Fibril organization and cellular microenvironment differentiate FTLD-TDP-A from FTLD-TDP-C aggregates in patients A) 2D TEM (first column) shows an example of the overall morphology of neuronal cytoplasmic inclusions from FTLD-TDP-A localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig.Figure 6A) 2D TEM (first column) shows an example of the overall morphology of neuronal cytoplasmic inclusions from FTLD-TDP-A localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV4D). 2D projections of the central slices of reconstructed 3D tomograms (middle column) taken at higher magnification within the aggregate show a dense accumulation of filaments seemingly enclosed by a boundary membrane (white arrowheads). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored, dots last column). B) STED imaging of nuclear structures (left column) and neuronal cytoplasmic inclusions found in a postmortem tissue of a patient with FTLD-TDP-A. LaminB1 (green) was used as a nuclear membrane marker along with TDP-43 (cyan) and TDP-43pS403/404 (magenta). C) Confocal imaging of dystrophic neurites found in a postmortem tissue of a patient with FTLD-TDP-C. Dystrophic neurites were identified using TDP-43 (cyan), and TDP-43pS403/404 (magenta). Neurite markers were used to visualize neuronal projections (MAP2 for dendrites, PNF for axons; yellow and orange, respectively). D) 2D TEM (first row) of dystrophic neurites from FTLD-TDP-C, localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV5D). 2D projections of the central slices of reconstructed 3D tomograms (middle row) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles (white asterisk). A similar arrangement of sparse filaments and vesicles is seen in a cross-section of a dystrophic neurite from FTLD-TDP-C (cross-section, also shown in Fig. EV5G). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored dots, last row). E) Representative 3D volumes resulting from the average of sampled filaments within tomograms are depicted for FTLD-TDP-A (FTLD-A), FTLD-TDP-C (FTLD-C) and cytoskeletal filaments (CF). No significant differences in morphology between the two FTLD subtypes were detected from the 3D averages, compared to the CFs which are visibly thinner and display a distinctive twist (dotted line). F) Average width of fibrils calculated from every particle cropped along the fibrils within each collected tomogram. The data points represent the average width calculated from the total number of particles extracted from multiple tomograms for the aggregates localized for FTLD-TDP-A (n=5 biological replicates) and FTLD-TDP-C, CF (n=6 biological replicates). The total number of particles extracted for each aggregate are shown in Appendix table S3. Error bars represent +/- standard deviation. The total average width calculated from all fibrils from FTLD-TDP-A is 7.8 nm (+/- 1.1 nm), 7.5 nm (+/- 1.3 nm) for FTLD-TDP-C and 5.2 nm (+/- 1.7 nm) for CF. G) The average distance between fibrils within each aggregate for FTLD-TDP-A and FTLD-TDP-C is shown. Data points represent the total combined average for all tomograms collected within a single aggregate (n=1 biological replicate). Error bars represent +/- the maximum standard deviation for each aggregate. P-value calculated from an unpaired t-test between averages of FTLD-TDP-A and FTLD-TDP-C.