Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment

Abstract

Tau aggregation is a hallmark of a group of neurodegenerative diseases termed Tauopathies. Reduction of aggregation-prone Tau has emerged as a promising therapeutic approach. Here, we show that an anti-aggregant Tau fragment (F3^ΔKPP, residues 258-360) harboring the ΔK280 mutation and two proline substitutions (I²⁷⁷P & I³⁰⁸P) in the repeat domain can inhibit aggregation of Tau constructs in vitro, in cultured cells and in vivo in a Caenorhabditis elegans model of Tau aggregation. The Tau fragment reduced Tau-dependent cytotoxicity in a N2a cell model, suppressed the Tau-mediated neuronal dysfunction and ameliorated the defective locomotion in C. elegans. In vitro the fragment competes with full-length Tau for polyanionic aggregation inducers and thus inhibits Tau aggregation. Our combined in vitro and in vivo results suggest that the anti-aggregant Tau fragment may potentially be used to address the consequences of Tau aggregation in Tauopathies.

Keywords: Aggregation; Alzheimer disease; Cell model; Life-span; Microtubules; Tau; Transgenic C.elegans; β-breaker peptides.

Fig. 1

Constructs of Tau. The top bar diagram represents the longest isoform of the human Tau40 (441 residues). The diagram below hTau40 shows the four-repeat construct Tau^RD. The two hexapeptides (₂₇₅VQIINK₂₈₀ and ₃₀₆VQIVYK₃₁₁) are the motifs with the highest β-propensity at the beginning of the 2nd and 3rd repeat domains. The construct Tau^RDΔK contains the FTDP-17 mutation ΔK280 that accelerates aggregation by promoting the β-structure (pro-aggregant mutant). The construct F3^ΔK is a proteolytic Tau fragment composed of aa. 258–360 [17, 18]. The construct F3^ΔK-PP harbors ΔK280 and has two proline mutations (I277P and I308P in the hexapeptide motifs) that inhibit aggregation by disrupting the β-structure (anti-aggregant mutant)

Fig. 2

F3^ΔKPP reduces Tau^RDΔK aggregation in vitro. Tau^RDΔK (10 μM, upper band) was induced to aggregate with heparin (2.5 μM) in the absence or presence of different concentrations of F3^ΔKPP (lower band) for up to 24 h. a The extent of aggregation as measured by the thioflavin S fluorescence assay. All the measurements were performed in triplicate, n = 3. b Pellet assay showing the distribution of soluble and aggregated Tau^RDΔK (10 μM) alone (lanes 1, 2) or in the presence of F3^ΔKPP 10 μM (lanes 3, 4), 20 μM (lanes 5, 6), 40 μM (lanes 7, 8) and 80 μM (lanes 9, 10) at the end of incubation (S denotes the soluble fraction, P is the insoluble pellet fraction). c Densitometry quantification of the soluble (blue bars) and the insoluble (red bars) Tau^RDΔK from the gel shown in (b). Note the suppression of Tau^RDΔK aggregation at higher F3^ΔKPP concentrations (40 μM & 80 μM, red bars 8, 10). The results are from 3 different gels. One-way ANOVA was applied for multiple comparisons. Error bars denote SD. (ns, non-significant, **p < 0.001). d In vitro aggregation of Tau^RDΔK (10 μM) visualized using AFM in the absence (top panel) or presence (middle panel) of F3^ΔKPP (80 μM). 1–2 μM protein was diluted in PBS and placed on mica for imaging. No filamentous structures are seen in the presence of F3^ΔKPP. F3^ΔKPP (80 μM) alone also does not form filamentous structures (bottom panel)

Fig. 3

F3^ΔKPP slightly reduces hTau40-induced microtubule assembly. Microtubule assembly was measured by light scattering at 350 nm in the absence or presence of hTau40 with or without different concentrations of F3^ΔKPP. a Tubulin and hTau40 concentration was 10 μM and 5 μM respectively. The ratios between the concentration of hTau40 and F3^ΔKPP were 1:1, 1:4, and 1:8. Note that F3^ΔKPP reduces hTau40-induced microtubule assembly by ~ 50% (curves 4 and 3, purple and blue). Tubulin without Tau (curve 1, green) or with F3^ΔKPP alone (20 μM; curve 2, ochre) does not assemble in these conditions either. b Microtubule (10 μM) assembly induced by Tau (5 μM) visualized by negative stain electron microscopy in the absence (left panel) or presence (right panel) of F3^ΔKPP (20 μM). Microtubules are reduced with F3^ΔKPP and become more fragile

