Effects of pharmacological modulators of α-synuclein and tau aggregation and internalization

Abstract

Parkinson's disease (PD) and Alzheimer's disease (AD) are common neurodegenerative disorders of the elderly and, therefore, affect a growing number of patients worldwide. Both diseases share, as a common hallmark, the accumulation of characteristic protein aggregates, known as Lewy bodies (LB) in PD, and neurofibrillary tangles in AD. LBs are primarily composed of misfolded α-synuclein (aSyn), and neurofibrillary tangles are primarily composed of tau protein. Importantly, upon pathological evaluation, most AD and PD/Lewy body dementia cases exhibit mixed pathology, with the co-occurrence of both LB and neurofibrillary tangles, among other protein inclusions. Recent studies suggest that both aSyn and tau pathology can spread and propagate through neuronal connections. Therefore, it is important to investigate the mechanisms underlying aggregation and propagation of these proteins for the development of novel therapeutic strategies. Here, we assessed the effects of different pharmacological interventions on the aggregation and internalization of tau and aSyn. We found that anle138b and fulvic acid decrease aSyn and tau aggregation, that epigallocatechin gallate decreases aSyn aggregation, and that dynasore reduces tau internalization. Establishing the effects of small molecules with different chemical properties on the aggregation and spreading of aSyn and tau will be important for the development of future therapeutic interventions.

Figure 1

Reconstitution of venus fluorescence upon co-culture of transfected cells indicates release and uptake of aSyn or tau proteins. (a) Representative scheme of the BiFC principle. Proteins tagged to the VN fragment of the venus protein interact with proteins tagged to the VC fragment of the venus protein leading to the reconstitution of the fluorophore. (b) Schematic of the co-culture process. Cells transfected with proteins tagged with the VC fragment of venus were trypsinized and mixed with cells transfected with proteins tagged to the VN fragment. Protein release and uptake by new cells results in an interaction and reconstitution of the fluorophore. (c) Epifluorescence microscopy showing positive cells. (i) Representative picture of cells cotransfected with aSyn BiFC constructs. (ii) Representative picture of aSyn co-cultured cells. (iii) Representative picture of cells cotransfected with tau BiFC constructs. (iv) Representative picture of tau co-cultured cells. Scale bar 50 µm. (d) Flow cytometry analysis of different cultures. (i) Representative histogram showing the number of cells versus fluorescence intensity for aSyn cotransfected cells. (ii) Representative histogram showing the number of cells versus fluorescence intensity for aSyn co-cultured cells. (iii) Representative histogram showing the number of cells versus fluorescence intensity for tau cotransfected cells. (iv) Representative histogram showing the number of cells versus fluorescence intensity for tau co-cultured cells. The average percentage of positive cells ± SD is shown in each histogram. Cells with a value of fluorescence intensity above 120 fluorescence units are considered positive. (e) Western blot showing the levels of all aSyn constructs. Full length blots are presented in Supplementary Fig. S6A. (f) Western blot showing the levels of all tau constructs. Full length blots are presented in Supplementary Fig. S6B. (g) Percentages of positive cells in co-cultures are significantly different from those obtained for the negative controls for the aSyn constructs. (h) Percentage of positive cells in co-cultures are significantly different from the percentage of positive cells in the negative controls for the tau constructs. ***P < 0.0005 from all other groups. ANOVA and subsequent paired t-test. Error bars represent SD.

Figure 2

Proteins are released to the cell culture media. (a) Western blot showing presence of different aSyn BiFC fragments in cell culture media. Full length blots are presented in Supplementary Fig. S6C. (b) Western blot showing presence of different tau BiFC fragments in cell culture media. Full length blots are presented in Supplementary Fig. S6D. (c) ELISA measurements of aSyn levels in cell culture media show presence of the protein in cell media (N = 4). (d) ELISA measurements of tau levels confirm the presence of the protein in the cell culture media (N = 5). *P < 0.05; **P < 0.005; ***P < 0.0005 in relation to empty vector. Error bars represent SD. ANOVA and subsequent paired t-test in relation to empty vector.

Figure 3

Conditioned media added to cells reports on protein release and uptake. (a) Schematics of the experimental setup. Cells are transfected with the different constructs, after 48 h, media are collected and added to either non-transfected cells or to cells transfected with a different construct. The two proteins interact inside the receptor cells leading to reconstitution of the fluorophore and fluorescence. (b) Representative pictures of the different combinations performed. In the negative cases, an immunocytochemistry was performed to assess the internalization of the proteins. Colors used for immunocytochemistry (pictures inside squares) are: green for aSyn and red for tau. Positive BiFC combinations yield green color. Mixes of aSyn and tau also yield green signal additionally confirming the interaction of the two proteins. Scale bar 50 µm.

