1. Cell Biology

Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2β

  1. Kanako Shinno
  2. Yuri Miura
  3. Koichi M Iijima
  4. Emiko Suzuki
  5. Kanae Ando  Is a corresponding author
  1. Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Japan
  2. Research Team for Mechanism of Aging, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Japan
  3. Department of Neurogenetics, National Center for Geriatrics and Gerontology, Japan
  4. Department of Experimental Gerontology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan
  5. Gene Network Laboratory, National Institute of Genetics and Department of Genetics, SOKENDAI, Japan
  6. Department of Biological Sciences, School of Science, Tokyo Metropolitan University, Japan
https://doi.org/10.7554/eLife.95576.5
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Cite this article
  1. Kanako Shinno
  2. Yuri Miura
  3. Koichi M Iijima
  4. Emiko Suzuki
  5. Kanae Ando
(2026)
Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2β
eLife 13:RP95576.
https://doi.org/10.7554/eLife.95576.5
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9 figures, 3 tables and 3 additional files

Figures

Figure 1 with 1 supplement
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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 was quantified from 918 to 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.

Figure 1—figure supplement 1
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Ubiquitinated proteins in brains with neuronal knockdown of milton at 1-day-old.

Brains dissected at 1-day-old were immunostained with an antibody against ubiquitin. Representative images (left) and quantitation of the number of ubiquitin-positive puncta (right) are shown. Scale bars of hemibrains, 100 µm, Means ± SE, n=8. N.S., p>0.05 (Student’s t-test).

Figure 2
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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).

Figure 2—source data 1

PDF file containing original western blots for Figure 2, indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig2-data1-v1.zip
Figure 2—source data 2

Original files for western blot analysis displayed in Figure 2.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig2-data2-v1.zip
Figure 3
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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 14 days old.

Figure 3—source data 1

PDF file containing original western blots for Figure 3 indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig3-data1-v1.zip
Figure 3—source data 2

Original files for western blot analysis displayed in Figure 3.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig3-data2-v1.zip
Figure 4
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milton knockdown upregulates eIF2β in young flies.

(A) Timing of proteome analysis and phenotypes observed in milton knockdown flies. (B) and (C) Volcano plots of the log2 abundance ratio (x-axis) against the -log10 p-value (y-axis) of proteins at 7 days old (B) and 21 days 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) Western blotting of head extracts of wild-type flies with an anti-eIF2β antibody. Flies were 7-, 21-, 35-, 49-, and 63-day-old. Representative blots (left) and quantitation (right) are shown. Tubulin was used as a loading control. Means ± SE, n=3, *p<0.05 (one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test).

Figure 4—source data 1

PDF file containing original western blots for Figure 4, indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig4-data1-v1.zip
Figure 4—source data 2

Original files for western blot analysis displayed in Figure 4.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig4-data2-v1.zip
Figure 5
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milton knockdown decreases phosphorylation of eIF2α.

(A, B) 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. (C) A schematic representation of the axon (Lobe tips), the cell body region (Kenyon cells), and dendritic region (Calyxes) in the fly brain. Scale bars, 100 µm. (D, E) Immunostaining with anti-eIF2α and anti-p-eIF2α antibodies. The mushroom body was identified by expression of mito-GFP. Scale bars, 20 µm. 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).

Figure 5—source data 1

PDF file containing original western blots for Figure 5 indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig5-data1-v1.zip
Figure 5—source data 2

Original files for western blot analysis displayed in Figure 5.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig5-data2-v1.zip
Figure 6
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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.

Figure 6—source data 1

PDF file containing original western blots for Figure 6, indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig6-data1-v1.zip
Figure 6—source data 2

Original files for western blot analysis displayed in Figure 6.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig6-data2-v1.zip
Figure 7 with 1 supplement
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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).

Figure 7—source data 1

PDF file containing original western blots for Figure 7 indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig7-data1-v1.zip
Figure 7—source data 2

Original files for western blot analysis displayed in Figure 7.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig7-data2-v1.zip
Figure 7—figure supplement 1
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Histology analysis of fly heads with eIF2β overexpression.

The morphology of the eye with eIF2β overexpression. The dotted lines indicate the retina. Vacuoles are indicated by arrows. Representative images (left) and quantification of vacuole area (right). The flies were 40-day-old. Means ± SE, n=5–13 N.S., p>0.05 (Student’s t-test).

Figure 8 with 1 supplement
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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).

