Fig. 9

Therapeutic enhancement of lysosomal proteostasis in NM-producing rats. a Ipsilateral SNpc sections from rats co-injected with AAV-hTyr/flagged-AAV-TFEB immunostained with Flag (blue, left) or hTyr (blue, right) at 5 m post-AAV. NM, brown. Arrowheads, nuclear Flag/TFEB. Scale bar, 25 μm. b Lamp1 immunolabeling (blue) in ipsilateral SNpc of AAV-TFEB-injected rats (12 m post-AAV). *p < 0.05, compared to contralateral (non-injected) SNpc (Mann–Whitney rank sum test). Scale bar, 12.5 μm. c NM-laden neurons with p62-positive MB (left) and PB/LB-like inclusions (right) in ipsilateral SNpc from AAV-hTyr- and AAV-hTyr/TFEB-injected rats (2 m post-AAV). *p < 0.05, compared to AAV-hTyr-injected animals (left, Mann–Whitney rank sum test; right, two-way ANOVA, Student–Newman–Keuls post-hoc test). d Intracellular NM density in ipsilateral SNpc TH-positive neurons from rats co-injected with AAV-TFEB/AAV-hTyr (12 m post-AAV). White arrowhead, Flag/TFEB-positive nucleus; black arrowhead, Flag/TFEB-negative nucleus. Scale bar, 12.5 μm. *p < 0.05, compared to Flag/TFEB-negative neurons (Mann–Whitney rank sum test). e SNpc TH-positive neurons at 12 m post-AAV/vehicle injections. f Striatal TH-positive fibers at 12 m post-AAV/vehicle injections. In e, f, *p < 0.05, compared to respective contralateral side; #p < 0.05, compared to ipsilateral vehicle- and AAV-TFEB-injected animals; §p < 0.05, compared to ipsilateral AAV-hTyr-injected animals (two-way ANOVA; Student–Newman–Keuls post-hoc test). g TH downregulation within NM-laden neurons (12 m post-AAV). *p < 0.05, compared to AAV-hTyr-injected animals (two-tailed t-test). h Total SNpc DA neurons at 12 m post-AAV/vehicle injections. *p < 0.05, compared to respective contralateral side; #p < 0.05, compared to ipsilateral vehicle- and AAV-TFEB-injected animals (two-way ANOVA; Student–Newman–Keuls post-hoc test). i Contralateral forepaw use in AAV-hTyr- and AAV-hTyr/TFEB-injected rats (12 m post-AAV). *p < 0.05, compared to AAV-hTyr (only)-injected animals (two-tailed t-test). Dashed line indicates average contralateral forepaw use in control-injected rats. In all panels, values are mean ± SEM. In b, n = 138 neurons from n = 4 non-injected rats and n = 203 neurons from n = 5 AAV-TFEB-injected rats. In c, n = 6 (hTyr), n = 8 (hTyr + TFEB) rats. In d, n = 132 nuclear flag- neurons and n = 173 nuclear flag + neurons from n = 4 AAV-hTyr + TFEB-injected rats. In e and h, n = 7 (vehicle), n = 4 (TFEB), n = 5 (hTyr), n = 4 (hTyr + TFEB) rats. In f, n = 7 (vehicle), n = 6 (TFEB), n = 4 (hTyr), n = 3 (hTyr + TFEB) rats. In g, n = 4 animals per group. In i, n = 12 (control), n = 3 (hTyr), n = 4 (hTyr + TFEB) rats. BF bright-field. Photomicrographs correspond to 5-μm-thick sections