cleared lysate was loaded onto the column, unbound was eluted off the column first with gentle centrifugation (2 min, 2000 rpm); the resin was washed to thoroughly remove unbound protein by twice loading 700 µl pre-equilibration buffer (see above) and centrifugation for 30 s, 2000 rpm, and then 2 min, 2000 rpm for the second wash. Finally, the pure protein was eluted in elution buffer (containing 50 mM NaH2PO4, 0.5 M NaCl, 0.5 M imidazole, pH 8). This was done by loading 100 µl elution buffer with a 30 s centrifugation at 2000 rpm followed by a second elution with 150 µl elution buffer and a 2 min centrifugation at 2000 rpm. Both fractions were pooled and checked for purity as below.

The purified STMN1 and HSP70 recombinant proteins were checked for purity by Coomassie staining after PAGE on NuPage Bis-Tris 12% gels. Protein identity was confirmed by mass spectrometry following a standard trypsinization protocol on a Waters Xevo-XS spectrometer as described in Manka et al., 2022.

Syntaxin-6 (STX6)

Syntaxin-6 (residues 38-318;accession number KU144824) was prepared according to Jackson et al., 1999 with modifications. Briefly, the DNA sequences encoding the syntaxin-6 protein in pQTEV were transformed into BL21 (DE3). BL21 cultures were grown in LB medium in the presence of 100 µg/ml ampicillin. Expression of the protein was induced using 1 mM IPTG and was purified from inclusion bodies under denaturing conditions using nickel superflow resin with an AKTA Pure (GE Healthcare Life Sciences). The protein was refolded on NiNTA resin and eluted from the column using an imidazole gradient. The eluted material was extensively dialyzed against 20 mM Tris, 2 mM EDTA, 10 mM DTT, 200 mM NaCl pH 8.0. DTT concentration was reduced to 2 mM in the final storage buffer. Syntaxin-6 was further purified by size-exclusion chromatography as detailed above, resulting in a protein band with an apparent molecular weight of ~28 kD (Figure 1—figure supplement 1A). The final concentration of the syntaxin-6 protein was determined by absorption measurement at 280 nm, ε = 31,970/M/cm. Aliquots were stored at −80°C until use.

Circular dichroism (CD) spectroscopy

CD data were recorded on a Jasco J-715 spectrophotometer equipped with a thermoelectric temperature controller. A 1 mm path length cuvette was used for all CD spectroscopy measurements. Wavelength measurements were recorded from 195 nm to 260 nm at 20°C using 0.36 mg/ml syntaxin-6 in 10 mM Na-phosphate, pH 8.

Native aggregation assay (NAA)

PrPC was filtered through a 100 kD membrane filter in 10 mM Bis-Tris pH 6.5 (Amicon ULTRA 0.5 ml 100K 96PK, UFC510096, Merck Life Science UK Ltd) to remove aggregates and then diluted into reaction buffer (50 mM Na-phosphate, pH 6.8, 150 mM NaCl, 0.01% Na-Azide, 20 µM ThT and 5 mM Bis-Tris pH 6.5) with the addition of 0.1% seed (w/w), unless indicated otherwise. The standard final concentrations of PrP and NaCl were 2.5 µM and 150 mM, pH 6.8 unless indicated otherwise. The seed consisted of aggregated PrP material from an unseeded reaction under the same conditions as above. It was diluted to 10% (w/w) in 5 mM Bis-Tris, pH 6.5 and sonicated in a water bath sonicator (GRANT, XUBA1) for 15 min prior to addition to the reaction mix. Aggregation assays for neuronal toxicity were set up in parallel to the standard assays for kinetic analysis, but here the ThT and Na-Azide was omitted from the reaction mixes.

The seed and post-aggregation samples were handled with pre-silanized tips, in pre-silanized non-binding tubes (Repel-silane ES, Sigma, 17-1332-01). The reaction mix (94 µl per well) was dispensed into low binding 96-well COSTAR (#3651) plates and three silanized zirconium beads (0.5 mm diameter, Figures 1—4; 1 mm diameter, Figure 5) were added to each well. A single glass bead (2 mm) was added instead of the Zr beads where indicated. ThT kinetics were recorded on a BMG ClarioStar plate reader at 42°C with a shaking speed of 700 rpm, set to shake for 100 s with an incubation time of 20 s between agitation. Focal height was set to 20 mm, top read, with excitation at 440 nm and emission set to 485 nm. Aggregation kinetics were fitted in MATLAB E2021b using the following equation:(1) F=f0+A1+e−k(t−t50)tlag=t50−2k

For Amylofit analysis (Meisl et al., 2016), kinetic traces were normalized to the first 5 hr of the plateau phase and noise in the fluorescence signal was smoothed by calculating a 6 hr moving average. Half-times were calculated in Amylofit and kinetics were analyzed at monomer concentrations of 2, 6, 10, 16, and 20 µM and a seed concentration of 2 nM (monomer equivalent) using the models for fragmentation, saturated elongation + fragmentation, secondary nucleation + fragmentation, and nucleated polymerization.

EM grid preparation

Endpoint samples (5–6 μl) from NAAs sonicated in a water bath for 10–15 s were loaded onto carbon-coated 300 mesh copper grids (Electron Microscopy Sciences) that had been glow-discharged for 40 s using an PELCO easiGLOW glow discharge unit (Ted Pella Inc, USA). Samples were left to bind for 30 min, blotted dry, washed in water (1 × 50 μl), blotted, and then stained with 10 μl Nano-W (methylamine tungstate) stain (Nanoprobes) for 1 min followed by 30 s (with blotting in between stain times). Images were acquired on a Talos electron microscope (FEI, Eindhoven, NL, now Thermo Fisher).

Immunogold labeling of PrP fibrils with NAPTA precipitation

40 µl of fibrils (completion at 115 hr) were dispensed into an RNase free pre-silanized tube with silanized tips and centrifuged for 1.5 hr (4°C, 16,100 rpm) after which the pellet was resuspended in 500 µl TBS containing 0.1% (w/v) sarkosyl with sonication for 30 s in a water bath sonicator (GRANT, XUBA1), and incubated with syntaxin-6 antibody (C34B2) Rabbit mAb 2869 (Cell Signaling Technology Europe B.V, 2869S; 1:100) for 16  hr at 25°C with gentle agitation.

The following day, the fibrils were precipitated with sodium phospho-tungstate (NaPTA) by addition of 40.5 µl of 4% (w/v) NaPTA (prepared in H2O; pH 7.4) and centrifuged at top speed for 30 min to recover a pellet which was resuspended in 10 µl total volume of 1:20 (v/v) Goat anti-Rabbit IgG conjugated to colloidal gold (10 nm) (Insight Biotechnology Ltd, GA1014) in TBS containing 5% (v/v) glycerol and incubated at 25°C for 3 hr with gentle agitation. The sample was pulse centrifuged for 5  s and 5 µl labeled sample was loaded onto a glow-discharged carbon-coated grids and stained with Nano-W as previously described without the 10–15 s pre-sonication step.

SR sample preparation

Eight-well chamber slides (IBL Baustoff, 2 20.140.082, C8-1.5-H-N, 8-well chambered cover glass with #1.5 high-performance cover glass, 0.170 ± 0.005 mm) were cleaned by soaking them overnight in a 2% solution of Hellmanex II detergent diluted in ultrapure water and then washed thoroughly in ultrapure water before rinsing in 100% methanol and then a final thorough rinse in ultrapure water and then allowing the slides to dry.

Once dry, the slides were glow-discharged for 40 s using a PELCO easiGLOW glow discharge unit (Ted Pella Inc). 10 µl sample was added to the center of a well and allowed to incubate for 1 hr for the fibrils to adhere to the glass. The well was then washed 5× with 500 µl of HPLC purified water before 200 µl buffer (which consists of 10 nM Nile Red; Sigma-Aldrich, 72485-1G in PBS or GLOX buffer). The wells were then sealed either with Nescofilm or TWINSIL before imaging on a custom-built TAB/dSTROM super-resolution microscope (Sun et al., 2023).

Enzymatic oxygen scavenger (GLOX, glucose oxidase with catalase) buffer consisted of two solutions. Solution A: Tris (50 mM, pH 8.3), NaCl (10 mM), glucose (10% w/v), and β-mercaptoethylamine (Sigma-Aldrich, 30070, 10 mM). Solution B: glucose oxidase (Sigma-Aldrich, G2133, 8 mg), and catalase (Sigma-Aldrich, C100, 38 μl, 21 mg/ml) in PBS (160 µl). Solutions A and B were mixed at the ratio of 99:1 (v/v) immediately before use.

Labeling recombinant protein with AlexaFluor dyes

AlexaFluor dyes (AlexaFluor 488 NHS Ester, A20000, AlexaFluor 647 NHS Ester, A20006, AlexaFluor 488 C5 Maleimide, A10254, https://www.thermofisher.com) were diluted in DMSO to a stock concentration of 10 mg/ml. The dyes were mixed with the recombinant protein constructs at a molar ratio of 2:1 dye:protein construct. The mixture of dye and protein constructs was protected from light by covering the vessel with foil and left to mix on a rotator overnight at 4°C. The following day, the unbound dye was removed by dialysis in 2.5 l for 2 × 1 hr, with 8 kD dialysis membrane (SpectraPor 7 8000 Dalton MWCO; 11425919, Fisher Scientific, UK). For syntaxin-6 the buffer used for dialysis was 10 mM Na-phosphate, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, pH 8; for PrP, the buffer used for dialysis was 10 mM Bis-Tris, pH 6.5. Once dialysis was complete, the labeled protein was recovered from the dialysis membrane and checked on a 4–12% Bis-Tris gel. To check labeling efficiency, an absorption spectrum measurement (200–700 nm) was taken and the final concentrations of dye and protein were calculated according to the manufacturer’s protocol. The labeled proteins were then aliquoted and stored at –80°C.