Figure 8—source data 1

PDF file containing original western blots for Figure 8, indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig8-data1-v1.zip
Figure 8—source data 2

Original files for western blot analysis displayed in Figure 8.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig8-data2-v1.zip
Figure 8—figure supplement 1
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Lowering the eIF2β level does not affect the levels of eIF2α and p-eIF2α.

(A, B) Blotting was performed 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. (C) 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-day-old. Means ± SE, n=3. N.S., p>0.05; *p<0.05 (Student’s t-test).

Figure 8—figure supplement 1—source data 1

PDF file containing original western blots for Figure 8—figure supplement 1 indicating the relevant bands.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig8-figsupp1-data1-v1.zip
Figure 8—figure supplement 1—source data 2

Original files for western blot analysis displayed in Figure 8—figure supplement 1.

https://cdn.elifesciences.org/articles/95576/elife-95576-fig8-figsupp1-data2-v1.zip
Figure 9
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The mitochondria-eIF2β axis in the axon maintains neuronal proteostasis during aging.

Aging is associated with a reduction in axonal transport of mitochondria. Our results suggest that the loss of axonal mitochondria leads to an increase in eIF2β, while the upregulation of eIF2β decreases autophagy-mediated protein degradation and promotes aging.

Tables

Table 1
Differentially expressed proteins in milton RNAi fly brains compared to control at 7 day-old detected by proteome analysis.
7-day-old
Accession*NameAbundance ratio:(7 days, milton KD)/ (7 days, control)Abundance ratio p-value: (7 days, milton KD) / (7 days, control)
Q9V751Attacin-B1001E-17
Q04448Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrial1001E-17
P22700Calcium-transporting ATPase sarcoplasmic/endoplasmic reticulum type1001E-17
Q9V558Cytochrome P450 4p11001E-17
P10552FMRFamide-related peptides1001E-17
P05661-19Isoform F of Myosin heavy chain, muscle1001E-17
Q9VE01Probable cytochrome P450 12a5, mitochondrial1001E-17
Q7KIN0Toll-like receptor 71001E-17
Q8MKN0Ubiquinone biosynthesis protein COQ9, mitochondrial1001E-17
Q9VJG0Xaa-Pro aminopeptidase ApepP1001E-17
Q9V8F5Bomanin Bicipital 14.9081E-17
P07701Salivary glue protein Sgs-52.8431.99252E-09
O76902Pleckstrin homology domain-containing family F member 1 homolog2.8362.22045E-16
P81641Alpha-amylase B2.6847.44847E-09
P19351-4Isoform 4 of Troponin T, skeletal muscle2.666.65563E-11
Q9VTJ8Mitochondrial import inner membrane translocase subunit TIM142.613.54205E-07
P41375Eukaryotic translation initiation factor 2 subunit 22.4653.38486E-09
Q9VYB0Selenoprotein BthD2.4622.25579E-08
B7Z0W9Proton channel OtopLc2.3821.71741E-06
Q9VLR5RNA polymerase II transcriptional coactivator2.2456.70245E-09
Q8IN44Protein Turandot A2.1278.38662E-13
P27779Pupal cuticle protein Edg-78E2.1131.00215E-08
Q9W1X8Probable GDP-L-fucose synthase0.4962.12601E-08
P5503526 S proteasome non-ATPase regulatory subunit 40.4875.50158E-11
Q9VHN639 S ribosomal protein L19, mitochondrial0.4870.000551337
Q9VPD2Cytosolic Fe-S cluster assembly factor NUBP2 homolog0.466.34514E-05
Q9VHD3Probable maleylacetoacetate isomerase 10.4324.50956E-06
Q94529Probable pseudouridine-5'-phosphatase0.4161.08802E-14
Q27606Cytochrome P450 4e20.3984.13128E-10
Q24388Larval serum protein 20.3781.33227E-14
Q9VKH6Lysosomal thioesterase PPT2 homolog0.3692.05225E-06
Q24114Division abnormally delayed protein0.3072.24406E-09
Q95NH6Attacin-C0.011E-17
P29993Inositol 1,4,5-trisphosphate receptor0.011E-17
Q94526Open rectifier potassium channel protein 10.011E-17
Q9Y115UNC93-like protein0.011E-17
21-day-old
Accession*NameAbundance ratio:(21 days, milton KD) /(21 days, control)Abundance ratio p-value:(21 days, milton KD) /(21 days, control)
Q10714Angiotensin-converting enzyme1001E-17
C0HKQ8Cecropin-A21001E-17
Q9V558Cytochrome P450 4p11001E-17
P51592E3 ubiquitin-protein ligase hyd1001E-17
Q9VMJ7Lysine-specific demethylase lid1001E-17
Q9VXP4Platelet-activating factor acetylhydrolase IB subunit beta homolog1001E-17
Q9VY28Probable 28 S ribosomal protein S25, mitochondrial1001E-17
Q9W391Probable phosphorylase b kinase regulatory subunit alpha1001E-17
Q9VUQ5Protein argonaute-21001E-17
P54359Septin-21001E-17
P24492Diptericin A15.7161E-17
Q9VVY3Glycogen-binding subunit 76 A8.9861E-17
Q70PY2Peptidoglycan-recognition protein SB16.6691E-17
Q9W0M1Centrosomal protein cep2906.5261E-17
P81641Alpha-amylase B5.7221E-17
P45884Attacin-A4.9971E-17
C0HL66Histone H3.3A4.7781E-17
P26675Protein son of sevenless4.6961E-17
P02515Heat shock protein 224.691E-17
Q95NH6Attacin-C4.351E-17
P17971-1Isoform A of Potassium voltage-gated channel protein Shal4.1951E-17
Q7K1U0Activity-regulated cytoskeleton associated protein 13.2711E-17
P14199Protein ref(2)P3.0141E-17
Q9VU02Probable small nuclear ribonucleoprotein Sm D12.434.91607E-13
Q9VD44Poly(A) RNA polymerase gld-2 homolog A2.2682.6084E-11
Q9V8F5Bomanin Bicipital 12.245.16671E-11
P22979Heat shock protein 67B32.2237.90048E-11
P27779Pupal cuticle protein Edg-78E2.1921.65944E-10
Q9NBK5Serine/threonine-protein kinase tricornered2.0594.15071E-09
Q8MLZ7Chitinase-like protein Idgf32.0554.61182E-09
Q9V751Attacin-B2.0386.79416E-09
Q9V8M5Probable 3-hydroxyisobutyrate dehydrogenase, mitochondrial0.4927.42952E-09
P84345ATP synthase protein 80.4211.809E-12
P33438Glutactin0.4146.13731E-13
Q8IN44Protein Turandot A0.2181E-17
Q8IN43Protein Turandot C0.1951E-17
Q9VFI9cGMP-specific 3',5'-cyclic phosphodiesterase0.011E-17
Q94526Open rectifier potassium channel protein 10.011E-17
Q9VHD3Probable maleylacetoacetate isomerase 10.011E-17
Q9W0A0Protein draper0.011E-17
A1Z7T0Serine/threonine-protein kinase N0.011E-17
  1. *

