Chaperoning activity of the cyclophilin family prevents tau aggregation

Abstract

Tauopathies, such as Alzheimer's disease, are characterized by the misfolding and progressive accumulation of the microtubule associated protein tau. Chaperones, tasked with maintaining protein homeostasis, can become imbalanced with age and contribute to the progression of neurodegenerative disease. Cyclophilins are a promising pool of underinvestigated chaperones with peptidyl-prolyl isomerase activity that may play protective roles in regulating tau aggregation. Using a Thioflavin T fluorescence-based assay to monitor in vitro tau aggregation, all eight cyclophilins, which include PPIA to PPIH prevent tau aggregation, with PPIB, PPIC, PPID, and PPIH showing the greatest inhibition. The low thermal stability of PPID and the strong heparin binding of PPIB undermines the simplistic interpretation of reduced tau aggregation. In a cellular model of tau accumulation, all cyclophilins, except PPID and PPIH, reduce insoluble tau. PPIB, PPIC, PPIE, and PPIF also reduce soluble tau levels with PPIC exclusively protecting cells from tau seeding. Overall, this study demonstrates cyclophilins prevent tau fibril formation and many reduce cellular insoluble tau accumulation with PPIC having the greatest potential as a molecular tool to mitigate tau seeding and accumulation.

Keywords: FRET biosensor cell; PPIase; Thioflavin T; cyclophilin; heparin binding; molecular chaperone; peptidyl-prolyl isomerase; tau; tau seeding; thermal stability.

FIGURE 1

Cyclophilin family domain architecture. Arrows indicate constructs designed for expression in E. coli. PPIG is segmented to indicate amino acids are not drawn to scale. The start of an individual protein region is indicated by the amino acid residue designated according to UniProt. RRM, RNA recognition motif; RS, arginine‐serine rich domain; SP, signal peptide sequence; TPR, tetratricopeptide repeat

FIGURE 2

Cyclophilins inhibit tau aggregation in a concentration dependent manner. (a) Workflow schematic of ThT assay and pellet analysis. In a centrifuge tube, a master mix of tau and cyclophilin is prepared at twice the final concentration. A small aliquot is removed to evaluate protein purity by Coomassie stained SDS‐PAGE gel. Next, a master mix of heparin, ThT, and DTT is prepared at twice the final concentration, then is equally combined with the tau plus cyclophilin master mix and aliquoted into a 96‐well plate to measure ThT fluorescence. Output ThT kinetic curves are then used to calculate the time to reach half maximum (T½) and the final ThT fluorescence (ThT_F). At the completion of the ThT assay, replicate wells containing identical conditions are removed from the 96‐well plate and combined in a single centrifuge tube, which is then centrifuged to yield a supernatant (S) and pellet (P) fraction. Figure created with biorender.com. (b) Average ThT_F at 72 hr from at least two independent experiments (except 1:10, tau:cyclophilin, from one independent experiment), with at least four to six replicates per condition. N/M represents not measured. (c) T½ at 1:1, tau:cyclophilin. (d) Relative intensity of tau in the pellet (insoluble tau) and supernatant (soluble tau) at 1:1, tau:cyclophilin compared to tau alone. Error bars represent standard error of the mean (SEM). Data analyzed by one‐way ANOVA with Dunnett's post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ThT, Thioflavin T

FIGURE 3

Multiple cyclophilins reduce soluble and insoluble tau levels. (a) Representative Western blot images of triton‐soluble and triton‐insoluble fractions from HEK293T cells co‐transfected with tau P301L/S320F and either an empty vector (EV) plasmid or cyclophilin at a plasmid mass ratio of 1:10, tau:cyclophilin. (b) Quantification of relative insoluble tau levels compared to EV from at least two independent experiments with two replicates per condition. (c) Quantification of relative soluble tau levels compared to EV from at least two independent experiments with two replicates per condition. (d) Representative Western blot images of total GFP levels from HEK293T cells co‐transfected with GFP and either an EV or cyclophilin at a plasmid mass ratio of 1:10, GFP:cyclophilin. (e) Quantification of relative GFP levels compared to EV from two replicates per condition. (f) Cell toxicity measured by LDH assay 48 hr post transfection of HEK293T cells co‐transfected with either an EV plasmid or tau with each cyclophilin or EV control at a plasmid mass ratio of 1:10, tau:cyclophilin. (g) Representative Western blot images of total tau levels from HEK293T cells co‐transfected with tau WT or tau P301L and either an EV or cyclophilin at a plasmid mass ratio of 1:10, tau:cyclophilin. (h) Quantification of relative tau, WT and P301L, levels compared to EV from three independent experiments. All intensities were normalized to GAPDH loading control prior to EV normalization. Error bars represent standard error of the mean (SEM). Data analyzed by one‐way ANOVA with Dunnett's post hoc test, **p < 0.01, ***p < 0.001, ****p < 0.0001. LDH, lactate dehydrogenase

FIGURE 4

PPIC reduces tau seeding. (a) Workflow schematic of tau FRET biosensor cell assay. Tau RD P301S FRET HEK293T cells, subcultured into 24‐well plates, are co‐transfected with mKate and cyclophilin or empty vector (EV) plasmids at a 1:4, mKate:cyclophilin plasmid ratio. Twenty‐four hours after transfection, cells are subcultured into 96‐well plates, then allowed to recover for 24 hr before seeding with sonicated recombinant human tau 4R0N P301L fibrils. After the addition of seeds, cells are housed in a BioTek BioSpa and imaged by a Cytation 3 every 12 hr for 60 hr using FRET and Texas Red cubes. Figure created with BioRender.com. (b) Relative positive FRET signal of tau seeding in the presence of cyclophilin compared to EV at 60 hr after addition of sonicated recombinant tau P301L fibrils. (c) Representative images of EV and PPIC are shown. Green represents tau FRET signal and red represents transfected mKATE positive cells. Scale bar is 20 μm. Data analyzed by two‐way ANOVA repeated measures with Dunnett's post hoc test, *p < 0.05 for PPIC at 60‐hr time point. ANOVA, analysis of variance