AXONAL distribution of mitochondria maintains neuronal autophagy during aging via eIF2β

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging.

Reviewer #2 (Public Review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of Milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria.

The manuscript has several weaknesses. The reader should take extra care while reading this manuscript and when acknowledging the findings and the model in this manuscript.

The defect in autophagy by the depletion of axonal mitochondria is one of the main claims in the paper. The authors should work more on describing their results of LC3-II/LC3-I ratio, as there are multiple ways to interpret the LC3 blotting for the autophagy assessment. Lysosomal defects result in the accumulation of LC3-II thus the LC3-II/LC3-I ratio gets higher. On the other hand, the defect in the early steps of autophagosome formation could result in a lower LC3-II/LC3-I ratio. From the results of the actual blotting, the LC3-I abundance is the source of the major difference for all conditions (Milton RNAi and eIF2β overexpression and depletion).

Another main point of the paper is the up-regulation of eIF2β by depleting the axonal mitochondria leads to the proteostasis crisis. This claim is formed by the findings from the proteome analyses. The authors should have presented their proteomic data with much thorough presentation and explanation. As in the experiment scheme shown in Figure 4A, the author did two proteome analyses: one from the 7-day-old sample and the other from the 21-day-old sample. The manuscript only shows a plot of the result from the 7-day-old sample, but that of the result from the 21-day-old sample. For the 21-day-old sample, the authors only provided data in the supplemental table, in which the abundance ratio of eIF2β from the 21-day-old sample is 0.753, meaning eIF2β is depleted in the 21-day-old sample. The authors should have explained the impact of the eIF2β depletion in the 21-day-old sample, so the reader could fully understand the authors' interpretation of the role of eIF2β on proteostasis.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging. To further support the authors' claims, several improvements are necessary, particularly in the methods of quantification and the points that should be demonstrated quantitatively. It is crucial to investigate the correlation between aging and the proteins eIF2β and eIF2α.

Thank you so much for your review and comments. We included analyses of protein levels of eIF2α, eIF2β, and eIF2γ at 7 days and 21 days (Figure 4D). The manuscript was revised as below;

Lines 242-245 ‘As for the other subunits of eIF2 complex, proteome analysis did not detect a significant difference in the protein levels of eIF2α and eIF2γ between milton knockdown and control flies at 7 and 21 days (Figure 4D).’

Reviewer #2 (Public Review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of Milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria.

The manuscript has several weaknesses. The reader should take extra care while reading this manuscript and when acknowledging the findings and the model in this manuscript.

The defect in autophagy by the depletion of axonal mitochondria is one of the main claims in the paper. The authors should work more on describing their results of LC3-II/LC3-I ratio, as there are multiple ways to interpret the LC3 blotting for the autophagy assessment. Lysosomal defects result in the accumulation of LC3-II thus the LC3-II/LC3-I ratio gets higher. On the other hand, the defect in the early steps of autophagosome formation could result in a lower LC3-II/LC3-I ratio. From the results of the actual blotting, the LC3-I abundance is the source of the major difference for all conditions (Milton RNAi and eIF2β overexpression and depletion). In the text, the authors simply state the observation of their LC3 blotting. The manuscript lacks an explanation of how to evaluate the LC3-II/LC3-I ratio. Also, the manuscript lacks an elaboration on what the results of the LC3 blotting indicate about the state of autophagy by the depletion of axonal mitochondria.

Thank you for pointing it out, and we apologize for an insufficient description of the result. We included quantitation of the levels of LC3-I and LC3-II in Figure 2A, 2D, 3D, 6B and 7B. As the reviewer pointed out, changes in the LC3-II/LC3-I ratio do not necessarily indicate autophagy defects. However, since p62 accumulation (Figure 2B, 2E, 3E, 6C, 7C in the original manuscript), these results collectively suggest that autophagy is lowered. We revised the manuscript to include this discussion as below:

