Fig. 4

Nonselective degeneration and reinnervation does not alter the susceptibility of the TA muscle to axonal dieback in the presence of pathological hTDP-43ΔNLS. a Schematic representation of a unilateral sciatic nerve crush. DRG dorsal root ganglion, DN distal nerve. b Representative fluorescence images of the TA muscle of nTg mice before and after nerve crush. Immunostaining for SV2 marks nerve endings (red), while labelling with BTX-488 marks motor endplates (green). Scale bar: 100 µm. c Representative image of TA-innervating MNs in the lumbar spinal cord of nTg mice backfilled with CTB-594 on the crushed side (left, red fluorescence) and with CTB-488 on the contralateral side (right, green fluorescence). Yellow arrows mark MNs reinnervating (crushed side) or innervating (non-crushed) motor pools in the TA. Scale bar: 100 µm. d Representative immunofluorescence staining for Mmp9, a marker of fast-fatigable (FF) MNs, in TA-reinnervating MNs (i.e., CTB-594 back-filled; red) on the ipsilateral side of the lumbar spinal cord of nTg mice 10 wks. after unilateral sciatic nerve crush. Scale bar: 100 µm. e Quantification of the proportion of reinnervated FF type MNs (Mmp9-positive) among all CTB-488/CTB-594-positive TA MNs on the ipsilateral and contralateral sides of nTg mice subjected to unilateral sciatic nerve crush. Three animals were assayed, with 30–40 MNs scored per animal. Paired t test, n. s., p = 0.2049. f The experimental approach used to evaluate whether recovery after unilateral sciatic nerve crush in rNLS8 mice ameliorates denervation during subsequent hTDP-43ΔNLS expression. Mice were subjected to unilateral sciatic nerve crush as illustrated in (a), then allowed to recover on Dox for 8–10 wks. Bilateral CMAP and TreadScan analysis were used to confirm full recovery, after which hTDP-43ΔNLS expression was induced by withdrawing Dox for 6 wks. MNs were counted and NMJs analyzed bilaterally at endpoint. g Quantitation of the number of MNs labelled with VaChT per ventral horn in rNLS8 mice analyzed at endpoint in the experiment outlined in (f). Three mice were assayed, with 10 spinal cord sections scored per animal. Paired t test, n. s., p = 0.054 h Quantitation of the proportion of intact NMJs (scored as coincident SV2-positive nerve endings and BTX-488/BTX-594-labeled motor endplates) relative to all BTX-488/BTX-594-labeled motor endplates in the TA muscles on the ipsilateral and contralateral sides of rNLS8 mice at the endpoint of the experiment outlined in (f). Five mice were assayed, with 600–1000 NMJs scored per animal. Paired t test, n. s., p = 0.391