Fig 2: Pathological inclusions identified in similar sections of the same cases examined with pTDP43 and iTDP43. NCIs were observed in the cortex on sections with pTDP43 (A), but not in the same region of sections with iTDP43 (B) in Patient #8. Moderate NCIs were observed in the dentate gyrus on sections with pTDP43 (C) whereas only mild NCIs were observed in the same region on sections with iTDP43 (D, arrows) in Patient #10. Compared to the NCIs observed in the upper cortices in Patient #6 with pTDP43 (E, black arrows), iTDP43 sections demonstrated less NCIs (G, black arrows) and a loss of TDP43 nuclear staining in surrounding neurons (G, white arrows). In Patient #1, NCIs were identified in the dentate gyrus in a case examined with pTDP43 (F), but not in sections with iTDP43 (H) although this antibody demonstrated mild loss of nuclear staining in surrounding neurons (white arrows). NIIs were observed in the cortices of four cases (Patients #1, 3, 4, 5) with pTDP43 (I) but were identified only in one case (Patient #1) with iTDP43 (J). Scale in A (equivalent for B,C,D,E,F,I and J), Scale in G (equivalent for H).

Fig 3: Effects of cell cycle gene mutations on D42-Gal4/UAS-TDP-43 fly lifespan.(A) Survival curves of D42-Gal4/UAS-TDP-43 flies in string and greatwall mutant backgrounds. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 26, N = 118; D42-Gal4/UAS-TDP-43, string EY12388 = 27, N = 106, p<0.0001; D42-Gal4/UAS-TDP-43, greatwall EP515 = 23, N = 67, p<0.0001. (B) Effects of the string EY12388 allele in males. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 23.5, N = 60; D42-Gal4/UAS-TDP-43, string EY12388 = 23, N = 52, p = 0.1831. (C) Effects of the string EY12388 allele in females. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 28, N = 58; D42-Gal4/UAS-TDP-43, string EY12388 = 32, N = 54, p<0.0001. (D) Survival curves of D42-Gal4 control flies with the string EY12388 mutant allele, divided by gender. Median survival (days) of males: D42-Gal4/+ = 58, N = 49; D42-Gal4/string EY12388 = 53.5, N = 60, p = 0.08. Median survival (days) of females: D42-Gal4/+ = 63, N = 53; D42-Gal4/string EY12388 = 60, N = 60, p = 0.43.

Fig 4: Aging negatively impacts SG assembly in neurons in vitro. (A) Representative micrographs of primary motor neurons aged in culture for 28 and 56 days and then subjected to 0.5 mM SA. SGs are marked with oligo(dT) (red) and neuronal processes marked with phosphorylated neurofilaments (SMI32, blue). Scale bar, 10 µm. Quantification of the number of SGs per cell and individual SG size in (B,C) motor neurons cultured for 28DIV and 56DIV (n = 3) and (D,E) cortical neurons cultured for 7DIV and 21DIV (n = 3). (B,D) Number of SGs per cell. (C,E) Size of individual SGs. Number and size were measured at two time points with ImageJ. (F) Whole cell lysates of cortical neurons at 7DIV and 21DIV immunoblotted for TDP-43. Bands of interest were cropped from unmodified images, quantified via densitometry and normalized to Actin. (Uncropped blots are in Supplemental Material.) A representative blot and the mean ± SEM of densitometric quantification of three independent experiments are shown. Student t test *p < 0.05.

Fig 5: TDP-43 is required for SG assembly in astrocytes. (A) Representative images of primary astrocytes transfected with TDP-43 siRNA (siTDP-43 #2) and exposed to SA with cytoplasmic SGs marked with an oligo(dT) probe. Scale bar, 10 µm. (B) Number of SGs per cell and (C) size of individual SGs measured at the indicated time points post-SA exposure (n = 3, N = 10). Data is expressed as the mean ± SEM; Student t test *p < 0.05, **p < 0.01, ns: not significant.