Lines 174-186 ‘During autophagy progression, LC3 is conjugated with phosphatidylethanolamine to form LC3-II, which localizes to isolation membranes and autophagosomes. LC3-I accumulation occurs when autophagosome formation is impaired, and LC3-II accumulation is associated with lysosomal defects(31,32). p62 is an autophagy substrate, and its accumulation suggests autophagic defects(31,32). We found that milton knockdown increased LC3-I, and the LC3-II/LC3-I ratio was lower in milton knockdown flies than in control flies at 14-day-old (Figure 2A). We also analyzed p62 levels in head lysates sequentially extracted using detergents with different stringencies (1% Triton X-100 and 2% SDS). Western blotting revealed that p62 levels were increased in the brains of 14-day-old of milton knockdown flies (Figure 2B). The increase in the p62 level was significant in the Triton X-100-soluble fraction but not in the SDS-soluble fraction (Figure 2B), suggesting that depletion of axonal mitochondria impairs the degradation of less-aggregated proteins.’

Line 189-190 : ‘At 30 day-old, LC3-I was still higher, and the LC3-II/LC3-I ratio was lower, in milton knockdown compared to the control (Figure 2D).’

Line 199-201: ‘However, in contrast with milton knockdown, Pfk knockdown did not affect the levels of LC3-I, LC3-II or the LC3-II/LC3-I ratio (Figure 3D).’

Line 275-281: ‘Neuronal overexpression of eIF2β increased LC3-II, while the LC3-II/LC3-I ratio was not significantly different (Figure 6A and B). Overexpression of eIF2β significantly increased the p62 level in the Triton X-100-soluble fraction (Figure 6C, 4-fold vs. control, p < 0.005 (1% Triton X-100)) but not in the SDS-soluble fraction (Figure 6C, 2-fold vs. control, p = 0.062 (2% SDS)), as observed in brains of milton knockdown flies (Figure 2B). These data suggest that neuronal overexpression of eIF2β accumulates autophagic substrates.’

Line 307-315: ‘Neuronal knockdown of milton causes accumulation of autophagic substrate p62 in the Triton X-100-soluble fraction (Figure 2B), and we tested if lowering eIF2β ameliorates it. We found that eIF2β heterozygosity caused a mild increase in LC3-I levels and decreases in LC3-II levels, resulting in a significantly lower LC3-II/LC3-I ratio in milton knockdown flies (Figure 7B). eIF2β heterozygosity decreased the p62 level in the Triton X-100-soluble fraction in the brains of milton knockdown flies (Figure 7C). The p62 level in the SDS-soluble fraction, which is not sensitive to milton knockdown (Figure 2B), was not affected (Figure 7C). These results suggest that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.’

Another main point of the paper is the up-regulation of eIF2β by depleting the axonal mitochondria leads to the proteostasis crisis. This claim is formed by the findings from the proteome analyses. The authors should have presented their proteomic data with much thorough presentation and explanation. As in the experiment scheme shown in Figure 4A, the author did two proteome analyses: one from the 7-day-old sample and the other from the 21-day-old sample. The manuscript only shows a plot of the result from the 7-day-old sample, but that of the result from the 21-day-old sample. For the 21-day-old sample, the authors only provided data in the supplemental table, in which the abundance ratio of eIF2β from the 21-day-old sample is 0.753, meaning eIF2β is depleted in the 21-day-old sample. The authors should have explained the impact of the eIF2β depletion in the 21-day-old sample, so the reader could fully understand the authors' interpretation of the role of eIF2β on proteostasis.

Thank you for pointing it out. We included plots of the results of 21-day-old proteome as a part of the main figure (Figure 4C). As the reviewer pointed out, eIF2β protein levels are reduced at the 21-day-old. Since a reduction in the eIF2_β_ ameliorated milton knockdown-induced locomotor defects in aged flies (Figure 7D), the reduction in eIF2β observed in the 21-day-old milton knockdown flies is not likely to negatively contribute to milton knockdown-induced defects. We included this discussion in the manuscript as below:

Lines 337-341:‘eIF2β protein levels are reduced at the 21-day-old; however, since a reduction in the eIF2β ameliorated milton knockdown-induced locomotor defects in aged flies (Figure 7), the reduction in eIF2β observed in the 21-day-old is not likely to negatively contribute to milton knockdown-induced defects.’

The manuscript consists of several weaknesses in its data and explanation regarding translation.

