Effect of AAV9-synuclein treatment on the LC.

<p>Quantification of TH immunohistochemistry in the locus coeruleus using unbiased stereological techniques. Graph shows that 4 months following α-synuclein gene transfer there is a significant loss of TH positive cells in the LC (2-way ANOVA did not show a significant interaction, but did reveal main effects of diet and treatment, and bonferonni post-hoc revealed a difference between the α-synuclein control and diet treated groups, p<0.01). Treatment with spirulina was able to prevent the loss of TH positive cells in the locus coeruleus. Asterisk denotes significance (**p<0.01 vs control GFP; *p<0.05 vs control α-synuclein).</p

Effect of spirulina on NeuN immunoreactive cells in the SNpc.

<p>NeuN positive cells in the SNpc after 1 or 4 months of α-synuclein expression. At 1 month, there was a decrease in NeuN positive cells in the SN, mirroring the loss of TH positive cells in Fig. 1. This confirms cell loss rather than loss of TH expression. The effect was similar at 4 months of expression (B). There was neuroprotection in the groups that received a diet enhanced with spirulina at both intervals. Two-way ANOVA yielded a significant interaction of diet and vector treatment at both time points [1 month F = 6.931, df = 1; 4 months F = 8.899 df = 1]. (*p<0.05; **p<0.01) after Bonferonni's post-hoc.</p

Effects of spirulina on CX3CR1.

<p>Spirulina diet increased expression of CX3CR1; inset of Western blot in upper right. When the data are analyzed across groups there is a significant increase in expression of CX3CR1 in the spirulina treatment groups. Asterisk denotes significance (p<0.05; Bonferroni post-hoc). Two-way ANOVA shows a main effect of diet (F = 19.90; df = 1) and no interaction or main effect of vector treatment.</p

Gene transfer efficiency is not affected by spirulina.

<p>Quantification of GFP positive cells in the SN. Stereological estimates of the number of GFP positive cells in the SN one month after gene transfer. There was no significant effect of the spirulina diet on numbers of GFP transduced cells (Student’s two-talied t-test).</p

Effect of spirulina on TH immunoreactive cells in the SNpc.

<p>TH positive cells in the SNpc after 1 or 4 months of α-synuclein expression. Cells were counted using unbiased stereological counting techniques. (A) One month after α-synuclein gene transfer, there was a significant decrease in TH positive cells as compared to GFP control (N = 12–18/group). The spirulina diet group lesioned with α-synuclein (N = 12) had greater numbers of TH positive cells compared to the control diet group lesioned with α-synuclein (N = 18). There was a diet by vector group interaction in the two way ANOVA analysis [F = 5.569, p<0.01]. (B) Results at four months were similar. There was a similar loss of TH positive cells and protective effect of spirulina. Two-way ANOVA yielded a main effect of diet (F = 81.3), and a main effect of vector group (F = 45.5; p<0.01), although without a significant interaction. Bonferonni post-hoc tests comparing NIH 31 GFP (N = 10) vs NIH 31 synuclein (N = 8) and NIH31 synuclein vs spirulina synuclein (N = 8) groups were significant). Asterisk denotes significance (*P<0.05; **p<0.01).</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

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

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

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

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