Table 1.
Primary Antibody Concentrations Used.
Figure 1.
Annonacin Causes an Upregulation of the 4R Isoforms of Tau.
A) LUHMES neurons were treated with different concentrations of annonacin for 48 h from day 8–10 post differentiation (n = 12). The MTT test, a measure for mitochondrial reducing function, and ATP concentration, are expressed as a relative percentage compared to untreated control cells. B) 4R isoform (exon 10) mRNA is upregulated with annonacin treatment. Quantitative PCR results showing the relative quantity of mRNA for different MAPT splicing variants in cells treated with 25 nM annonacin for 48 h from day 8–10 post differentiation compared to untreated cells (dotted line). 3 biological repeats with 3 technical repeats each. ***: p<0.001, *: p<0.05 vs. untreated cells (2-way ANOVA with Sidak's post-hoc test). C) 4R isoform protein is upregulated with annonacin treatment. Western blot for 3R and 4R isoforms of tau protein, as well as total tau (detected with the HT7 antibody). LUHMES cells were either left untreated or treated with 25 nM annonacin. Actin was used as loading control. D) Quantification of figure 1C. Results show the relative quantity (fold-change) compared to untreated control cells (relative quantity = 1, represented by dotted line). 3 biological repeats. ***: p<0.001 vs. untreated cells (2-way ANOVA with Sidak's post-hoc test).
Table 2.
Overview of the splicing factors known to influence MAPT exon 10 alternative splicing.
Figure 2.
SRSF2 is a Critical Player in Annonacin Mediated Tau Alternative Splicing.
A) Quantitative PCR results for 9 different splicing factors known to have an effect on exon 10 alternative splicing (table 2). Data shown are relative quantities compared to untreated cells (dotted line). Only SRSF2 was elevated significantly with annonacin treatment. All other splicing factors tested were not significantly elevated. 3 biological repeats with 3 technical repeats each. ***: p<0.001, *: p<0.05 vs. untreated control (2-way ANOVA with Sidak's post-hoc test). B) Quantitative PCR results for LUHMES cells on day 10 post differentiation treated with SRSF2 knockdown siRNA for 10 days and/or with annonacin for 48 h. 3 biological repeats with 3 technical repeats each. ***: p<0.001 vs. untreated control (dotted line); ###: p<0.001 (2-way ANOVA with Sidak's post-hoc test). C) Quantitative PCR results for the 4 splicing factors known to increase MAPT exon 10 inclusion in locus coeruleus tissue of four PSP patients and five controls without neurodegenerative diseases. 3 biological repeats with 3 technical repeats each. #: p<0.05, ##: p<0.01 (2-way ANOVA with Sidak's post-hoc test).
Table 3.
Overview of Human Tissue Used.
Figure 3.
The 4R Isoform Shift Can Be Reproduced with Another Complex I Inhibitor but not with the Oxidative Stressor 6-OHDA.
A) ATP concentration and MTT cell viability in LUHMES cells as measured by the MTT assay for different concentrations of 6-OHDA. Treatment was for 48 h from day 8–10 post differentiation. n = 12. B) ATP concentration and MTT cell viability in LUHMES cells as measured by the MTT assay for different concentrations of MPP+. Treatment was for 48 h from day 8–10 post differentiation. n = 12. C) Quantitative PCR results of MAPT splicing variants for LUHMES cells treated with 10 µM MPP+ for 48 h from day 8–10 post differentiation. 3 biological repeats with 3 technical repeats each. **: p<0.01 vs. untreated cells (dotted line), (2-way ANOVA with Sidak's post-hoc test). D) Quantitative PCR results of MAPT splicing variants for LUHMES cells treated with 20 µM 6-OHDA for 48 h from day 8–10 post differentiation. 3 biological repeats with 3 technical repeats each. *: p<0.05 vs. untreated cells (dotted line), (2-way ANOVA with Sidak's post-hoc test). E) Quantitative PCR results of MAPT splicing variants for LUHMES cells starved of nutrients and glucose for 24 h from day 8–9 post differentiation. 3 biological repeats with 3 technical repeats each. *: p<0.05 vs. untreated cells (dotted line), (2-way ANOVA with Sidak's post-hoc test). F) Quantitative PCR results of SRSF2 for LUHMES cells treated with 10 µM MPP+ or 20 µM 6-OHDA for 48 h from day 8–10 post differentiation or starved for 24 h from day 8–9 post differentiation. 3 biological repeats with 3 technical repeats each. *: p<0.05, **: p<0.01 vs. untreated cells (dotted line), (2-way ANOVA with Sidak's post-hoc test).
Figure 4.
The 4R Isoform was upregulated in the protein level with MPP+ treatment.
A) 4R isoform protein is upregulated with MPP+ treatment. Western blot for 3R and 4R isoforms of tau protein, as well as total tau (detected with the HT7 antibody). LUHMES cells were either left untreated or treated with 10 µM MPP+. Actin was used as loading control. B) Quantification of figure 1G. Results show the relative quantity compared to the untreated control cells (dotted line). 3 biological repeats. ***: p<0.001 vs. untreated cells (2-way ANOVA with Sidak's post-hoc test).