Primary neuronal culture and toxicity analysis

Primary neuronal cultures were derived from brains of unmodified inbred FVB/N mice. Hippocampi of male and female E17 mouse brains from a single litter (7–9 embryos) were dissected in HBSS (Thermo Fisher Scientific) supplemented with 1% L-glutamine, 1% HEPES, and 1% pen-strep. Cells were dissociated using 0.25% trypsin + 0.04% benzonase, triturated mechanically, and counted using a Neubauer haemocytometer. Cells were plated in DMEM supplemented with 10% horse serum (#26050-88, Invitrogen) at 10K/well to the inner 60 wells of poly-l-lysine-coated 96-well plates (Greiner, 655936). At 1 hr post-plating, DMEM was aspirated and exchanged for Neuralbasal medium (21103049, Thermo Fisher Scientific) supplemented with 0.25% GlutaMAX (35050061, Thermo Fisher Scientific) 2% Gibco B27 supplement (17504044, Thermo Fisher Scientific) and incubated at 37°C (20% O2, 5% CO2). FVB/N neurons were maintained in culture for 11–12 days prior to a 96 hr treatment. Images of live cells were taken on an IncuCyte S3 reader (Sartorius) with a ×20 objective in phase contrast. Four views were captured in each well every 4 hr. PrP samples were diluted 1:10 into fresh media and added at the 24 hr mark.

Neurite lengths were evaluated using the NeuroTrack module of the IncuCyte S3 software package (rev 2019A) using the following parameters: cell body cluster segmentation 0.7; cleanup 0; cluster filter 0; neurite sensitivity 0.25; and neurite width 1 μm. Detected neurite masks are highlighted in pink in images. Neurite length data were normalized to the initial (0 hr) value for each well and means ± standard deviation were calculated from quintuplicate sample wells. Average neurite lengths at the 80–100 hr time interval were visualized in a box plot.

The number of live neurons was counted after 0- and 4-day incubation as a secondary toxicity assay. Data represent averages from four images under each condition and time point, normalized to the number of neurons present at the time of protein addition in each field of view. ANOVA statistical analysis was performed in OriginPro 2019.

FRET imaging

500 µl of 50,000 cells/ml (uninfected S7 and infected iS7 subclones of PK1 N2a neuroblastoma cells) were plated in eight-well chambered glass coverslips (Thermo Scientific [155411]) and incubated at 37°C/5% CO2 for 3 days. Cells were then fixed for 15 min at RT with 3.7% formaldehyde diluted in Dulbecco’s phosphate-buffered saline (DPBS; Gibco [14190-094]). After washing once with DPBS, cells were treated with ice-cold acetone for 1 min. Cells were washed before treatment with 3.5 M GdnSCN for 10 min. Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C. Antibodies were removed, and following one wash imaging was performed with a Zeiss LSM 710 laser-scanning microscope with oil-immersion ×63/1.4 NA objective.

Four images were acquired for each sample: (a) donor (excitation 561 nm, emission 580–610 nm); (b) acceptor (633 nm excitation, 670–800 nm emission); (c) FRET excitation 561 nm, emission 670–800 nm, all using the MBS 488/561/633 triple dichroic mirror; and (d) DAPI (405 nm excitation, 440–460 nm emission). Laser power, photomultiplier gain, and pinhole size were adjusted as to not exceed the PMT dynamic range. Identical parameters were used for all image acquisition. Three biological replicates were imaged, with n = 3–6 images each.

FRET data were analyzed using the ImageJ PixFRET plugin according to the developer’s manual (Feige et al., 2005). Syntaxin-6-only-stained iS7 cells were used as acceptor bleed-though control and 5B2/6D11/8H4-only stained cells were used as donor bleed-through controls, respectively. FRET images were calculated at 1 pixel Gaussian blur and rendered using the Parula HDR lookup table at a range of 0–500.

Cell lines

Neuro2a (N2a) cell line and derivatives

N2a cells (male donor) were sourced from ATCC (CCL-131). Prion susceptible subclones of N2a cells were derived as described in Klöhn et al., 2003; Marbiah et al., 2014 and authenticated by transcriptomic analysis, where gene expression differences between original N2a cells and prion-susceptible subclones (PK1 S7, iS7) were investigated and documented (Marbiah et al., 2014). All actively used N2a sublines are tested for mycoplasma contamination at least once every 2 years. No mycoplasma contaminations have been reported in the MRC Prion Unit in the last 10 years.

PMCA using Stx6+/+ and Stx6-/- mouse brain homogenate

Tissue collection

Brains for the PMCA substrate were derived from juvenile Stx6+/+ and Stx6-/- male C57BL/6N mice at 3 months of age. Animals were sacrificed by CO2 asphyxiation and immediately perfused with 20 ml ice-cold perfusion buffer (1× DPBS + 5 mM EDTA). The perfused brain was then removed and stored frozen at –70°C until use.

Substrate preparation

9% (w/v) mouse brain homogenates were prepared in ice-cold conversion buffer (1× DPBS, 150 mM NaCl, 1% [v/v] Triton X-100) with protease inhibitors (1x Protease Complete with EDTA) with a dounce, glass homogenizer. Debris was removed by centrifuging the homogenates at 1200 × g for 2 min, with the supernatant subsequently being collected and stored at –70°C until use.

PMCA

0.8 µl of seed (I6200 10% [w/v] brain homogenate from terminal RML-infected CD-1 mice) was spiked into 0.2 ml PCR tubes containing 79.2 µl of Stx6+/+ or Stx6-/- substrate containing three 0.5-mm-diameter zirconium beads. Samples were subjected to PMCA in an automated sonication bath (QSONICA) at 35°C for 96 cycles (50% amplitude, 30 s sonication every 30 min) for a total of 48 min sonication over 48 hr. Following sonication, the samples were briefly centrifuged before storage at –80°C. Control reactions were also prepared, which were directly frozen.

Immunoblotting

Immunoblotting was performed as previously described with minor modifications. Briefly, each reaction was incubated with proteinase K (Roche, Cat# 3115887001) at 50 μg/ml for 30 min at 37°C. 20 µl of PK-digested material was electrophoresed and gels electroblotted. Membranes were probed with 200 ng/ml ICSM35 anti-PrP antibody (D-Gen Ltd) in PBST for 1 hr at RT or overnight at 4°C. After washing, the membranes were probed with a 1:10,000 dilution of alkaline-phosphatase-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich, A2179) in PBST. Blots were incubated for 5  min with CDP-Star Chemiluminescent Substrate (Thermo Scientific, T2147) and visualized on Biomax MR film (Kodak) or visualized on a LiCor Odyssey imager using anti-mouse pAb-IRdye800CW (1:5000) as secondary antibody.

RML prion rods for immunoblotting were prepared as described in Wenborn et al., 2015. Endpoint NAA samples (160 µl, 2.5 µM hPrP23 monomer equivalent) were digested with proteinase K (50 µg/ml) at 37°C for 30 min, pelleted with 4% (w/v) NaPTA as described above, resuspended in 10 µl water, and sonicated for 15 min in a water bath sonicator (GRANT, XUBA1) before SDS-PAGE and blotting.

Work with animals was performed under the license granted by the UK Home Office (Project Licenses 70/6454 and 70/7274) and conformed to University College London institutional and ARRIVE guidelines.

Funding Information

This paper was supported by the following grants:

http://dx.doi.org/10.13039/100000065 National Institute of Neurological Disorders and Stroke 1R21NS101588-01A1 to Jan Bieschke.

http://dx.doi.org/10.13039/501100000265 Medical Research Council MC_UU_00024/6 to Daljit Sangar, Mark Batchelor, Graham S Jackson, Jan Bieschke.

http://dx.doi.org/10.13039/501100000265 Medical Research Council MRC Prion Unit Graduate Programme to Elizabeth Hill, Kezia Jack, Beenaben Mistry.

Acknowledgements

We thank Adam Wenborn and Dr. Jonathan Wadsworth, MRC Prion Unit, for providing mouse scrapie material and mass spectrometry; Dr. Peter Klöhn for providing PK1 S7 and iS7 cell lines; Prof. Erich Wanker, MDC-Berlin, Germany, for providing E. coli clones for protein expression; and the staff of the MRC Prion Unit at UCL Biological Services Facility for animal husbandry and care. We thank Dr. Georg Meisl, University of Cambridge, for helpful discussions. The research was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health grant number 1R21NS101588-01A1, MRC grant MC_UU_00024/6 to JB, and the MRC Prion Unit at UCL graduate programme. The Stx6-/- mice were obtained from the MRC Harwell Institute, which distributes these mice on behalf of the European Mouse Mutant Archive (https://www.infrafrontier.eu/emma/). The MRC Harwell Institute is also a member of the International Mouse Phenotyping Consortium (IMPC) and has received funding from the Medical Research Council for generating and/or phenotyping the Stx6-/- mice. The research reported in this publication is solely the responsibility of the authors and does not necessarily represent the official views of the Medical Research Council. Funding and associated primary phenotypic information may becan be found at https://www.mousephenotype.org.

Additional information

Competing interests

Author contributions

Ethics

Additional files

Reporting standard 1. ARRIVE E10 checklist.

MDAR checklist

Data availability

All data generated or analysed during this study and all used analysis scripts have been uploaded to Mendeley data and are available to the public under https://doi.org/10.17632/yggpkrgnx8.1.

The following dataset was generated:

Bieschke J 2024 Syntaxin 6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates Mendeley Data 10.17632/yggpkrgnx8.1

10.7554/eLife.83320.sa0
Editor's evaluation
Mapa Koyeli Reviewing Editor https://ror.org/05aqahr97 Department of Life Sciences, School of Natural Sciences, Shiv Nadar University India

The current study presents an important discovery about Syntaxin 6 (Stx6)'s anti-chaperoning activity on PrP. The authors provide compelling evidence that the anti-chaperone activity arises as Stx6 delays PrP fibril formation and in the presence of Syntaxin 6, the amorphous aggregates of PrP are more toxic to neuronal cells. This study provides a critical molecular link between PrP aggregation and neurotoxicity.

10.7554/eLife.83320.sa1
Decision letter
Mapa Koyeli Reviewing Editor https://ror.org/05aqahr97 Department of Life Sciences, School of Natural Sciences, Shiv Nadar University India

Chakraborty Kausik Reviewer https://ror.org/05ef28661 CSIR- Institute of Genomics and Integrative Biology India

Maji Samir K Reviewer https://ror.org/02qyf5152 Indian Institute of Technology Bombay India

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Syntaxin 6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Kausik Chakraborty (Reviewer #2); Samir K Maji (Reviewer #3).

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife. As you will see below, reviewers 1 and 2 were critical with respect to the physiological relevance of interaction between Stx6 and PrP in addition to raising several other technical points. Reviewer 3 also asked many pertinent questions, as elaborated below. The time that it would take to satisfactorily address these concerns may be substantial, which was the basis for declining the work in its current form.

