Parkinson’s disease-associated Pink1 loss disrupts vesicle trafficking in Ensheathing glia causing dopaminergic neuron synapse loss

Reviewer #1 (Public review):

Summary:

This study investigates the impact of Pink1 loss on glial function and neuronal health in a Drosophila model, highlighting the role of mitochondria-organelle contacts and key genes such as Ccz1, Vps13, Mon1, and Rab7. The work provides insights into cellular processes underlying neurodegenerative diseases, with a focus on glia-neuron interactions. While the findings are promising, the study lacks critical controls, detailed mechanistic evidence, and explanatory figures to strengthen its claims.

Strengths:

(1) The study addresses an important topic in neuroscience, exploring the mechanisms of Pink1 loss, which has implications for Parkinson's disease and neurodegeneration.

(2) The focus on mitochondria-organelle contacts and their regulation by Rab7-mediated pathways is novel and provides a potential mechanism for neuronal dysfunction.

(3) The identification of key genes (Ccz1, Vps13, Mon1, Rab7) and their potential roles in Pink1-related pathways adds valuable knowledge to the field.

(4) The manuscript uses a combination of genetic tools, Drosophila models, and functional assays to approach the problem from multiple angles.

Weaknesses:

(1) Specificity of Mz-Gal4: The study lacks validation of Mz-Gal4 specificity, as it may also drive expression in a few neurons or other types of glia. Additional control experiments using nls-GFP with Elav, Repo, or Draper antibody staining or alternative glial drivers would be helpful.

(2) DLG staining is central to the story but is not well-supported by high-resolution Z-stack imaging, which should be included in the supplementary figures.

(3) The manuscript does not confirm whether the candidate RNAi (Ccz1, Vps13, Mon1, Rab7) directly influence Rab7-mediated membrane trafficking or mitochondria-lysosome contacts in Pink1 mutants.

(4) Using ERG as a readout for EG effects in the antenna is not a direct or appropriate assay. Alternative functional assays relevant to antenna glia should be considered.

(5) A graphical explanation of the interactions and functions of the candidate genes in Pink1 KO mutants is missing. This would greatly enhance the manuscript's clarity.

(6) The study lacks details on sample sizes, effect sizes, and reproducibility, which are necessary for robust conclusions.

(7) There are repeated words on page 3 ("olfactory Olfactory Receptor Neurons") and a lack of explanation in Figure 3C regarding the most up-regulated and down-regulated genes and the significance of large red dots.

Reviewer #2 (Public review):

Summary: This study proposes a novel role for ensheathing glia (EG) in a Pink1-model of Parkinson's disease and shows that this cell population exibits the highest number of DEG in a pre-symptomatic stage. In the olfactory system, there seems to be morphological changes in this cell-type that resembles an 'activated' state and the authors further show that the neuronal loss of Pink1 is responsible for this defect. The authors go on to show that manipulation of Pink1 in EG also leads to some defects in the visual system and in the dopaminergic neurons (DAN) that innervate the mushroom body (MB), and performed a screen based on the 'on-transient' defect of the ERG to identify potential genes that may modulate the function of EG in synaptic regulation. They focus on several genes related to Rab7/Vps13, and performed some additional experiments in the visual system and MB to propose the role of vesicle/lipid trafficking in EG as a important factor for PD pathogenesis.

Strengths: The study proposes functional and mechanistic connections between several genes that have been linked to PD (PINK1, VPS13A/C). I feel that the data presented in Figure 1 and Fig3A-C are performed with rigor and are convincing/novel. The selection of Drosophila to study the questions is also a strength and the lab has extensive experiences in this field and model organism.

Weaknesses: There is one fundamental concern I have with the genetic experiments performed in this paper (especially in Fig 3D and Fig4, see major issue #1), and I feel that there is a bit of a disconnect between the EG 'activation' phenotype the author show in the olfactory system and the other two neuronal systems (visual system, MB DAN) that the authors investigate see major issue #2). Also, there are quite a bit of information that is not provided in the manuscript (see major issues #3 and #4), which makes me difficult to judge the rigor and interpretation of several experiments.

