1. α-synuclein Induces Mitochondrial Dysfunction through Spectrin and the Actin Cytoskeleton 
Dalila G. Ordonez, Michael K. Lee, Mel B. Feany 
Neuron 
Volume 97, Issue 1, Pages e6 (January 2018)
DOI: /j.neuron
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2. Figure 1 Drosophila Model of Diffuse α-synucleinopathy
(A) Western blot showing α-synuclein levels in human (Hu) brain homogenate, the previous Parkinson’s disease Drosophila model (PD) and the current α-synucleinopathy Drosophila model (LBD). Flies are 1 day old.
(B) Behavioral analysis of motor deficits using the climbing test indicates loss of locomotor ability with age in α-synuclein transgenic flies. n = 60 per genotype.
(C and D) Hematoxylin and eosin staining showing vacuolization in the medulla of 10-day-old α-synuclein transgenic flies (arrows). Scale bar, 50 μm. Quantification (D) reveals an increase in vacuole formation with age in the brains of 1-, 10- and 20-day-old α-synuclein transgenic flies. n = 6 per genotype.
(E) Quantification of cortical neurons in the anterior medulla showing decreased neuronal density in 10-day-old α-synuclein transgenic flies. n = 6 per genotype.
(F and G) Caspase activation in neurons (marked by elav) in 10-day-old α-synuclein transgenic flies (arrow) and quantification with aging (G). Scale bar, 7 μm. n = 6 per genotype. Control is elav-GAL4; UAS-CD8-PARP-Venus/Syb-QF2.
(H and I) α-synuclein aggregates (arrows) in the brain of 10-day-old α-synuclein transgenic flies detected by immunofluorescence. Scale bar, 7 μm. Quantification (I) shows increased numbers of inclusions with age in 1-, 10- and 20-day-old flies. n = 6 per genotype.
Asterisks indicate ∗∗∗p < and ∗∗∗∗p < , two-way ANOVA with Student-Newman-Keuls test. Control in (A)–(E), (H), and (I) is Syb-QF2/+. Controls are shown in green and α-synuclein transgenics in red in all bar graphs in (D), (E), (G), and (I). Data are represented as mean ± SEM.
See also Figure S1.
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3. Figure 2
Actin Cytoskeletal Abnormalities in the Brains of α-synuclein Transgenic Flies, Mice, and in α-synucleinopathy Patients
(A and B) F-actin staining of whole mount brains of α-synuclein transgenic flies compared to control flies. Scale bar, 50 μm. Quantification (B) of fluorescence intensity shows an increase in F-actin in α-synuclein transgenic flies. n = 6 per genotype.
(C and D) Actin fractionation by high-speed centrifugation and western blotting (C) and ELISA (D) show increased F-actin levels in α-synuclein transgenic flies compared to control flies. n = 10 (C) and n = 3 (D) per genotype. WCL, whole-cell lysate
(E and F) Immunofluorescent staining for actin in tissue sections and quantification (F) reveals numerous actin-rich rods (arrows) in tissue sections from brains of α-synuclein transgenic flies. Scale bar, 20 μm. n = 6 per genotype.
(G and H) Immunofluorescent staining of actin-rich rods (arrows) and quantification (H) in brainstem sections of ∼11- to 14-month-old A53T α-synuclein transgenic mice reveals the presence of numerous actin-rich rods. Scale bar, 10 μm. n = 4 per genotype.
(I–K) Immunofluorescent staining of actin-rich rods (arrows) in brainstem sections of A53T α-synuclein transgenic mice shows co-labeling with cofilin (I) and localization within MAP2-positive neurons (J and K). Scale bar, 5 μm in (I) and 10 μm in (J). n = 4 per genotype.
(L–O) Immunofluorescent staining of actin-rich rods (arrows) in cingulate cortex of patients with diffuse neocortical Lewy body pathology (arrows) reveals co-labeling with phalloidin (M and O) and cofilin (N and O). Scale bar, 10 μm in (L) and (M), left and 5 μm in (N) and (O).
Asterisks indicate ∗∗p < and ∗∗∗p < , unpaired t test. Data are represented as mean ± SEM. Flies are 10 days old in (A)–(F) and mice are ∼11–14 months old (G)-(K). Control in (A)–(F) is Syb-QF2/+ and in (G)–(K) is non-transgenic littermates.