Reviewer #1 (Recommendations for the authors):

The suggested experiments can be addressed by the authors to make their claim stronger:

1) Authors should check in vivo interaction of PrP and Stx6 in different conditions that favors prion pathogenesis. If there is no interaction between Stx6 and Prp, the whole hypothesis of Stx6-mediated enhancement of prion pathogenesis would be baseless.

2) Figure 2D: What is the assay end point? Is it 115h? If it is 115h, then figure 2E should also show the status of aggregates at the same time point rather than at 65h.

3) Figure 5: The reduction in relative neurite length by Prp-Stx6 aggregates compared to PrP fibrils as an indicator of neuro-toxicity although significant but very small in % change. Is this much of change in neurite length with Stx6-PrP aggregates justify the enhanced toxicity model by Stx6? Can authors use some other assays like cell death assays that can show the enhanced toxicity more clearly?

Reviewer #2 (Recommendations for the authors):

In this paper, Sangar.et al. discuss an in vitro assay to generate toxic PrP aggregates under near native conditions using purified human and murine PrP (Prpc) which allows them to characterize protein-amyloid interactions. Interestingly, they find that interaction of STX6 (Syntaxin-6) leads to formation of amorphous PrP aggregates instead of fibrillar aggregates which prolongs toxicity in primary neurons. There is novelty in the work but has not been sufficiently explored.

Details:

1) Does Stx6 bind to preformed prions, or do they bind to the prions only while they are forming? If they do bind to preformed prions their effect on aggregation kinetics may have a different explanation (please see the comment below).

2) Stx6 seems to only increase the lag phase (with a minor effect on the elongation rate) – does it interfere with the nucleation kinetics by binding to the seeds, or do they bind to the folded/unfolded PrP to change the equilibrium and hence alter the nucleation kinetics?

Without understanding these basics, it is hard to conclusively state the mechanism of Stx6's effect on PrP aggregation.

3) Is stx6 secreted when they are overexpressed? Stx6 overexpression is a risk factor for CJD, so how is the in vivo expression level related to prion formation?

4) How do the authors connect the expression level correlation to PrP toxicity to formation of PrP/Stx6 mixed aggregates?

5) Is there any evidence that PrP/Stx6 forms mixed amyloids in vivo?

6) How do the authors deconvolute the toxicity of PrP and Stx6 in the PrP/Stx6 mixed amyloids? The toxicity could be due to Stx6 or PrP aggregates/amyloids?

7) The toxicity differences are very modest given stark differences in aggregate morphology in the presence or absence of Stx6. How do the authors explain this?

Other comments:

1) Apart from the development of an in vitro setup to generate toxic PrP aggregates, a major finding of the manuscript is the interaction between STX6 and PrP which prevents formation of fibrillar aggregates supported by ThT fluorescence assay, immunogold-EM analysis, and high-resolution microscopy and cytotoxicity data using primary neurons. From the data shown here we can convincingly conclude the following:

a) Recombinant PrP forms fibrillar aggregates in the assay conditions described.

b) STX6 and PrP interact in vitro.

c) This interaction prevents formation of fibrillar aggregates.

2) However, the data to show that STX6 interacts with PrP in-vivo (both in its monomeric and aggregated form) is lacking. The co-localization of Syntaxin-6 in the PrP aggregates isolated from clinical samples (patient samples) would be a crucial validation of the observations made in-vitro i.e, Interaction of Syntaxin-6 with PrP aggregates. While this may be complicated, it will be difficult to interpret the observations made in vitro in the absence of in vivo data.

3) And more importantly, the article could be further strengthened by more robust biochemical analysis into the mechanism of how STX6 interacts with PrP.

4) Following up on point 3, the authors may investigate whether STX6 interaction facilitates release of PrP from cells similar to tau (Lee et al. 2021).

5) In the TEM micrographs, it is very hard to locate the aggregates (Figure 2F and S4B). But for the ease of understanding of general reader, it is important to show presence of both the aggregate and the colocalization of STX6 in the immunogold staining.

6) Does treatment of primary neurons with 1:1 stoichiometry of STX6 and PrP prevent PrP induced neurotoxicity? Concentration dependent analysis may be done. This will further validate the finding observed in Figure S2(A).

7) While this may be out of scope of the study, authors may look into the effects of overexpression or knockdown of syntaxin-6 on PrP aggregate formation.

Reviewer #3 (Recommendations for the authors):

The objectives of the project are well-addressed and the discussion part is nicely illustrated. Some of the results look very interesting, leading to some exciting predictions (e.g., refolding of monomer, formation of off-pathway intermediates, etc.) based on the initial result. However, some additional experiments are required for a better understanding of the present results. Hereby, the authors are requested to address the following concerns and perform additional experiments that are mentioned in the following section.

Major points:

• Although the fibrillation assay used in this manuscript does not use denaturant or detergent, however, the PrP proteins are kept at 42{degree sign}C, pH 6.8, under agitation with zirconium 86 beads. How a condition like agitation can be claimed as a native environmental condition and will ensure that the assay condition is not facilitating protein unfolding, unlike the other reported fibrillation assays?

• In line 121, the authors wrote that "formation of aggregates with more amorphous morphology, which seems to consist of short fibrils" this is inconsistent comment as proteins will form amorphous or amyloids. Amorphous protein aggregates can't compose of short fibrils?

• The Thioflavin T aggregation profile looks extremely noisy (e.g., Figure 1A, 2A) with huge fluctuation in the Y axis. The authors should explain the reason for this much fluctuation in the ThT signal and fit the kinetic profile taking care of the standard deviation.

• The authors have calculated lag times (t50) and fibril elongation rate (k) in different experimental conditions throughout the manuscript. However, the authors did not show the calculation performed to determine these two parameters in the method section. The authors should incorporate that. Moreover, the authors should perform a web-based global fitting software interface, such as Amylofit (Meisl et al., 2016), to precisely evaluate different kinetic parameters for better comparison.

• The quality of TEM images of hPrP (Figure 1C) and moPrP (Figure S2C) is of poor quality. The authors are requested to change these with high-resolution images. Moreover, the present immune-gold staining image (Figure 2F) does not show the fibril where immune-gold labeled Stx6 is incorporated. The authors should provide a better-quality immune-gold staining image with a clear fibril image so that the location of incorporated Stx6 (immune-gold labeled) is clearly understood.

• The authors have shown that full-length PrP proteins form amyloid fibrils under near-native conditions. The authors should centrifuge to isolate the fibril fraction and show the proteinase-K digestion of the fibril, which is a known property of PrP amyloid fibril. The authors also should compare this PK resistance data with native PrP amyloid isolated from mouse prion strains ME7 and RML that the authors have used. Further, the authors should also perform the same with the fibril bound to Stx6, Hspa1a and Stathmin 1 to understand the nature of the aggregates in the presence of the additives.

• The authors have speculated the formation of the "off-pathway" intermediate based on the observation that the lag phase of aggregation has increased and ThT amplitude has dropped at high monomer concentration. The author should isolate the intermediate through centrifugation at the particular aggregation condition where the predicted off-pathway intermediate populates the most. To precisely comment on the "off-pathway" intermediate, the authors should add the isolated intermediate fraction during the fibrillation kinetics in a dose-dependent manner to see the concentration effect of the intermediate in fibrillation kinetics that will provide direct evidence to support the statement.

• To check the interaction with PrP protein in super-resolution microscopy, the authors have chosen only Stx6 among HSPA1A, STMN1, and STX6. The authors should justify the specific reasons behind choosing Stx6 over the others.

• The authors have observed that the interaction sites of Stx6 were often located at fibril ends or kinks in the fibril. Thus, the authors have proposed a potential role of Stx6 in fibril breakage or secondary nucleation. Firstly, the authors should reconfirm the Stx6 binding location in PrP from the clear immune-gold staining images. Secondly, the authors should isolate the Stx6-bound fibrils and perform a seeding reaction (prion amplification) with only the PrP monomer to see whether Stx6-bound PrP fibrils are competent for elongation or not. If Stx6 binds to the fibril end, it might not elongate in the presence of PrP monomer unless sonication is done. Also, authors should comment on whether Stx6 fibrils are capable of surface-catalyzed secondary nucleation.

• Although the highlight of the paper is the formation of toxic aggregation intermediates in the presence of Stx6 that prolongs the fibril formation, the report does not describe much about the nature of toxic intermediates. The authors should isolate the intermediate through centrifugation and structurally characterize the intermediate through biophysical experiments (e.g., mass spectrometry, CD, DLS, FTIR, TEM, and Native-PAGE). The authors should perform an MTT assay to check the toxicity of the intermediate apart from the already shown experiment of time-dependent relative neurite length of mouse primary neuron.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Syntaxin-6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates" for further consideration by eLife. Your revised article has been evaluated by David Ron (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) The authors have not addressed the issue of extremely noisy ThT aggregation kinetics correctly, although it is likely that the lag-phase data is robust. However, as the graph is noisy indicating that the aggregation assays are non-standard and do not replicate the behavior of similar assays reported earlier, further explanation from the authors is required.

2) The author's reply to usage of Amylofit fitting software to determine the kinetic parameters is not well justified and the previous concern remains. Authors can regenerate better quality of kinetics data for proper fitting by Amylofit or may explain the limitations of their dataset to such fitting software.

3) It is still unclear whether the physiological prions formed in mouse brains are similar in properties to the ones formed in vitro in NAA conditions by the authors. While the fibrils formed in vivo are quite resistant to PK digestion, can a similar assay be performed on the in vitro formed fibrils? Without any experimental proof, it is difficult to validate if the structure of the fibrils formed in vitro under NAA conditions would be similar to the fibril structures found naturally.

4) The suggested experiment to prove if the intermediate is off-pathway was not performed. The authors are requested to perform this crucial experiment.

10.7554/eLife.83320.sa2
Author response
[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

Reviewer #1 (Recommendations for the authors):

The suggested experiments can be addressed by the authors to make their claim stronger:

1) Authors should check in vivo interaction of PrP and Stx6 in different conditions that favors prion pathogenesis. If there is no interaction between Stx6 and Prp, the whole hypothesis of Stx6-mediated enhancement of prion pathogenesis would be baseless.

