Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2β (2024)

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Depletion of axonal mitochondria by knockdown of milton or Miro causes protein accumulation in the axon

In Drosophila, mitochondrial transport is mediated by milton and Miro, which attaches mitochondria to microtubules via kinesin heavy chain^26,27 (Figure 1A). It has been reported that expression of milton RNAi in neurons via pan-neuronal elav-GAL4 driver reduced milton protein levels in Drosophila head lysate to 40% and mito-GFP signals in axons to 50%²⁹.

To test how loss of axonal mitochondria affects proteostasis in neurons, we first examined the accumulation of ubiquitinated proteins. At 14 day-old, more ubiquitinated proteins were deposited in the brains of milton knockdown flies than in those of age-matched control flies (Figure 1B, p < 0.005 between control RNAi and milton RNAi). Knockdown of Miro, a partner of milton, also resulted in more ubiquitin-positive puncta, but the difference was not significant (Figure 1B, p = 0.452 between control RNAi and Miro RNAi). These data suggest that the depletion of axonal mitochondria leads to accumulation of ubiquitinated proteins before neurodegeneration occurs.

It has been reported that ubiquitinated proteins accumulate with aging³⁰; thus, we analyzed the accumulation of ubiquitinated proteins in aged brains (30 day-old) with milton knockdown. The number of puncta of ubiquitinated proteins did not significantly differ between control and milton knockdown flies or between control and Miro knockdown flies (Figure 1C, p > 0.05). Together with the effect of milton knockdown in 14-day old flies, this result suggests that depletion of axonal mitochondria accelerates the onset of age-related accumulation of ubiquitinated proteins.”

We examined the ultrastructure of synaptic terminals and cell bodies in photoreceptor neurons with milton knockdown by transmission electron microscopy in 27 day-old flies (Figure 1D). As previously reported²⁴, axonal swelling was observed in these neurons (Figure 1E, asterisks). Some swollen axons in milton knockdown neurons contained dense materials, indicating abnormal protein accumulation in the axon (Figure 1F, arrowheads). In contrast, dense materials were not observed in cell bodies in the milton knockdown retina (Figure 1G). These results indicate that the depletion of axonal mitochondria induces protein accumulation in the axon.

Depletion of axonal mitochondria impairs protein degradation pathways

Since abnormal proteins were accumulated in milton knockdown brains, we next examined if protein degradation pathways were suppressed. We analyzed autophagy via western blotting of the autophagy markers LC3 and p62³¹. During autophagy progression, LC3 is conjugated with phosphatidylethanolamine to form LC3-II, which localizes to isolation membranes and autophagosomes. The LC3-II/LC3-I ratio was lower in the head lysate of milton knockdown flies than in that of control flies (Figure 2A). We also analyzed an autophagy substrate p62. Brain lysates were sequentially extracted using detergents with different stringencies (1% Triton X-100 and 2% SDS), and p62 was detected by western blotting³². The p62 level was increased in brains of 14 day-old milton knockdown flies (Figure 2B), indicating that autophagy is decreased in these brains. The increase in the p62 level was more prominent in the Triton X-100-soluble fraction than in the SDS-soluble fraction (Figure 2B), suggesting that depletion of axonal mitochondria impairs the degradation of less-aggregated proteins. We also analyzed proteasome activity. At 14 day-old, 20S proteasome activity was significantly decreased in brains with neuronal knockdown of milton (Figure 2C, p < 0.005). Autophagy was also reduced in the brains of milton knockdown flies at 30 day-old (Figure 2D). At this age, milton knockdown increased p62 in both fractions significantly (Figure 2E). Proteasome activities were also decreased in milton knockdown flies at 30 day-old (Figure 2F). These results indicate that depletion of axonal mitochondria impairs protein degradation pathways.

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Figure 2.

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

milton knockdown downregulates ATP in the axon³³. To examine whether the disruption of protein degradation pathways by milton knockdown is due to ATP deprivation, we investigated the effects of knocking down phosphofructokinase (Pfk), a rate-limiting enzyme in glycolysis, on protein degradation pathways. Neuronal knockdown of Pfk was reported to lower ATP levels in brain neurons³³. Pfk knockdown and milton knockdown decreased ATP to similar levels (Figure 3A–C). However, in contrast with milton knockdown, Pfk knockdown did not affect the LC3-II/LC3-I ratio (Figure 3D) and decreased p62 level (Figure 3E), suggesting that autophagy is promoted. On the other hand, proteasome activity was decreased by Pfk knockdown (Figure 3F). These results suggest that the downregulation of axonal ATP upon depletion of axonal mitochondria decreases proteasome activity, but not autophagy.

