Fig. 7: UXT prevents SOD1(A4V)-induced progressive loss of motor ability in Xenopus.

a SOD1 and/or UXT in vitro-transcribed capped RNA were expressed in the central nervous system by targeted microinjection. Motor function was assessed by touch-induced swimming responses at stages 28 and 4. b Each tadpole was touched three times on its tail. Each trace in a circle indicates swimming trajectory of the same tadpole within 3 s (time after the touch is color-coded). The best response of three trials was used to classify the swimming ability. c Injecting SOD1 mutant genes did not result in distinguishable morphological changes. Scale bar, 2 mm. d Swimming responses of tadpoles injected with SOD1 mutant and/or UXT (wild type or L50A/L59A mutant) in vitro-transcribed capped RNA are shown. The behavior of tadpoles was recorded in three independent experiments. The Y-axis indicates the percentage of tadpoles showing a normal swimming response, and the X-axis indicates developmental stages. e Quantification of d at stage 43 with the number of tadpoles in each category is shown. Cyan, yellow, and orange bars represent normal, reduced, and no swimming responses, respectively. The Y-axis indicates the percentage of tadpoles. Ctl control; ***p < 0.001 (two-sided Fisher’s exact test). f, g Effect of UXT expression on SOD1(G93A)-GFP-induced motor deficit was tested as in d and e. +p = 0.0968; **p < 0.01 (two-sided Fisher’s exact test). h, i Effect of a p62 translation-blocking antisense morpholino (MO) on swimming responses was analyzed as in d and e. j Quantification of i at stage 43 as in e. ***p < 0.001 (two-sided Fisher’s exact tests). ns not significant. k SOD1(A4V)-GFP was visualized by immunohistochemistry in cell bodies of lower motor neurons localized in the ventral horn of the spinal cord. Red and green arrows indicate SOD1(A4V)-GFP aggregation and SOD1(A4V)-GFP, respectively. Co-injecting UXT completely prevents SOD1(A4V)-GFP aggregation indicated by white arrows. In cells co-injected with UXT(L50A/L59A (L/A)), the SOD1(A4V)-GFP aggregation is often observed as indicated by yellow arrows. Numbers in parenthesis indicate the number of cells with aggregation/the number of transfected cells analyzed. Scale bar, 10 µm.