We agree with the reviewer that the interaction of syntaxin-6 with PrP in the cellular context is a key piece of evidence to support the physiological relevance of our data. We have therefore analysed the interaction between PrP and STX6 in a cell model of prion disease, both in non-infected (PK1) cells and in persistently infected (iF11) cells by FRET imaging. In all cases PrP and STX6 interacted in a perinuclear compartment, most likely the trans Golgi network (TGN). In prion infected cells, we observed additional FRET signals indicating interactions between PrP and STX6 at or near the cell membrane, supporting a role of STX6 in the prion infection process (new Figure 4A, Figures 4S1, 4S2). We also analysed the role of cellular STX6 in PMCA prion replication assays by using brain homogenates of Stx6 -/- knockout mice (new Figure 4B). Here, the deletion of Stx6 did not alter the amplification of RML prions, suggesting a role of syntaxin-6 in the initiation of PrP misfolding and/or in the toxicity of misfolded PrP assemblies rather than in the fragmentation / elongation mechanism of prion replication, which corresponds well to our data from experiments in vitro. We have added these points to the discussion of possible mechanisms by which syntaxin-6 alters initial prion formation, prion replication and prion-associated toxicity.

2) Figure 2D: What is the assay end point? Is it 115h? If it is 115h, then figure 2E should also show the status of aggregates at the same time point rather than at 65h.

We have replaced the EM images in Figure 2D and E to better match the endpoint of aggregation of hPrP23 without STX6 (115 h) and to improve contrast in immuno-EM.

3) Figure 5: The reduction in relative neurite length by Prp-Stx6 aggregates compared to PrP fibrils as an indicator of neuro-toxicity although significant but very small in % change. Is this much of change in neurite length with Stx6-PrP aggregates justify the enhanced toxicity model by Stx6? Can authors use some other assays like cell death assays that can show the enhanced toxicity more clearly?

In the new Figure 5S1, we have added neuronal survival measured as % of live neurons compared to t = 0 h as an additional measure of toxicity. Neuronal survival data closely mirror the results from the neurite length assay and are statistically highly significant. It should be noted that the observed 30% drop in neurite length corresponded to a very substantial fraction of neuronal loss. This is likely due to a technical limitation of the automated neurite length assay, in which fragmented neurites of dead neurons are misidentified by the algorithm.

Reviewer #2 (Recommendations for the authors):

In this paper, Sangar.et al. discuss an in vitro assay to generate toxic PrP aggregates under near native conditions using purified human and murine PrP (Prpc) which allows them to characterize protein-amyloid interactions. Interestingly, they find that interaction of STX6 (Syntaxin-6) leads to formation of amorphous PrP aggregates instead of fibrillar aggregates which prolongs toxicity in primary neurons. There is novelty in the work but has not been sufficiently explored.

Details:

1) Does Stx6 bind to preformed prions, or do they bind to the prions only while they are forming? If they do bind to preformed prions their effect on aggregation kinetics may have a different explanation (please see the comment below).

Our immuno-EM and SR-microscopy data indicate that STX6 can both bind to preformed PrP fibrils (Figure 3) and co-aggregate with PrP when present at the outset of the assay (Figure 3S1). When added to preformed fibrils, the protein does not coat the fibril but seems to bind at kinks and fibril ends. We performed a new secondary seeding experiment, using the PrP co-aggregated with STX6 as seeds for a new fibril formation assay (Figure 5S2, now Figure 2S3). The assay revealed that STX6 bound to fibrillar aggregates formed in the plateau phase of aggregation did not significantly impact seeding competence. Similarly, deletion of STX6 from the brain homogenate substrate of PMCA reactions did not alter the amplification of preformed prion seeds (Figure 4B). We therefore conclude that the role of STX6 must lie in the nucleation of fibril formation, as is suggested by the kinetic data the reviewer highlighted. It should be noted that prion replication and PrP fibril formation are not identical processes. Both may be orthogonal in vivo, so that inhibition of PrP amyloid can increase the presence of toxic aggregation intermediates while not affecting prion replication per se. We have added this aspect to the discussion of the revised manuscript.

2) Stx6 seems to only increase the lag phase (with a minor effect on the elongation rate) – does it interfere with the nucleation kinetics by binding to the seeds, or do they bind to the folded/unfolded PrP to change the equilibrium and hence alter the nucleation kinetics?

Without understanding these basics, it is hard to conclusively state the mechanism of Stx6's effect on PrP aggregation.

Our interpretation of the new seeding data (Figure 5S2, now Figure 2S3) and PMCA prion replication data (Figure 4B) strongly suggest a role in nucleation rather than seed replication through prion mechanisms (fragmentation / elongation) as discussed above. This does not preclude that STX6 may facilitate secondary nucleation and branching as suggested by our imaging data (Figures 2E, 3, 3S1-3), but does imply that these processes do not dominate the kinetics under our assay conditions. This interpretation matches the finding in vivo, where STX6 is a risk gene specifically for sporadic, not acquired, prion disease.

3) Is stx6 secreted when they are overexpressed? Stx6 overexpression is a risk factor for CJD, so how is the in vivo expression level related to prion formation?

The reviewer raises very relevant questions on the role of STX6 in sporadic Creutzfeldt Jakob disease. Genetic studies have identified two point mutations, which increase STX6 expression, as genetic risk factors for sCJD. Specifically, STX6 mRNA levels are higher in cells implicated in prion replication in vivo. Our collaborator Simon Mead has found that Stx6 knockout moderately delays onset of prion disease in RML-infected mice (Jones et al. bioRxiv, https://doi.org/10.1101/2023.01.10.523281) and will explore questions of STX6 expression and trafficking in vivo in forthcoming manuscripts.

4) How do the authors connect the expression level correlation to PrP toxicity to formation of PrP/Stx6 mixed aggregates?

As we discuss in the manuscript and in our response to the reviewer’s overall assessment, STX6 delays the formation of mature amyloid fibrils, which coincides with a prolonged presence of neurotoxic PrP aggregates. The nature of the neurotoxic species in prion disease – and for that matter in Alzheimer’s and other amyloid diseases – is the subject of intense research in our institute and elsewhere. We cannot claim to know its exact nature and we did not mean to imply that the co-aggregates of PrP and STX6 are necessarily toxic themselves. Our argument follows the well-established toxic oligomer hypothesis (see: Haass & Selkoe 2007), so our data imply that by delaying formation of mature amyloid fibrils, STX6 causes toxic aggregation intermediates to persist. We have edited our discussion to make this point more clearly.

5) Is there any evidence that PrP/Stx6 forms mixed amyloids in vivo?

Our new FRET interaction data from prion-infected cell models (Figures 4A, 4S1, 4S2) imply that both proteins interact in the trans Golgi network and, specifically in prion infected cells, in a membrane associated compartment, which does suggest interaction of STX6 with misfolded PrP species in these infected cell models. In their forthcoming paper, our collaborator Prof. Simon Mead presents immuno-histological data from scrapie infected mice to further assess STX6 deposition in vivo (Jones et al. bioRxiv, https://doi.org/10.1101/2023.01.10.523281).

6) How do the authors deconvolute the toxicity of PrP and Stx6 in the PrP/Stx6 mixed amyloids? The toxicity could be due to Stx6 or PrP aggregates/amyloids?

As we discussed in response to point 4 above, the nature of the toxic species in prion and other neurodegenerative protein misfolding diseases is yet unknown. However, from our in vitro experiments (Figure 5 and 5S1) we can exclude the hypothesis that STX6 itself is neurotoxic under our assay conditions.

7) The toxicity differences are very modest given stark differences in aggregate morphology in the presence or absence of Stx6. How do the authors explain this?

Please refer to our response to reviewer 1, point 3. In brief, the neurite length assay underreports the differences in toxicity. We added neuronal survival data (Figure 5S1) from the same experiment, which shows that PrP species from early aggregation time points are highly toxic and kill up to 80% of neurons. Neurotoxicity decreases with time as amyloid fibrils form, but persists longer in the presence of STX6 than in its absence.

Other comments:

1) Apart from the development of an in vitro setup to generate toxic PrP aggregates, a major finding of the manuscript is the interaction between STX6 and PrP which prevents formation of fibrillar aggregates supported by ThT fluorescence assay, immunogold-EM analysis, and high-resolution microscopy and cytotoxicity data using primary neurons. From the data shown here we can convincingly conclude the following:

a) Recombinant PrP forms fibrillar aggregates in the assay conditions described.

b) STX6 and PrP interact in vitro.

c) This interaction prevents formation of fibrillar aggregates.

We agree with the reviewer’s interpretation, but would amend that STX6 delays rather than completely prevents fibril formation at sub-stoichiometric ratios.

2) However, the data to show that STX6 interacts with PrP in-vivo (both in its monomeric and aggregated form) is lacking. The co-localization of Syntaxin-6 in the PrP aggregates isolated from clinical samples (patient samples) would be a crucial validation of the observations made in-vitro i.e, Interaction of Syntaxin-6 with PrP aggregates. While this may be complicated, it will be difficult to interpret the observations made in vitro in the absence of in vivo data.

We agree with the reviewer that in vivo interaction data are key to demonstrating the physiological relevance of STX6 in prion disease. As discussed above, we have therefore performed FRET imaging in scrapie-infected murine cells to demonstrate interaction between both proteins in vivo. The forthcoming corresponding study from the Mead laboratory (https://doi.org/10.1101/2023.01.10.523281) shows histopathological data of STX6 deposition in scrapie infected mice. A histopathological analysis of brain autopsies of CJD patients lies beyond the scope of the biophysical study presented in this manuscript.

3) And more importantly, the article could be further strengthened by more robust biochemical analysis into the mechanism of how STX6 interacts with PrP.

We fully agree with the reviewer on this point. The native aggregaton assay developed in this manuscript now provides us with the tools to do so, and a NMR-based study on the molecular interaction between STX6 and PrP will be the subject of a forthcoming manuscript.

4) Following up on point 3, the authors may investigate whether STX6 interaction facilitates release of PrP from cells similar to tau (Lee et al. 2021).

This is an excellent suggestion. The cell to cell transfer of prions is still poorly understood and is an active topic of ongoing research in the MRC Prion Unit.

5) In the TEM micrographs, it is very hard to locate the aggregates (Figure 2F and S4B). But for the ease of understanding of general reader, it is important to show presence of both the aggregate and the colocalization of STX6 in the immunogold staining.