See also Figure S2.
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4. Figure 3
Genetic Manipulation of the Actin Cytoskeleton Rescues α-synuclein Neurotoxicity
(A–C) Genetic modification of locomotor dysfunction (A) and neuronal degeneration (B and C) in α-synuclein transgenic flies showing rescue by reducing actin in animals heterozygous for Act5CG0010 and enhancement with overexpression of actin (UAS-Act5C). Scale bar, 20 μm. n = 60 (A) and n = 6 (C) per genotype.
(D) Reduction in F-actin levels in head homogenates of α-synuclein transgenic flies heterozygous for Act5CG0010 and further increase in F-actin levels with overexpression of actin (UAS-Act5C) as determined by F-actin ELISA. n = 3 per genotype.
(E and F) Immunostaining of actin-rich rods (E) and quantification (F) showing reduction in actin rods in the brains of α-synuclein transgenic flies heterozygous for Act5CG0010 and further increase in actin rods with overexpression of actin (UAS-Act5C) as determined by immunostaining sections for actin. Scale bar, 5 μm. n = 6 per genotype.
(G–I) Genetic rescue of locomotor dysfunction (G) and neuronal degeneration (H and I) in α-synuclein transgenic flies expressing transgenic RNAi directed to Fhos. Scale bar, 20 μm. n = 60 (G) and n = 6 (I) per genotype.
(J) Reduction in F-actin levels in head homogenates of α-synuclein transgenic flies expressing transgenic RNAi directed to Fhos as determined by F-actin ELISA. n = 3 per genotype.
(K and L) Immunostaining of actin-rich rods (K) and quantification (L) showing reduction in actin rods in the brains of α-synuclein transgenic flies expressing transgenic RNAi directed to Fhos as determined by immunostaining sections for actin. Scale bar, 5 μm. n = 6 per genotype.
Asterisks indicate ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < , and ∗∗∗∗p < , two-way ANOVA in (A) and (G) and one-way ANOVA in (C), (D), (F), (I), (J), and (L) with Student-Newman-Keuls test. Data are represented as mean ± SEM. The same control and α-synuclein samples were used for Act5C and Fhos analysis. Flies are 10 days old in (D), (F), (J), and (L) and 20 days old in (C) and (I). Control (ctrl) is elav-GAL4/+; Syb-QF2/+.
See also Figure S3.
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5. Figure 4 α-synuclein Expression Induces Mitochondrial Abnormalities
(A) 3D reconstruction of immunofluorescence-stained mitochondria showing mitochondrial enlargement in central brain neurons of α-synuclein transgenic flies compared to control flies. Scale bar, 5 μm.
(B) Electron microscopic images of the fly brain cortex showing mitochondrial enlargement in neurons of α-synuclein transgenic flies compared to control flies (arrows). Scale bar, 500 nm.
(C and D) Measurements of mitochondrial diameter and elongation show changes in mitochondrial morphology in α-synuclein transgenic flies. n = 6 per genotype.
(E and F) Oxidation of mitochondrial protein is elevated in whole-mount brain preparations of α-synuclein transgenic flies compared with controls as monitored by the transition from green to red fluorescence in transgenic MitoTimer protein. Scale bar, 100 μm. n = 6 per genotype. Control is elav-GAL4/+; UAS-MitoTimer/Syb-QF2.
(G and H) MitoSOX Red fluorescence (G) and quantification (H) in whole-mount brain preparations from α-synuclein transgenic and control flies. Scale bar, 50 μm. n = 6 per genotype.
(I and J) Mitochondrial (mito-GFP, green) colocalization with Drp1 (red) is reduced in neurons from α-synuclein transgenic fly brains. Scale bar, 5 μm. n = 6 per genotype.
(K and L) Loss of mitochondrial localization of Drp1 as revealed by structured illumination microscopy. Scale bar, 1 μm.
(M and N) Western blot of mitochondria subcellular fractionation probed for Drp1 (M) and quantification (N) showing reduction in Drp1 levels in the mitochondria fraction of α-synuclein flies compared to control. Control is elav-GAL4/+; HA-Drp1/Syb-QF2.