    UniProt accession number.

Table 2
Molecule networks based on “Interaction search” of KeyMolnet.
7-day-old
RankNameScoreScore (p)*Score (v)†Score (c)‡
1Autophagy-related protein signaling pathway50.3946.76E-160.1590.11
2Calcium signaling pathway47.5834.75E-150.1460.117
3Transcriptional regulation by SMAD44.0125.64E-140.1460.095
4GABA signaling pathway40.7065.58E-130.1220.123
5estrogen signaling pathway37.5075.12E-120.110.13
6Sirtuin signaling pathway36.877.96E-120.1220.095
7Transcriptional regulation by AP-134.8743.18E-110.110.107
8Arrestin signaling pathway32.841.30E-100.110.092
9G protein (Gq/11) signaling pathway30.8895.03E-100.0850.149
10Kainate receptor signaling pathway30.0499.00E-100.0730.214
11Transcriptional regulation by C/EBP29.51.32E-090.0980.093
12Calpain signaling pathway28.5972.46E-090.110.066
13Phospholipase D signaling pathway28.3442.94E-090.0980.084
14HSP90 signaling pathway27.1886.54E-090.0850.104
14CYP family27.1886.54E-090.0850.104
16Kir3 channel signaling pathway26.4951.06E-080.0610.25
17Estrogen biosynthesis26.1071.38E-080.0610.238
18CaSR signaling pathway25.392.27E-080.0610.217
19PI3K signaling pathway24.9273.14E-080.0730.122
20PAF receptor signaling pathway24.5554.06E-080.0490.4
21Transcriptional regulation by PPARa24.3984.52E-080.0730.115
21BTK signaling pathway24.3984.52E-080.0730.115
23Transcriptional regulation by STAT24.0765.66E-080.0850.077
24G protein (Gi/o) signaling pathway24.0635.71E-080.0730.111
25PARP signaling pathway23.7427.13E-080.0730.107
25mGluR signaling pathway23.7427.13E-080.0730.107
27Free fatty acid signaling pathway23.4338.83E-080.0730.103
28Kir channel signaling pathway23.3389.43E-080.0610.167
29Oxytocin signaling pathway23.3279.51E-080.0490.333
30Transcriptional regulation by MEF222.991.20E-070.0730.098
31S100 family signaling pathway22.4341.77E-070.0730.092
32Transcriptional regulation by FOXO22.3011.94E-070.0730.091
33P2Y signaling pathway22.1722.12E-070.0610.143
34Transcriptional regulation by SRF21.1744.23E-070.0610.125
34ATF4/ATF6/IRE1 signaling pathway21.1744.23E-070.0610.125
36Chemerin signaling pathway21.0824.50E-070.0490.235
36Vasopressin signaling pathway21.0824.50E-070.0490.235
38Serotonin signaling pathway20.8545.28E-070.0730.077
39Transcriptional regulation by HIF20.8345.35E-070.0980.043
40Leukotriene receptor signaling pathway20.7245.78E-070.0490.222
40CART signaling pathway20.7245.78E-070.0490.222
42MAPK signaling pathway20.6935.90E-070.0850.055
43Transcriptional regulation by RB/E2F20.5436.55E-070.0980.042
44NAD metabolism20.4686.89E-070.0610.114
45ERK signaling pathway20.4257.11E-070.0730.073
46Adenylyl Cyclase signaling pathway20.3037.73E-070.0610.111
47Bile acid signaling pathway20.1418.65E-070.0610.109
21-day-old
RankNameScoreScore (p)*Score (v)†Score (c)‡
1Histone demethylation84.1984.51E-260.1020.425
