Extended Data Fig. 5: Quantitative proteomic and molecular analysis of tNeurons identify signatures of pathogenic mutations in the PSEN1 gene in AD patient neurons.
From: Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer’s disease
(a) IF images and quantification of proteostasis- and disease-associated protein markers in tNeurons from healthy young donors as well as AD patients with aged/sAD and fAD-PSEN1. White dash line represents the nuclear region (N). p62/SQSTM1: n = 62 (young), 66 (aged/sAD) and 49 (fAD-PSEN1) cells; ubiquitin: n = 46 (young), 66 (aged/sAD) and 50 (fAD-PSEN1) cells; total Aβ: n = 111 (young), 114 (aged/sAD) and 57 (fAD-PSEN1) cells; Aβ42: n = 103 (young), 93 (aged/sAD) and 68 (fAD-PSEN1) cells; pTau: n = 91 (young), 114 (aged/sAD) and 107 (fAD-PSEN1) cells; pTDP-43: n = 91 (young), 95 (aged/sAD) and 82 (fAD-PSEN1) cells; N-to-C ratio of TDP-43: n = 85 (young), 103 (aged/sAD) and 109 (fAD-PSEN1) cells. Scale bar: 20 μm. (b) Detection of endogenous Aβ42 in total cell lysates of tNeurons using sandwich ELISA assay. The values are revealed by a fold change relative to young tNeurons. n = 4 (young), 4 (aged/sAD) and 4 (fAD-PSEN1) independent replicates. (c) IF quantification of small heat shock protein HspB1 in young, aged/sAD and fAD-PSEN1 tNeurons. n = 80 (young), 134 (aged/sAD) and 136 (fAD-PSEN1) cells. (d) Top-ranked proteins (rows) changing with aged/sAD and fAD-PSEN1 (columns) based on log2-FC. (e) Network analysis of tNeuron proteins in the top-ranked pathways associated with aged/sAD and fAD-PSEN1. Comparison between aged/sAD (n = 6 individuals) and fAD-PSEN1 (n = 2 individuals) at PID 40. Coloured circles represent the enrichment of identified proteins revealing by log2FC: increase in red and decrease in blue. (f) GO analysis of the differentially expressed proteins across aged/sAD and fAD-PSEN1 tNeurons. Circle sizes reflect the number of proteins. (g) Lists of top-ranked proteins (rows) changing with fAD-PSEN1 and young (columns) based on log2FC. (h) Network analysis and differential expression of proteins detected in tNeurons from healthy young donors (n = 3 individuals) and patients with fAD-PSEN1 (n = 2 individuals) at PID 40. The top-ranked pathways for fAD-PSEN1 proteome are analysed using GO databases. Coloured circles represent the enrichment of identified proteins revealing by log2FC: increase in red and decrease in blue. (i) GO analysis of the differentially expressed proteins across fAD-PSEN1 and young tNeurons. Circle sizes reflect the number of proteins. In panels a, b and c, the boxes show median and 1st and 3rd quartile and the whiskers extending 1.5 times the interquartile range from the boxes. Data show box-and-whisker plots of two to three independent experiments and cells from independent HC and AD patients. Statistical analysis is performed using One-Way ANOVA followed by Bonferroni post-hoc analysis. *P < 0.05, **P < 0.01 and ***P < 0.001. Source numerical data are provided.
