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

In this manuscript, Marahori et al. describe the importance of mitochondrial transport and degradation, mitostasis, at the synapses of motor neurons in the Thy1-mito-XFP mouse model. Prior to this, the molecular events that target dysfunctional mitochondria for site specific capture at synapses remained unresolved. Using ex vivo optical ‘pulse-chase’ experiments to study mitochondrial volume flux, they identified a pathway near synaptic terminals responsible for mitochondrial degradation. Interestingly, they discovered a local degradative pathway in which retrograde-moving mitochondria are captured by optineurin, independent of the PINK1 and parkin mitophagy pathway. Overall, the study is thorough integrating quantitative live imaging, mitochondrial flux measurements, ultrastructural validation, multiple genetic perturbations and disease models to elucidate the location and mechanism of mitophagy in mature motor axons. Addressing the following would strengthen the study:

Major concerns:

1.     The authors convincingly show that captured mitochondria are rounder and exhibit dysfunctional characteristics. However, it is unclear to me whether the capture signal is triggered during transport or at the exit point? Additionally, the authors could talk about in their discussion what molecular events precede the optineurin-dependent capture event?

2.     In figure 5, although optineurin conditional knockout reduces capture and increases passing flux, the cellular localization remains unclear. It would be beneficial if the authors could discuss optineurin enrichment in neurons. Does the loss of optineurin alter ubiquitination at these sites?  Does it colocalize with LC3-positive structures? Elaborating on these questions could clarify the interpretation of optineurin’s role as the primary protein involved in capture prior to autophagosome engulfment.

3.     In Figure 2, the authors measured mitochondrial membrane potential to study organelle function in ChAT-Cre⁺ x ROSA-mito-GFP mice. The authors could measure reactive oxidative species (ROS), similar to data presented in Figure 4g, to further support the evidence that dysfunctional mitochondria moving retrograde are more likely to be captured than healthy mitochondria.

4.     In Figure 5 results show mitochondrial capture is independent of the PINK1/parkin mitophagy pathway. The authors state there was no significant difference in the mitochondrial capture in Pink1 KO compared to Pink1 WT. However, in Figure 5a the Pink1 knockout appears to show a visibly higher capture proportion than WT. Could a different statistic be beneficial for the interpretation of the data? The authors could elaborate on whether additional replicates or increased sample size might resolve this discrepancy.  

Minor concerns:

1.     The authors provide movies illustrating capture and passing behaviors of mitochondria. However, the resolution makes it difficult to confidently follow individual mitochondrial events. Perhaps zooming in more and reducing the frame rate could help with the resolution of the videos. For example, in Movie 5, zooming into a single region of interest rather than displaying multiple regions simultaneously would allow the readers to easily distinguish between structures and better capture the dynamics.

2.     In Figures 1 and 3, it is difficult to understand the different internodes being studied. The authors could define and clarify the different internodes in Figure 1F and label them in the schematic of Figure 1a. Additionally, the authors could elaborate more on how important the Nodes of Ranvier for mitochondrial capture are.

3.     In figure 1d the authors show representative images of the presynaptic motor terminal with Dendra-tagged mitochondria. Including a zoomed out representative image of the entire neuromuscular junction would allow readers to evaluate the Thy1-mito-RFP expression is consistent throughout the axon.

4.     The capture/passing mitochondria shown in the pie charts of Figure 5b appear reversed or inconsistent with the numerical flux data. The authors could improve clarity by verifying if pie charts are correctly labeled.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.