2CDK inhibitor signaling pathway56.4979.83E-180.0780.295
3Transcriptional regulation by RB/E2F46.5989.39E-150.1080.095
4Mst(Hippo) signaling pathway46.3431.12E-140.090.133
5Transcriptional regulation by androgen receptor46.0781.35E-140.0780.178
6p160 SRC signaling pathway45.8091.62E-140.0780.176
7Transcriptional regulation by SMAD43.9615.84E-140.090.119
8Autophagy-related protein signaling pathway41.0634.35E-130.0840.119
9Transcriptional regulation by HIF39.5271.26E-120.0960.086
10Nucleophosmin signaling pathway38.4172.72E-120.0540.273
11HSP90 signaling pathway37.8873.93E-120.0660.164
12PAF metabolism37.5624.93E-120.0420.5
13Transcriptional regulation by STAT37.2766.01E-120.0720.132
14Bcl-2 family signaling pathway36.1571.31E-110.0720.124
15Sirtuin signaling pathway34.7823.39E-110.0720.114
16Transcriptional regulation by C/EBP33.8196.60E-110.0660.128
17PIN1 signaling pathway33.1721.03E-100.060.149
18RSK signaling pathway30.5666.29E-100.060.125
19Transcriptional regulation by High mobility group protein29.8731.02E-090.0540.148
20BET family signaling pathway29.6561.18E-090.0540.145
21Transcriptional regulation by Myc28.8382.08E-090.0660.093
22Transcriptional regulation by FOXO28.8272.10E-090.0540.136
23PSD-95 family signaling pathway26.1541.34E-080.0480.14
24AKT signaling pathway25.1692.65E-080.0480.129
25Arginine methylation24.7993.43E-080.0480.125
26gp130 signaling pathway24.255.01E-080.0540.096
27Transcriptional regulation by CREB23.8586.58E-080.0660.067
28Gene regulation by microRNAs (metastasis)23.8526.60E-080.0540.093
29HDAC signaling pathway23.5368.22E-080.0360.207
30Calpain signaling pathway23.0921.12E-070.060.074
31Transcriptional regulation by IRF22.7381.43E-070.0540.085
322-Oxoglutarate signaling pathway22.6731.50E-070.0480.104
3214-3-3 signaling pathway22.6731.50E-070.0480.104
34Transcriptional regulation by POU domain factor22.6011.57E-070.060.071
35Transcriptional regulation by BLIMP-122.4741.72E-070.0420.132
36Gene regulation by microRNAs (metabolism)22.391.82E-070.0540.083
37Fatty acid beta oxidation22.0962.23E-070.0420.127
38Transcriptional regulation by RXR22.082.26E-070.0360.176
39ERK signaling pathway21.962.45E-070.0480.098
40PARP signaling pathway21.9132.53E-070.0420.125
41Transcriptional regulation by VDR21.6183.11E-070.0540.078
42Transcriptional regulation by p5321.1684.24E-070.0720.05
43Acetylcholine metabolism21.1524.29E-070.0240.444
44Gene regulation by microRNAs (embryonic stem cells)21.084.51E-070.0360.158
45mTOR signaling pathway21.0484.61E-070.0420.115
46Gene regulation by microRNAs (cancer)21.044.64E-070.0480.09
47Transcriptional regulation by Ets-1/220.7245.77E-070.0420.111
48MAPK signaling pathway20.4117.18E-070.0540.07
49Gene regulation by microRNAs (cell cycle)20.4047.21E-070.0360.146
50Transcriptional regulation by p7320.2597.97E-070.0420.106
  1. *

    Score(p) indicates p-value of the pathway.

