p62 Pathology Model in the Rat Substantia Nigra with Filamentous Inclusions and Progressive Neurodegeneration

<div><p>One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.</p></div

Tyrosine hydroxylase staining of dopaminergic neurons in the nigrostriatal pathway.

<p>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.</p

TDP-43 Filament Assembly after CKII Phosphorylation.

<p>A) Unphosphorylated rTDP-43 was prepared for electron microscopy immediately after high-speed clarification and no large aggregates or filamentous structures were observed. B) After 4 hours of incubation at 37°C without phosphorylation, no filamentous structures were not observed C) Similarly, soluble rTDP-43 samples phosphorylated with CKI for 4 hours did not form filaments, but D) the rTDP-43 incubated with CKII for 4 hours did polymerize. E) These filaments formed rapidly after CKII phosphorylation and could be detected at T = 5 minutes, and F) they could be immuno-gold labeled (arrows) with TDP-43 primary antibodies specific for the carboxy-terminus of TDP-43 (10 nm gold). G) The most common filaments observed after CKII phosphorylation of soluble rTDP-43 resemble randomly oriented FTLD 10–12 nm filaments (double arrows) shown above without a granular coating though numerous pore-like flowerette structures are also observed (arrowheads). H) Additionally smaller protofibrils (single arrows) and wider filaments (triple arrows) are occasionally found. The scales bar is in nanometers.</p

HSPs Inhibits CKII Mediated TDP-43 Filament Assembly.

<p>Hsp90 can inhibit the polymerization of tau, and TDP-43 is a known substrate for Hsp90. To examine the effects of Hsp90 on CKII mediated rTDP-43 filament assembly, 2.5 µM TDP-43 was polymerized for 60 minutes with CKII in the presence of 0.0–2.5 µM Hsp90. A) The number of TDP-43 filaments was dramatically reduced with increasing Hsp90, and this led to B) a substantial decrease in the total polymer mass even as C) the average length of the TDP-43 filaments was only slightly lowered. These data also confirmed Image Pro Plus (darker gray) provided reliable counting compared to the NIH Image J (lighter gray) allowing for larger fields to be counted. D) To confirm that these effects were not species dependent, GST-tagged recombinant human Hsp90 at 1.25 µM was incubated with 2.5 µM TDP as before and again the filament number and total filament length were reduced. Here the # indicates p<0.10 and the * indicates p<0.01 significance. Additionally, Hsp70 has been found to constitutively bind TDP-43 and is released after heat shock leading to TDP-43 aggregation. Hsp70 effects on CKII mediated rTDP-43 assembly support this observation as both the number of TDP-43 filaments (E) and the total filament mass (F) were decreased with increasing Hsp70.</p

TDP-43 Polymerization into Structures Resembling FTLD Filaments.

<p>A) Numerous pore-like structures are detected before and after CKII phosphorylation. These putative pores have an average outer diameter of 13.5 +/− 1.2 nm and an inner opening of 6.2 +/− 0.8 nm. These were observed in all the reactions. B) Single filament protofibrils are less commonly observed, and they have an average diameter of 4.2 +/− 0.6 nm. E) The mostly commonly observed filaments are 9.9 +/− 0.9 nm and appear most similar to those detected in FTLD-TDP patients. These appear to be composed of two smaller tracks resembling the protofilaments, but the vast majority of the structures have clean ends indicating that are not formed by assembly of the smaller preformed protofibrils. F) Similarly, the larger 14.5 +/− 2.0 triple filament structures are rarely observed with splayed ends displaying smaller two track filaments or protofibrils. G) The double track 9.9 +/− 0.9 nm structures accounted for nearly 90% of the filaments detected in images captured for filaments characterization. H) The near integral multiples of the widths of these filaments indicate they may share a common structural protofilament even if the larger diameter filaments do not assemble from the smaller fibrils.</p

p62 inclusions.

<p>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.</p

Functional Validation of Soluble rTDP-43.

<p>To verify that the rTDP-43 was functional, its ability to specifically bind repeated (TG)12 single stranded DNA compared to (AC)12 DNA was examined. A) Nitrocellulose was spotted with reactions of increasing concentrations of rTDP43 and fixed DNA probe to bind proteins as demonstrated by the Ponceau S staining. This nitrocellulose was placed on top of a DNA binding Hy-Bond N+ membrane which trapped the free biotinylated (TG)12 or (AC)12. B) The nitrocellulose retained the (TG)12 sequences indicating they were binding the rTDP-43 while C) biotinylated (AC)12 probes passed through to the Hy-Bond N+ for each amount of rTDP-43 spotted onto the nitrocellulose and the (TG)12 decreased in a dose dependent manner. Phosphorylation reactions demonstrate that the soluble rTDP-43 is a functional substrate for two known kinases. D) Phosphorylation with CKI leads to retardation of the monomeric rTDP-43 mobility in SDS-PAGE gels (single arrow) with little oligomer formation (double arrow) while CKII does not shift the apparent molecular weight of the monomer, but it leads to increased oligomer formation when total TDP-43 is monitored. E) Analysis of the phosphorylation by these kinases confirms both label the S409/410 sites, and CKI is more efficient than CKII.</p

p62 impairs mitochondrial function in transfected cells: decreased oxidative phosphorylation and increased glycolysis.

<p>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.</p

Development of a turning bias over time in rats overexpressing p62 unilaterally in the substantia nigra.

<p>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).</p

Specific, robust dissociation of the double-tagged LC3 autophagy reporter protein by p62.

<p>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.</p