Fig. 2: Splicing is dysregulated in aged neurons due to mislocalization of spliceosome components.

a, Confocal fluorescence images of transdifferentiated (top row, blue) and iPSC-derived (middle row, green) neurons stained for the indicated spliceosome proteins in magenta with DAPI in blue; the average fraction of nuclear protein was calculated for each cell type (n = 3 replicates) and is plotted (bottom row) where the centerline denotes the median, the box bounds encompass the 25th–75th percentile values and the error bars denote the range of data values. Statistics were calculated using a two-tailed Welch’s t test. Scale bar = 10 μm. b, Volcano plot of AP-MS of TDP-43 (n = 2 replicates) in the indicated neurons (n = 2 replicates). Detected proteins in the indicated KEGG terms are highlighted. The P value was calculated using a two-tailed Welch’s t test. c, Airyscan fluorescence images of transdifferentiated neurons treated with sodium arsenite for 1 h and stained for G3BP1 and TDP-43. White scale bar = 10 μm; yellow scale bar = 2 μm. d, Fraction of TDP-43-bound RNA windows detected by eCLIP (n = 2 replicates) within each of the indicated transcript elements for transdifferentiated and iPSC-derived neurons. e, The top RNA-binding motif for TDP-43 eCLIP in the indicated cell types/conditions (n = 2 replicates). f, Exon inclusion odds ratio for transcripts detected by RNA-seq (n = 3 replicates) at the indicated loci around each exon with a TDP-43 binding site in iPSC-derived neurons, where *P < 0.05 calculated by a likelihood-ratio test. g, Same as f, but for exon exclusion. h, TDP-43 binding and RNA-seq intensity tracks for the AGRN locus in the indicated cell types. The orange highlighted region is depicted in the lower panel. i, Schematic representation of the mouse brain used for IF and eCLIP. j, Confocal fluorescence images of TDP-43 (magenta) and DAPI (blue) in the motor cortex of 1.5-, 6- and 24-month-old mice; the yellow dashed box indicates the inset region. White scale bar = 10 μm; yellow scale bar = 2 μm. k, Same as d, but for the indicated mouse ages (n = 2 mice per cohort). l, Same as e, but for the 1.5-month-old mouse brain. IF, immunofluorescence.