Figures and data
Knockdown of milton or Miro causes protein accumulation in the axon
(A) Schematic representation of the mitochondrial transport machinery. Knockdown of milton, an adapter protein for mitochondrial transport, depletes mitochondria in the axon. (B, C) Ubiquitinated proteins in brains with neuronal knockdown of milton or Miro. Brains dissected at 14-day-old (B) or 30-day-old (C) were immunostained with an antibody against ubiquitin. Firefly luciferase RNAi was used as a control. Representative images (left) and quantitation of the number of ubiquitin-positive puncta (right) are shown. Scale bars of hemibrains, 100 µm, Scale bars of high magnifications, 10 µm. Means ± SE, n = 8. N.S., p > 0.05; ***p < 0.005 (one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) post hoc test). (D) Cross sections in the lamina and in the retina were used to analyze the ultrastructure of synapses and cell bodies, respectively. milton RNAi was expressed in the retina and neurons via a combination of GAL4 drivers, a pan-retinal gmr-GAL4 and pan-neuronal elav-GAL4. (E) Quantitation of the number of mitochondria in a presynaptic terminal from transmission electron micrographs. 180 presynaptic terminals from cross-sections of the lamina from three brains were analyzed. ***p < 0.005 (Chi-square test). (F, G) Presynaptic terminals of photoreceptor neurons of control and milton knockdown flies. Photoreceptor neurons are highlighted in blue. Swollen presynaptic terminals (asterisks in (F)), characterized by the enlargement and higher circularity, were found more frequently in milton knockdown neurons. Arrowheads indicate presynaptic terminals with dense materials. Scale bars, 2 µm. Representative images (Left) and quantitation (Right) are shown. 918–1118 from three heads were quantified for the percentage of swollen presynaptic terminals, and 180 presynaptic terminals from three heads were quantified for the size of presynaptic terminals. Mean ± SE, **p < 0.01, ***p < 0.005 (Student’s t-test). (G) Dense materials (arrowheads in (G)) in the presynaptic terminals of milton knockdown neurons. Scale bars, 2 µm. The ratio of presynaptic terminals containing dense materials were quantified from 918–1118 presynaptic terminals from three heads. Mean ± SE, ***p < 0.005 (Student’s t-test). (H) Cell bodies of photoreceptor neurons of control and milton knockdown flies. Scale bars, 2 µm. Flies were 27-day-old.
milton knockdown impairs protein degradation pathways
(A, B) Western blotting of head extracts of control and milton knockdown flies with antibodies against LC3 (A) and Ref2P, the fly homolog of mammalian p62 (B). For the analyses of p62 levels, heads were extracted with 1% Triton X-100 or 2% SDS (B). Flies were 14-day-old. Representative blots (left) and quantitation (right) are shown. Actin was used as a loading control. Means ± SE, n = 6 (LC3), n = 3 (p62). (C) Proteasome activity in head extracts of control and milton knockdown flies was measured by hydrolysis of Suc-LLVY-AMC at 14-day-old. Means ± SE, n = 3. (D, E) Western blotting of head extracts of 30-day-old control and milton knockdown flies. Blotting was performed with anti-LC3 (D) and anti-p62 (E) antibodies. Representative blots (left) and quantitation (right) are shown. Actin was used as a loading control. Means ± SE, n = 6 (LC3), n = 3 (p62). (F) Proteasome activity in head extracts of 30-day-old control and milton knockdown flies. Means ± SE, n = 3. N.S., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.005 (Student’s t-test).
ATP deprivation does not impair autophagy
(A-C) ATP levels in brain extracts of control and milton knockdown flies (A) and control and Pfk knockdown flies (B) and comparison of the effects of milton knockdown and Pfk knockdown on ATP levels (C). Flies were 14 day-old. Means ± SE, n = 3. (D, E) Western blotting of head extracts of flies with neuronal expression of control or Pfk RNAi. Blotting was performed with anti-LC3 (D) and anti-p62 (E) antibodies. For analyses of p62 levels, heads were extracted with 1% Triton X-100 or 2% SDS. Representative blots (left) and quantitation (right) are shown. Actin was used as a loading control. Means ± SE, n = 6 (LC3), n = 3 (p62). (F) Proteasome activity in head lysates of flies with neuronal expression of control or Pfk RNAi was measured by hydrolysis of Suc-LLVY-AMC. Means ± SE, n = 3. N.S., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.005 (Student’s t-test). Flies were at 14 day-old.
