Knockdown of milton or Miro causes protein accumulationin the axon

(A) Schematic representation of the mitochondrial transport machinery. (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 ubiquitinated proteins. 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 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, F) Transmission electron micrographs of presynaptic terminals of photoreceptor neurons of control and milton knockdown flies. Photoreceptor axons are highlighted in blue. Swollen axons (asterisks in (E)) are observed in milton knockdown neurons. Scale bars, 2 µm. Representative images and quantitation are shown. In total, 918–1118 cells from cross-sections of the lamina from each half of the head were analyzed. Mean ± SE, n = 3. **p < 0.001 (Student’s t-test). (F) Dense materials (arrowheads) in synaptic terminals of milton knockdown neurons. Scale bars, 2 µm. (G) 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 assessment 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 (chymotrypsin-like activity) 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 was measured by hydrolysis of Suc-LLVY-AMC (chymotrypsin-like activity). Means ± SE, n = 3. N.S., p > 0.05; *p < 0.05; **p < 0.001; ***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 assessment 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 (chymotrypsin-like activity). Means ± SE, n = 3. N.S., p > 0.05; *p < 0.05; **p < 0.001; ***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) Age of proteome analysis and phenotypes of milton knockdown flies. (B) A scatter plot of the log2 abundance ratio (x-axis) against the -log10 p-value (y-axis) of proteins at 7 day-old. (C) 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. (D) eIF2β mRNA levels quantified by qRT-PCR. Means ± SE, n = 4. (E) 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. (F, G) 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. (H) Representative polysome traces of head lysates of control and milton knockdown flies. (I) 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. N.S., p > 0.05; *p < 0.05; ***p < 0.005 (Student’s t-test).

Differentially expressed proteins in milton RNAi fly brains compared to control at 7 day-old detected by proteome analysis

Molecule networks based on “Interaction search” of KeyMolnet

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 2–3 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) Climbing assay revealed early-onset 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).

A schematic representation of the mitochondria-eIF2β axis in the axon to maintain neuronal proteostasis during aging

Western blot analysis of eIF2α and p-eIF2α with milton knockdown.

Western blotting of head extracts with anti-eIF2α (A) and anti-p-eIF2α (B) 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. N.S., p > 0.05; *p < 0.05 (Student’s t-test).

Western blot analysis of eIF2α and p-eIF2α with eIF2β.

overexpression Western blotting of head extracts with anti-eIF2α (A) and anti-p-eIF2α (B) 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. N.S., p > 0.05; ***p < 0.005 (Student’s t-test).

Lowering the eIF2β level does not affect the levels of eIF2α and p-eIF2α.

(A-C) Blotting was performed with anti-eIF2α (A) and anti-p-eIF2α (B, C) 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. (D) eIF2β gene disruption does not affect the knockdown efficiency of milton. milton mRNA levels in head extracts were quantified by qRT-PCR. Flies were 2–3 day-old. Means ± SE, n = 3. N.S., p > 0.05; *p < 0.05 (Student’s t-test).