(1) The authors are likely misunderstanding the effect of phosphorylation of eIF2α on translation. The P-eIF2α is inhibitory for translation initiation. However, the authors seem to be mistaken that the down-regulation of P-eIF2α inhibits translation.

We are sorry for our insufficient explanation in the previous version. As the reviewer pointed out, it is well known that the phosphorylated form of eIF2α inhibits translation initiation. Neuronal knockdown of milton caused a reduction in p-eIF2α (Figure 4J and K), and it also lowered translation (Figure 5); the relationship between these two events is currently unclear. We do not think that a reduction in the p-eIF2α suppressed translation; rather, we propose that the unbalance of expression levels of the components of eIF2 complexes negatively affects translation. We revised discussion sections to describe our interpretation more in detail as below:

Line 368-378: ‘eIF2β is a component of eIF2, which meditates translational regulation and ISR initiation. When ISR is activated, phosphorylated eIF2α suppresses global translation and induces translation of ATF4, which mediates transcription of autophagy-related genes(39,40). Since ISR can positively regulate autophagy, we suspected that suppression of ISR underlies a reduction in autophagic protein degradation. We found neuronal knockdown of milton reduced phosphorylated eIF2α, suggesting that ISR is reduced (Figure 4). However, we also found that global translation was reduced (Figure 5). It may be possible that increased levels of eIF2β disrupt the eIF2 complex or alter its functions. The stoichiometric mismatch caused by an imbalance of eIF2 components may inhibit ISR induction. Supporting this model, we found that eIF2β upregulation reduced the levels of p-eIF2α (Figure 6).’

We have revised the graphical abstract and removed the eIF2 complex since its role in the loss of proteostasis caused by milton knockdown has not been elucidated yet.

(2) The result of polysome profiling in Figure 4H is implausible. By 10%-25% sucrose density gradient, polysomes are not expected to be observed. The authors should have used a gradient with much denser sucrose, such as 10-50%.

Thank you for pointing it out. It was a mistake of 10-50%, and we apologize for the oversight. It was corrected (Figure 5).

(3) Also on the polysome profiling, as in the method section, the authors seemed to fractionate ultra-centrifuged samples from top to bottom and then measured A260 by a plate reader. In that case, the authors should have provided a line plot with individual data points, not the smoothly connected ones in the manuscript.

Thank you for pointing it out. We revised the graph (Figure 5).

(4) For both the results from polysome profiling and puromycin incorporation (Figure 4H and I), the difference between control siRNA and Milton siRNA are subtle, if not nonexistent. This might arise from the lack of spatial resolution in their experiment as the authors used head lysate for these data but the ratio of Phospho-eIF2α/eIF2α only changes in the axons, based on their results in Figure 4E-G. The authors could have attempted to capture the spatial resolution for the axonal translation to see the difference between control siRNA and Milton siRNA.

Thank you for your comment. We agree that it would be an interesting experiment, but it will take a considerable amount of time to analyze axonal translation with spatial resolution. We will try to include such analyses in the future. For this manuscript, we revised the discussion section to include the reviewer's suggestion as below;

Lines 351-353: ‘Further analyses to dissect the effects of milton knockdown on proteostasis and translation in the cell body and axon by experiments with spatial resolution would be needed.’

Recommendations for the authors:

From the Reviewing Editor:

As the Reviewing Editor, I have read your manuscript and the associated peer reviews. I have concerns about publishing this work in its current form. I think that your manuscript cannot claim to have found a novel function of eIF2beta because of technical uncertainties and conceptual problems that should be addressed.

Thank you so much for your review and comments. We addressed all the concerns raised by the reviewers. Point-by-point responses are listed below.

First, your manuscript is based partly on what appears to be a mistaken understanding of the mechanistic basis of the ISR. Specifically, eIF2 is a heterotrimeric complex of alpha, beta, and gamma subunits. When eIF2a is phosphorylated, the heterotrimer adopts a new conformation. This conformation directly binds and inhibits eIF2B, the decameric GEF that exchanges the GDP bound to the gamma subunit of the eIF2 complex for GTP. Unless I misunderstood your paper, you seem to propose that decreasing levels of phospho-eIF2a will inhibit translation, but this is backward from what we know about the ISR.