We have replaced EM micrographs in Figures 2, 3 and S4 (now: Figure 2S1) to improve contrast, which hopefully resolves the issue raised by the reviewer. Interaction between both proteins can also be clearly seen in super-resolution fluorescence microscopy images (Figures 3 and 3S1) and in the FRET data (Figures 4, 4S1, 4S2).

6) Does treatment of primary neurons with 1:1 stoichiometry of STX6 and PrP prevent PrP induced neurotoxicity? Concentration dependent analysis may be done. This will further validate the finding observed in Figure S2(A).

Unfortunately, it was not possible to perform the toxicity assay on primary neurons at 1:1 stoichiometry under our assay conditions, since STX6 itself became toxic at these very high concentrations. We did, however, add data on the number of live neurons as an additional marker of neurotoxicity to validate our results (Figure 5S1). We found that neuronal survival data fully correlated to neurite length data and demonstrated a high degree of neuronal loss (~80%) when incubated with early PrP aggregation intermediates.

7) While this may be out of scope of the study, authors may look into the effects of overexpression or knockdown of syntaxin-6 on PrP aggregate formation.

We have included data from in vitro prion amplification assays (PMCA) in the revised manuscript, in which we compare amplification of RML mouse prions in a substrate of WT mouse brain homogenate with that of Stx6 -/- mice. Interestingly, Stx6 knockout did not alter the amplification of pre-existing prion seed (Figure 4B) under these conditions. This hypothesis is supported by the new experimental data of a secondary seeding assay, in which we assessed the seeding competence of PrP assemblies at different incubation times in the presence and absence of STX6 (Figure 5S2, now Figure 2S3). The incubation times and seed samples were identical to those used in neurotoxicity assays. We found that, analogous to its effect on toxicity and ThT fluorescence, syntaxin-6 delayed, but did not prevent the formation of seeding competent PrP assemblies. These data support our interpretation that syntaxin-6 likely alters primary nucleation and prolongs the presence of early, non-fibrillar aggregation intermediates, while it has little influence on the fibril fragmentation/elongation step of prion replication.

Reviewer #3 (Recommendations for the authors):

The objectives of the project are well-addressed and the discussion part is nicely illustrated. Some of the results look very interesting, leading to some exciting predictions (e.g., refolding of monomer, formation of off-pathway intermediates, etc.) based on the initial result. However, some additional experiments are required for a better understanding of the present results. Hereby, the authors are requested to address the following concerns and perform additional experiments that are mentioned in the following section.

• Although the fibrillation assay used in this manuscript does not use denaturant or detergent, however, the PrP proteins are kept at 42{degree sign}C, pH 6.8, under agitation with zirconium 86 beads. How a condition like agitation can be claimed as a native environmental condition and will ensure that the assay condition is not facilitating protein unfolding, unlike the other reported fibrillation assays?

The reviewer raises a valid point that there is no direct physiological correlate to agitation by stirring beads in vivo. There have been arguments that parts of the chaperone system, i.e. Hsp104 in yeast, which can fragment amyloid fibrils, have a similar function in vivo to the physical fragmentation through stirring beads. Systematic studies involving agitation (eg. Cohen et al. PNAS 2013; https://doi.org/10.1073/pnas.1218402110) have concluded that increasing agitation shifts secondary replication mechanisms to a prion-like fragmentation-elongation mechanism, which is compatible with our observations and with the results from PMCA prion amplification assays, which we have now included in Figure 4B. While it is difficult to deconvolute aggregation from protein misfolding, we see no evidence for PrP denaturation as response to agitation under our assay conditions. We furthermore find that, while PrP didn’t form fibrils in the course of a typical experiment when agitated without stirring beads, fibril formation did not depend on the specific surface of the bead, as glass beads were as effective, or more, as Zr beads in facilitating fibril formation (new Figure S2C).

• In line 121, the authors wrote that "formation of aggregates with more amorphous morphology, which seems to consist of short fibrils" this is inconsistent comment as proteins will form amorphous or amyloids. Amorphous protein aggregates can't compose of short fibrils?

We apologize for the misleading wording. We were trying to express that PrP formed aggregate clusters rather than single amyloid fibrils. These clusters retained a fibril-like substructure and exhibited ThT fluorescence. We have clarified this in the text. (see: Figures 2D and E, Figures 3, 3S2, 3S3).

• The Thioflavin T aggregation profile looks extremely noisy (e.g., Figure 1A, 2A) with huge fluctuation in the Y axis. The authors should explain the reason for this much fluctuation in the ThT signal and fit the kinetic profile taking care of the standard deviation.

The noise of the aggregation curves is most likely due to the formation of very large aggregates and the presence of the stirring beads. However, our kinetic data all resulted from the individual fitting of replicate wells. While the fluctuations may add some uncertainty to the ThT fluorescence amplitude, they had very little effect on the precision of lag phase determination, which was our primary readout.

• The authors have calculated lag times (t50) and fibril elongation rate (k) in different experimental conditions throughout the manuscript. However, the authors did not show the calculation performed to determine these two parameters in the method section. The authors should incorporate that. Moreover, the authors should perform a web-based global fitting software interface, such as Amylofit (Meisl et al., 2016), to precisely evaluate different kinetic parameters for better comparison.

We apologize for the omission. Parameters were determined by a simple sigmoidal fit; we have added the fitted equation to the method section. We also attempted to analyse the kinetics through the AmyloFit global fitting model and discussed our results with Dr Georg Meisl, one of the lead developers of AmyloFit. Unfortunately, at the moment the AmyloFit framework is not suited to the analysis of folded proteins, which have a pre-equilibrium of a native and an aggregation competent state, so that we could not use it to further analyse or data.

• The quality of TEM images of hPrP (Figure 1C) and moPrP (Figure S2C) is of poor quality. The authors are requested to change these with high-resolution images. Moreover, the present immune-gold staining image (Figure 2F) does not show the fibril where immune-gold labeled Stx6 is incorporated. The authors should provide a better-quality immune-gold staining image with a clear fibril image so that the location of incorporated Stx6 (immune-gold labeled) is clearly understood.

We apologize for the suboptimal quality of immuno-EM images. We have replaced micrographs in Figures 2, 3 and 2S1 with higher contrast images.

• The authors have shown that full-length PrP proteins form amyloid fibrils under near-native conditions. The authors should centrifuge to isolate the fibril fraction and show the proteinase-K digestion of the fibril, which is a known property of PrP amyloid fibril. The authors also should compare this PK resistance data with native PrP amyloid isolated from mouse prion strains ME7 and RML that the authors have used. Further, the authors should also perform the same with the fibril bound to Stx6, Hspa1a and Stathmin 1 to understand the nature of the aggregates in the presence of the additives.

We performed several PK digestion assays on fibrils formed in NAA when seeded either with recombinant fibrils from a previous round of NAA or with prion rods isolated from RML or ME7 scrapie infected mice. PrP fibrils formed via NAA showed a substantial (~50%) resistance to PK digestion in ThT assays and a PK-resistant PrP fragment band when digested with PK at 5 µg/mL concentration. (see Author response image 1).

Interestingly, we found that PrP fibrils formed under NAA conditions proved to be highly resistant to SDS denaturation. Unfortunately, this prevented us from characterizing their PK resistance via SDS-PAGE in a more thorough fashion, since we could not be sure that PrP bands in SDS-PAGE were representative of the NAA fibril population as a whole. We are therefore presenting these data here in Author response image 1 for the reviewer’s information rather than including them in the revised manuscript. The structural characterization of NAA fibrils when compared to authentic prion rods will be the subject of a forthcoming study.

Author response image 1. Murine (mPrP23) fibrils formed in NAA show limited proteinase K resistance.

(A) Baseline-corrected ThT fluorescence of endpoint NAA fibrils digested by PK (5 µg / mL). NAA fibrils had been formed in NAA assays seeded with recombinant fibrils (NAA), RML or ME7 prion rods. A ThT fluorescence baseline was recorded for 1.5 h, PK was added and AEBSF protease inhibitor was then added after 5 h incubation. Curves represent means ± SD of triplicate experiments. (B) Silver stained SDS-PAGE of the samples from panel A.

• The authors have speculated the formation of the "off-pathway" intermediate based on the observation that the lag phase of aggregation has increased and ThT amplitude has dropped at high monomer concentration. The author should isolate the intermediate through centrifugation at the particular aggregation condition where the predicted off-pathway intermediate populates the most. To precisely comment on the "off-pathway" intermediate, the authors should add the isolated intermediate fraction during the fibrillation kinetics in a dose-dependent manner to see the concentration effect of the intermediate in fibrillation kinetics that will provide direct evidence to support the statement.

The reviewer raises a very valid point, which we addressed in two new experiments: (1) Figure 2S2 analyzes the solubility of rPrP23 at different time points of the native aggregation assay corresponding to the time points at which neurotoxicity was assessed. We found that the presence of soluble PrP intermediates coincides with the time points of maximal toxicity. Syntaxin-6 prolongs the presence of toxic PrP species as it prolongs the presence of soluble intermediates, but, unlike Hsp70, it did not prevent PrP aggregation. (2) We assessed the seeding competence of PrP aggregation intermediates in a secondary seeding assay (Figure 5S2, now Figure 2S3). Here, syntaxin-6 delayed the formation of seeding competent assemblies. The formation of seeding competent assemblies coincided with a decrease in toxicity. Taken together, these data support our interpretation that STX6 delays the formation of fibrillar seeding competent assemblies and prolongs the presence of pre-fibrillar toxic PrP species.

• To check the interaction with PrP protein in super-resolution microscopy, the authors have chosen only Stx6 among HSPA1A, STMN1, and STX6. The authors should justify the specific reasons behind choosing Stx6 over the others.

We chose STX6 as our main protein of interest and as a test case for our newly developed native aggregation assay because it was recently identified as a risk factor for sporadic CJD. We included Hsp70 (HSPA1A) as a known inhibitor of amyloid fibril formation. STMN1 was included as a negative control. It has a highly α-helical structure, similar to STX6 and was previously identified in our lab as a potential inhibitor of α-synuclein fibril formation. This rationale has now been stated explicitly in the text.

