Fig. 6. Interfering peptides attenuate both USP10 overexpression or Aβ-triggered Tau aggravation in primary cells.

a IF micrographs of FITC (green), MAP2(red) and DAPI (blue) in the primary neurons incubated with 25 μM FITC-tagged Tau307-326K or FITC-tagged Tau341-378K for 24 h. The scale bar represents 100 μm. b The primary cells viability was evaluated with CCK8 by pretreating cells with different concentrations of peptides; n = 6 independent experiments. c Upper panel was a schematic of experimental process. HEK293Tau cells overexpressing full-length USP10 with the vehicle were pretreated with 50 μM of Tau 307-326 K, Tau341-378K, or Tau 307-326 K + Tau341-378K for 48 h. WB showed the effects of interfering peptides on USP10-Tau interaction, Tau ubiquitination, and the level of WCL’s USP10, Tau5, ubi, pS199, and β-actin. d Graphs showing the normalized USP10 and Tau ubiquitination level following immunoprecipitation with Tau5 antibody, and the WCL Tau5 level. e–h Rat primary neurons were infected with AAV9-USP10 for 5 days and co-incubated with 50 μM of Tau 307-326 K + Tau341-378K or Tau 307-326Q + Tau341-378Q for 48 h. Cells lysates were collected and analyzed by WB. Interfering peptides abrogated the USP10-OE induced p-Tau upregulation, while non-ubiquitination mimics failed. i–l Rat primary neurons preincubated with 50 μM Tau 307-326 K + Tau341-378K or Tau 307-326Q + Tau341-378Q for 12 h, then supplemented with 2 μM Aβ oligomers for 24 h. CCK-8 was used to detect the cell viability of primary neurons as indicated (i). Lysates were analyzed by WB (j). Interfering peptides abrogate the Aβ₄₂ oligomers-induced upregulation of p-Tau at Ser199 (k) and Tau5 (l), while non-ubiquitination mimics could not. All data are presented as mean ± SEM, p-value significance is calculated from one-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.