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König et al. explore the relationship between peroxisomes and neuropeptides using the insulin-producing cells (IPCs) in Drosophila melanogaster. By utilizing a very clean assay it is evident that Dilp2 accumulates in some of the IPCs in conditions of starvation and is no longer after feeding. This release of neuropeptide is not present if the animal has loss-of- function mutations in genes key for peroxisome biogenesis or a loss-of-function mutation in proteins required for Golgi-peroxisome lipid transfer but is still present in animals with loss-of-function mutations in a peroxisome enzyme maintenance protein. This indicates that peroxisome biogenesis, as well as lipid transfer from the Golgi to peroxisomes, is required for neuropeptide secretion. The authors further support this using lipidomics. Interestingly, the authors also determine that neuronal activity is not altered by the presence of peroxisome biogenesis proteins, indicating a dissociation between neuronal activity and neuropeptide

The strengths of this paper are that their primary assay for monitoring Dilp2 release has a very strong and clean phenotype, and there is no doubt that they are monitoring the release of Dilp2. The absence then, of Dilp2 release in the pex19/pex3/wat-deficient animals is therefore very convincing that the aberrations to their normal function are impacting neuropeptide release. The authors build a compelling picture of the wide- ranging effects that impacting peroxisome biogenesis causes, such as the organism-wide altered lipid profile, the prevention of lipid release from the fat body and gut, and the increased retention of peroxisomal matrix proteins.

While the authors provide some compelling images showing the interactions of Golgi and peroxisome markers in fed, starved, and refed conditions, seeing the presence of Dilp2 in these peroxisomes would better associate this interaction as the reason neuropeptides are being retained during starvation and increase the strength of the conclusions of the paper. Furthermore, seeing these interactions impacted in the pex19/pex3 peroxisomal mutants vs those in the non-peroxisome biogenesis mutants pex2 would provide strong evidence that these interactions are causing the decrease in Dilp2 release post-feeding.

Points for the authors

Major Points

1.        The authors show that the peroxisomes and Golgi are near each other in Figure 5 and Supp Figure 3, and they argue in the discussion (line 405) that this interaction enables the secretion of insulin-like peptides. While they do show this interaction and show that neuropeptides are not released, more evidence is needed to support the claim that this interaction is the reason for this. By doing the experiments shown in Figure 3 with their antibody against Dilp2, they could support their claim by showing Dilp2 accumulation in peroxisomes at the Golgi. Additionally, showing that the Golgi-peroxisome connection across fed-states does not mean that this connection is what is aberrant in the peroxisome biogenesis mutant lines. Using the same experimental parameters used in the Figure 3 experiments, but in a pex19/pex3 mutant background to show the increase in Golgi-peroxisome connections, and in a pex2 mutant background to show no change in Golgi- peroxisome connections, would make their claim on line 404 very strong.

2.        The manuscript utilizes a variety of RNAi lines and antibodies against different proteins. However, it is unclear whether these reagents were validated, either in the current study or through referenced literature. Providing information on reagent efficiency and potential off-target effects would lend greater confidence to the study’s conclusions.

3.        A particularly exciting result is that neuronal activity appears uncoupled from neuropeptide release. Figure 2 shows that pex19 mutant animals exhibit “normal” activity levels following starvation and refeeding but do not release neuropeptides. However, this interpretation would benefit from comparison to control animals under the same conditions. Furthermore, in the standard diet condition for pex19 mutants, the activity distribution is difficult to interpret due to the clustering of data points near zero. Additional replicates or clearer quantification may help clarify the interpretation.

Minor Points

1.        In Figure 1A, it should be explicitly stated in the figure that the image depicts Dilp2 antibody staining so that the readers understand this is not a transgene.

2.        In Figure 1E, it would be helpful to elaborate on what occurs to pex19/3/16 during peroxisome maturation, and to clarify where Pex2/1 come from. Adding this to the schematic would improve clarity for the reader.

3.        The manuscript shows Dilp2 release from IPC somas, but it remains unclear whether the peptides are being directly secreted from the soma or trafficked along axons to synapses. Clarification on the route of peptide transport would strengthen the narrative.

4.        Line 143: Figure 2C is cited, but the corresponding data appears to be in Figure 1C. This should be corrected.

5.        Line 147: The hemocyte rescue of pex19 mutants appears more robust than the IPC rescue. Could the authors elaborate on this finding, especially since the role of pex19 has primarily been discussed in the context of IPCs?

6.        Lines 153–157: The term “depletion of peroxisomes” would benefit from clarification—does it refer to the loss of lipids, membrane proteins, matrix proteins, or all the above? The definition of matrix proteins (presumably lumenal proteins) should also be made more explicit. Additionally, the term “ghosts” is unclear—do these refer to empty peroxisomes, or to aggregates of membrane proteins? Further explanation would be appreciated to clarify what change is actually occurring within the peroxisomes.

7.        The same figure panel appears in both Figure 3A'' and Supplemental Figure 2A (chart 3, PI), which may lead to confusion. It should be shown in only one location to avoid redundancy.

8.        Line 263: BODIPY-Cer is referred to as a Golgi marker, yet later described as labeling the plasma membrane. This inconsistency should be addressed, and the appropriate designation clarified. Additionally, Figure 4E: The saturated images help highlight signal accumulation, but make it difficult to determine whether accumulation occurs at the Golgi. Including an unsaturated version of the image in the supplement would help address this.

9.        Line 292: The px-mCherry tool, as well as other transgenic Golgi markers, are novel and useful. However, it is important to demonstrate that they do not perturb Golgi- peroxisome interactions. Using the Dilp2 release assay (from Figure 1) in these transgenic backgrounds would provide reassurance.

10.  Figures 5A/C: It is not immediately clear which label corresponds to the Golgi and which to peroxisomes. A consistent color scheme across panels or individual legends for each would aid comprehension.

11.  In Figure 5, the Golgi appears to enlarge upon refeeding. If quantified, this result should be shown. If not, the authors may wish to consider quantification, as an increase in Golgi size could contribute to increased interaction probability.

12.  Line 363: The BioGRID database may not be familiar to all readers. A brief explanation of what BioGRID is and how it was used in this context would be helpful.

13.  Line 440: The manuscript states that GM130 accumulation in pex19 mutants suggests missorting of membrane proteins. However, as GM130 is a matrix protein, this conclusion seems inconsistent. It may be more accurate to suggest the mislocalization of matrix proteins or to revise this statement.

14.  Line 659: The 500nm threshold used to define dense-core vesicles (DCVs) appears quite large, as typical DCV sizes range from 100–150nm. The authors should clarify the rationale behind this threshold.

15.  The relevance of Figures 1E and 6G to Dilp2 biology is unclear, as the peptide is not shown. In Figure 1E, the diagram seems to depict peroxisome stages rather than function. Please revise to better illustrate Dilp2 biology or clarify their intended message.

Reviewer Disclosure:

Language in the Minor and Major comments of this review was refined with the assistance of OpenAI's ChatGPT to improve clarity and tone.

Competing interests

The author declares that they have no competing interests.