Fig. 4

Expression of the N-terminal projection domain of hTau dramatically decreases protein synthesis and ribosomal biogenesis. a Schematic of the hTau regions separately expressed in HEK293 cells and investigated in the study: hTau’s N-terminal projection domain (AA 1-AA 224, Proj-dom hTau), microtubule binding region (AA 225-AA 380, MTBR hTau), and carboxy-terminal region (AA 381- AA 441, C-term hTau). b Protein synthesis is reduced by the expression of the N-terminal projection domain of hTau in HEK293 cells. 4 mM AHA was used to label newly synthesised proteins for 16 h in cells expressing either Emerald, Proj-dom hTau, MTBR hTau, or C-term hTau. FUNCAT-WB analysis revealed that de novo protein synthesis was only decreased in Proj-dom hTau expressing cells after 24 h of expression. The abundance of RPL5, RPS14, RPS6 and RPL22 was found to be unaltered in these cells. Expression of the various domains of hTau was detected using the FLAG tag fused to these domains. Protein abundance was normalised to the total protein stain REVERT. n = 3 wells, one-way ANOVA, Tukey’s MCT. c Polysome profiling reveals that the abundance of polysomes, monosomes and the 60S ribosomal subunit is decreased by 24 h of Proj-dom hTau expression. Transfected cells were treated with 100 ug/mL CHX for 5 min in order to prevent the dissociation of bound ribosomes from mRNA, and following lysis, ribosomal complexes were separated on a 10–50% linear sucrose gradient via ultracentrifugation. 40S and 60S ribosomal subunits, along with monosome and polysomes were detected via their absorbance at 260 nm, with the area under the curve (AUC) of these peaks being used for quantification. The abundance of the 60S ribosomal subunit, monosomes and polysomes was only decreased in Proj-dom hTau expressing cells. n = 3 wells, one-way ANOVA, Tukey’s MCT