Thank you for your insightful comment, and we are sorry for the confusion. We did not mean to propose that decreasing levels of phospho-eIF2_a_ inhibits translation. We apologize for our insufficient explanation, which might have caused a misunderstanding (Lines 312-318 in the original version). We agree with the reviewer that ‘mismatch due to elevated eIF2-beta could change the behavior of the ISR’. We revised the text in the result section as follows:

Lines 259-264 (in the Result section) ‘Phosphorylation of eIF2α induces conformational changes in the eIF2 complex and inhibits global translation(36). To analyze the effects of milton knockdown on translation, we performed polysome gradient centrifugation to examine the level of ribosome binding to mRNA. Since p-eIF2α was downregulated, we hypothesized that milton knockdown would enhance translation. However, unexpectedly, we found that milton knockdown significantly reduced the level of mRNAs associated with polysomes (Figure 5A and B).’

Lines 368-378 (in the Discussion section): ‘eIF2β is a component of eIF2, which meditates translational regulation and ISR initiation. When ISR is activated, phosphorylated eIF2α suppresses global translation and induces translation of ATF4, which mediates transcription of autophagy-related genes(39,40). Since ISR can positively regulate autophagy, we suspected that suppression of ISR underlies a reduction in autophagic protein degradation. We found neuronal knockdown of milton reduced phosphorylated eIF2α, suggesting that ISR is reduced (Figure 4). However, we also found that global translation was reduced (Figure 5). It may be possible that increased levels of eIF2β disrupt the eIF2 complex or alter its functions. The stoichiometric mismatch caused by an imbalance of eIF2 components may inhibit ISR induction. Supporting this model, we found that eIF2β upregulation reduced the levels of p-eIF2α (Figure 6).’

It may be possible that a stoichiometric mismatch due to elevated eIF2-beta could change the behavior of the ISR, but your paper doesn't adequately address the expression levels of all three eIF2 subunits: alpha, beta, and gamma. The proteomic data shown in Fig 4B is unconvincing on its own because the changes in the beta subunit are subtle. The Western blot in Figure 4C suggests that the KD changes the mass or mobility of the beta subunit, and most importantly, there are no Western blots measuring the levels of eIF2a, eIF2a-phospho, or eIF2-gamma.

We appreciate the reviewer’s comment and agree that the stoichiometric mismatch due to elevated eIF2β may interfere with ISR. We found overexpression of eIF2β lowered p-eIF2 alpha (Figure S2 in V1), which supports this model. We included this data in the main figure in the revised manuscript (Figure 6D) and revised the text as below:

Lines 279-281: ‘Since milton knockdown reduced the p-eIF2α level (Figure 4K), we asked whether an increase in eIF2β affects p-eIF2α. Neuronal overexpression of eIF2β did not affect the eIF2α level but significantly decreased the p-eIF2α level (Figure 6D, E).’

Expression data of eIF2α and eIF2γ from proteomic analyses has been extracted from proteome analyses and included as a table (Figure 4D). Western blots of phospho-eIF2a (Figure S1 in V1) in the main figure (Figure 4G). The result section was revised as below;

Lines 242-245: ‘As for the other subunits of eIF2 complex, proteome analysis did not detect a significant difference in the protein levels of eIF2α and eIF2γ between milton knockdown and control flies at 7 and 21 days (Figure 4D).’

Reviewer #1 (Recommendations For The Authors):

L125-128: In this section, while the efficiency of Milton knockdown is referenced from a previous publication, it is necessary to also mention that the Miro knockdown has been similarly reported in the literature. Additionally, the Methods section lacks details on the Miro RNAi line used, and Table 2 does not include the genotype for Miro RNAi. This information should be included for clarity and completeness.