Fig. 4

F3^ΔKPP reduces Tau^RDΔK aggregation and cytotoxicity in N2a cells. N2a cells were transfected with Tau^RDΔK or co-transfected with Tau^RDΔK and F3^ΔKPP for 2 days. a Thioflavin S (ThS) staining of Tau aggregates in N2a cells. Tau was monitored by immunostaining using a pan-Tau antibody K9JA (red panel 2 and 5). b Quantification of the ThS positive cells in relation to the Tau-expressing cells shown in (a). F3^ΔKPP strongly reduces ThS positive cells. (t test, n = 3; * p < 0.05). c Western blot analysis (17% PAGE, Tau antibody K9JA) of sarkosyl soluble (S) and insoluble (P) Tau^RDΔK in the absence (lanes 2, 3) or presence (lanes 4, 5) of F3^ΔKPP. d Densitometry quantification of insoluble Tau^RDΔK (lanes 3 and 5) of the blot shown in (c). Note the strong reduction (~ 60%, red bar) of aggregated Tau^RDΔK by co-expression of F3^ΔKPP. (unpaired t test, n = 6; p = 0.0624). e Cell death monitored by nuclear staining with Ethidium Homodimer (EthD). Tau expression was determined by immunolabeling with antibody K9JA (panel 3 and 7), Tau aggregation by ThS staining (green), and cell death by EthD staining (blue). Note: that cell death (blue) was dramatically reduced by the co-expression of F3^ΔKPP (t test, SD, *p < 0.05). f Quantification of cells positive for EthD staining shown in E. Cell death was reduced by the co-expression of F3^ΔKPP (t test, SD, *p < 0.05)

Fig. 5

F3^ΔKPP at higher levels (F3^ΔKPP-hi) improves the motor deficits in a C. elegans Tau aggregation model (T^VM). F3^ΔKPP was expressed pan-neuronally in worms transgenic for human 1N4R-Tau^V337M. T^VM expresses human 1N4R-Tau^V337M pan neuronally. T^VM;F3^ΔKPP-lo and T^VM;F3^ΔKPP -hi are doubly transgenic for human 1N4R-Tau^V337M and F3^ΔKPP at low and high levels respectively. a Western blot of the total worm lysates from synchronized 1-day-old adults using pan-Tau antibody K9JA. Tubulin served as internal control. b Quantification of the total Tau levels. One-way ANOVA with Tukey’s test (n = 3, error bars denote SEM. ns, non-significant). c Micrographs showing tracks left behind by 1-day-old adults of the single and double transgenic worms. Non-transgenic (non-tg) served as control. d Body bending frequency (thrashes) of synchronized 1-day-old adults in liquid. Non-tg served as control, n = 40. One-way ANOVA with Tukey’s test was applied for multiple comparisons. Error bars denote SEM. (ns, non-significant, **p < 0.01). e Representative survival curves of single- T^VM, double- T^VM;F3^ΔKPP-lo, and T^VM;F3^ΔKPP-hi transgenic worms, non-tg served as control. Mantel-Cox log-rank test was performed to determine the statistical differences between genotypes

Fig. 6

F3^ΔKPP-hi reduces morphological defects and suppresses the Tau aggregation in T^VM. a Fluorescence micrographs of GABAergic motor neurons in non-tg (1), T^VM (2), T^VM;F3^ΔKPP-lo (3), and T^VM;F3^ΔKPP-hi (4). Animals have ventral side oriented up. Arrowheads show gaps in the ventral and dorsal cord. b Number of gaps quantified in the neural cords of 1-day-old adults. Error bars denote SD. For comparison, one-way ANOVA with Tukey’s test was applied (n = 25, ns, not significant, **P < 0.01). c Sequentially extracted Tau from worm lysates of mixed stage animals resolved on 17% PAGE and immunoblotted using pan-Tau K9JA antibody. Tubulin served as a loading control. d Densitometry quantification of the insoluble Tau. T^VM;F3^ΔKPP-hi (insoluble panel, lane 4) shows reduced insoluble Tau (~ 50%). One-way ANOVA with Tukey’s test (n = 3, error bars denote SEM. ns, non-significant, ***P < 0.001)

