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The preprint from Robertson et al. focuses on exploring the consequences of two DNM1L mutations (G32A and R403C) on DRP1 protein functions. DRP1 is a GTPase regulating organelle morphology by inducing fragmentation events. Since DRP1 is localized both at mitochondria and at peroxisomes, the authors ultimately wish to determine how the G32A and the R403C mutations impact mitochondrial and peroxisomal functions in patient-derived cells. These mutations are clinically relevant, as they were previously retrieved in patients with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1). EMPF1 is a severe neurodevelopmental syndrome leading to death during childhood, for which no treatment currently exists.

The authors combine super-resolution microscopy techniques, (SIM), electron microscopy approaches, and the analysis of cellular glycolysis and mitochondrial respiration rates in patient-derived cells.

The main results presented in the paper are as follows:

1.    The G32A and R403C mutations induce a significant elongation of the mitochondrial network and an increase in mitochondrial volume;

2.    The effect of the mutants on mitochondrial elongation seems to be due to a stalling in fission mechanisms, exemplified by a lowering of the abundance of fusion proteins and resulting in an incomplete fission;

3.    Mitochondria in mutant cells are more uncoupled than in control cells, and have a metabolic switch towards glycolysis. This could potentially be explained both in terms of metabolism and in terms of organellar ultrastructure. Metabolically, mutant cells show an impairment in the transporter of pyruvate inside mitochondria, while ultrastructurally, mitochondria from mutant cells show shorter cristae than those found in control cells.

4.    The G32A and R403C mutations also induce a significant elongation of the peroxisomal network and an increase in the volume of these organelles as well;

5.    Blocking fatty acid oxidation (FAO) pharmacologically does not change the ability of mitochondria from mutant cells to use this pathway. The authors conclude that, compared to control cells, mutant cells have adapted their metabolism to use this pathway as a constitutively-active source of ATP.

This is a very timely and interesting paper. The writing style is concise, straightforward and easy to read. The authors make an extensive effort in quantifying super-resolution and TEM data. This allows the reader to appreciate data variability and the reproducibility of the experiments. In addition, the images presented are of high quality and representative of the graphs shown. The discussion is very interesting and opens up exciting questions that could be addressed in a follow-up study.

The materials and methods section could be improved to ensure a better reproducibility from independent groups. In particular:

-      Where did the authors get the cells from (i.e. cohort of patients, collaboration…);

-      Why all the independent points in super-resolution and TEM quantifications are merged in the same graph and plotted as independent n? As the authors state, they are obtained from a given number of independent cells in (at least) 3 independent replicates. The notion of replicates is absent from the graphs, and should instead be considered as it could affect the overall statistical outcome.

-      Image analysis pipelines are not extensively described. The authors state that they use “Fiji macros”, but it is unclear whether these are home-made macros, or previously-published ones. The analysis steps and parameters should be clearly defined and/or previous publications should appropriately be cited, so that other groups can use them to reproduce these results or for their own applications.

Concerning about the conclusions reached by the authors, I have some specific questions:

-      The authors conclude that the G32A and R403C mutations have a dominant-negative effect. These mutations show almost no impairment in the capacity of pSer616 to be phosphorylated, and of pDRP1 to shuttle to mitochondria. However, mitochondrial elongation is boosted. Did the authors try to verify the stability of these two mutant proteins, or that their effect on mitochondrial elongation is induced by an impaired interaction with DRP1 receptors such as MFF or Mid49/51? These possibilities should also be considered, and could potentially give the same results as the ones shown in the paper without G32A and R403C being dominant-negative mutations.

-      On the same point, the authors state that “in the patient fibroblast, the interaction of mutant DRP1 with mitochondria seems to be less transient”. Have they considered doing some live microscopy to support this conclusion? Globally, it seems that there is no difference in the capacity of pDrp1 to localize to mitochondria, which is not in agreement with the WB data (which shows less pDRP1 for the G32A). So, how do they assess the transient-like feature of DRP1 interaction with mitochondria?

-      Would you comment on the fact that the R403C mutation induces the appearance of mitochondrial "rings" or “forks” in the pictures shown? They are quite a widespread feature, so these are potentially of functional relevance?

-      Could it be that the lower number of cristae shown in TEM data are due to the fact that mutant mito are longer than ctrl ones, and the authors quantify cristae number as a ratio of mito length? Is the absolute number of cristae different in mutants vs controls?

-      Higher TMRM levels could simply be due to higher quantity of mitos in the specific field of view shown in the paper. Could you verify this possibility by quantify cells in multiple fields of view/cells/replicates? And how do you reconcile the fact that you have such huge differences in TMRM fluorescence over time: could TMRM be more readily incorporated into fused mito? Could you create another model of fusion (i.e. overexpression of mitofusins) to compare the mutants with?

-      The peroxisomal phenotype is really convincing! Would you consider monitoring whether it is a cause or a consequence for the mitochondrial one? Basically, who comes first: the mito phenotype or the peroxisomal phenotype, or are they happening simultaneously (which would hint at the presence of two different DRP1 pools)?

-      The authors state that “peroxisomes and mitochondria are hubs for FAO”, so they try to correlate the peroxisomal phenotype with defects in mitochondrial enzymes contributing to FAO. I would suggest to better explain the rationale of this correlation, which may be unclear to an unfamiliar readership. Besides, do the mitochondrial enzymes acting in FAO have a peroxisomal counterpart? Or do they shuttle among compartments?