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Figure 3.

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.

Proteome analysis suggests that depletion of axonal mitochondria causes disruption of autophagy and premature aging

To identify the pathways that mediate the decrease in autophagy in milton knockdown brains, we performed proteome analysis to systematically detect differentially expressed proteins upon neuronal knockdown of milton. We analyzed flies at 7 and 21 day-old, the age before autophagic defects are detected and the age just before the onset of neurodegeneration, respectively. (Figure 4A). 1039 proteins were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Expression of 36 proteins was significantly increased (22 proteins) or decreased (14 proteins) by milton knockdown at 7 day-old (Tables 1 and S1). At 21 day-old, the expression of 41 proteins (31 upregulated and ten downregulated proteins) was significantly altered in milton knockdown flies compared with control flies (Tables 2 and S1). The “Interaction search” algorithm using KeyMolnet showed that proteins whose expression was significantly altered in the brains of milton knockdown flies at both 7 and 21 day-old were closely associated with the autophagic pathway (Tables 3 and 4, respectively). Proteins involved in pathways characteristic of aging, such as the immune response (Transcriptional regulation by STAT), cancer (Transcriptional regulation by SMAD, Transcriptional regulation by myc), longevity (Transcriptional regulation by FOXO, Sirtuin signaling pathway), and stress responses (HSP90 signaling pathway, MAPK signaling pathway)^34,35, were enriched in the proteome profiles of milton knockdown flies compared with those of control flies (Tables 3 and 4), suggesting that depletion of axonal mitochondria accelerates aging in the brain.

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Figure 4.

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).

Depletion of axonal mitochondria upregulates eIF2β and decreases phosphorylation of eIF2α and translation

Differentially expressed proteins in 7 day-old flies may reflect alterations that are causal for autophagic defects. We noticed that the expression level of eIF2β was 2.465-fold higher in the brains of milton knockdown flies than in those of control flies (Figure 4B). Upregulation of eIF2β in the brains of milton knockdown flies was confirmed by western blotting. milton knockdown upregulated eIF2β protein levels more than twice (Figure 4C), but did not change the level of eIF2β mRNA (Figure 4D). eIF2β is a subunit of the eukaryotic initiation factor 2 (eIF2) complex, which is critical for translation initiation and the integrated stress response (ISR)⁴. eIF2 is a heterotrimer of α, β, and γ subunits, and during the ISR, eIF2α is phosphorylated⁶. To characterize the effect of milton knockdown on the eIF2α, we analyzed the levels of eIF2α and p-eIF2α in the brain. Western blotting of brain lysates showed that milton knockdown reduced eIF2α levels, but p-eIF2α levels were not significantly affected (Figure S1). To analyze local changes of eIF2α and p-eIF2α, we carried out immunostaining. We focused on the mushroom body, where axons, dendrites, and cell bodies can be easily identified (Figure 4E). Both eIF2α and p-eIF2α were downregulated in the cell body (Kenyon cells) and dendritic (Calyxes) regions of the brains of milton knockdown flies (Figure 4F). In axons (lobe tips), milton knockdown did not downregulate eIF2α (Figure 4G, p = 0.271) but significantly downregulated p-eIF2α (Figure 4G). The ratio of p-eIF2α to eIF2α was lower in the axon but not in the soma or dendritic region.

Downregulation of p-eIF2α alters global translation³⁶. We performed polysome gradient centrifugation to examine the level of ribosome binding to mRNA. In the brains of milton knockdown flies, the level of mRNAs associated with polysomes was reduced (Figure 5H), as expected upon downregulation of p-eIF2α. We also compared the level of translation between the brains of control and milton knockdown flies by assessing the incorporation of puromycin (Figure 4I). Puromycin incorporation was lower in the brains of milton knockdown flies than in those of control flies (Figure 4I, indicated by a bracket). These data indicate that depletion of axonal mitochondria disrupts eIF2 functions in axons and suppresses global translation.

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Figure 5.

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).

eIF2β upregulation reduces the level of p-eIF2α, impairs autophagy, and decreases locomotor function

We were motivated to ask if eIF2β upregulation mediates autophagic defects caused by milton knockdown. If so, neuronal overexpression of eIF2β would also induce autophagy impairment. Neuronal overexpression of eIF2β did not affect the LC3-II/LC3-I ratio (Figure 5A and ​andB).B). However, overexpression of eIF2β significantly increased the p62 level in the Triton X-100-soluble fraction (Figure 5C, 4-fold vs. control, p < 0.005 (1% PBST)). The p62 level in the SDS-soluble fraction was not significantly affected (Figure 5C, 2-fold vs. control, p = 0.062 (2% SDS)), as observed in brains of milton knockdown flies (Figure 2B). Neuronal overexpression of eIF2β did not affect the eIF2α level but significantly decreased the p-eIF2α level (Figure S2).