Major Concern #1: A number of lines used in this study are referred to as "RNAi" lines but when I look at the actual genotypes of reagents listed in the table in the METHODS section, many are actually NOT RNAi lines. Quite a few lines, including lines that the authors use as RNAi against Ccz1, Rab7 and Mon1, are gRNA lines for the TKO (TRiP-CRISPR knockout) system. While these reagents can theoretically knock-out these genes in somatic cells if used in combination with UAS-Cas9, there is no mention that UAS-Cas9 was used in this work throughout the manuscript. Hence, when these lines are just crossed to GAL4 with or without the Pink1 mutant, they shouldn't be having any effects. Similarly, the strongest hit from their screen was a TOE (TRiP-CRISPR Over Expression) gRNA against PIG-A, which could allow overexpression of PIG-A if there is a UAS-dCas9::VP64. However, I also do not see any mention that such activator was introduced into the crossing scheme. Considering that 3 of the 4 'hits' from their screen are not RNAi lines, I am quite skeptical of the study. Similarly, except for Vps13, all reagents used in Fig4 are TKO gRNA lines. Therefore, if this experiment was conducted without an UAS-Cas9, most of the data shown here are problematic. Also, note that several of the 'RNAi' lines listed in the Table in the METHODS section are actually MiMIC alleles. While some MiMIC lines could function as strong LOF alleles (if they are inserted in the exon or in an intron of the gene in the same orientation as the gene), some of the lines are not expected to affect gene function (e.g. FASN2 and CG17712, MiMICs are in introns and face the opposite orientation). Hence, the rationale of including these reagents in the screen doesn't make much sense. The description of the modifier screen should be much more detailed in the RESULTS and METHODS section and if the UAS-Cas9/dCas9::VP64 transgenes were not introduced when the TKO/TOE reagents were utilized, what can be concluded?

In addition, for the 4 genes that the authors further study in Fig4, there are many other reagents that the authors can use, including mutant alleles, previously characterized RNAi lines (e.g. Vps13) and dominant negative/constitute active lines (e.g. especially for Rab7). The authors should validate their results with independent reagents to really convincingly show that the same conclusions can be drawn for the Vps13/Rab7 related genes since this is the key takeaway message of this paper.

Also, they do not show whether the manipulation of these genes in a wild-type background (they only show what happens in Pink1 mutants) affect ERG and MB DAN synapse morphology. If these manipulations alone dramatically affect these phenotypes, it would be very difficult to interpret their data.

Major Concern #2: In Figure 1, the authors show some morphological evidence that EG are 'activated' in Pink1 mutants, but whether the same phenomenon occurs in the visual system and in the MB is not shown. Since all of the studies in Fig3D and Fig4 are done in the visual system and MB, it is not clear whether the visual system and MB phenotypes are related to 'activation' of EG.

Also, in the RNA-seq data in Fig1A and Fig3C, is there any molecular evidence that EG are indeed 'activated'? The only evidence that the authors show to state that EG are 'activated' in young Pink1 null animals is based on increased CD8::GFP staining in the olfactory system.

The authors cannot draw a strong conclusion that indeed EG are 'activated' based on these data (e.g. perhaps the expression level of CD8::GFP is just increased). Additional evidence that the EG are 'activated' could be provided by looking at the increase in Draper intensity (as reported by Doherty et al. and MacDonald et al. that the authors cite), not only in the olfactory system, but also in the visual system and in the MB. It would also be informative if the authors can look at morphology of the EG in the visual system and MB to convincingly that the data shown in Fig4 is relevant to EG 'activation'.

Major Concern #3: In Fig3, there is no clear explanation why they focus on the ON transients and ignore the OFF transients, and also why the difference in the depolarization is not quantified in Fig4.

Major Concern #4: While the authors claim that mz709-GAL4 is a EG specific driver, do the authors know that this is indeed true in the tissues and stages that are studied here? The Ito et al,. paper that is cited in the METHOD section has only looked at the expression of this reporter in embryonic and larval stages. The authors need to that the authors should validate their findings with an additional EG specific driver and/or provide additional data that mz709-GAL4 is indeed specific to EG in the adult fly brain and eye. If mz709-GAL4 is expressed in other cell-types, the interpretation of many of the data in this paper becomes quite questionable. I believe the data in Fig3B is suggesting that mz709-GAL4 is indeed specific to glia cells and not expressed in neurons, but whether this driver is truly specific to EG (and not in other glial types), especially in the visual system (including the lamina as well as in the eye), is not obvious.

Author response:

We would like to thank the reviewers and the editors for carefully reading and commenting our manuscript and plan to prepare a revised manuscript. Particularly, we want to thank reviewer 2 for spotting a major oversight regarding the use of the TKO (TRiP-CRISPR knockout) and TOE (TRiP-CRISPR Over Expression) systems and the MiMIC alleles. As the reviewer pointed out, these lines were not used as intended, therefore our results and conclusions regarding the genetic interactions between Pink1 and several of genes in the paper (PIG-A, Rab7, Ccz1, CG10646, Mon1, FASN2, CG17712) that we attempted to target, are incorrect and based on a technical mistake. These results need to be removed from the manuscript.