Thank you for pointing it out. Knockdown efficiency with this strain has been reported (Iijima-Ando et al., PLoS Genet, 2012). We revised the text to include citation and knockdown efficiency as follows:

Lines 139-147: ‘There was no significant increase in ubiquitinated proteins in milton knockdown flies at 1-day old, suggesting that the accumulation of ubiquitinated proteins caused by milton knockdown is age-dependent (Figure S1). We also analyzed the effect of the neuronal knockdown of Miro, a partner of milton, on the accumulation of ubiquitin-positive proteins. Since severe knockdown of Miro in neurons causes lethality, we used UAS-Miro RNAi strain with low knockdown efficiency, whose expression driven by elav-GAL4 caused 30% reduction of Miro mRNA in head extract(24). Although there was a tendency for increased ubiquitin-positive puncta in Miro knockdown brains, the difference was not significant (Figure 1B, p>0.05 between control RNAi and Miro RNAi). These data suggest that the depletion of axonal mitochondria induced by milton knockdown leads to the accumulation of ubiquitinated proteins before neurodegeneration occurs.’

L132-L136: The current phrasing in this section suggests an increase in ubiquitinated proteins for both Milton and Miro knockdowns. However, since there is no significant difference noted for Miro, it is incorrect to state an increase in ubiquitin-positive puncta. Furthermore, combining the results of Milton knockdown to claim an increase in ubiquitinated proteins prior to neurodegeneration is misleading. At the very least, the expression here needs to be moderated to accurately reflect the findings.

Thank you for pointing it out. We revised the text as above.

L137-L141: Results in Figure 1 indicate that Milton knockdown leads to an increase in ubiquitinated proteins at 14 days, while Miro knockdown shows no difference from the control at either 14 or 30 days. Conversely, both the control and Miro exhibit an increase in ubiquitinated proteins with aging, but this trend does not seem to apply to Milton knockdown. This observation suggests that Milton KD may not affect the changes in protein quality control associated with aging. It implies that Milton's function might be more related to protein homeostasis in younger cells, or that changes due to aging might overshadow the effects of Milton knockdown. These interpretations should be included in the Results or Discussion sections for a more comprehensive analysis.

Thank you for your insightful comment. We revised the text to include those points as follows:

Lines 152-153: ‘These results suggest that depletion of axonal mitochondria may have more impact on proteostasis in young neurons than in old neurons.’

Lines 355-362: ‘The depletion of axonal mitochondria and accumulation of abnormal proteins are both characteristics of aged brains(37,38). Our results suggest that the loss of axonal mitochondria is an event upstream of proteostasis collapse during aging. Neuronal knockdown of milton had more impact on proteostasis in young neurons than the old neurons (Figure 1). Proteome analyses also showed that age-related pathways, such as immune responses, are enhanced in young flies with milton knockdown (Table 2). The reduction in axonal transport of mitochondria may be one of the triggering events of age-related changes and accelerates the onset of aging in the brain.’

L143 : Please remove the erroneously included quotation mark.

Thank you for pointing it out. We corrected it.

L145-L147:

- While it is understood that Milton knockdown results in a reduction of mitochondria in axons, as reported previously and seemingly indicated in Figure 1E, this paper repeatedly refers to axonal depletion of mitochondria. Therefore, it would be beneficial to quantitatively assess the number of mitochondria in the axonal terminals located in the lamina via electron microscopy. Such quantification would robustly reinforce the argument that mitochondrial absence in axons is a consequence of Milton knockdown.

Thank you for pointing it out. We included quantitation of the number of mitochondria in the synaptic terminals (Figure 1E).

The text and figure legend was revised accordingly:

Lines 156-157: ‘As previously reported(24), the number of mitochondria in presynaptic terminals decreased in milton knockdown (Figure 1E).’

- The knockdown of Milton is known to reduce mitochondrial transport from an early stage, but what about swelling? By observing swelling at 1 day and 14 days, it may be possible to confirm the onset of swelling and discuss its correlation with the accumulation of ubiquitinated proteins.

Quantitation of axonal swelling has also been included (Figure 1F).

