Avalilação PREreview de A retrograde transit filter mediated by optineurin controls mitostasis in distal axons

Summary

In this manuscript, Marahori et al., discuss mitochondrial turnover in the axons of motor neurons and its relation to ‘mitostasis’. This is a topic of ongoing debate regarding whether mitochondrial turnover can occur outside the soma and whether sufficient resources are available there to support this process. Using confocal and airy scan microscopy, they demonstrate that mitochondria are captured at the proximal end of the neuromuscular junction of motor neurons where there is a large population of lysosomes that can degrade the dysfunctional mitochondria. These dysfunctional mitochondria are characterized and seem to be captured based on lower membrane potential and rounded morphology. Additionally, they show that loss of Optn impedes capture of these mitochondria, but loss of key autophagy factors such as pink1 and parkin do not. Atg7 knockout did not lower capture rates, suggesting autophagy is not involved. Overall, the manuscript uses several techniques to address their questions, which supports their hypotheses, but several experiments and quantifications will improve the quality of the manuscript:

Major concerns:

1.     While the authors have previously published triangularis sterni muscle explant preparations in thy1-XFP mice (PMID: 18833201) and provide appropriate references, the current study introduces new mouse lines carrying different reporter systems. For readers to confidently interpret the data, it would be very helpful to include a clearer characterization of reporter expression in these new lines. Specifically, I recommend adding images or panels that illustrate the baseline expression patterns of the reporters in explants, similar to what was shown in (PMID: 18833201). In the current version, the confocal images in Figure 1 and Extended Figure 1 are difficult to interpret without such context, making it challenging to understand precisely what structures or cell types are being visualized. Providing these reference images would greatly improve clarity and reproducibility for readers.

2.     Figure 1g suggests that mitochondrial volume flux into and out of the NMJ is approximately balanced, implying that similar amounts of mitochondria enter and exit these synapses. However, the text also notes that a substantial proportion of mitochondria within NMJs are stationary. This raises an important question: what percentage of the total mitochondrial population in NMJs is stationary versus motile? It would greatly improve clarity if the authors could:

a.     Provide explicit quantification of the stationary mitochondrial population at NMJs (for example, proportion or percentage of total mitochondrial volume or count).

b.     Clarify how this stationary pool relates to the observed bidirectional flux; is the data suggesting that, despite a large stationary population, the motile mitochondria consistently show equal anterograde and retrograde movement?

c.     Provide additional explanation, or a simple schematic, to help readers reconcile the high proportion of stationary mitochondria with the apparent directional balance observed in the flux analysis.

3.     In Figure 2, the ALS mouse model is used to assess whether the mitochondrial “capture” phenotype also occurs under disease-related conditions. However, it is unclear whether the mitochondria that become captured in this model exhibit the same morphological features reported earlier in the manuscript. For consistency and interpretability, it would be very helpful if the authors could provide additional characterization of the captured mitochondria in the ALS model. Demonstrating whether these mitochondria share the same structural or functional properties as those in the wild-type context will allow readers to better understand how generalizable the capture phenotype is, which would ultimately strengthen the authors’ conclusions.

4.     The data from Figure 4 is strong and shows that Atg7-cKO results in the accumulation of organelles due to the loss of Atg7, which degrades the organelles at these capture sites. However, the text and the figure were difficult to follow and some of the data chosen to support conclusions in the text could benefit from further explanation or data.

a.     For example, Figure 4e provides useful information showing that anterograde mitochondrial movement is not altered in the Atg7-cKO mouse model. However, the use of the term “passing” in relation to the data in Figure 4e may be misleading. The retrograde/passing definition could be provided early and defined earlier to help the reader follow along with the interpretation of the data. The authors could also provide additional analysis showing passing vs captured mitochondria, like Figure 5i-j.

5.     To help readers interpret Supplemental Figure 3d-e more easily, it would be helpful to state explicitly in the figure legends whether the quantified mitochondrial movements are anterograde or retrograde. Although this information appears to be included in the main text, having it directly in the legend would improve clarity for readers. In addition, the terms “proximal” and “distal” would benefit from clearer definition. If these are meant to be relative to the soma (or another reference point), stating this explicitly would avoid potential confusion.

a.     More broadly, a simple schematic, similar to Figure 1F or adjusted to match the specific measurements here, could greatly assist readers by illustrating axonal orientation, the position of the NMJ or soma, and the direction of mitochondrial transport. If all measurements were made within a specific distance from the NMJ or soma, noting this would also be very helpful. Alternatively, if distances vary or are defined relatively to something, clarifying this would strengthen the data’s interpretability.

6.     In the discussion, the authors note that the source of retrograde-moving mitochondria is undetermined. While the argument in this paragraph is sound, its structure makes it difficult to follow. To help, it can be specified that when discussing “retrogradely-traveling mitochondria” in this paragraph, you are discussing mitochondria observed in the stem axon, not the NMJs.

Minor concerns:

1.     In Figure 2c, 2f, and 2h, the stacked sum data for mitochondria are difficult to see at their current size. Enlarging these panels (or adjusting the layout to increase their visual clarity) would make the data easier to interpret and improve the overall readability of the figure.

2.     The statistical significance bars in Figure 3d and 3h are not applied consistently, which makes it difficult to determine exactly which groups are being compared. Clarifying or standardizing the placement and style of these significance indicators would greatly improve readability and ensure that readers can accurately interpret the statistical comparisons.

3.     It would help for readers to understand why ATG7 was chosen explicitly for testing autophagy, given that multiple factors contribute to the autophagy pathway. Providing a brief rationale, such as ATG7’s role in autophagosome formation or its relevance to the specific experimental context, would clarify the choice and strengthen the generalization that autophagic filters not directly capturing mitochondria at synaptic exit points based on the ATG7 data.

a.     Providing immunostaining against ubiquitin in the ATG7-cKO mice would support the authors argument that upstream of Atg7 is responsible for capture of the mitochondria.

4.     The statistical analysis used for Supplemental Figure 3d and 3e is not specified. Please indicate which statistical test(s) were applied to these data in either the figure legend or the Methods section. Including this information is important for transparency and for allowing readers to properly interpret the significance of the results.

5.     Supplementary Figure 4 contains a particularly strong result that could merit inclusion in the main text. This figure is important because it supports the hypothesis that the junction is capable of degrading captured mitochondria, and the data showing once captured mitochondria enter the junction, they do not leave. Moving the main finding to the primary figures would help highlight its importance and increase visibility to readers.

6.     The authors show data supporting Optn is involved in the capture of damaged mitochondria. It would aid the authors’ argument if immunostaining for Optn were performed to show that Optn colocalizes with either mitochondria or lysosomes. It would provide more context on where Optn is active in the retrograde movement of mitochondria and when these mitochondria might be targeted for capture and degradation.

7.     The supplementary videos would benefit from additional labeling to help viewers orient themselves, particularly for identifying the full node structure. For readers who may be less familiar with this specific anatomy, the current video quality makes it challenging to follow mitochondrial movements and to determine their exact location when they briefly disappear from view.

8.     Many of the figure legends use “≥” notation to indicate the number of mice, axons, or mitochondria quantified. Because the statistical analyses may be influenced by the variability in sample sizes, it would be helpful to provide more precise information. I recommend replacing the “≥” values with the exact n for each analysis or reporting the range from the smallest to the largest n used for each statistical test. This clarification would improve transparency and allow readers to better assess the robustness of the statistical interpretations.

9.     Figures utilizing red and green exclusively should be made color-blind friendly. A suggestion is to make red channels magenta instead.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.