Fig 2: Immunoblotting of spinal cord in sALS and fALS. a Western blot of healthy donors, sALS and fALS spinal cords for neuronal marker TUJ1, TDP-43, ELAVL3, and GAPDH. b Quantifications of ELAVL3 and TDP-43 normalized to GAPDH in western blot

Fig 3: ELAVL3 downregulation in sALS motor neurons. a Volcano plots of RNAs from laser captured motor neurons comparing sALS and control showing significant ELAVL3 downregulation in two independent studies. Raw expression values for ELAVL3 (b) and TARDBP (c). Validation by qPCR of bulk tissue (left) and by ALS subcategories (right) of ELAVL3 (d) and TARDBP (e). Immunofluorescence of motor neurons of human spinal cords in control (f) and sALS (g). h Pie chart of frequency of ELAVL3 nuclear location and depletion. i Expression of other ELAVLs family members. Scale bar 20 µm

Fig 4: ELAVL3 abnormalities are upstream of TDP-43. Double-labeled immunofluorescence of ELAVL3 and TDP-43 in spinal motor neurons. a Control motor neuron showing ELAVL3 is present in the nucleus and diffuse in the cytoplasm and TDP-43 is nuclear. Motor neurons from sALS (b), C9orf72 (c), and SOD1 (d) show ELAVL3 nuclear depletion is observed when TDP-43 is nuclear (b–d). e Statistical comparison of ELAVL3 and TDP-43 abnormalities in the same neurons. f Statistical analysis of ELAVL3 abnormalities in ALS overall and in sorted groups. g Statistical analysis of TDP-43 abnormalities in ALS overall and in sorted groups. h Comparison and variance of ELAVL3 and TDP-43 abnormalities in each nervous system sorted by groups. ELAVL3 green, TDP-43 red, DAPI blue, scale bar 20 µm

Fig 5: Immunofluorescence of spinal motor neurons in fALS. a Control motor neurons show nuclear and cytoplasmic ELAVL3. b, c SALS with intermediate length expansions in ATXN2 showing nuclear depletion and cytoplasmic inclusions. d, f C9orf72 ALS motor neurons showing nuclear depletion and occasional fibrillary-like (e) and dot-like inclusions (f) are seen. g–i SOD1 ALS motor neurons showing nuclear depletion and dot-like inclusion (i). j Pie chart of frequency of ELAVL3 nuclear location and depletion in fALS. ELAVL3 green, DAPI blue, scale bar 20 µm