• The authors have observed that the interaction sites of Stx6 were often located at fibril ends or kinks in the fibril. Thus, the authors have proposed a potential role of Stx6 in fibril breakage or secondary nucleation. Firstly, the authors should reconfirm the Stx6 binding location in PrP from the clear immune-gold staining images. Secondly, the authors should isolate the Stx6-bound fibrils and perform a seeding reaction (prion amplification) with only the PrP monomer to see whether Stx6-bound PrP fibrils are competent for elongation or not. If Stx6 binds to the fibril end, it might not elongate in the presence of PrP monomer unless sonication is done. Also, authors should comment on whether Stx6 fibrils are capable of surface-catalyzed secondary nucleation.

We thank the reviewer for this suggestion. We have performed both experiments, as detailed above (Figures 2S2 and 5S2) and included higher contrast immuno-EM images (Figure 3F). In addition we performed PMCA amplification of infectious RML prion seeds in brain homogenate substrates from WT mice and from Stx6 -/- knockout mice (Figure 4B). In all cases, STX6 had no influence on the seeding competence of fibrillar aggregates. So, while STX6 seems to preferentially bind to the ends of fibrils, it does not seem to prevent elongation, but rather affects early aggregate formation of PrP. Our imaging data suggest that binding of STX6 may also facilitate branching and secondary nucleation. However, prion-like replication (fragmentation / elongation) dominates under PMCA conditions, so that deletion of cellular Stx6 had no effect on the replication efficiency. We have included these aspects in our revised discussion to narrow down the mechanistic implications of our results.

• Although the highlight of the paper is the formation of toxic aggregation intermediates in the presence of Stx6 that prolongs the fibril formation, the report does not describe much about the nature of toxic intermediates. The authors should isolate the intermediate through centrifugation and structurally characterize the intermediate through biophysical experiments (e.g., mass spectrometry, CD, DLS, FTIR, TEM, and Native-PAGE). The authors should perform an MTT assay to check the toxicity of the intermediate apart from the already shown experiment of time-dependent relative neurite length of mouse primary neuron.

We have assessed the relative numbers of live neurons as an orthogonal toxicity assay and added these data in the new Figure 5S1. Toxicity data from neuronal survival closely mirrored those from the neurite length assay.

While we appreciate that the lack of solid structural and biophysical data on the nature of the toxic species in prion disease (and other protein misfolding diseases) is frustrating, characterizing the nature of the toxic species is well beyond the scope of the current manuscript. The reviewer is doubtlessly aware that it is one of the key unsolved questions in all amyloid disease and therefore one of the top research priorities for the MRC Prion Unit, as well as many other institutes around the world. If we are successful in our characterization, we will certainly publish these results in a prominent journal in a timely manner.

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) The authors have not addressed the issue of extremely noisy ThT aggregation kinetics correctly, although it is likely that the lag-phase data is robust. However, as the graph is noisy indicating that the aggregation assays are non-standard and do not replicate the behavior of similar assays reported earlier, further explanation from the authors is required.

Thank you for highlighting this issue. Fluctuations appeared to be caused by the formation of large aggregate clusters and were more prominent at high PrP concentrations. These experiments also saw a faster drop-off in fluorescence signal after the plateau phase was reached (see: Figure 2 —figure supplement 2 vs. Figure 2- FS 3) We did explore strategies to suppress the fluctuations in ThT fluorescence traces in the additional secondary seeding experiments performed in response to comment 4 (Figure 2 – FS 4 and 5). Here, fluctuations could be minimized by optimizing the height of fluorescence detection in the plate. To address the concern whether fluorescence fluctuations would change basic kinetic parameters such as lag time, we performed a moving average filtering of the kinetic data in the concentration dependent aggregation (Figure 1E) before analysis in Amylofit (Figure 1 – FS 4). As the reviewer correctly predicted, the noise had no effect on the analysis of lag times when comparing the filtered (Figure 1 – FS 4B) and unfiltered data (Figure 1F), so that we decided to keep the original kinetic data rather than repeating experiments for a large part of the study. We now discuss fluorescence fluctuations in ll. 119-126 of the revised manuscript.

2) The author's reply to usage of Amylofit fitting software to determine the kinetic parameters is not well justified and the previous concern remains. Authors can regenerate better quality of kinetics data for proper fitting by Amylofit or may explain the limitations of their dataset to such fitting software.

At the reviewer’s suggestion, we performed a global fitting analysis of concentration-dependent aggregation in Amylofit (new Figure 1 —figure supplement 4). Unlike previous versions of the software, Amylofit can now incorporate a saturated elongation process into the fitting model, which made it feasible to analyse our data in that framework. As we had predicted, all kinetic models that did not include saturated elongation completely failed to fit our experimental data. In contrast a simple fragmentation model with saturated elongation fitted our data adequately (Figure 1 – FS 4C-F). We did test more elaborate models that included saturated elongation, but they did not improve the fit and were therefore not included in the manuscript. In conclusion, Amylofit supported our conclusion that hPrP23 aggregation kinetics under native conditions are rate-limited by a 0th order process with regard to PrP concentration, most likely the structual rearrangement from PrPC to the amyloid conformer.

3) It is still unclear whether the physiological prions formed in mouse brains are similar in properties to the ones formed in vitro in NAA conditions by the authors. While the fibrils formed in vivo are quite resistant to PK digestion, can a similar assay be performed on the in vitro formed fibrils? Without any experimental proof, it is difficult to validate if the structure of the fibrils formed in vitro under NAA conditions would be similar to the fibril structures found naturally.

We thank the reviewer for the suggestion and we have now added a proteinase K digestion experiment under conditions used for PrPSc detection (50 µg/ml, 30 min, 37°C; Figure 1D, Figure 1 —figure supplement 2H). We found NAA fibrils to be as resistant to PK digestion as authentic RML prion rods isolated from scrapie-infected mice. However, PK digestion resulted in a fragment of ~ 12 kD, which is considerably smaller than the PK-resistant fragment of PrPSc. Whether the fibril structure of NAA products is identical to a part of the prion rod structure will be determined in a future cryo-EM study.

4) The suggested experiment to prove if the intermediate is off-pathway was not performed. The authors are requested to perform this crucial experiment.

To precisely comment on the "off-pathway" intermediate, the authors should add the isolated intermediate fraction during the fibrillation kinetics in a dose-dependent manner to see the concentration effect of the intermediate in fibrillation kinetics that will provide direct evidence to support the statement.

We have added two secondary seeding assays to probe the nature of the aggregates formed in the presence of syntaxin-6. The second assay is the one suggested by the reviewer, from which we can conclude that any PrP aggregates formed in the presence of syntaxin-6 prior to the formation of ThT-positive fibrils are reduced in seeding capacity by at least three orders of magnitude when compared to untreated PrP and that soluble aggregation intermediates are not seeding competent (ll. 112 -126):

“To probe the nature of PrP aggregates formed in the presence of syntaxin-6, we performed two secondary seeding assays. In the first assay, we tested the seeding capacity of PrP samples harvested at different incubation times (20, 40, 60, 90 h) in the presence and absence of syntaxin-6 in a NAA seeded with 0.1% fibrillar hPrP23 (Figure 2 —figure supplement 3). Samples harvested during the lag phase of either reactions were not seeding competent, but both aggregates formed in the presence and absence of syntaxin-6 (1:10) were seeding competent after the formation of ThT-positive aggregates, indicating that, at sub-stoichiometric ratios, syntaxin-6 delays, but does not prevent the formation of seeding competent PrP fibrils. We then generated seeds in a second experiment, in which the primary seed was added at a lower concentration (0.01%), which prolonged the delay in fibril formation by syntaxin-6 (Figure 2 —figure supplement 4A). Seed preparations harvested after 70 h incubation were then separated into total, soluble and insoluble fractions and added to secondary seeding assays at 10-3 to 10-8 molar ratio monomer equivalents (Figure 2 —figure supplement 4B-E). Both in syntaxin-6 and in untreated samples seeding activity was only found in the total and insoluble fractions. The presence of syntaxin-6 lowered the amount of seeding competent aggregates by at least three orders of magnitude (Figure 2 —figure supplement 4C-E and 5).”

No competing interests declared.

Formal analysis, Investigation, Writing - review and editing.

Formal analysis, Investigation, Methodology, Writing - review and editing.

Formal analysis, Investigation, Writing - review and editing.

Formal analysis, Investigation.

Investigation.

Resources, Methodology.

Formal analysis, Supervision, Investigation, Methodology, Writing - review and editing.

Supervision, Writing - review and editing.

Conceptualization, Formal analysis, Funding acquisition, Writing - original draft, Writing - review and editing.

Work with animals was performed under the licence granted by the UK Home Office (Project Licences 70/6454 and 70/7274) and conformed to University College London institutional and ARRIVE guidelines.
==== Refs
References