milton knockdown upregulates eIF2β and decreases phosphorylation of eIF2α and translation
(A) Timing of proteome analysis and phenotypes observed in milton knockdown flies. (B, C) Scatter plots of the log2 abundance ratio (x-axis) against the −log10 p-value (y-axis) of proteins at 7-day-old (B) and 21-day-old (C). (D) eIF2 subunit protein levels from proteome analysis of milton knockdown flies compared to those of control flies. (E) Western blotting of head extracts of flies expressing control or milton RNAi in neurons with an anti-eIF2β antibody. Flies were 14-day-old. Representative blots (left) and quantitation (right) are shown. Tubulin was used as a loading control. Means ± SE, n = 6. (F) eIF2β mRNA levels quantified by qRT-PCR. Means ± SE, n = 4, (G, H) Western blotting of head extracts with anti-eIF2α (G) and anti-p-eIF2α (H) antibodies. Flies were 14-day-old. Representative blots (left) and quantitation (right) are shown. Tubulin was used as a loading control. Means ± SE, n = 6. (I) A schematic representation of the axon (Lobe tips), the cell body region (Kenyon cells), and dendritic region (Calyxes) in the fly brain. Scale bars, 100µm. (J, K) Immunostaining with anti-eIF2α and anti-p-eIF2α antibodies. The mushroom body was identified by expression of mito-GFP. Scale bars, 20µm. The signal intensities of eIF2α and p-eIF2α in axons, dendrites, and cell bodies were quantified and are shown as ratios relative to the control. Means ± SE, n=12. N.S., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.005 (Student’s t-test).
milton knockdown suppressed global translation
(A) Representative polysome traces of head lysates of control and milton knockdown flies. (B) Quantitation of polysome fraction. The relative ratio of area under the curve (AUC) of polysome fractions (sedimentation 28%-50%). Means ± SE, n = 3. ***p < 0.005 (Student’s t-test) (C) Western blotting of head lysates of control and milton knockdown flies fed puromycin alone or puromycin and cycloheximide (CHX) with an anti-puromycin antibody. Flies were 14-day-old. Actin was used as a loading control. Representative blots (left) and quantitation (right) are shown. Means ± SE, n = 3. Student’s t-test.
eIF2β upregulation impairs autophagy and decreases locomotor function
(A) eIF2β mRNA levels in head extracts of flies with UAS-eIF2β driven by elav-Gal4 (eIF2β OE) or UAS-GFP driven by elav-Gal4 (control) were quantified by qRT-PCR. Flies were 2-day-old. Means ± SE, n=4. (B, C) Western blotting of head extracts with anti-LC3 (B) and anti-p62 (C) antibodies. Flies were 14-day-old. Representative blots (left) and quantitation (right) are shown. Tubulin and actin were used as loading controls. Means ± SE, n = 3 (p62), n = 5 (LC3). (D, E) Western blotting of head extracts with anti-eIF2α (D) and anti-p-eIF2α (E) antibodies. Flies were 14-day-old. Representative blots (left) and quantitation (right) are shown. Tubulin was used as a loading control. Means ± SE, n = 6. (F) Climbing assay revealed early-onset of age-dependent locomotor defects in eIF2β-overexpressing flies. Means ± SE, n = 5. N.S., p > 0.05; ***p < 0.005 (Student’s t-test).
Lowering eIF2β rescues autophagic impairment and locomotor dysfunction induced by milton knockdown
(A) eIF2β mRNA levels with one disrupted copy of the eIF2β gene (eIF2βSAstopDsRed/+ (eIF2β -/+)). Head extracts of flies 2–3 day-old were analyzed by qRT-PCR. Means ± SE, n = 3. (B, C) Western blotting of head extracts of flies with neuronal expression of milton RNAi with or without eIF2β heterozygosity with anti-LC3 (B) and anti-p62 (C) antibodies. Flies were 14-day-old. Representative blots (left) and quantitation (right) are shown. Actin was used as a loading control. Means ± SE, n = 5 (LC3), n = 3 (p62). (D) The climbing ability of 20-day-old flies expressing milton RNAi with or without eIF2β heterozygosity. Means ± SE, n = 15. N.S., p > 0.05; *p < 0.05; ***p < 0.005 (Student’s t-test).
