In vitro cross‐seeding between FapC fragments (FapCS) and Aβ. A) ThT assay of Aβ (50 × 10⁻⁶m, incubated at 37 °C) in the presence or absence of FapCS, FapC monomers or preformed FapC fibrils (5 × 10⁻⁶m) (n = 3). B‐D) ThT kinetic parameters of fibrillization rate constant k (B), half‐life t_1/2 (C) and lag time (D) (n = 3). FapCS significantly increased (*, p < 0.05) k of Aβ, while the parameter was significantly suppressed (**, p < 0.005) with FapC monomer and fibrils. t_1/2 and lag time of Aβ were significantly shortened (**, p < 0.005) by FapCS. E) Static light scattering (SLS) indicating a rapid growth in the size of Aβ fibrils, immediately after mixing with FapCS (n = 3). F) TEM images of Aβ (at 12 h showing prefibrillar species) and G) FapCS (scale bar: 100 nm). H) TEM and I) AFM images of Aβ incubated with FapCS at 12 h (scale bar: 100 nm). J) CD spectra and K) secondary structure of Aβ alone and with FapCS (n = 3). After 12 h of incubation, the negative peaks of Aβ at 215 and 198 nm were slightly increased and decreased in intensity, respectively, indicating a transition from random coil (decreased from 34 ± 6 to 28 ± 1.5%) to β‐sheet (slightly increased from 28 ± 9 to 33 ± 6%). At 48 h, a strong negative peak appeared at 201 nm representing formation β‐sheets rich (54 ± 4.5%) fibrils. However, similar β‐sheets rich structure (59 ± 6%) were observed in Aβ + FapCS sample at 12 h time point. L) Enthalpy (ΔH) and free energy (ΔG), M) entropic factor (TΔS) for binding between FapCS and Aβ monomers or oligomers (Aβo) (n = 3). N) Immunolabeling of Aβ with β‐amyloid specific antibodies (scale bar: 10 × 10⁻⁶m). O) Quantification of green fluorescence intensity from (N) (n = 3). Significantly higher immune‐recognition (**, p < 0.005) was observed with Aβ + FapCS than Aβ alone, after 12 h incubation. P) STED microscopy of Aβ + FapCS (scale bar: 1 × 10⁻⁶m). FapCS labeled with Alexa 647 appeared to be adsorbed onto or integrated into Aβ fibrils that were labeled with ThT.

DMD simulations of molecular interactions between FapC and Aβ. A) The averaged binding frequency of each FapC with Aβ42 monomer (top) and the corresponding β‐sheet structure propensity (bottom). The results were obtained from binding simulations of Aβ42 with 10‐residue FapC with overlapping sequences. B) Mass‐weighted average cluster or aggregate sizes (top left) and β‐sheet contents (top right) obtained from aggregation simulations of six tested peptides, including three Aβ‐binding hotspot fragments and three controls for comparison. Typical snapshots of these cases formed β‐sheet rich aggregates (bottom). FapC was individually colored. C) Overlaying of final snapshots from ten independent cross‐seeding simulations, where preformed nanofibril by FapC41‐50 was shown as cartoon with molecular surface colored according to each residue's binding probability with Aβ from low (blue) to high (red). Aβ42 atoms were shown in wheat spheres. D) The intermolecular center‐of‐mass (COM) distance distribution between Aβ42 and the FapC nanofibril. Representative structures as cartoon were shown for each peak as inset with FapCS colored in gray and Aβ42 in rainbow.

FapCS accelerated Aβ fibrillization and associated pathology in zebrafish larvae. Aβ (100 × 10⁻¹⁵m), FapCS (10 × 10⁻¹⁵m) or Aβ + FapCS at a 10:1 molar ratio (100:10 × 10⁻¹⁵m) were injected to the cerebroventricular space of one week old zebrafish larvae. Congo red (100 × 10⁻¹⁵m) was injected at 2 d or one week post Aβ injection to stain and monitor the Aβ fibrillization. A) The fluorescence images recorded via the brightfield and red fluorescence channel of a microscope for the whole‐mount dorsal and lateral side of larvae. B) Quantitative measurement of the relative red fluorescence intensity, indicative of Congo‐red‐stained Aβ plaques, from the cerebral region of larvae (n = 10). Aβ injected together with FapCS presented significantly stronger (**, p < 0.005) fluorescence and thus elevated Aβ fibrillization compared to Aβ alone or buffer, at 2 d postinjection. FapCS did not show any retention of Congo red fluorescence in the brain. C,D) Aβ‐induced behavioral pathology in terms of movement frequency (C) and total distance traveled by zebrafish larvae (D) (n = 10 per group and three groups per sample). The measurements were made for the observation period of 1 h at 2 d and one week postinjection. FapCS significantly aggravated (**, P < 0.005) Aβ toxicity and reduced the movement frequency and total travel distance of the larvae at 2 d postinjection. E) ROS generation in the brain homogenates of zebrafish larvae presented as relative DFC fluorescence. H₂O₂ was used as positive control. Similar to behavioral toxicity, Aβ + FapCS significantly enhanced ROS production in the larval brain (n = 10 per group and 3 groups per sample).