Depletion of axonal mitochondria causes an age-dependent decline in locomotor function²⁴, which we also observed in flies with neuronal overexpression of eIF2β (Figure 5D). Locomotor functions were significantly impaired in those flies at 20 day-old and worsened further during aging (Figure 5D, compare 4, 20, and 30 day-old).

These data indicate that upregulation of eIF2β in neurons phenocopies depletion of axonal mitochondria, including suppression of autophagy, and age-dependent locomotor dysfunction, suggesting that upregulation of eIF2β mediates these phenotypes downstream of loss of axonal mitochondria.

Lowering eIF2β rescues autophagic impairment and locomotor dysfunction induced by milton knockdown

Finally, we investigated whether suppression of eIF2β rescues autophagy impairment and locomotor dysfunction caused by neuronal knockdown of milton. Null mutants and flies with RNAi-mediated knockdown of eIF2β in neurons did not survive. Flies lacking one copy of the eIF2β gene survived without any gross abnormality, and the level of eIF2β mRNA in these flies was about 80% of that in control flies (Figure 6A). eIF2β heterozygosity did not affect the eIF2α and p-eIF2α levels (Figure S3 A and B). However, the LC3-II/LC3-I ratio was significantly lower in milton knockdown flies with eIF2β heterozygosity than in flies with milton knockdown alone (Figure 6B). Reduction of eIF2β expression decreased the p62 level in the Triton X-100-soluble fraction, but not the SDS-soluble fraction, in the brains of milton knockdown flies (Figure 6C). Given that milton knockdown increased the p62 level in the Triton X-100-soluble fraction but not in the SDS-soluble fraction (Figure 2B), these results indicate that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.

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Figure 6.

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).

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eIF2β heterozygosity also rescued locomotor dysfunction induced by milton knockdown. milton knockdown flies with eIF2β heterozygosity exhibited better locomotor function than milton knockdown alone (Figure 6D). The milton mRNA level was not increased in these flies, indicating that the rescue effect in the eIF2β heterozygous background was not mediated by an increased milton mRNA level (Figure S3D). These data suggest that eIF2β upregulation mediates autophagy impairment and locomotor dysfunction caused by depletion of axonal mitochondria.

Fly stocks and husbandry

Flies were maintained in standard cornmeal medium (10% glucose, 0.7% agar, 9% cornmeal, 4% yeast extract, 0.3% propionic acid and 0.1% n-butyl p-hydroxybenzoate) at 25°C under light–dark cycles of 12:12 h. The flies were transferred to fresh food vials for every 2–3 days. UAS-milton RNAi (v41508) was from VDRC and outcrossed to [w1118] for five generations in our laboratory. Transgenic fly lines carrying UAS-luciferase RNAi control for milton RNAi) was reported previously²⁴. GMR-gal4, Elav-gal4, UAS-Pfk RNAi (Bloomington stock center #36782), UAS-luciferase RNAi (Bloomington stock center #31603) (control for Pfk RNAi), UAS-GFP (used for control for UAS-eIF2β) and UAS-eIF2β (eIF2β^EY08063, Bloomington stock center #17425) were from the Bloomington stock center. eIF2β heterozygous strain (PBac{SAstopDsRed} LL07719, DGRC#142114) was from KYOTO Drosophila Stock Center. UAS-mitoGFP was a kind gift from Drs. W. M. Saxton (University of California, Santa Cruz).

Immunohistochemistry and image acquisition

Fly brains were dissected in PBS and fixed for 45 minutes in formaldehyde (4% v/v in PBS) at room temperature. After incubation in PBST containing 0.1% Triton X-100 for 10 min for three times, samples were incubated for 1h at room temperature in PBST containing 1% normal goat serum (Wako, Cat# 143-06561) and then incubated overnight with the primary antibody (anti-ubiquitin antibody Ubi-1 (1:50), anti-eIF2α (1:50) and anti-p-eIF2α (1:50)) diluted in 1% NGS/PBST at 4 °C. Samples were then washed for 10 min with PBST including 0.1% Triton X-100 three times and incubated with the secondary antibody for overnight at 4° C. Brains were mounted in Vectashield (Vectorlab Cat#H-1100) and analyzed under a confocal microscope (Nikon).

