Fig 1: Enhancement of UPR by guanabenz after SCI restores autophagy flux by modulating TFEB.A Representative immunoblots and densitometric box-and-whisker plots showing the total TFEB and TFE3 levels in CTRL, SCI-sal and SCI-Guana groups (N = 4 rats/group, m/f = 2/2; one-way ANOVA p < 0.0001 TFEB; one-way ANOVA p = 0.1368); **p < 0.01 vs CTRL; §p < 0.05 vs SCI-sal. B Representative immunoblots and densitometric box-and-whisker plots showing the TFEB cytosolic level (normalized to GAPDH) and nuclear level (normalized to H3) in CTRL, SCI-sal and SCI-Guana groups (N = 4 rats/group, m/f = 2/2; one-way ANOVA p = 0.0001); ***p < 0.001 vs CTRL; *p < 0.05 vs CTRL; §§§p < 0.001 vs SCI-sal. C Representative confocal images of TFEB immunofluorescence showing the subcellular compartimentalization of TFEB immunostaining in RN neurons of CTRL, SCI-sal and in SCI-Guana groups (scale bar = 20 µm; inset = 5 µm). D Box and whisker plots showing the percentage of neurons of RN with nuclear expression of TFEB in CTRL, SCI-sal and SCI-Guana (n = 5 sections/rat; N = 5 rats/group; m/f = 3/2; one-way ANOVA p < 0.0001). The process was made off-line and only neurons identified by a clear nuclear profile were included for analysis. Cells presenting nuclear TFEB expression were expressed as percentage of the total number of RN neurons. ***p < 0.001 vs CTRL; §§p < 0.001 vs SCI-sal. E The experimental protocol used for assessing the role of TFEB in restoring the SCI-induced effects. Adult rats underwent spinal cord injury (SCI) received compound C1 (10 mg/Kg i.p. once a day) or saline for 5 days. At day 5 animals were sacrificed and the red nucleus (RN) contralateral to the lesion side was extracted and processed for biochemical analyses or analyzed on fixed-brain sections. F Time course of functional recovery measured by Beam-walking test showing the score of SCI-sal, SCI-Guana and SCI-CompC1 rats (N = 6 mice/group, m/f = 4/2; Two-way RM ANOVA, (time × treatment: time p < 0.0001; treatment p = 0.0002; Interaction: time × treatment p < 0.0001) ***p < 0.001 SCI-sal vs SCI-Guana; §§p < 0.001 SCI-sal vs SCI-CompC1; §§§p < 0.001 SCI-sal vs SCI-CompC1. G Box-and-whisker plots showing the percentage of surviving neurons in RN of SCI-sal, SCI-Guana and SCI-CompC1 rats measured by stereological analysis (N = 5 rats/group, m/f = 3/2; one-way ANOVA p = 0.0003) ***p < 0.0001, **p < 0.001 vs SCI-sal. H Representative confocal images TFEB immunofluorescence from RN showing the compartimentalization of TFEB immunostaining in neurons of SCI-sal, SCI-Guana and SCI-CompC1 groups (scale bar = 20 µm; inset = 5 µm). I Box and whisker plots showing the percentage of neurons of RN with nuclear expression of TFEB in SCI-sal, SCI-Guana, and SCI-CompC1 (n = 5 sections/rat; N = 5 rats/group; m/f = 3/2; one-way ANOVA p < 0.0001) ***p < 0.001 vs SCI-sal. J Representative western blot and densitometric box-and-whisker plots from the SCI-sal, SCI-Guana and SCI-CompC1 groups showing the levels (expressed as % of CTRL) of LC3, p62, LAMP1, and LAMP2 normalized to GAPDH used as loading control (N = 5 rats per group; m/f = 3/2; one-way ANOVA p = 0.4094 LC3-II; p < 0.0001 p62; p < 0.0001 LAMP1; p < 0.001 LAMP2) ***p < 0.0001 vs SCI-sal. §§p < 0.01 vs SCI-Guana.