Figure 4

Effect of inhibition of tau internalization. (a) Schematic representation of the experimental setup. Cells were co-cultured and treated with 10 µM dynasore. Inhibiting this internalization should lead to no fluorescence. (b) Representative immunofluorescence pictures showing the expression of dynamin 1 and dynamin 2. Both dynamin 1 and 2 are detected in HEK293 cells by immunocytochemistry. Scale bar 25 µm. (c) Representative pictures of vehicle and dynasore-treated co-cultures. Tau is present in dynasore-treated cells. (d) Flow cytometry results for vehicle and dynasore-treated co-cultures showing the average percentages of positive cells ± SD. (e) Flow cytometry results show that dynasore-treated co-cultured cells show a significant decrease in percentage of positive cells in comparison with vehicle-treated co-cultured cells (N = 4). (f) Flow cytometry results show that dynasore-treated co-cultured cells show no significant decrease in fluorescence intensity in comparison with vehicle-treated co-cultured cells (N = 4). (g) Western blot results show that dynasore-treated co-cultured cells show no significant decrease in protein expression levels in comparison with vehicle-treated co-cultured cells (N = 3). (h) Western blot picture of the vehicle-treated and dynasore-treated co-cultured cells. Full length blots are presented in Supplementary Fig. S6E. Data are shown as mean ± SD. *P < 0.05, t test.

Figure 5

Anle138b, fulvic acid and EGCG inhibit protein aggregation. (a) RT-QuiC graph of the monomeric aSyn amplification in the presence and absence of 100 nM anle138b (N = 4). Protein amplified in presence of 100 nM anle138b shows lower ThT fluorescence intensity than in absence of anle138b. Orange: only monomeric protein added; grey: monomeric protein and DMSO added; blue: monomeric protein and anle138b (dissolved in DMSO) added. (b) RT-QuiC results show a significant decrease in monomer incorporation rate per cycle when amplification in the presence of 100 nM anle138b. Orange: only monomeric protein added; grey: monomeric protein and DMSO added; blue: monomeric protein and anle138b (dissolved in DMSO) added. (c) RT-QuiC graph of the monomeric aSyn amplification in the presence and absence of 10 nM EGCG. Protein amplified in presence of 10 nM EGCG shows lower ThT fluorescence intensity than in absence of EGCG. Orange: only monomeric protein added; blue: monomeric protein and EGCG added. (d) RT-QuiC results show a significant decrease in monomer incorporation rate per cycle in the presence of 10 nM EGCG (N = 4). Orange: only monomeric protein added; blue: monomeric protein and EGCG added. (e) RT-QuiC graph of the tau MBD (K18) amplification in the presence and absence of 37 µM fulvic acid (N = 4). Protein amplified in presence of fulvic acido shows lower ThT fluorescence intensity than in the absence of fulvic acid. Orange: only monomeric protein added; grey: monomeric protein and methanol added; blue: monomeric protein and fulvic acid (dissolved in methanol) added. (f) RT-QuiC assay shows a significant decrease in monomer incorporation rate per cycle in the presence of 37 µM fulvic acid. Orange: only monomeric protein added; grey: monomeric protein and DMSO added; blue: monomeric protein and anle138b (dissolved in DMSO) added. (g) Flow cytometry results of co-cultured cells treated with anle138b (1 µM) show a significant decrease in the percentage of positive cells in comparison with cells treated with vehicle (N = 3). (h) HTRF measurements of aSyn-transfected and co-cultured cells treated with anle138b (1 µM) show significant differences in aggregation (N = 4). (i) Flow cytometry results of co-transfected cells treated with the aggregation inhibitor EGCG (0.1 µM) show statistically significant differences in the percentage of positive cells in relation to cells treated with vehicle (N = 3). (j). HTRF results of aSyn-transfected cells treated with EGCG (0.1 μM) show significant differences in aggregation (N = 4). (k) Flow cytometry results of cells treated with fulvic acid (37 μM) show no significant differences in the percentage of positive cells in comparison with cells treated with vehicle (N = 3). (l) *P < 0.05 in comparison with control-treated cells. Error bars represent SD.

Figure 6

Fulvic acid and anle138b inhibit protein aggregation. (a) Flow cytometry results of co-cultured cells treated with anle138b (1 μM) show a significant decrease in the percentage of positive cells in comparison with cells treated with vehicle (N = 6). (b) Tau levels after PK digestion of tau-transfected and co-cultured cells treated with anle138b (1 μM) show significant differences in comparison with cells treated with vehicle (N = 3). (c) Tau levels after PK digestion of tau-transfected cells treated with anle138b (1 μM) show significant differences in comparison with cells treated with vehicle (N = 3). (d) HTRF measurements of tau-transfected cells treated with anle138b (1 μM) show significant differences in aggregation (N = 3). (e) Flow cytometry results of co-cultured cells treated with EGCG (0.1 μM) show no differences between treated and untreated cells. (f) Tau levels after PK digestion of tau-transfected and co-cultured cells show no differences between treated and untreated cells (N = 3). (g) Tau levels after PK digestion of tau-transfected cells show no differences between treated and untreated cells (N = 3). (h) HTRF measurements of tau-transfected cells treated with EGCG (0.1 μM) show no significant differences in aggregation (N = 4 for co-cultures). (i) Flow cytometry results of co-cultured cells treated with fulvic acid (37 μM) show a significant decrease in the percentage of positive cells in comparison with cells treated with vehicle ( N = 6). (j) aSyn levels after PK digestion of aSyn-transfected and co-cultured cells treated with fulvic acid (37 μM) show significant differences in comparison with cells treated with vehicle ( N = 3). (k) aSyn levels after PK digestion of aSyn-transfected cells treated with fulvic acid (37 μM) show no significant differences in comparison with cells treated with vehicle (N = 3). (l) HTRF measurements of aSyn-transfected and co-cultured cells treated with fulvic acid show significant differences in aggregation in comparison with cells treated with vehicle (N = 6). *P < 0.05 in comparison with vehicle-treated cells. Error bars represent SD.