FapCS accelerated Aβ‐induced cognitive and neuronal pathology in adult zebrafish. Aβ (1 µL, 40 × 10⁻⁶m) was injected to the cerebroventricular space of 10 months old adult zebrafish. A) Representative movement trajectories of adult fish after injection with FapCS, Aβ, or Aβ + FapCS. B) The quantitative measurements of movement frequency and total distance traveled by the fish (n = 3 per group and three groups per sample). Aβ + FapCS significantly suppressed (**, p < 0.005) the swimming behavior of adult fish, compared to Aβ alone at 4 d posttreatment. C) Cognitive memory function of the adult fish. The swimming tank was divided into arenas 1 and 2. Arena 2 was labeled with red paper from the bottom and fish was trained to avoid swimming into arena 2. The fish was shocked (9 V) whenever it swam into arena 2. D) Quantitative measurement of movement frequency and distance traveled in arena 1 versus arena 2. Before training, fish were able to freely swim in both arenas and no difference per arena was observed. After training, buffer injected and Aβ (4 d postinjection) fish were able to cognitively avoid swimming into arena 2. However, FapCS + Aβ and Aβ alone at 4 d and 2 weeks postinjection, respectively, were unable to avoid arena 2 and no significant difference in the swimming activities was observed in arena 1 versus arena 2 (n = 3 per group and 3 groups per sample). E) IHC for Aβ deposition in the brain of zebrafish. F) Quantitative measurement of green fluorescence for antibody staining (n = 3). G) Synaptophysin positive cells, H) quantitative measurement of anti‐synaptophysin antibodies labeling (n = 3), I) TUNEL assay and J) quantitative measurement for antibodies labeling in TUNEL assay (n = 3). Aβ alone at 2 weeks and Aβ + FapCS at 4 d presented a similar level of Aβ burden, neurodegeneration of synaptophysin positive cells and neuronal cell death in the fish brain, that were significantly different (**, p < 0.005) than Aβ alone at 4 d postinjection.

P. aeruginosa biofilm fragments elevated the Aβ pathology in adult zebrafish. ThT assay of Aβ (50 × 10⁻⁶m, 37 °C) in the presence or absence of biofilm fragments (5 × 10⁻⁶m, with respect to protein contents) (n = 3). Like FapCS, biofilm fragments significantly accelerated Aβ fibrillization. B) TEM images of Aβ fibrillized with biofilm fragments. The fragments appeared to be adsorbed onto Aβ fibrils. C) Movement trajectories and D) quantification of behavioral pathology, at 4 d postinjection, when Aβ was injected together with biofilm fragments (n = 3 per group and 3 groups per sample). Biofilm fragments enhanced Aβ toxicity and significantly (**, p < 0.005) suppressed the swimming behavior of adult zebrafish. E) Movement trajectory and F) quantification of the cognitive behavior of the fish in arena 1 versus arena 2 of the swimming tank (n = 3 per group and 3 groups per sample). Fish injected with biofilm fragments + Aβ presented deteriorated cognitive memory function at 4 d postinjection while biofilm fragments alone did not induce any behavioral toxicity. At 4 days postinjection, adult fish brains were subjected to IHC. H) Aβ deposition and I) quantification of Aβ deposition as green fluorescence of antibody labeling (n = 3), J) synaptophysin positive cells, K) quantification of antibody labeling of synaptophysin positive cells (n = 3), L) TUNEL assay and M) quantification of cell death in TUNEL assay (n = 3). Like FapCS, biofilm fragments enhanced Aβ deposition, accelerated the depletion of synaptophysin positive neurons and induced cell death in the brain of adult zebrafish.

Cross‐seeding of Aβ and FapC fragments (FapCS), involving complementary in vitro and in silico techniques as well as in vivo assays.