Electron microscopy

Proboscis was removed from decapitated heads, which were then incubated in primary fixative solution (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer) at R.T. for 2 hours. After washing heads with 3% sucrose in 0.1 M sodium cacodylate buffer, fly heads were post-fixed for 1 hour in secondary fixation (1% osmium tetroxide in 0.1 M sodium cacodylate buffer) on ice. After washing with H₂O, heads were dehydrated with ethanol and infiltrated with propylene oxide and Epon mixture (TAAB and Nissin EM) for 3 hours. After infiltration, specimens were embedded with Epon mixture at 70°C for 2~3 days. Thin-sections (70 nm) of laminas were collected on copper grids. The sections were stained with 5% uranyl acetate in 50% ethanol and Reynolds’ lead citrate solution. Electron micrographs were obtained with a CCD Camera mounted) on a JEM-1400 plus electron microscope (Jeol Ltd.).

SDS–PAGE and immunoblotting

Western blotting was performed as reported previously²⁴. Briefly, heads of 10-20 Drosophila were homogenized with SDS-Tris-Glycine sample buffer (0.312M Tris, 5% SDS, 8% glycerol, 0.0625% BPB, 10% β-mercaptoethanol, 10μ/mL leupeptin, 0.4 μM Pefabloc, 10mM β-glycerphosphate, 10mM NaF) and after boiling at 95°C for 2 minutes, it was centrifuged at 13,200 rpm, and the supernatant was used as a sample. To analyze p62 levels, fly heads were homogenized with 1% PBST and after centrifugation at 13,200 rpm, the supernatant was mixed 1:1 SDS-Tris-Glycine sample buffer, and boiled at 95°C for 2 minutes. The pellet was dissolved with 2% SDS in PBS, then centrifuged again at 13,200 rpm. The supernatant was mixed 1:1 SDS-Tris-Glycine sample buffer and then boiled at 95°C for 2 minutes. SDS–PAGE for western blotting was performed using 15%(w/v) (LC3), 7.5%(w/v) (p62), 10% (w/v) (eIF2α, β, and p-eIF2α) polyacrylamide gels. After electrophoresis, they were transferred to PVDF membrane (Merck Millipore) using a transfer device (BIO-RAD). After transfer, the membrane was blocked with 5% skim milk/TBST (50 mM Tris (pH 7.5), 0.15 M NaCl, 0.05% Tween20) for 1 hour and incubated with primary antibody listed below overnight at 4° C. Membranes were rinsed twice with TBST containing 0.65M NaCl and once with TBST containing 0.15M NaCl. After incubation with the secondary antibody at room temperature for 1 hour, membranes were rinsed twice with TBST containing 0.65M NaCl and once with TBST containing 0.15M NaCl. After incubation with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore), chemiluminescent signals were detected with Fusion FX (Vilber). Experiments were repeated at least 3 times with independent cohorts of flies. Primary antibodies: anti-LC3 antibody Atg8 (Merck Millipore #ABC975) (1:1000), anti-p62 antibody Ref2P (abcam #ab178440) (1:750), anti-eIF2β antibody (1:1500), anti-eIF2α antibody (abcam #ab26197) (1:1000), anti-p-eIF2α antibody (Cell signaling #3398S) (1:2000), anti-actin antibody (SIGMA A2066) (1:3000) and anti-β tubulin antibody (Sigma #T9026) (1:800,000). Polyclonal anti-eIF2β antibody was raised against a synthetic peptide (CGLEDDTKKEDPQDEA) corresponding to the C-terminal residues 29–43 of Drosophila eIF2β (1:1500).

Secondary antibodies: Peroxidase-conjugated goat anti-mouse IgG antibody (Dako #P0447) (1:2000), peroxidase-conjugated pig anti-rabbit IgG antibody (Dako #P0399) (1:2000)

Proteasome assay

Heads from the 10 flies were homogenized in 150 μl of buffer B (25 mM Tris-HCl [pH 7.5], 2 mM ATP, 5 mM MgCl2, and 1 mM dithiothreitol). Proteasome peptidase activity in the lysates was measured with a synthetic peptide substrate, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (Suc-LLVY-AMC) (Cayman). Luminescence was measured on an multimode plate reader 2300 Enspire (PerkinElmer). Experiments were repeated at least 3 times with independent cohorts of flies.