Amin L Harris DA 2021 Aβ receptors specifically recognize molecular features displayed by fibril ends and neurotoxic oligomers Nature Communications 12 3451 10.1038/s41467-021-23507-z 34103486
Atarashi R Moore RA Sim VL Hughson AG Dorward DW Onwubiko HA Priola SA Caughey B 2007 Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein Nature Methods 4 645 650 10.1038/nmeth1066 17643109
Atarashi R Sano K Satoh K Nishida N 2011 Real-time quaking-induced conversion: a highly sensitive assay for prion detection Prion 5 150 153 10.4161/pri.5.3.16893 21778820
Balch WE Morimoto RI Dillin A Kelly JW 2008 Adapting proteostasis for disease intervention Science 319 916 919 10.1126/science.1141448 18276881
Benilova I Reilly M Terry C Wenborn A Schmidt C Marinho AT Risse E Al-Doujaily H Wiggins De Oliveira M Sandberg MK Wadsworth JDF Jat PS Collinge J 2020 Highly infectious prions are not directly neurotoxic PNAS 117 23815 23822 10.1073/pnas.2007406117 32900920
Bieschke J Weber P Sarafoff N Beekes M Giese A Kretzschmar H 2004 Autocatalytic self-propagation of misfolded prion protein PNAS 101 12207 12211 10.1073/pnas.0404650101 15297610
Bieschke J Zhang Q Powers ET Lerner RA Kelly JW 2005 Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation Biochemistry 44 4977 4983 10.1021/bi0501030 15794636
Bieschke J Russ J Friedrich RP Ehrnhoefer DE Wobst H Neugebauer K Wanker EE 2010 EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity PNAS 107 7710 7715 10.1073/pnas.0910723107 20385841
Bieschke J Herbst M Wiglenda T Friedrich RP Boeddrich A Schiele F Kleckers D Lopez del Amo JM Grüning BA Wang Q Schmidt MR Lurz R Anwyl R Schnoegl S Fändrich M Frank RF Reif B Günther S Walsh DM Wanker EE 2012 Small-molecule conversion of toxic oligomers to nontoxic β-sheet–rich amyloid fibrils Nature Chemical Biology 8 93 101 10.1038/nchembio.719
Bongiovanni MN Aprile FA Sormanni P Vendruscolo M 2018 A rationally designed hsp70 variant rescues the aggregation-associated toxicity of human iapp in cultured pancreatic islet β-cells International Journal of Molecular Sciences 19 1443 10.3390/ijms19051443 29757200
Bove-Fenderson E Urano R Straub JE Harris DA 2017 Cellular prion protein targets amyloid-β fibril ends via its C-terminal domain to prevent elongation The Journal of Biological Chemistry 292 16858 16871 10.1074/jbc.M117.789990 28842494
Brundin P Melki R Kopito R 2010 Prion-like transmission of protein aggregates in neurodegenerative diseases Nature Reviews. Molecular Cell Biology 11 301 307 10.1038/nrm2873 20308987
Buell AK Galvagnion C Gaspar R Sparr E Vendruscolo M Knowles TPJ Linse S Dobson CM 2014 Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation PNAS 111 7671 7676 10.1073/pnas.1315346111 24817693
Castle AR Gill AC 2017 Physiological functions of the cellular prion protein Frontiers in Molecular Biosciences 4 19 10.3389/fmolb.2017.00019 28428956
Choudhury A Marks DL Proctor KM Gould GW Pagano RE 2006 Regulation of caveolar endocytosis by syntaxin 6-dependent delivery of membrane components to the cell surface Nature Cell Biology 8 317 328 10.1038/ncb1380 16565709
Cohen E Bieschke J Perciavalle RM Kelly JW Dillin A 2006 Opposing activities protect against age-onset proteotoxicity Science 313 1604 1610 10.1126/science.1124646 16902091
Cohen SIA Linse S Luheshi LM Hellstrand E White DA Rajah L Otzen DE Vendruscolo M Dobson CM Knowles TPJ 2013 Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism PNAS 110 9758 9763 10.1073/pnas.1218402110 23703910
Cohen SIA Arosio P Presto J Kurudenkandy FR Biverstal H Dolfe L Dunning C Yang X Frohm B Vendruscolo M Johansson J Dobson CM Fisahn A Knowles TPJ Linse S 2015 A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers Nature Structural & Molecular Biology 22 207 213 10.1038/nsmb.2971 25686087
Collinge J Clarke AR 2007 A general model of prion strains and their pathogenicity Science 318 930 936 10.1126/science.1138718 17991853
Collinge J 2016 Mammalian prions and their wider relevance in neurodegenerative diseases Nature 539 217 226 10.1038/nature20415 27830781
Corbett GT Wang Z Hong W Colom-Cadena M Rose J Liao M Asfaw A Hall TC Ding L DeSousa A Frosch MP Collinge J Harris DA Perkinton MS Spires-Jones TL Young-Pearse TL Billinton A Walsh DM 2020 PrP is a central player in toxicity mediated by soluble aggregates of neurodegeneration-causing proteins Acta Neuropathologica 139 503 526 10.1007/s00401-019-02114-9 31853635
Du D Murray AN Cohen E Kim HE Simkovsky R Dillin A Kelly JW 2011 A kinetic aggregation assay allowing selective and sensitive amyloid-β quantification in cells and tissues Biochemistry 50 1607 1617 10.1021/bi1013744 21268584
Ehrnhoefer DE Bieschke J Boeddrich A Herbst M Masino L Lurz R Engemann S Pastore A Wanker EE 2008 EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers Nature Structural & Molecular Biology 15 558 566 10.1038/nsmb.1437 18511942
Evans CG Wisén S Gestwicki JE 2006 Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro The Journal of Biological Chemistry 281 33182 33191 10.1074/jbc.M606192200 16973602
Feige JN Sage D Wahli W Desvergne B Gelman L 2005 PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs Microscopy Research and Technique 68 51 58 10.1002/jemt.20215 16208719
Fernandez-Funez P Sanchez-Garcia J de Mena L Zhang Y Levites Y Khare S Golde TE Rincon-Limas DE 2016 Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila PNAS 113 E5212 E5221 10.1073/pnas.1608045113 27531960
Freir DB Nicoll AJ Klyubin I Panico S Mc Donald JM Risse E Asante EA Farrow MA Sessions RB Saibil HR Clarke AR Rowan MJ Walsh DM Collinge J 2011 Interaction between prion protein and toxic amyloid β assemblies can be therapeutically targeted at multiple sites Nature Communications 2 336 10.1038/ncomms1341 21654636
Friedrich RP Tepper K Rönicke R Soom M Westermann M Reymann K Kaether C Fändrich M 2010 Mechanism of amyloid plaque formation suggests an intracellular basis of Abeta pathogenicity PNAS 107 1942 1947 10.1073/pnas.0904532106 20133839
Frost B Diamond MI 2010 Prion-like mechanisms in neurodegenerative diseases Nature Reviews. Neuroscience 11 155 159 10.1038/nrn2786 20029438
Glover JR Lindquist S 1998 Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins Cell 94 73 82 10.1016/s0092-8674(00)81223-4 9674429
Goold R Rabbanian S Sutton L Andre R Arora P Moonga J Clarke AR Schiavo G Jat P Collinge J Tabrizi SJ 2011 Rapid cell-surface prion protein conversion revealed using a novel cell system Nature Communications 2 281 10.1038/ncomms1282 21505437
Grassmann A Wolf H Hofmann J Graham J Vorberg I 2013 Cellular aspects of prion replication in vitro Viruses 5 374 405 10.3390/v5010374 23340381
Haass C Selkoe DJ 2007 Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid β-peptide Nature Reviews Molecular Cell Biology 8 101 112 10.1038/nrm2101 17245412
Hartl FU 1996 Molecular chaperones in cellular protein folding Nature 381 571 580 10.1038/381571a0 8637592
Hartl FU 2011 Chaperone-assisted protein folding: the path to discovery from a personal perspective Nature Medicine 17 1206 1210 10.1038/nm.2467 21989011
Hegde RS Mastrianni JA Scott MR DeFea KA Tremblay P Torchia M DeArmond SJ Prusiner SB Lingappa VR 1998 A transmembrane form of the prion protein in neurodegenerative disease Science 279 827 834 10.1126/science.279.5352.827 9452375
Hill E 2023 Knockout of sporadic creutzfeldt-jakob disease risk gene stx6 in mice extends prion disease incubation time bioRxiv 10.1101/2023.01.10.523281
Höglinger GU Melhem NM Dickson DW Sleiman PMA Wang L-S Klei L Rademakers R de Silva R Litvan I Riley DE van Swieten JC Heutink P Wszolek ZK Uitti RJ Vandrovcova J Hurtig HI Gross RG Maetzler W Goldwurm S Tolosa E Borroni B Pastor P Cantwell LB Han MR Dillman A van der Brug MP Gibbs JR Cookson MR Hernandez DG Singleton AB Farrer MJ Yu C-E Golbe LI Revesz T Hardy J Lees AJ Devlin B Hakonarson H Müller U Schellenberg GD PSP Genetics Study Group 2011 Identification of common variants influencing risk of the tauopathy progressive supranuclear palsy Nature Genetics 43 699 705 10.1038/ng.859 21685912
Honda RP Kuwata K 2017 The native state of prion protein (PrP) directly inhibits formation of PrP-amyloid fibrils in vitro Scientific Reports 7 562 10.1038/s41598-017-00710-x 28373719
Hosszu LLP Wells MA Jackson GS Jones S Batchelor M Clarke AR Craven CJ Waltho JP Collinge J 2005 Definable equilibrium states in the folding of human prion protein Biochemistry 44 16649 16657 10.1021/bi051277k 16342955
Jackson GS Hill AF Joseph C Hosszu L Power A Waltho JP Clarke AR Collinge J 1999 Multiple folding pathways for heterologously expressed human prion protein Biochimica et Biophysica Acta 1431 1 13 10.1016/s0167-4838(99)00038-2 10209273
Jain S Udgaonkar JB 2008 Evidence for stepwise formation of amyloid fibrils by the mouse prion protein Journal of Molecular Biology 382 1228 1241 10.1016/j.jmb.2008.07.052 18687339
Jarrett JT Lansbury PT 1993 Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell 73 1055 1058 10.1016/0092-8674(93)90635-4 8513491
Jin S Kedia N Illes-Toth E Haralampiev I Prisner S Herrmann A Wanker EE Bieschke J 2016 Amyloid-β(1-42) aggregation initiates its cellular uptake and cytotoxicity The Journal of Biological Chemistry 291 19590 19606 10.1074/jbc.M115.691840 27458018
