Extended Data Fig. 5: TDP-43 mRNA localization and local translation in motor neuron axons.

a) Relative mRNA transcript levels in axons versus cell-bodies (soma) of Tardbp, Elavl1, Stau1, and Actb. Data are shown as fold change (F.C) of normalized counts in axons over soma for each mRNA transcript. Raw data reanalysis of axonal and soma RNA-seq from primary motor neurons (Rotem et al., 2017). b) Representative images of smFISH for β-actin and TDP-43 mRNAs compared with no probe control in primary motor neuron cell bodies. Gray indicates RNA FISH, green indicates phalloidin/HB9:GFP, blue indicates nuclei (DAPI). Red dashed line marks proximal axon shown in magnified insets. Scale bar: 15 µm. Experiment performed in 3 neuronal cultures. c-d) Representative images (c) and quantitative analysis (d) of TDP-43 immunolabeling of distal axons in MFC 4 days after localized transfection with TDP-43 siRNA mix or with control siRNA. Magenta indicates TDP-43, green indicates NFH. Scale bar: 10 µm. Data are shown in violin density plots with markings of first, median and third quartiles of mean number of TDP-43 puncta in 100 µm axons. Two-tailed unpaired student’s t-test. ****p < 1 × 10⁻¹⁴. n = 94;87 axons from 3 independently grown co-cultures. e) Representative immunofluorescent images of TDP-43 puro-PLA in a growth cone at a premature NMJ, and in a mature NMJ in healthy co-cultures. Blue indicates NFH, gray indicates BTX, yellow indicates TDP-43 puro-PLA, white dashed line indicates skeletal muscle margins (in NMJ panel, the entire region is over a skeletal muscle) Scale bar: 5 µm. Experiment repeated in 3 neuromuscular co-cultures.