D-peptide-magnetic nanoparticles fragment tau fibrils and rescue behavioral deficits in a mouse model of Alzheimer's disease

Abstract

Amyloid fibrils of tau are increasingly accepted as a cause of neuronal death and brain atrophy in Alzheimer's disease (AD). Diminishing tau aggregation is a promising strategy in the search for efficacious AD therapeutics. Previously, our laboratory designed a six-residue, nonnatural amino acid inhibitor D-TLKIVW peptide (6-DP), which can prevent tau aggregation in vitro. However, it cannot block cell-to-cell transmission of tau aggregation. Here, we find D-TLKIVWC (7-DP), a d-cysteine extension of 6-DP, not only prevents tau aggregation but also fragments tau fibrils extracted from AD brains to neutralize their seeding ability and protect neuronal cells from tau-induced toxicity. To facilitate the transport of 7-DP across the blood-brain barrier, we conjugated it to magnetic nanoparticles (MNPs). The MNPs-DP complex retains the inhibition and fragmentation properties of 7-DP alone. Ten weeks of MNPs-DP treatment appear to reverse neurological deficits in the PS19 mouse model of AD. This work offers a direction for development of therapies to target tau fibrils.

Fig. 1.. 7-DP inhibits tau aggregation and fragments tau fibrils in vitro and in cells.

(A) ThT assays of 20 μM tau K18⁺ monomer incubated alone or in the presence of 5, 10, 20, 50, and 100 μM 7-DP. (B) ThT fragmentation assay of 20 μM tau K18⁺ fibrils with H₂O, 50 μM 6-DP, or 50 μM 7-DP added at 16 hours. (C) TEM image of 20 μM recombinant tau K18⁺ fibrils before and after 24 hours of incubation with 50 μM 7-DP or 50 μM 6-DP at 37°C. Scale bars, 200 nm. (D) CD spectra of tau K18⁺ monomer, tau K18⁺ fibrils before and after incubation with various concentrations of 7-DP for 48 hours at 37°C. (E) Representative TEM images of AD-extracted tau fibrils (indicated by red arrows) before and after 48 hours of incubation with 500 μM 7-DP or 500 μM 6-DP at 37°C. (F) Quantification of AD tau fibrils in TEM images (n = 10 images). (G) Western blotting analysis of cell lysate from HEK293 cells expressing 1N4R tau treated with 20 μM or 50 μM 7-DP, using Dako tau antibody. β-Actin was used as a loading control. a.u., arbitrary units.

Fig. 2.. 7-DP blocks tau seeding and reduces tau-induced cell toxicity.

(A) Representative fluorescent images of HEK293 cells expressing YFP-labeled tau K18 transfected with AD tau fibril seeds in various concentrations of 7-DP. Fluorescent puncta were marked by white arrows. Scale bars, 100 μm. (B) Quantification of puncta formed in tau biosensor cells seeded with AD tau fibrils with various concentrations of 7-DP. (C) Cell viability of N2a cells treated with tau K18⁺ monomer incubated alone or in various concentrations of 7-DP, measured by MTT assay. 7-DP treatment of tau K18⁺ monomer rescues cell viability. (D) Cell viability of N2a cells treated with tau K18⁺ fibrils in various concentrations of 7-DP, measured by MTT assay. 7-DP treatment of tau K18⁺ fibrils rescues cell viability. (E) N2a cells treated with a range of 7-DP concentrations shows no toxicity. Results shown as mean + SD of triplicate wells. Statistical significance was analyzed by one-way analysis of variance (ANOVA) (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant).

Fig. 3.. MNPs-DP retain the properties of 7-DP to inhibit tau aggregation and fragment tau fibrils in vitro.

