A powerful yeast model to investigate the synergistic interaction of α-synuclein and tau in neurodegeneration

Abstract

Several studies revealed consistent overlap between synucleinopathies and tauopathies, demonstrating that α-synuclein (ASYN) and tau co-localize in neurofibrillary tangles and in Lewy bodies from Alzheimer's and Parkinson's disease patients and corresponding animal models. Additionally, it has been shown that ASYN can act as an initiator of tau aggregation and phosphorylation and that these two proteins directly interact. Despite these evidences, the cellular pathway implicated in this synergistic interaction remains to be clarified. The aim of this study was to create a yeast model where the concomitant expression of ASYN and tau can be used to perform genome wide screenings for the identification of genes that modulate this interaction, in order to shed light into the pathological mechanism of cell dysfunction and to provide new targets for future therapeutic intervention. We started by validating the synergistic toxicity of tau and ASYN co-expression in yeast, by developing episomal and integrative strains expressing WT and mutant forms of both proteins, alone or in combination. The episomal strains showed no differences in growth delay upon expression of ASYN isoforms (WT or A53T) alone or in combination with tau 2N/4R isoforms (WT or P301L). However, in these strains, the presence of ASYN led to increased tau insolubility and correlated with increased tau phosphorylation in S396/404, which is mainly mediated by RIM11, the human homolog of GSK3β in yeast. On the other hand, the integrative strains showed a strong synergistic toxic effect upon co-expression of ASYN WT and tau WT, which was related to high levels of intracellular ASYN inclusions and increased tau phosphorylation and aggregation. Taken together, the strains described in the present study are able to mimic relevant pathogenic features involved in neurodegeneration and are powerful tools to identify potential target genes able to modulate the synergistic pathway driven by ASYN and tau interaction.

Figure 1. Cytotoxic growth effects upon high-copy plasmid expression of ASYN alone or in combination with Tau.

A) High expression level of ASYN (WT and A53T) and tau (WT and P301L), detectable by western blot upon expression with pESC-LEU bidirectional high-copy (2µ) episomal vector. Whereas ASYN levels (WT and A53T) are similar when either expressed alone or in combination with tau (WT or P301L), tau (WT and P301L) levels are strongly decreased when expressed in combination with ASYN (WT or A53T), (***p<0,001). GAPDH was used as loading control. B) Strong growth delay observed by dot spot at 37°C after 72 hours of incubation upon expression of ASYN (WT and A53T) alone or in combination with tau (WT and P301L). Expression of tau (WT or P301L) alone has no effect on cells vitality whereas when expressed in combination with ASYN (WT and A53T) cytotoxicity is observed. A quantitative plot of the dot spots is shown. Results are representative of at least three independent experiments. C) No differences observed upon expression of ASYN and tau isoforms alone or in combination by growth ability tests performed in liquid media using 96 well plates at both 30°C and 37°C. All results are representative of at least three independent experiments.

Figure 2. ASYN increases Tau insoluble aggregation state.

A) Immunofluorescence with an anti-ASYN antibody showed no significant differences between the percentage of cells that contain ASYN (WT and A53T) intracellular inclusions when expressed alone or in combination with tau (WT and P301L). No yeast cells with ASYN big inclusions were observed in the strain expressing ASYN A53T in combination with tau P301L For statistical analysis at least 800 cells were counted. B) Both ASYN (WT and A53T) and tau (WT and P301L) form intracellular sarkosyl insoluble aggregates when expressed either alone or in combination, which are detectable by western blot. GAPDH was used as loading and soluble protein control. *corresponds to an unspecific band. Results are representative of three independent experiments.

Figure 3. ASYN increases Tau phosphorylation in S396/404 via RIM11.

A) Relative percentage of phosphorylated tau (WT and P301L) in S396/404 labelled by the antibody AD2 was increased in the presence of ASYN (WT and A53T). (*p<0,05; **p<0,01). GAPDH was used as loading control. B) No phosphorylated tau (WT or P301L) detected in ΔRIM11 mutant by western blot when expressed alone or in combination with ASYN (WT or A53T). GAPDH was used as loading control. C) Lack of tau (WT and P301L) phosphorylation in S396/404 doesn’t alter cytotoxicity observed in solid media when tau (WT or P301L) and ASYN (WT and A53T) are expressed alone or in combination in ΔRIM11 as compared to expression in BY4741 WT.A quantitative plot of the dot spots is shown (*p<0,05; **p<0,01; ***p<0,001). Results are representative of at least three independent experiments.

Figure 4. Strong synergistic growth effect upon genome integration of ASYN WT and Tau WT.

A) ASYN protein expression levels detectable by western blot, remain generally constant, displaying a reduction upon co-expression of either tau isoform. Total tau expression levels remain constant whereas the percentage of tau WT phosphorylated in S396/404 increases only in the presence of ASYN WT. (*p<0,05; **p<0,01). GAPDH was used as loading control. B) Slight growth delay observed by dot spot at 30°C upon expression of ASYN WT, high growth delay with ASYN A53T and strong synergistic cytotoxicity observed when ASYN WT and tau WT are co-expressed. At 37°C a total growth arrest is observed for the strains expressing either ASYN (WT or A53T) alone or expressing ASYN WT in combination with tau WT. Tau P301L seems to rescue cytotoxic effect mediated by ASYN (WT and A53T) as well as tau WT when in combination with ASYN A53T. A quantitative plot of the dot spots is shown (*p<0,05; ***p<0,001). Results are representative of at least three independent experiments. C) Strong growth delay also observed in liquid growth analysis at 30°C upon co-expression of ASYN (WT and A53T) alone or ASYN WT in combination with tau WT. As in solid media tau P301L seems to rescue cytotoxic effect mediated by ASYN (WT and A53T) as well as tau WT when in combination with ASYN A53T. All results are representative of at least three independent experiments.

Figure 5. High percentage of ASYN cytoplasmatic inclusions and insoluble Tau correlate with the observed synergistic growth effect.

A) Percentage of cells showing ASYN intracellular aggregates, detectable by immunofluorescence with anti-ASYN antibody, is similar when ASYN WT is expressed alone or in combination with tau WT, while when it is in combination with tau P301L it is highly reduced. Co-expression of ASYN A53T with tau (WT or P301L) also shows a decreased percentage of cells with intracellular aggregates. (*p<0,05; ***p<0,001). For statistical analysis at least 800 cells were counted. B) ASYN inclusions localization analyzed after 16 h, by immunofluorescence with anti-ASYN antibody, shows that in the strain expressing ASYN (WT or A53T) alone or ASYN WT in combination with tau WT inclusions are mainly cytoplasmatic. In the strains co-expressing ASYN A53T in combination with tau (WT or P301L) both small membrane associated and cytoplasmatic inclusions are detectable, whereas only small membrane associated inclusions can be observed when ASYN WT is expressed in combination with tau P301L For statistical analysis at least 800 cells were counted. C) ASYN (WT and A53T) alone or in combination with tau (WT or P301L) is detectable by western blot in the sarkosyl insoluble fraction, whereas tau is detectable in the sarkosyl insoluble fraction only when co-expressed with ASYN WT. GAPDH was used as loading and soluble protein control. *corresponds to an unspecific band. Results are representative of at least three independent experiments.