Fig. 7

F3^ΔKPP-hi improves the mitochondrial distribution in C. elegans. a Schematic representation of neurons with a normal and abnormal mitochondrial distribution. b Representative images of GFP tagged mitochondria in the mechanosensory neurons of non-tg reporter strain, T^VM and T^VM;F3^ΔKPP-hi animals at day 1 of adulthood. c Representative images of GFP-tagged mitochondria in the mechanosensory neurons of non-tg reporter strain, T^VM and T^VM;F3^ΔKPP-hi animals at day 3 of adulthood. d Average number of mitochondria quantified in the proximal axon (~ 80 μm axonal part adjacent to cell body) at days 1 and 3. Student’s t-test for comparison (error bars denote SEM. **P < 0.01). e Average number of mitochondria quantified in the mid-region of the axon (beyond ~ 80-μm length from cell body) at days 1 and 3. Student’s t test for comparison (error bars denote SEM. ***P < 0.001)

Fig. 8

F3^ΔKPP does not interact directly with Tau40 or Tau^RDΔK to prevent aggregation. N2a cells were co-transfected with F3^ΔKPP and Tau^RDΔK-His or hTau40 for 2 days. Antibody anti-His (blot A) and DA9 (epitope: aa. 112–129) (blot B) were used to immunoprecipitate Tau^RDΔK-His or hTau40 in the cell lysates respectively, using non-specific IgG as control (A, B, lane 2). a Anti-His pulled down Tau^RDΔK-His but not F3^ΔKPP (blot 1, lane 3). Similarly, DA9 pulled down hTau40 but not F3^ΔKPP (blot 2, lane 3), indicating that there is no direct interaction between F3^ΔKPP and hTau40. b Recombinant hTau40 (50 μM) and F3^ΔKPP (200 μM) were incubated at 37 °C for 48 h in the presence (blot 1) or absence (blot 2) of 12.5 μM heparin (M.W. 16 K) in BES buffer. Antibody DA9 (blots 1, 2, lane 3) was used to immunoprecipitate hTau40, using non-specific IgG as control (Ctrl-IgG, blots 1, 2, lane 2). Note that in the presence of heparin, DA9 pulls down both hTau40 and F3^ΔKPP (blot 1, lane 3). However, in the absence of heparin, DA9 pulls down only htau40 but not F3^ΔKPP (blot 2, lane 3), indicating an absence of a direct interaction between F3^ΔKPP and hTau40. c Heparin binds and pulls down F3^ΔKPP and hTau40 in the absence of antibody. Recombinant F3^ΔKPP and hTau40 at the same concentrations as described above were incubated at 37 °C for 48 h in the presence or absence of heparin (16 K) in BES buffer and Dynabeads Protein G added to the reaction mixtures afterwards. Note that hTau40 and F3^ΔKPP can be pulled down by heparin without requiring an antibody (blots 1, 2, 3, lane 2). In the absence of heparin, neither of the proteins is pulled down (blot 1, 2, 3, lane 4). Note the thick bands (red circle) corresponding to F3^ΔKPP in the pull-down lanes (blot 2, 3, lanes 2). This set of experiments shows (i) heparin is able to bind F3^ΔKPP and hTau40, (ii) heparin is able to bind the beads and thereby pull down both the proteins either individually or in combination, and (iii) the affinity is higher for F3^ΔKPP than hTau40. Hence, a direct interaction between F3^ΔKPP and hTau40 is absent, but the two interact indirectly via an aggregation inducer like heparin

Fig. 9

Model of Tau aggregation and competition with anti-aggregant F3^ΔKPP. Tau aggregation can be induced in vitro or in cells by cofactors such as heparin, RNA, or other polyanions (a). In such a scenario, F3^ΔKPP can compete with Tau molecules by preferentially binding and sequestering the aggregation inducers. This might prevent the formation of early oligomers (dimers, trimers etc.) (b). At low F3^ΔKPP levels, sufficient inducers are available so that the aggregation of Tau may not be disturbed. With increasing F3^ΔKPP levels, the inducers available for Tau are reduced and the aggregation of Tau is retarded