A Dual Pathogenic Mechanism Links Tau Acetylation to Sporadic Tauopathy

Transcription

1 A Dual Pathogenic Mechanism Links Tau Acetylation to Sporadic Tauopathy Hanna Trzeciakiewicz, Jui-Heng Tseng, Connor M. Wander, Victoria Madden, Ashutosh Tripathy, Chao-Xing Yuan, and Todd J. Cohen Supplementary Information (Figures S1-S6)

2 Trzeciakiewicz et al., Supplementary Figure S1 WT tau CBP CBP-LD WT tau CBP CBP-LD WT tau CBP CBP-LD AT8 AT270 tau S262 AT180 T46 Full- length blots from Fig. 1a

3 Trzeciakiewicz et al., Supplementary Figure S1 _ wt KK 4KQ 4KR +CBP wt KK 4KQ 4KR +HDAC6 wt KK 4KQ 4KR +GSK3 wt KK 4KQ 4KR +PP2A wt KK 4KQ 4KR AT8 AT8 tau1 tau Full- length blots from Fig. 1b T46 T46

4 Trzeciakiewicz et al., Supplementary Figure S1 OA wt KK 2KQ 4KQ 4KR _ AT S262 Full- length blots from Fig. 1c T46

5 Trzeciakiewicz et al., Supplementary Figure S2 Trzeciakiewicz et al., Supplementary Figure 1 Mass spectrometry analysis of purified tau expressed in cells identified the doubly acetylated K280/K281 peptide. Full-length wild-type Tau-T40 was acetylated by co-expression with CBP followed by immunoprecipitation with anti-tau T14+T46 antibodies, separated by SDS-PAGE, gel excised, and analyzed by mass spectrometry. Listed in the table are the individual ion scores that correspond to the m/z spectrum depicted in Figure 2a. Statistically significant ion scores in red and blue confirm the doubly acetylated K280/K281 peptide, 275VQIINKKLDLSNVQSK290. These data were generated using Mascot software (Matrix Science).

6 Trzeciakiewicz et al., Supplementary Figure S3 K18 K18 KK K18 K19 autoradiography Coomassie K18 vs K18- ΔKK K18 vs K19-10 Full- length gels from Fig. 2b and 2c

7 Trzeciakiewicz et al., Supplementary Figure S4 Tau K18 fragment a K18 WT 0 1h 2h 4h 8h MW S P S P S P S P S P b K18 K280Q 0 1h 2h 4h 8h MW S P S P S P S P S P c K18 P301L 0 1h 2h 4h 8h MW S P S P S P S P S P d K18 K280R 0 1h 2h 4h 8h MW S P S P S P S P S P Full- length tau T40 a T40 WT 0 1d 2d 3d S P S P S P S P b T40 K280Q 0 1d 2d 3d S P S P S P S P MW MW MW c T40 P301L 0 1d 2d 3d S P S P S P S P Full- length gels from Fig. 3

8 Trzeciakiewicz et al., Supplementary Figure S5 WT P301L 2KQ 2KR WT P301L 2KQ 2KR WT P301L 2KQ 2KR Soluble FracMon (Triton extracted) ps396 AT8 Total tau (K9JA) Insoluble FracMon (SDS- extracted) ps396 AT8 Total tau (K9JA) Full- length blots from Fig. 6a

9 Trzeciakiewicz et al., Supplementary Figure S5 ** ** ns ** Tau acetyla*on mimics enhance seed- dependent tau aggrega*on in cells with minimal phospho- tau accumula*on. QuanMficaMon of immunoblots in Fig. 6a was performed by protein band densitometry. Error bars indicate s.d. of the mean. P- values indicate stamsmcal significance (**, p- value < 0.01).

10 Trzeciakiewicz et al., Supplementary Figure S6 Tau-K18 WT Tau-K18 WT Tau-K18 WT MB: S P S P MB: S P S P MB: S P S P 10-0 hr 2 hr 4 hr Tau-K18-K280Q Tau-K18-K280Q Tau-K18-K280Q MB: MB: MB: S P S P S P S P S P S P 10-0 hr 0.5 hr 2 hr Full- length gels from Fig. 7a

11 Trzeciakiewicz et al., Supplementary Figure S6 Control Tau-K18 WT 3MA+Ars MB+3MA+Ars Tau-K18 WT Tau-K18 WT Control 3MA+Ars MB+3MA+Ars Control 3MA+Ars MB+3MA+Ars 75- Soluble fraction- Total Tau (K9JA) Insoluble fraction- Total Tau (K9JA) Soluble fraction- Hsc70 Tau-K18-K280Q Tau-K18-K280Q Tau-K18-K280Q Control 3MA+Ars MB+3MA+Ars Control 3MA+Ars MB+3MA+Ars Control 3MA+Ars MB+3MA+Ars 75- Soluble fraction- Total Tau (K9JA) Insoluble fraction- Total Tau (K9JA) Full- length blots from Fig. 7b Soluble fraction- Hsc70

12 Trzeciakiewicz et al., Supplementary Figure S6 a 3MA+Ars MB K18 P301L c K18 P301L - CON ATPZ MB [ 14 C]-Ac-tau 15 kda ac-k kda p-s262 total tau Hsc total tau Ac-tau/ total tau (%) b Densitometry P301L ac-k280 tau ns * * Densitometry P301L p-s262 tau * ** * Densitometry P301L total tau ** ** ** 0 Control 3MA+Ars MB+ 3MA+Ars 0 Control 3MA+Ars MB+ 3MA+Ars 0 Control 3MA+Ars MB+ 3MA+Ars Methylene blue modulates tau- P301L solubility and acetyla*on status. a) Immunoblot analysis with indicated anmbodies of cells expressing K18- P301L treated with 3MA, Ars, or MB, where indicated. Hsc70 was included as a loading control. QuanMficaMon of immunoblots in (b). Error bars indicate s.d. of the mean (*, p < 0.05; **, p < 0.01). c) Autoradiography and Coomassie blue staining of in vitro auto- acetylated recombinant K18- P301L protein incubated with 20 μm inacmve control, ATPZ, or MB compounds. The raw intensity ramo of acetylated to total tau protein is presented as a percentage and set relamve to untreated control reacmons.

13 Trzeciakiewicz et al., Supplementary Figure S6 * ac-k280 ps262 Total tau Full- length blots from Supplementary Fig. S6

14 Trzeciakiewicz et al., Supplementary Figure S6 K18 P301L - CON ATPZ MB K18 P301L - CON ATPZ MB 10- Coomassie autoradiography Full- length gels from Supplementary Fig. S6