We appreciate reviewer’s comments on the correlation between the accumulation of ubiquitinated proteins and axonal swelling. Axonal swelling was not observed at 3-days-old (Iijima-Ando et al., PLoS Genetics, 2012), indicating that axonal swelling is an age-dependent event. Dense materials are found in swollen axons more often than in normal axons, suggesting a positive correlation between disruption of proteostasis and axonal damage. It would be interesting to analyze the time course of events further; however, we feel it is beyond the scope of this manuscript. We revised the text as below to include this discussion:

Lines 157-159: ‘The swelling of presynaptic terminals, characterized by the enlargement and roundness, was not reported at 3-day-old(24) but observed at this age with about 4% of total presynaptic terminals (Figure 1F, asterisks).’

Lines 162-167: ‘Dense materials are rarely found in age-matched control neurons, indicating that milton knockdown induces abnormal protein accumulation in the presynaptic terminals (Figure 1G and H). In milton knockdown neurons, dense materials are found in swollen presynaptic terminals more often than in presynaptic terminals without swelling, suggesting a positive correlation between the disruption of proteostasis and axonal damage (Figure 1G).’

Lines 362-365: ‘Disruption of proteostasis is expected to contribute neurodegeneration(38), and it would be interesting to analyze the sequence of protein accumulation and axonal degeneration in milton knockdown ((24,29) and Figure 1) in detail with higher time resolution.’

L147-L151: Though Figures 1F and 1G provide qualitative representations, it is advisable to quantitatively assess whether dense materials significantly accumulate. Such quantitative analysis would be required to verify the accumulation of dense materials in the context of the study.

Thank you for pointing it out. We included quantitation of the number of neurons with dense material (Figure 1G). We revised the manuscript as follows:

Line 161-163: ‘Dense materials are rarely found in age-matched control neurons, indicating that milton knockdown induces abnormal protein accumulation in the presynaptic terminals (Figure 1G and H).’

Regarding Figure 1B, C:

- Even though the count of puncta in the whole brain appears to be fewer than 400, the magnification of the optic lobe suggests a substantial presence of puncta. Please clarify in the Methods section what constitutes a puncta and whether the quantification in the whole brain is based on a 2D or 3D analysis. Detail the methodology used for quantification.

Thank you for your comment. We revised the method section to include more details as below:

Lines 434-437: ‘Quantitative analysis was performed using ImageJ (National Institutes of Health) with maximum projection images derived from Z-stack images acquired with same settings. Puncta was identified with mean intensity and area using ImageJ.’

- What about 1-day-old specimens? Does Milton knockdown already show an increase in ubiquitinated protein accumulation at this early stage? Investigating whether ubiquitin-protein accumulation is involved in aging promotion or is already prevalent during developmental stages is a necessary experiment.

Thank you for your comment. We carried out immunostaining with an anti-ubiquitin antibody in the brains at 1-day-old. No significant difference was detected between the control and milton knockdown. This result has been included as Figure S1 in the revised manuscript. The result section was revised as below:

Line 136-139 ‘There was no significant increase in ubiquitinated proteins in milton knockdown flies at 1-day old, suggesting that the accumulation of ubiquitinated proteins caused by milton knockdown is age-dependent (Figure S1).’

For Figure 1E: In the Electron Microscopy section of the Methods, define how swollen axons were identified and describe the quantification methodology used.

Thank you for your comment. Swollen axons are, unlike normal axons, round in shape and enlarged. We revised the text as below;

Lines 157-160: ‘The swelling of presynaptic terminals, characterized by the enlargement and roundness, was not reported at 3-day-old(24) but observed at this age with about 4% of total presynaptic terminals (Figure 1F, asterisks).’

Lines 683-684, Figure 1 legend: ‘Swollen presynaptic terminals (asterisks in (F)), characterized by the enlargement and higher circularity, were found more frequently in milton knockdown neurons.’

L218-L219: Throughout the text, the expression 'eIF2β is "upregulated" in response to Milton knockdown' is frequently used. However, considering the presented results, it might be more accurate to interpret that under the condition of Milton knockdown, eIF2β is not undergoing degradation but rather remains stable.

Thank you for pointing it out. We replaced ‘upregulated’ with ‘increased’ throughout the text.

L234-L235: On what basis is the conclusion drawn that there is a reduction? Given that three experiments have been conducted, it would be possible and more convincing to quantify the results to determine if there is a significant decrease.