(O–Q) Immunofluorescent staining for mitochondria (ATPVα) and Drp1 (O) shows mitochondrial enlargement (P) and reduced Drp1 colocalization (Q) in ∼11- to 14-month-old A53T human α-synuclein transgenic mice compared to non-transgenic littermates. Scale bar, 5 μm. n = 4 per genotype.
Asterisks indicate ∗∗p < 0.001, ∗∗∗p < , and ∗∗∗∗p < Unpaired t test. Data are represented as mean ± SEM. Flies are 10 days old in (A)–(L) and 1 day old in (M) and (N). Control in (A)–(D), (G), and (H) is elav-GAL4/+; Syb-QF2. Control in (I)–(L) is elav-GAL4/+; UAS-mito-GFP/+; HA-Drp1/Syb-QF2.
See also Figure S4.
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6. Figure 5
Overexpressing Drp1 Rescues α-synuclein-Induced Mitochondrial Dysfunction and Neurodegeneration
(A–C) Rescue of locomotor deficits (A) and neuronal loss (B and C) by overexpression of Drp1 (UAS-Drp1) in α-synuclein transgenic flies. Scale bar, 20 μm. n = 60 (A) and n = 6 (C) per genotype. Control (ctrl) is elav-GAL4/+; Syb-QF2/+.
(D and E) Mitochondrial length is normalized by overexpression of Drp1 (UAS-Drp1) in α-synuclein transgenic flies. Scale bar, 10 μm. n = 6 per genotype. Control (ctrl) is elav-GAL4/+; UAS-mito-GFP/+; Syb-QF2/+.
(F) Mitochondrial protein oxidation as monitored by the transition from green to red fluorescence in MitoTimer protein is rescued by Drp1 expression (UAS-Drp1) in α-synuclein transgenic flies. n = 6 per genotype. Control (ctrl) is elav-GAL4/+; UAS-MitoTimer/Syb-QF2.
(G and H) MitoSOX red fluorescence is reduced by Drp1 overexpression (UAS-Drp1) in α-synuclein transgenic flies. Scale bar, 50 μm. n = 6 per genotype. Control (ctrl) is elav-GAL4/+; Syb-QF2/+.
(I and J) Drp1 localization to the mitochondria, as monitored by HA-tagged Drp1 (HA-Drp1), is restored following Drp1 overexpression (UAS-Drp1) in α-synuclein transgenic flies (arrows). Scale bar, 5 μm. n = 6 per genotype. Control is elav-GAL4/+; UAS-Drp1, mito-GFP/+; HA-Drp1/Syb-QF2.
Asterisks indicate ∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p < 0.002, two-way ANOVA in (A) and one-way ANOVA in (C), (E), (F), (H), and (J) with Student-Newman-Keuls test. Data are represented as mean ± SEM. Flies are 10 days old except for (C) in which flies are 20 days old.
See also Figure S5.
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7. Figure 6
Abnormal F-actin Stabilization Drives Mitochondrial Pathology in α-synucleinopathy
(A–C) Mitochondrial morphology (A), mitochondrial protein oxidation as assessed by fluorescence of the MitoTimer protein (B), and MitoSOX red fluorescence (C) are all normalized in α-synuclein transgenic flies heterozygous for the Act5CG0010 mutation. n = 6 per genotype.
(D–F) Mitochondrial morphology (D), mitochondrial protein oxidation as assessed by fluorescence of the MitoTimer protein (E), and MitoSOX Red fluorescence (F) are all normalized in α-synuclein transgenic flies expressing RNAi directed to Fhos. n = 6 per genotype.
(G–I) Drp1 localization to mitochondria is partly restored by heterozygosity for Act5CG0010 (H) or expressing Fhos RNAi (I). Scale bar, 5 μm. n = 6 per genotype.
