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. 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.

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.

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.

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.

(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%.

(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.

(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.

Author Response

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 comments. We will further investigate the correlation between aging and the proteins eIF2β and eIF2α and include the results in the revised version.

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.

We agree with the reviewer that multiple ways exist to interpret the LC3 blotting for the autophagy assessment. Thus, we analyzed the levels of p62, an autophagy substrate, and found that milton knockdown caused elevated levels of p62 (Figure 2B). Together, these results suggest that autophagic degradation is lowered.

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 your comments. We will include more analyses of the proteomic data in the next version of our manuscript. In this study, we aimed to elucidate the mechanisms by which depletion of axonal mitochondria induces proteostasis disruption prematurely. Thus, we did not investigate the roles of differentially expressed proteins in proteostasis at 21-day-old in milton knockdown. Aging disrupts proteostasis via multiple pathways: eIF2β levels may be lowered by feedback of earlier changes or via interaction with other age-related changes at 21-day-old. We will include more discussion in the next version of our manuscript.

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. Thank you for your comment. We understand that the phosphorylation of eIF2α is inhibitory for translation initiation, as we described in page 9, Line 312-314. We propose a model in which autophagic defects caused by milton knockdown is mediate by upregulation of eIF2β, however, we are not arguing that the translational suppression in milton knockdown is caused by a reduction in p-eIF2α. We found that milton knockdown causes an increase in eIF2β, and overexpression of eIF2β copied phenotypes of milton knockdown such as autophagic defects (Figure 5 and 6). We also found that the increase in eIF2β reduces the level of p-eIF2α (Supplemental Figure 2), thus, eIF2α phosphorylation in milton knockdown may be caused by an increase in eIF2β. However, the effects of upregulation of eIF2β on the function of eIF2 complex is not fully understood. The translational suppression in milton knockdown may be caused by disruption of eIF2 complex, while it is also possible that it is mediated by a function of eIF2β that is yet-to-be-determined, or mediated by the pathways other than eIF2. We will include more details in the revised version.

(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. We are sorry, it was a mistake. The gradient was actually 10-50%, and we described it wrong. We will correct it in the revised version.

(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 will replace the graph.

(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. A new set of experiments with technical challenges will be required to capture the spatial resolution for the axonal translation. We will work on it and hope to achieve it in the future.