Thank you for pointing it out. We quantified the AUC of polysome fraction and carried out statistical analysis. There is a significant decrease in polysome in milton knockdown, and this result has been included in Figure 5B. We revised the figure and the legend accordingly.

L236: 5H-> 4H

Thank you for pointing it out, and we are sorry for the confusion. We corrected it.

L238-L239: Since there is no significant difference observed, it may not be accurate to interpret a reduction in puromycin incorporation.

Thank you for pointing it out. As described above, quantification of polysome fractions showed that milton knockdown significantly reduce polysome (Figure 5B). We revised the manuscript as below;

Lines 263-264: ‘However, unexpectedly, we found that milton knockdown significantly reduced the level of mRNAs associated with polysomes (Figure 5A and B).’

Figure 5D and Figure 6D: Climbing assays have been conducted, but I believe experiments should also be performed to examine whether overexpression or heterozygous mutants of eIF2β induce or suppress degeneration.

Thank you for pointing it out. We analyzed the eyes with eIF2_β_ overexpression for neurodegeneration. Although there was a tendency of elevated neurodegeneration in the retina with eIF2_β_ overexpression, the difference between control and eIF2_β_ overexpression did not reach statistical significance (Figure S2). This result has been included as Figure S2 in the revised manuscript, and the following sentences have been included in the text:

Lines 288-293: ‘We asked if eIF2β overexpression causes neurodegeneration, as depletion of axonal mitochondria in the photoreceptor neurons causes axon degeneration in an age-dependent manner(24). eIF2β overexpression in photoreceptor neurons tends to increase neurodegeneration in aged flies, while it was not statistically significant (p>0.05, Figure S2).’

L271-L272: The results in Figure 6B are surprising. I anticipated a greater increase compared to the Milton knockdown alone. While p62 appears to be reduced, it is not clear why these results lead to the conclusion that lowering eIF2β rescues autophagic impairment. Please add a discussion section to address this point.

Thank you for pointing it out. We apologize for the unclear description of the result. Milton knockdown flies show p62 accumulation (Figure 2), and deleting one copy of eIF2beta in milton knockdown background reduced p62 accumulation (Figure 7C). We revised the text as below:

Lines 307-315: ‘Neuronal knockdown of milton causes accumulation of autophagic substrate p62 in the Triton X-100-soluble fraction (Figure 2B), and we tested if lowering eIF2β ameliorates it. We found that eIF2β heterozygosity caused a mild increase in LC3-I levels and decreases in LC3-II levels, resulting in a significantly lower LC3-II/LC3-I ratio in milton knockdown flies (Figure 7B). eIF2β heterozygosity decreased the p62 level in the Triton X-100-soluble fraction in the brains of milton knockdown flies (Figure 7C). The p62 level in the SDS-soluble fraction, which is not sensitive to milton knockdown (Figure 2B), was not affected (Figure 7C). These results suggest that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.’

L369: Please specify the source of the anti-ubiquitin antibody used.

Thank you for pointing it out. We included the antibody information in the method section.

Figure 7: While the relationship between Milton knockdown and the eIF2β and eIF2α proteins has been elucidated through the authors' efforts, I would like to see an investigation into whether eIF2β is upregulated and eIF2α phosphorylation is reduced in simply aged Drosophila. This would help us understand the correlation between aging and eIF2 protein dynamics.

Thank you for your comment. We agree that it is an important question, and we are working on it. However, we feel that it is beyond the scope of the current manuscript.

L645-L646: If the mushroom body is identified using mito-GFP, then include mito-GFP in the genotype listed in Supplementary Table 2.

We are sorry for the oversight. We corrected it in Supplementary Table 2.

Additionally, while it is presumed that the mito-GFP signal decreases in axons with Milton RNAi, how was the lobe tips area accurately selected for analysis? Please include these details along with a comprehensive description of the quantification methodology in the Methods section.

Thank you for your comment. Although the mito-GFP signal in the axon is weak in the milton knockdown neurons, it is sufficient to distinguish the mushroom body structure from the background. We revised the method section to include this information in the method section:

Line 437-438: ‘For eIF2α and p-eIF2α immunostaining, the mushroom body was detected by mitoGFP expression.’