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First published online 3 July 2008
doi: 10.1242/dev.020644

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Reactive oxygen species act remotely to cause synapse loss in a Drosophila model of developmental mitochondrial encephalopathy

¹ Department of Neurobiology, 299 W. Campus Drive, Stanford University, Stanford, CA 94305, USA.
² Department of Pathology, Stanford University School of Medicine, Palo Alto, CA 94304, USA.

View larger version (68K): [in this window] [in a new window]   Fig. 1. Mutations in SdhA block SDH complex activity. (A) Schematic illustration of the electron transport chain complexes I-V. IMS, intermembrane space; IM, inner membrane; M, matrix; Ub, ubiquinone; CytC, cytochrome C. (B) Schematic of the SdhA protein structure denoting identified mutations. (C-E) Eye-specific mosaic third instar larval eye discs, visualizing the enzymatic activity of the SDH complex. (C) Wild type. Arrowheads demarcate the morphogenetic furrow. (D) SdhA mutant. (E) Wild-type disc, stained in the presence of the competitive inhibitor malonate.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Quantification of the degeneration of SdhA mutant R cell structure in the retina and lamina. The mean number of photoreceptors with intact rhabdomes (A) and the relative intensity of vesicle staining in R cell synaptic terminals (B) in control (gray) and SdhA homozygous mutants (blue) across experimental conditions. Numbers above columns indicate the number of ommatidia scored in A; the number of laminas in B.

View larger version (117K): [in this window] [in a new window]   Fig. 3. SdhA mutant R cell synaptic terminals develop normally, and then degenerate. (A-H) Cross sections of the lamina neuropil containing small negatively marked clones in wild-type (A,B,E,F) and SdhA1110 mosaic (C,D,G,H) animals, at (A-D) 0 days after eclosion, and (E-F) 5 days after eclosion. Homozygous tissue is marked by the absence of GFP (green). Photoreceptor terminals are stained with the synaptic vesicle marker CSP (magenta; A,C,E,G), or the R cell-specific antibody mAb24B10 (red; B,D,F,H). Insets show single cartridges. Scale bar: 10 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 4. Rhabdomeres degenerate in SdhA mutant retinas. (A-F) Cross sections of the retina in wild-type (A-C), and Sdh1110 eye-specific mosaic (D-F) animals, at 44 hours APF (A,D), 0 days after eclosion (B,E), and 5 days after eclosion (C,F). R cells are visualized using mAb24B10 (A,D), or by using phase contrast in plastic sections (B,C,E,F). Insets show single ommatidia. Scale bar: 10 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 5. Mitochondria are lost from SdhA mutant terminals. (A-P) Cross section of the lamina neuropil in wild type (A-D,I-L), and SdhA1110 mutant eye-specific mosaic (E-H,M-P) animals, stained with the R cell-specific antibody mAb24B10 (A,E,I,M), for mitochondrially localized GFP (B,F,J,N), or for the mitochondrial complex V subunit alpha (C,G,K,O), at 0 (A-H) or 5 days (I-P) after eclosion. Merged images are shown in D,H,L,P. Insets show single synaptic cartridges. Scale bar: 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Mitochondrial volume and structure are abnormal in SdhA mutant R cell terminals. (A-E') Ultrastructural analysis of wild-type (A,A',D,D') or SdhA1110 mutant (B,B',C,C',E,E') R cell terminals in the lamina on the day of eclosion (A-C) or 5 days after eclosion (D,E). Asterisk marks a multilamellar body. (A',B',C',D',E') Schematic tracings of R cell terminals (black lines, R), lamina neurons (blue lines, L), and mitochondrial profiles within R cell terminals (magenta), or unidentifiable cells (gray). Insets show profiles of single mitochondria. Specialized glial invaginations called capitate projections are denoted (cp). Scale bar: 1 µm; in inset, 0.1 µm. (F) Quantification of mitochondrial area per terminal area in control (gray) and SdhA mutants (blue).

View larger version (64K): [in this window] [in a new window]   Fig. 7. R cells are not depleted of ATP. (A) The average luminance and s.e.m., per retina, as determined using luciferin/luciferase-based ATP assays performed on retinas taken from wild-type (gray) or SdhA1110 mutant mosaic (blue) flies at 0 and 5 days after eclosion. (B-I) Cross sections of the laminas (B,C,F,G) or retinas (D,E,H,I) from animals raised in complete darkness. Laminas are stained with the R cell-specific antibody mAb24B10 (green) and CSP (magenta). (B,D,F,H) Wild type. (C,E,G,I) SdhA1110 mutant. (B-E) 0 days after eclosion. (F-I) 5 days after eclosion.

View larger version (112K): [in this window] [in a new window]   Fig. 8. Blocking ROS suppresses synaptic terminal degeneration in SdhA mutant R cells. (A-O) Cross sections of the lamina neuropil (A-J) and retinas (K-O). (A,F,K) 5-day-old wild-type flies grown on food treated with 200 µg/ml -tocopherol. (B,G,L) 5-day-old SdhA1110 mutant flies treated with vehicle alone. 5-day-old (C,H,M) SdhA1110 and (D,I,N) SdhA1404 mutants treated with 200 µg/ml -tocopherol. (E,J,O) SdhA1110 mutant overexpressing CuZnSOD. Laminas are stained (A-E) for R cells (green) and vesicles (magenta). (F-J) Single channel images stained for vesicles of 5-day laminas. Insets show single synaptic cartridges (A-E) or ommatidia (K-O). Scale bars: 20 µm.

View larger version (129K): [in this window] [in a new window]   Fig. 9. Mitochondria restricted to the cell body are sufficient to induce synaptic degeneration. (A-L) Cross sections of the lamina neuropil in wild-type (A,E,I), SdhA1110 mutant (B,F,J), Miro (C,G,K) and SdhA1110; Miro double mutant eye-specific mosaic (D,H,L) animals, at 0 days after eclosion (A-D) or 5 days after eclosion (E-L). R cells (green) and CSP (magenta). (I-L) Single channel images stained for vesicles of 5-day laminas. (M) Schematic describing the logic of this experiment. Mitochondria are normally found in both cell bodies and axons. Thus, in SdhA mutants, excess ROS could be produced in both compartments. However, transport of mitochondria from cell bodies to axons is dependent upon Miro. To test whether synapse loss was induced by ROS locally in the terminal, we blocked axonal transport of SdhA mutant mitochondria using mutations in Miro, thereby restricting ROS production to the cell body. Retinas in control flies were made homozygous for two FRT chromosomes using two cell-lethal mutations. SdhA and Miro single homozygote retinas were also heterozygous for Miro and SdhA, respectively. Scale bar: 20 µm.

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