ATP assay

Heads from the 10 flies were homogenized in 50 μl of 6 M guanidine-HCl in extraction buffer (100 mM Tris and 4 mM EDTA, pH 7.8) to inhibit ATPases. Samples were boiled for 5 min and centrifuged. Supernatant was diluted 4% with extraction buffer and mixed with a reaction solution (ATP Determination kit, Invitrogen). Luminescence was measured on a multimode plate reader 2300 Enspire (PerkinElmer). The relative ATP levels were calculated by dividing the luminescence by the total protein concentration, which was determined by the Bradford method. Experiments were repeated at least 3 times with independent cohorts of flies.

Proteomic assay and pathway analysis

RNA extraction and quantitative real-time PCR analysis

Heads from more than 25 flies were mechanically isolated, and total RNA was extracted using ISOGEN (NipponGene) followed by reverse-transcription using PrimeScript RT reagent kit (Takara). The resulting cDNA was used as a template for PCR with THUNDERBIRD SYBR qPCR mix (TOYOBO) on a Thermal Cycler Dice real-time system TP800 (Takara). Expression of genes of interest was standardized relative to rp49. Relative expression values were determined by the ΔΔCT method (Livak and Schmittgen, 2001). Experiments were repeated three times, and a representative result was shown. Primers were designed using DRSC FlyPrimerBank (Harvard Medical School). Primer sequences are shown below;

eIF2β for 5′-GGACGACGACAAGAGCGAAG -3′

eIF2β rev 5′-CGGTCGCATCACGAACTTTG -3′

milton for 5′-GGCTTCAGGGCCAGGTATCT-3′

milton rev 5′-GCCGAACTTGGCTGACTTTG-3′

Polysome gradient centrifugation

30 heads were homogenized in 150 μl of lysis buffer (25 mM Tris pH 7.5, 50 mM MgCl2, 250 mM NaCl, 1mM DTT, 0.5 mg/ml cycloheximide, 0.1 mg/ml heparin). The lysates were centrifuged at 13,200 rpm at 4 °C for 5 minutes and the supernatant was collected. The samples containing 38μ of RNA was layered gently on top of a 10–25% w/w sucrose gradient (50 mM Tris pH 7.5, 50 mM MgCl2, 250 mM NaCl, 0.1 mg/ml heparin, 0.5 mg/ml cycloheximide in 5 ml polyallomer tube) and centrifuged at 37,000 rpm at 4°C for 150 minutes in a himac CP-NX ultracentrifuge using a P50AT rotor. Samples were fractionated from top to bottom, and absorbance at OD260 nm was analyzed by a Plate reader (EnSpire). Experiments were repeated at least 3 times with independent cohorts of flies.

Puromycin analysis

13 day-old flies were starved for 6 hr and fed 600 μM puromycin (Sigma) or 600 μM puromycin/35 mM cycloheximide (Sigma) in 5% sucrose solution for 20 hr. Incorporated puromycin was quantified by western blot with anti-puromycin antibody and normalized with actin. Experiments were repeated at least 3 times with independent cohorts of flies.

Climbing assay

The climbing assay was performed as previously described²⁴. Flies were placed in an empty plastic vial (2.5 cm in diameter × 10 cm in length). The vial was gently tapped to knock the flies to the bottom, and the number of flies reached to the top, middle and bottom areas of the vials in 10 s was counted. Experiments were repeated 10 times, and the mean percentage of flies in each area and standard deviations was calculated. Experiments were repeated with independent cohorts more than three times, and a representative result was shown.

Statistics

The number of replicates, what n represents, precision measurements, and the meaning of error bars are indicated in Figure Legends. Data are shown as means ± SEM. For pairwise comparisons, Student’s t-test was performed with Microsoft Excel (Microsoft). For multiple comparisons, data were analyzed using one-way ANOVA with Tukey’s HSD multiple-comparisons test in the GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA). Results with a p-value of less than 0.05 were considered to be statistically significant.

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1

The authors thank the Bloomington stock center; TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947); the Kyoto Drosophila Stock Center and the Vienna Drosophila RNAi Center for fly stocks. The authors thank Dr. Masayuki Miura from Department of Pharmaceutical Science, University of Tokyo, for proteasome activity assay protocol; Drs. Shin-ichi Hisanaga and Adam Weitemier from the Department of Biological Sciences, Tokyo Metropolitan University, for critical comments; Dr. Taro Saito, Dr. Akiko Asada from the Department of Biological Sciences, Tokyo Metropolitan University, and Dr. Michiko Sekiya from Department of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology, for technical supports.

Declaration of interests

The authors declare no competing interests.

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