(A) ThT assays of 20 μM tau K18⁺ monomer incubated alone or in the presence of MNPs-DP (0.005, 0.01, 0.02, and 0.04 mg/ml). The concentration of 7-DP on the surface of MNPs is 21.5, 43, 86, 172 μM, respectively. (B) TEM images of tau K18⁺ incubated alone or in the presence of MNPs-DP (0.005 and 0.04 mg/mL) for 48 hours. Scale bars, 200 nm. (C) ThT fragmentation assay of 20 μM tau K18⁺ fibrils with H₂O, MNPs (0.02 mg/ml), MNPs-DP (0.02 mg/ml; 86 μM 7-DP), or 50 μM 7-DP added at 10 hours. (D) ThT fragmentation assay of 20 μM tau K18⁺ fibrils with MNPs-DP (0.005, 0.01, and 0.02 mg/ml) added at 16 hours. (E) TEM images of tau K18⁺ fibrils before and after 24 hours of incubation with MNPs-DP (0.02 mg/ml). Scale bars, 100 nm. (F) Representative TEM images of AD-extracted tau fibrils (indicated by red arrows) before and after 48 hours of incubation with MNPs (0.02 mg/ml) or MNPs-DP (0.02 mg/ml; 86 μM 7-DP) at 37°C. (G) Quantification of AD tau fibrils in TEM images (n = 10 images).

Fig. 4.. MNPs-DP block tau seeding and reduce tau-induced cell toxicity in cells.

(A) Representative fluorescence images of HEK293 cells expressing YFP-labeled tau K18 transfected with AD tau fibril seeds in various concentrations of MNPs-DP. Fluorescent puncta were marked by white arrows. Scale bars, 100 μm. (B) Quantification of puncta formed in tau biosensor cells seeded with AD tau fibrils in various concentrations of MNPs-DP. (C) Cell viability of tau K18⁺ monomer incubated alone or in various concentrations of MNPs-DP, assessed by MTT assay. (D) Cell viability of N2a cells treated with tau K18⁺ fibrils, in various concentrations of MNPs-DP measured by MTT assay. Results shown as mean + SD of triplicate wells. Statistical significance was analyzed by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 5.. MNPs-DP rescues memory deficits in PS19 mice without causing obvious toxicity.

(A) Body weight of WT and PS19 mice treated with PBS, MNPs and MNPs-DP, respectively (n = 13 per group, eight male and five female mice for each group). (B) Total latency time to find the escape hole in the learning period for each group of mice (n = 13 per group, eight male and five female mice for each group). Statistical significance was analyzed by two-way ANOVA (P < 0.05; *MNPs-DP versus PBS; #MNPs-DP versus MNPs). (C) Primary latency for finding escape hole in the probe trial for each group of mice. (D) Entry number to target hole in probe test for each group of mice. (E) Representative track of probe test for each group of mice. (F) Time spent during the exploration of the familiar and novel objects in novel object recognition test (n = 13 per group, eight male and five female mice for each group). (G) Discriminatory index obtained in novel object recognition test. (H) Serum aspartate aminotransferase (AST) levels for each group of mice (n = 13 per group, eight male and five female mice for each group). Results shown as mean + SD (n = 13). Statistical significance was analyzed by two-way ANOVA (*P < 0.05; **P < 0.01).

Fig. 6.. MNPs-DP reduce phosphorylated tau in the hippocampus of PS19 mice.

(A) Representative immunohistochemical images of the hippocampus (CA1 and CA3 regions) of WT and PS19 mice that treated with PBS, MNPs, and MNPs-DP, respectively, at ×10 magnification, stained with AT8 antibody for phosphorylated tau at residues Ser²⁰²/Thr²⁰⁵. The neurofibrillary tangles were indicated by red arrows. (B) Quantification of AT8-positive staining in the hippocampus CA1. (C) Quantification of AT8-positive staining in the hippocampus CA3. Representative Western blot for AT8 of (D) soluble and (E) insoluble hippocampus homogenates from WT and PS19 mice treated with PBS, MNPs, and MNPs-DP, respectively. β-Actin was used as a loading control. Quantification of (F) soluble phosphorylated tau (ptau) and (G) insoluble ptau normalized to β-actin (n = 8 per group, four male and four female PS19 mice for each group). Statistical significance was analyzed by two-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 7.. Hypothesized schematic mechanism of fragmentation of tau fibrils.

(A) Scheme illustrates the interaction of tau fibrils with 7-DP. (B) A metastable interface forms between 7-DP and tau fibrils because of self-assembly of 7-DP along tau fibrils. (C) A gap opens between tau molecules as the 7-DP assembly changes polymorph to attain a lower energy state and tau strains to maintain its interface with the new polymorph of 7-DP assembly. (D) Water invades the newly formed gap to fragment tau fragments.