Jones E Hummerich H Viré E Uphill J Dimitriadis A Speedy H Campbell T Norsworthy P Quinn L Whitfield J Linehan J Jaunmuktane Z Brandner S Jat P Nihat A How Mok T Ahmed P Collins S Stehmann C Sarros S Kovacs GG Geschwind MD Golubjatnikov A Frontzek K Budka H Aguzzi A Karamujić-Čomić H van der Lee SJ Ibrahim-Verbaas CA van Duijn CM Sikorska B Golanska E Liberski PP Calero M Calero O Sanchez-Juan P Salas A Martinón-Torres F Bouaziz-Amar E Haïk S Laplanche J-L Brandel J-P Amouyel P Lambert J-C Parchi P Bartoletti-Stella A Capellari S Poleggi A Ladogana A Pocchiari M Aneli S Matullo G Knight R Zafar S Zerr I Booth S Coulthart MB Jansen GH Glisic K Blevins J Gambetti P Safar J Appleby B Collinge J Mead S 2020 Identification of novel risk loci and causal insights for sporadic Creutzfeldt-Jakob disease: a genome-wide association study The Lancet Neurology 19 840 848 10.1016/S1474-4422(20)30273-8 32949544
Jung JJ Inamdar SM Tiwari A Choudhury A 2012 Regulation of intracellular membrane trafficking and cell dynamics by syntaxin-6 Bioscience Reports 32 383 391 10.1042/BSR20120006 22489884
Klement K Wieligmann K Meinhardt J Hortschansky P Richter W Fändrich M 2007 Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer’s abeta(1-40) amyloid fibrils Journal of Molecular Biology 373 1321 1333 10.1016/j.jmb.2007.08.068 17905305
Klöhn P-C Stoltze L Flechsig E Enari M Weissmann C 2003 A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions PNAS 100 11666 11671 10.1073/pnas.1834432100 14504404
Kraus A Hoyt F Schwartz CL Hansen B Artikis E Hughson AG Raymond GJ Race B Baron GS Caughey B 2021 High-resolution structure and strain comparison of infectious mammalian prions Molecular Cell 81 4540 4551 10.1016/j.molcel.2021.08.011 34433091
Lam HT Graber MC Gentry KA Bieschke J 2016 Stabilization of α-synuclein fibril clusters prevents fragmentation and reduces seeding activity and toxicity Biochemistry 55 675 685 10.1021/acs.biochem.5b01168 26799377
Lashuel HA Hartley D Petre BM Walz T Lansbury PT 2002 Neurodegenerative disease: amyloid pores from pathogenic mutations Nature 418 291 10.1038/418291a 12124613
Lee WS Tan DC Deng Y van Hummel A Ippati S Stevens C Carmona-Mora P Ariawan D Hou L Stefen H Tomanic T Bi M Tomasetig F Martin A Fath T Palmer S Ke YD Ittner LM 2021 Syntaxins 6 and 8 facilitate tau into secretory pathways The Biochemical Journal 478 1471 1484 10.1042/BCJ20200664 33769438
Legname G Baskakov IV Nguyen H-OB Riesner D Cohen FE DeArmond SJ Prusiner SB 2004 Synthetic mammalian prions Science 305 673 676 10.1126/science.1100195 15286374
Manka SW Zhang W Wenborn A Betts J Joiner S Saibil HR Collinge J Wadsworth JDF 2022 2.7 Å cryo-EM structure of ex vivo RML prion fibrils Nature Communications 13 4004 10.1038/s41467-022-30457-7 35831275
Marbiah MM Harvey A West BT Louzolo A Banerjee P Alden J Grigoriadis A Hummerich H Kan H-M Cai Y Bloom GS Jat P Collinge J Klöhn P-C 2014 Identification of a gene regulatory network associated with prion replication The EMBO Journal 33 1527 1547 10.15252/embj.201387150 24843046
Mead S Lloyd S Collinge J 2019 Genetic factors in mammalian prion diseases Annual Review of Genetics 53 117 147 10.1146/annurev-genet-120213-092352
Meisl G Kirkegaard JB Arosio P Michaels TCT Vendruscolo M Dobson CM Linse S Knowles TPJ 2016 Molecular mechanisms of protein aggregation from global fitting of kinetic models Nature Protocols 11 252 272 10.1038/nprot.2016.010 26741409
Meissner B Kallenberg K Sanchez-Juan P Ramljak S Krasnianski A Heinemann U Eigenbrod S Gelpi E Barsic B Kretzschmar HA Schulz-Schaeffer WJ Knauth M Zerr I 2009 MRI and clinical syndrome in dura mater-related Creutzfeldt-Jakob disease Journal of Neurology 256 355 363 10.1007/s00415-009-0026-z 19159063
Milto K Michailova K Smirnovas V 2014 Elongation of mouse prion protein amyloid-like fibrils: effect of temperature and denaturant concentration PLOS ONE 9 e94469 10.1371/journal.pone.0094469 24747600
Muchowski PJ Wacker JL 2005 Modulation of neurodegeneration by molecular chaperones Nature Reviews. Neuroscience 6 11 22 10.1038/nrn1587 15611723
Nicoll AJ Panico S Freir DB Wright D Terry C Risse E Herron CE O’Malley T Wadsworth JDF Farrow MA Walsh DM Saibil HR Collinge J 2013 Amyloid-β nanotubes are associated with prion protein-dependent synaptotoxicity Nature Communications 4 2416 10.1038/ncomms3416 24022506
Post K Pitschke M Schäfer O Wille H Appel TR Kirsch D Mehlhorn I Serban H Prusiner SB Riesner D 1998 Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation Biological Chemistry 379 1307 1317 10.1515/bchm.1998.379.11.1307 9865603
Powers ET Powers DL 2008 Mechanisms of protein fibril formation: nucleated polymerization with competing off-pathway aggregation Biophysical Journal 94 379 391 10.1529/biophysj.107.117168 17890392
Prusiner SB 1998 Prions PNAS 95 13363 13383 10.1073/pnas.95.23.13363 9811807
Reilly M Benilova I Khalili-Shirazi A Schmidt C Ahmed P Yip D Jat PS Collinge J 2022 A high-content neuron imaging assay demonstrates inhibition of prion disease-associated neurotoxicity by an anti-prion protein antibody Scientific Reports 12 9493 10.1038/s41598-022-13455-z 35680944
Rosas PC Nagaraja GM Kaur P Panossian A Wickman G Garcia LR Al-Khamis FA Asea AAA 2016 Hsp72 (HSPA1A) prevents human islet amyloid polypeptide aggregation and toxicity: a new approach for type 2 diabetes treatment PLOS ONE 11 e0149409 10.1371/journal.pone.0149409 26960140
Sandberg MK Al-Doujaily H Sharps B Clarke AR Collinge J 2011 Prion propagation and toxicity in vivo occur in two distinct mechanistic phases Nature 470 540 542 10.1038/nature09768 21350487
Sandberg MK Al-Doujaily H Sharps B De Oliveira MW Schmidt C Richard-Londt A Lyall S Linehan JM Brandner S Wadsworth JDF Clarke AR Collinge J 2014 Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked Nature Communications 5 4347 10.1038/ncomms5347 25005024
Schmidt C Fizet J Properzi F Batchelor M Sandberg MK Edgeworth JA Afran L Ho S Badhan A Klier S Linehan JM Brandner S Hosszu LLP Tattum MH Jat P Clarke AR Klöhn PC Wadsworth JDF Jackson GS Collinge J 2015 A systematic investigation of production of synthetic prions from recombinant prion protein Open Biology 5 150165 10.1098/rsob.150165 26631378
Shi Z Sachs JN Rhoades E Baumgart T 2015 Biophysics of α-synuclein induced membrane remodelling Physical Chemistry Chemical Physics 17 15561 15568 10.1039/C4CP05883F 25665896
Spehar K Ding T Sun Y Kedia N Lu J Nahass GR Lew MD Bieschke J 2018 Super-resolution imaging of amyloid structures over extended times by using transient binding of single thioflavin T molecules Chembiochem 19 1944 1948 10.1002/cbic.201800352 29953718
Stewart RS Harris DA 2005 A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons The Journal of Biological Chemistry 280 15855 15864 10.1074/jbc.M412298200 15671025
Stöhr J Weinmann N Wille H Kaimann T Nagel-Steger L Birkmann E Panza G Prusiner SB Eigen M Riesner D 2008 Mechanisms of prion protein assembly into amyloid PNAS 105 2409 2414 10.1073/pnas.0712036105 18268326
Sun Y Jack K Ercolani T Sangar D Hosszu L Collinge J Bieschke J 2023 Direct observation of competing prion protein fibril populations with distinct structures and kinetics ACS Nano 17 6575 6588 10.1021/acsnano.2c12009 36802500
Swietnicki W Morillas M Chen SG Gambetti P Surewicz WK 2000 Aggregation and fibrillization of the recombinant human prion protein huPrP90-231 Biochemistry 39 424 431 10.1021/bi991967m 10631004
Terry C Wenborn A Gros N Sells J Joiner S Hosszu LLP Tattum MH Panico S Clare DK Collinge J Saibil HR Wadsworth JDF 2016 Ex vivo mammalian prions are formed of paired double helical prion protein fibrils Open Biology 6 160035 10.1098/rsob.160035 27249641
Thackray AM Lam B McNulty EE Nalls AV Mathiason CK Magadi SS Jackson WS Andréoletti O Marrero-Winkens C Schätzl H Bujdoso R 2022 Clearance of variant Creutzfeldt-Jakob disease prions in vivo by the Hsp70 disaggregase system Brain 145 3236 3249 10.1093/brain/awac144 35446941
Wacker JL Zareie MH Fong H Sarikaya M Muchowski PJ 2004 Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer Nature Structural & Molecular Biology 11 1215 1222 10.1038/nsmb860 15543156
Wenborn A Terry C Gros N Joiner S D’Castro L Panico S Sells J Cronier S Linehan JM Brandner S Saibil HR Collinge J Wadsworth JDF 2015 A novel and rapid method for obtaining high titre intact prion strains from mammalian brain Scientific Reports 5 10062 10.1038/srep10062 25950908
Wendler F Tooze S 2001 Syntaxin 6: the promiscuous behaviour of a SNARE protein Traffic 2 606 611 10.1034/j.1600-0854.2001.20903.x 11555414
Willbold D Strodel B Schröder GF Hoyer W Heise H 2021 Amyloid-type protein aggregation and prion-like properties of amyloids Chemical Reviews 121 8285 8307 10.1021/acs.chemrev.1c00196 34137605
Wingo AP Liu Y Gerasimov ES Gockley J Logsdon BA Duong DM Dammer EB Robins C Beach TG Reiman EM Epstein MP De Jager PL Lah JJ Bennett DA Seyfried NT Levey AI Wingo TS 2021 Integrating human brain proteomes with genome-wide association data implicates new proteins in Alzheimer's disease pathogenesis Nature Genetics 53 143 146 10.1038/s41588-020-00773-z 33510477
Yamasaki T Suzuki A Hasebe R Horiuchi M 2018 Retrograde transport by clathrin-coated vesicles is involved in intracellular transportpersistently prion-infected c of PrPSc in Persistently Prion-Infected Cells Scientific Reports 8 12241 10.1038/s41598-018-30775-1 30115966
Yim YI Park BC Yadavalli R Zhao X Eisenberg E Greene LE 2015 The multivesicular body is the major internal site of prion conversion Journal of Cell Science 128 1434 1443 10.1242/jcs.165472 25663703
Ziaunys M Sakalauskas A Mikalauskaite K Snieckute R Smirnovas V 2021 Temperature-dependent structural variability of prion protein amyloid fibrils International Journal of Molecular Sciences 22 5075 10.3390/ijms22105075 34064883
