Phosphorylation of TDP-43 occurred sequentially from N- to C-terminal sites and showed a subtype-specific timeline A) Analysis of neoaggregation in HEK cells (similar method as ) using Imaris.Figure 3A) Analysis of neoaggregation in HEK cells (similar method as Figure 1 and Appendix Fig S3) using Imaris. Images represent field of view for FTLD-TDP-A seeded cells (left) and FTLD-TDP-C seeded cells (right) and labelled with DAPI (transparent blue), neoaggregates phosphorylated at S403/404 (NeoAggpS404, orange) and neoaggregates phosphorylated at S409/410 (NeoAggpS409, light purple). Bottom panel shows neoaggregates bearing double phosphorylation S403/404 and S409/410 (NeoAggpS404-409, yellow). All images are presented at the same scale, 5 μm as indicated in FTLD-TDP-C panel. B) Bar graphs representing the number of NeoAggpS403 only (bars filled in light orange), NeoAggpS409 only (bars filled in light purple) and double phosphorylated NeoAggpS403-409 (unfilled bars) for each condition. Individual patients are labelled in different colored dots (n=3 biological replicates for each condition, graph bar showing mean ± SEM). Two-way ANOVA repeated measures, with post-hoc Fisher's LSD test, F(24.54)=3.799, p<0.0001, Day 4: non-neurodegenerative control pS403/404 vs pS409/410, p<0.0001, non-neurodegenerative control pS403/404 vs bi-phosphorylated, p<0.0001, FTLD-TDP-A pS403/404 vs pS409/410, p<0.0001, FTLD-TDP-A pS403/404 vs bi-phosphorylated, p<0.0001 FTLD-TDP-C pS403/404 vs pS409/410, p<0.0001, FTLD-TDP-C pS403/404 vs bi-phosphorylated, p<0.0001. Day 5: non-neurodegenerative control pS403/404 vs bi-phosphorylated, p=0.0219, FTLD-TDP-A pS403/404 vs double, p=0.0650, FTLD-TDP-C pS403/404 vs double, p=0.0157. Day 6: Control bi-phosphorylated vs FTLD-TDP-A bi-phosphorylated, p=0.5096, control bi-phosphorylated vs FTLD-TDP-C, p=0.1010. Non-neurodegenerative control pS403/404: day 3 vs 4, p=0.0559, day 3 vs 6, p=0.0369. FTLD-TDP-A pS404/404, day 3 vs 4, p=0.0075, day 4 vs 5, p=0.0714. FTLD-TDP-C pS403-404, day3 vs 4, P=0.0759, day 4 vs 6, 9=0.0305. FTLD-TDP-A bi-phosphorylated: day 3 vs 5, P=0.0577, day 3 vs 6, p=0.0609. FTLD-TDP-C bi-phosphorylatede: day 3 vs 4, p=0.0522. C) Bar graph representing the percentage of NeoAggpS403/404 bearing double phosphorylation (left side, bars filled in light orange) and percentage of NeoAggpS409 bearing double phosphorylation (right side, bars filled in light purple). Each individual patient is labelled in different colored dots. N=3 biological replicates per condition, graph bar showing mean ± SEM. Two-way ANOVA repeated measures, with post-hoc Fisher's LSD test, pS403/404 also double positive: F(3,6)=6.567, p=0.0253: FTLD-TDP-A: Day 3 vs 5, p=0.0491, day 4 vs 6, p=0.0321, FTLD-TDP-C: ay 4 vs 6, p=0.037. pS409/419 also double positive: F(3,6)=1.246, p=0.3731, FTLD-TDP-C: Day 4 vs 5, p=0.0144. D) Violin graph representing the area of NeoAgg404 (filled in light orange) and NeoAgg409 (filled in light purple) at day 3 and day 6 post seeding with FTLD-TDP-A and C seeds. Full bar represent median values, dashed lines represent quartiles. Two-way ANOVA with post hoc Fisher's LSD test, day 3: FTLD-TDP-A vs FTLD-TDP-C NeoAggpS404 p<0.0001, FTLD-TDP-A vs FTLD-TDP-C NeoAggpS409 p=0.0342, FTLD-TDP-A NeoAggpS409 vs FTLD-TDP-C NeoAggpS404 p=0.0047, FTLD-TDP-C NeoAggpS404 vs NeoAggpS409 p<0.0001; Day 6: FTLD-TDP-A vs FTLD-TDP-C NeoAggpS404 p<0.0001, FTLD-TDP-A NeoAggpS409 vs FTLD-TDP-C NeoAggpS404 p<0.0001, FTLD-TDP-A NeoAggpS404 vs FTLD-TDP-C NeoAggpS409 p<0.0001, FTLD-TDP-A NeoAggpS404 vs NeoAggpS409 p<0.0001, FTLD-TDP-C NeoAggpS404 vs NeoAggpS409 p<0.0001. E) Overexpression of GFP-TDP-43 variants with different mutations that either block (CC and AA mutation, red labels) or mimic phosphorylation (DD and EE, green label) at S403/404 or S409/410 in motor neuron-like NSC-34 cells. Western blot showed a specific signal for total TDP-43 (top panel), TDP-43pS403/404 (middle panel) and TDP-43p409/410 (lower panel) and load control SOD1 (bottom panel). F) Schematic of the extreme C-terminal serine phosphorylation found in pathological TDP-43.