  2. †

    Score(v) indicates the ratio of ‘Count’ to total molecules associated with the loaded list.

  3. ‡

    Score(c) indicates the ratio of ‘Count’ to total molecules contained in the pathway.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Drosophila)UAS-milton RNAiVienna Drosophila Resource Center (VDRC)VDRC:v41508, FLYB:FBst0464139
Strain, strain background (Drosophila)UAS-Miro RNAiIijima-Ando et al., 2012
Strain, strain background (Drosophila)UAS-luciferase RNAiIijima-Ando et al., 2012
Strain, strain background (Drosophila)UAS-Pfk RNAiBloomington Drosophila Stock CenterBDSC:36782, FLYB:FBti0146432
Strain, strain background (Drosophila)UAS-luciferase RNAiBloomington Drosophila Stock CenterBDSC:31603, FLYB:FBti0130444
strain, strain background (Drosophila)UAS-eIF2βBloomington Drosophila Stock CenterBDSC:17425, FLYB:FBti0038792
Strain, strain background (Drosophila)UAS-GFPBloomington Drosophila Stock CenterBDSC:1521, FLYB:FBti0003040
Strain, strain background (Drosophila)eIF2β[PBac{SAstopDsRed} LL07719]KYOTO Drosophila Stock Center (DGRC)DGRC:142114, FLYB:FBgn0004926
Strain, strain background (Drosophila)w1118Vienna Drosophila Resource Center (VDRC)VDRC:60000
Strain, strain background (Drosophila)UAS-mitoGFPM. Saxton, University of California, Santa Cruz
Strain, strain background (Drosophila)elav-GAL4Bloomington Drosophila Stock CenterBDSC:458,
FLYB:FBti0002575
Strain, strain background (Drosophila)GMR-gal4Bloomington Drosophila Stock CenterBDSC:1104,
FLYB:FBti0002994
Antibodyanti-ubiquitin antibody
Ubi-1		Thermo FisherCat#:13–1600,
RRID:AB_2533002		IHC:1:50
Antibodyanti-LC3 antibody Atg8Merck MilliporeCat#:ABC974,
RRID:AB_2939040		WB:1:1000
Antibodyanti-p62 antibody Ref2PAbcamCat#:ab178440,
RRID:AB_2938801		WB:1:750
Antibodyanti-eIF2αAbcamCat#:ab26197,
RRID:AB_2096478		IHC:1:50
Antibodyanti-p-eIF2αCell signalingCat#:3398 S,
RRID:AB_2096481		IHC:1:50
Antibodyanti-Drosophila eIF2βThis paperWB:1:1500
Antibodyanti-puromycinEnzoCat#:CAC-CAC-PEN-MA001, RRID:AB_2620162WB:1:1000
Antibodyanti-actinSigmaCat#:A2066, RRID:AB_476693WB:1:3000
Antibodyanti-β tubulinSigmaCat#:T9026, RRID:AB_477593WB:1:10000
Antibodyperoxidase-conjugated goat anti-mouse IgG antibodyDakoCat#:P0447, RRID:AB_2617137WB:1:2000
Antibodyperoxidase-conjugated
pig anti-rabbit IgG antibody		DakoCat#:P0399, RRID:AB_2617141WB:1:2000
Commercial Assay
or Kit		20 S Proteasome Substrate (SUC-LLVY-AMC)CaymanCat#:10011095
Commercial Assay
or Kit		ATP Determination KitInvitrogenCat#:A22066

Additional files

Supplementary file 1

Excel file containing a list of proteins detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

https://cdn.elifesciences.org/articles/95576/elife-95576-supp1-v1.xlsx
Supplementary file 2

Excel file containing a list of fly genotypes used in this study.

https://cdn.elifesciences.org/articles/95576/elife-95576-supp2-v1.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/95576/elife-95576-mdarchecklist1-v1.docx

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  1. Kanako Shinno
  2. Yuri Miura
  3. Koichi M Iijima
  4. Emiko Suzuki
  5. Kanae Ando
(2026)
Axonal distribution of mitochondria maintains neuronal autophagy during aging via eIF2β
eLife 13:RP95576.
https://doi.org/10.7554/eLife.95576.5