Fig 1.
Tyrosine hydroxylase staining of dopaminergic neurons in the nigrostriatal pathway.
A) Substantia nigra from an uninjected side of the brain. B) Substantia nigra from an AAV9 GFP injected side after 12 weeks after gene transfer. C) Substantia nigra from an AAV9 p62 injected side at 3.5 weeks after gene transfer. D) Substantia nigra from an AAV9 p62 injected side at 12 weeks after gene transfer. There is a noticeable hypertrophy of the neurons on the p62 side at both intervals yet an apparently progressive loss of cells between the two time points in the p62 group. E) Forebrain from a GFP animal at 12 weeks after gene transfer. F) Forebrain from a p62 animal at 12 weeks after gene transfer. There is a loss of striatal tyrosine hydroxylase on the side where AAV9 p62 was injected into the substantia nigra. Bar in D = 134 μm; same magnification in A-C. Bar in E = 536 μm; same magnification in F.
Fig 2.
Development of a turning bias over time in rats overexpressing p62 unilaterally in the substantia nigra.
Amphetamine-stimulated rotations occur when there is a large side-to-side difference in dopamine levels in the nigrostriatal pathway in rats, i.e., when there is a large loss of dopamine neurons on one side. A) In the p62 group, the behavioral phenotype of ipsilateral turning bias developed by 12 weeks (N = 7, P < 0.05, t-test), but not at earlier times in the p62 group. B) Turning bias did not manifest in a group of rats expressing the control protein GFP (N = 4).
Fig 3.
A, B) p62 immunoreactivity was induced on one side of the ventral midbrain. White rectangle in (A) shows the area of enlargement in (B). C, D) The overexpression induced numerous inclusions within the transduced neurons. E, F) Visualization of inclusions in p62 expressing cells using co-expression of a green and red double-tagged LC3 reporter protein. The p62 caused severe dissociation of the two tags. G) In a control neuron expressing the LC3 reporter without the p62, the two tags remain together and superimpose. The time point in these samples was 21–25 days. Bar in A = 536 μm; bar in B = 134 μm; bar in D = 14 μm; same magnification in C; bar in E = 14 μm; bar in F = 10 μm; same magnification in G.
Fig 4.
p62-, ubiquitin-, and ubiquilin-2-positive inclusions.
p62, ubiquitin, and ubiquilin-2 are involved in protein degradation pathways. A, B) p62-positive inclusions. C, D) ubiquitin- or ubiquilin-2 positive inclusions on adjacent sections (thin paraffin sections). The recombinant p62 expression led to deposition of endogenous rat ubiquitin and ubiquilin-2 into aggregates. The time point was 21 days after gene transfer. Bar in D = 20 μm; same magnification in A-C.
Fig 5.
Electron microscopy: filamentous inclusions and disruption of mitochondria induced by p62 gene transfer.
A, B) Consistent with the p62-positive inclusions observed by light microscopy, there were numerous inclusions (I) viewed within neurons on the AAV9 p62 injected side, but not found on the uninjected side. The filaments were densely packed in non-membrane bound inclusions and had a width of approximately 10 nm (right panel in B). B, C) In cells with the inclusions, the mitochondria (M) were grossly abnormal with the disruption of cristae structure and the formation of vacuoles within the mitochondria. Arrows point to small vesicles. D) A sample from the uninjected side of the brain shows normal mitochondria. Time point of 9 days after gene transfer in A, and 21 days after gene transfer in B-D. Bar in A = 1 μm; bar in B left = 0.2 μm, bar in B right = 50 nm; bar in C, D = 0.1 μm.
Fig 6.
p62 impairs mitochondrial function in transfected cells: decreased oxidative phosphorylation and increased glycolysis.
HEK 293T cells were transfected with a plasmid for p62 or control plasmids (GFP and empty). A) Basal oxygen consumption, i.e., oxidative phosphorylation, was decreased in the p62 group compared to the two control groups (ANOVA/Bonferroni, p < 0.001). There was also a small decrease in oxygen consumption in the GFP group relative to the empty group (ANOVA/Bonferroni, p < 0.001). B) Glycolysis and glycolytic reserve were increased in the p62 group compared to the two controls as evaluated by the extracellular acidification rate (ANOVA/Bonferroni, p < 0.001). C) Lactate, a by-product of glycolysis, was increased in the p62 group compared to the two controls (ANOVA/Bonferroni, p < 0.001). N is indicated in parentheses, asterisk indicates significance compared to the empty vector group.
Fig 7.
Specific, robust dissociation of the double-tagged LC3 autophagy reporter protein by p62.
A green and red fluorescent tagged LC3 was co-expressed with p62, or GFP. The columns from left to right are the green channel, the red channel, and the merger. A-C) With p62, the two tags strongly separate with both red-only and green-only labeling. The formation of the red only puncta (B) is consistent with the progression of the expressed LC3 to the autolysosome. In stark contrast, when LC3 is expressed by itself (D-F) or co-expressed with GFP (G-I), the two tags on the LC3 do not dissociate and superimpose in the merger (yellow). The time point was 22 days after gene transfer. Bar in A = 42 μm. Same magnification in all panels.