1. Age- and α-Synuclein-Dependent Degeneration of Dopamine and Noradrenaline Neurons in the Annual Killifish Nothobranchius furzeri 
Hideaki Matsui, Naoya Kenmochi, Kazuhiko Namikawa 
Cell Reports 
Volume 26, Issue 7, Pages e6 (February 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 1727-1733.e6DOI: (10.1016/j.celrep.2019.01.015)
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3. Figure 1 Degeneration of TH+ Neurons in Nothobranchius furzeri
(A) In N. furzeri, the number of dopaminergic neurons in the posterior tuberculum (DC2 and DC4) and of noradrenergic neurons in the locus ceruleus significantly decreased at 3 months. These neurons further decreased at 5 months (ANOVA: dopaminergic [DA]: F = , p < ; noradrenergic [NE]: F = , p < 0.0001). A degeneration of the dopaminergic or noradrenergic neurons is not observed even at 24 months in zebrafish (Danio rerio) or medaka (Oryzias latipes). n = 12 fish per group. The bar represents SE.
(B) (B1) and (B3) are the representative images of the noradrenergic neurons in the locus ceruleus of N. furzeri at 1 and 5 months, respectively. (B2) and (B4) are the magnified images of the red square regions shown in (B1) and (B3), respectively. (B5–B8) (B5) and (B7) are the representative images of the dopaminergic neurons in the posterior tuberculum at 1 and 5 months, respectively. (B6) and (B8) are the magnified images of the red square regions shown in (B5) and (B7), respectively. Each image is a representative optical section of a thick vibratome specimen.
(C) Quantification of TH in the brain of N. furzeri at 1, 3, and 5 months (ANOVA: F = , p = ). n = 10 fish per group. The bar represents SE. Quantification of th mRNA in the brain of N. furzeri at 1 and 5 months. n = 4 fish per group. The bar represents SE.
(D) Serotonin-positive neurons in the median raphe. n = 12 fish per group. The bar represents SE.
See also Figures S1–S3 and Table S1.
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4. Figure 2 α-Synuclein Pathology in Nothobranchius furzeri
(A) Ubiquitin-positive (A1) and α-synuclein-positive (A2) inclusion bodies are observed. (A3–A6) Ubiquitin-positive aggregates were restricted only to the medulla through the spinal cord at 1 month (A3) and progressed throughout the brain at the later stages (A5 and A6). (A4) shows a magnified image of the red square region shown in (A3). (A7–A9) Ubiquitin-positive aggregates are plotted as black points next to the original images. (A10–A13) α-Synuclein-positive aggregates were restricted only to the medulla through the spinal cord at 1 month (A10) and progressed throughout the brain at the later stages (A12 and A13). (A11) is a magnified image of the red square region shown in (A10). (A14–A16) α-Synuclein-positive aggregates are plotted as black points next to the original images.
(B) Quantitative analysis of α-synuclein signals in the hindbrain (Hind), midbrain + diencephalon (Mid + Di), and telencephalon (Tel). α-Synuclein signals were less in the rostral site of the brain at 1 month (ANOVA: F = , p < ) and gradually became abundant in the entire brain at 5 months (ANOVA: F = , p < ). n = 5 fish per group. The bar represents SE.
(C) (C1) Amount of high-molecular-weight α-synuclein in the SDS-insoluble fraction increased in the brain of 5-month-old N. furzeri. The graph shows quantification of the high-molecular α-synuclein in the SDS-insoluble fraction (ANOVA: F = , p < ). n = 8 fish per group. The bar represents SE. (C2) Dot-blot analysis of α-synuclein fibrils in the brain of N. furzeri. A conformation-specific antibody (rabbit monoclonal anti-α-synuclein filament antibody [MJFR ]; Abcam) was used to detect α-synuclein fibrils in the brain of N. furzeri. The blot on the left shows specificity of the signals. The single signal disappeared in α-synuclein KO N. furzeri. The blot on the right shows increased fibrils in the brain of 5-month-old N. furzeri. The graph shows quantification of the dot blot (ANOVA: F = , p < ). n = 6 fish per group.
(D–F) Progression of α-synuclein pathologies.
(D) Schematic illustration of the lysate injection assay. See also Lysate injection assay in STAR Methods.
(E) 3,3′-Diaminobenzidine tetrahydrochloride (DAB) immunostaining of N. furzeri and human α-synuclein. N. furzeri α-synuclein (E1) and human α-synuclein (E2) in vehicle-injected wild-type (WT) zebrafish. N. furzeri α-synuclein (E3) and human α-synuclein (E4) in vehicle-injected zebrafish expressing human α-synuclein. N. furzeri α-synuclein (E5) and human α-synuclein (E6) in lysate-injected WT zebrafish. N. furzeri α-synuclein (E7) and human α-synuclein (E8) in lysate-injected zebrafish expressing human α-synuclein. (E9) Number of human α-synuclein inclusion bodies per sagittal section (10 μm). n = 4 fish for each group (lysate-injected zebrafish expressing human α-synuclein versus vehicle-injected zebrafish expressing human α-synuclein). The bar represents SE.
(F) Fluorescence immunostaining of N. furzeri α-synuclein (green) and human α-synuclein (magenta). N. furzeri α-synuclein (F1) and human α-synuclein (F2) in vehicle-injected zebrafish expressing human α-synuclein (control). (F3) Merged figure of (F1) and (F2). N. furzeri α-synuclein (F4) and human α-synuclein (F5) in lysate-injected zebrafish expressing human α-synuclein. (F6) Merged figure of (F4) and (F5). White arrows indicate inclusion bodies containing human α-synuclein. N. furzeri α-synuclein (F7) and human α-synuclein (F8) in lysate-injected WT zebrafish (control). (F7) Merged figure of (F8) and (F9).
See also Figure S4.
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5. Figure 3 Rescued Pathologies by α-Synuclein Depletion
(A) Profile of α-synuclein-deficient (knockout [KO]) N. furzeri. (A1) Sequences of wild-type (WT) and α-synuclein KO fish (c.40_59del, α-synuclein Δ20/Δ20) are shown. The red square or red characters indicate the lesion with a 20-base pair deletion. (A2) Immunohistochemistry and western blotting show complete depletion of α-synuclein protein in α-synuclein KO (3-month-old) fish. (A3) Kaplan-Meier survival curves of α-synuclein WT and KO N. furzeri. Each fish was separated in one tank 30 days after hatching. n = 12 fish per group. Statistical analysis for the survival curves was performed by log-rank test. The life span of α-synuclein KO N. furzeri was comparable to that of the wild-type.
(B) The number of dopaminergic neurons in the posterior tuberculum and of noradrenergic neurons in the locus ceruleus at 1 month (n = 6 fish per group) and 5 months (n = 10 fish per group). The bar represents SE.
(C) (C1) and (C3) are the representative images of dopaminergic neurons in the posterior tuberculum in 5-month-old WT and KO fish. (C2) and (C4) are the magnified images of the red square regions shown in (C1) and (C3), respectively. (C5) and (C7) are the representative images of the noradrenergic neurons in the locus ceruleus in the 5-month-old WT and KO fish. (C6) and (C8) are the magnified images of the red square regions in (C5) and (C7), respectively.
(D) Western blotting of TH protein in the brain of α-synuclein KO fish (at 1 and 5 months). n = 6 (1 month) and 5 (5 months) fish per group.
See also Figures S2 and S5.
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