Asterisks indicate ∗∗p < and ∗∗∗p < 0.002, one-way ANOVA with Student-Newman-Keuls test. Data are represented as mean ± SEM. Flies are 10 days old in all panels. Control (ctrl) in (A) and (D) is elav-GAL4/+; UAS-mito-GFP/+; Syb-QF2/+. Control (ctrl) in (B) and (E) is elav-GAL4/+; UAS-MitoTimer/Syb-QF2. Control (ctrl) in (C) and (F) is elav-GAL4/+; Syb-QF2/+. Control in (G)–(I) is elav-GAL4/+; UAS-mito-GFP/+; HA-Drp1/ Syb-QF2.
See also Figure S6.
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8. Figure 7
Spectrin Induces Actin Stabilization and Subsequent Neurodegeneration
(A and B) Rescue of locomotor deficits (A) and neuronal loss (B) by overexpression of α-spectrin (UAS-α-spectrin) in α-synuclein transgenic flies. n = 60 (A) and n = 6 (B) per genotype.
(C) Reduction in F-actin levels in head homogenates of α-synuclein transgenic flies overexpressing α-spectrin as determined by F-actin ELISA. n = 3 per genotype.
(D) Reduction in actin rods in the brains of α-synuclein transgenic flies overexpressing α-spectrin as determined by immunostaining sections for actin. n = 6 per genotype.
(E) Mitochondrial length is normalized by overexpression of α-spectrin in α-synuclein transgenic flies. n = 6 per genotype. Control (ctrl) is elav-GAL4/+; UAS-mito-GFP/+; Syb-QF2/+.
(F and G) Rescue of Drp1 localization to the mitochondria by overexpression of α-spectrin in α-synuclein transgenic flies. Scale bar, 5 μm, n = 6 per genotype. Control (ctrl) is elav-GAL4/+; UAS-mito-GFP/+; Syb-QF2/HA-Drp1.
(H) Immunofluorescence staining with an antibody to α-spectrin shows loss of normal subplasmalemmal organization in α-synuclein transgenic flies. Overexpression of α-spectrin restores normal organization. Inset indicates the area of the brain analyzed in (H) and subsequently; elav immunostain identifies neuronal nuclei. Scale bar, 100 μm (inset, 5 μm). Control is elav-GAL4/+; UAS-α-spectrin /+.
(I) Structured illumination microscopy (SIM) immunostaining showing colocalization of α-spectrin with α-synuclein in Kenyon cells of α-synuclein transgenic flies compared to control flies. Scale bar, 1 μm.
(J) Fluorescence spectral z-profile showing association between α-spectrin (blue) and α-synuclein (red) in α-synuclein transgenic flies compared to control flies.
(K) SIM immunostaining showing colocalization of α-spectrin with mitochondria and F-actin in Kenyon cells of α-synuclein transgenic flies compared to control flies. Scale bar, 1 μm.
(L) Fluorescence spectral z-profile shows association between α-spectrin (blue) and F-actin (red) in control flies. In α-synuclein flies, spectrin is disassociated with F-actin (red).
(M) Pearson’s correlation coefficient indicating infrequent association of α-spectrin with actin and increased association with mitochondria in α-synuclein transgenic flies compared to control flies. n = 3 per genotype.
(N) F-actin precipitation with biotinylated phalloidin showing biochemical interaction of F-actin with α-spectrin and α-synuclein in α-synuclein transgenic flies compared to control. n = 10 per genotype.
(O and P) Immunostaining and quantification (P) of α-synuclein aggregates shows increased numbers of α-synuclein aggregates following α-spectrin overexpression. Scale bar, 3 μm. n = 6 per genotype.
(Q and R) Western blot of soluble and insoluble fractions prepared from fly heads (Q) and quantification (R) showing significantly increased α-synuclein levels in insoluble fractions from heads of α-synuclein transgenic flies overexpressing α-spectrin. Both soluble and insoluble fractions were normalized to α-synuclein transgenic flies and compared to α-synuclein transgenic flies overexpressing α-spectrin. Statistical significance was determined based on percent change. n = 10 per genotype.
Asterisks indicate ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < , and ∗∗∗∗p < , two-way ANOVA in (A) and one-way ANOVA in (B)–(E), (G), and (P) with Student-Newman-Keuls test. Data are represented as mean ± SEM. Flies are 10 days old in all panels. Control (ctrl) in (A)–(D) and (I)–(R) is elav-GAL4/+; Syb-QF2/+.
See also Figures S7 and S8.
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