Decreasing pdzd8-mediated mitochondrial-ER contacts in neurons improves fitness by increasing mitophagy

  1. Victoria L. Hewitt
  2. Leonor Miller-Fleming
  3. Simonetta Andreazza
  4. Francesca Mattedi
  5. Julien Prudent
  6. Franck Polleux
  7. Alessio Vagnoni
  8. Alexander J. Whitworth

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The complex cellular architecture of neurons combined with their longevity makes maintaining a healthy mitochondrial network particularly important and challenging. One of the many roles of mitochondrial-ER contact sites (MERCs) is to mediate mitochondrial quality control through regulating mitochondrial turn over. Pdzd8 is a newly discovered MERC protein, the organismal functions of which have not yet been explored. Here we identify and provide the first functional characterization of the Drosophila melanogaster ortholog of Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila . The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion and reduces lifespan. We also show that depletion of pdzd8 rescues the locomotor defects characterizing an Alzheimer’s disease (AD) fly model over-expressing Amyloidβ 1–42 (Aβ 42 ) and prolongs the survival of flies fed with mitochondrial toxins. Together, our results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

Article activity feed

  5. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Response to Reviewers

    We thank the reviewers for their careful reading of our manuscript and their valuable suggestions and comments. To address the reviewers’ concerns and improve our manuscript, we will complete the additional experiments and further revise the text as described below.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    **Summary:**

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2. The authors present an in vivo analysis of pdzd8 (CG10362) and a synthetic ER-mitochondria tether in the regulation of locomotor activity, lifespan, and mitochondrial turnover of Drosophila melanogaster, using basic bioinformatics, RNAi, SPLICS, imaging and microscopies observations (i. e. TEM, SIM), fly lines, and a representative AD fly disease model, etc. The research methodologies were detailed in good order. The model system employed was suitable to address the research topic. The manuscript was written in a clear language and statistical analysis were correctly applied.

    **Major comments:**

    *-Are the key conclusions convincing?*

    Yes. The results/conclusions are logical and provide an overview of Pdzd8 in the regulation of mitochondrial quality control and neuronal homeostasis.

    *-Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.*

    No. Experiments were generally well performed, and all the data support the conclusions.

    *-Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.*

    No suggested experiments needed.

    *-Are the data and the methods presented in such a way that they can be reproduced?*

    Yes. The authors have followed proper experimental design and methods have been described in sufficient detail.

    *-Are the experiments adequately replicated and statistical analysis adequate?*

    Yes, they are.

    **Minor comments:**

    *-Specific experimental issues that are easily addressable.*

    No comment.

    *-Are prior studies referenced appropriately?*

    Yes. The relevant literatures have been cited appropriately.

    *-Are the text and figures clear and accurate?*

    1.Please pay attention to the correct spelling of the described protein name (Pdzd8) and gene name (should be in 'italic') throughout the manuscript, i. e. line 36, 98, and 556, etc.

    As this is the first published characterization of the fly homolog of the mammalian Pdzd8 We have decided to name the fly protein pdzd8, using the lower case “p” to distinguish it from the mammalian protein. We have checked and corrected our use of italics for the gene name as noted in track changes.

    2.In figure 1C and its figure legend, please state what the numbers "201" and "195" stand for.

    We have added the text “numbers on bars indicate number of mitochondria analysed” to the figure legend.

    3.Your data needs to be converted the lowercase letter "x" to math symbol "×" when representing times sign, i. e. line 523, 5x, etc.

    Corrected

    *-Do you have suggestions that would help the authors improve the presentation of their data and conclusions?*

    No comment.

    Reviewer #1 (Significance (Required)):

    *-Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.*

    Discoveries from this study include 1) characterization of the tethering protein Pdzd8 in Drosophila melanogaster, and 2) shed light on a possible way on how to enhance mitochondrial quality control and to help promote healthy aging of neurons by manipulating MERCs.

    *-Place the work in the context of the existing literature (provide references, where appropriate).*

    With this manuscript, the authors present a straightforward but sound piece of scientific research, with the intent to illustrate the consequences of neuronal depletion of pdzd8 in Drosophila melanogaster. Since Pdzd8 plays specific functions in ER-mitochondrial tethering complexes and dysregulations of MERCs are damaging to neurons, this protein represents a good potential target. In this context the characterization of Pdzd8 should represent an interesting starting point. To this purpose, the gene was knockdown and the tether construct was recombinantly produced. The fly lines were then subjected to analysis both at the organismal and at the cellular level.

    *-State what audience might be interested in and influenced by the reported findings.* Audience might include those who are in the field of neuroscience and pharmaceutical, and benefit from an awareness of this research.

    *-Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.*

    Key words in my field of expertise: Ageing, neurodegenerative diseases, Alzheimer's disease, mitophagy, NAD+, neuroprotection. My group is investigating the molecular mechanisms of ageing and age-related neurodegeneration (especially AD) using cross-species model systems, ranging from human brain samples, iPSCs, C. elegans, Drosophila melanogaster, and mice, therefore I have sufficient expertise to evaluate this paper.

    **Referees Cross-commenting**

    To this reviewer the key novelty of this paper was the study of the regulation of the mitochondrial-ER contact sites (MERCs) in life and health. The data indicate that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial homeostasis, neuronal function, and lifespan. In a transitional perspective, this reviewer would ask to check whether this mechanism conserves in rodents or not (e.g. to to memory in the AD mice and to run lifespan in mitochondrial toxin condition). This may be to much. But will depend on the standard of the journal. We thank the reviewer for their input, evaluation and interest. We too are keen to know whether this mechanism is conserved and hope to investigate this in our ongoing work including characterizing a mouse mutant, but the current work already represents a substantial investment of resources and a worthy study in its own right as the first description of the in vivo role of pdzd8, so we feel it is beyond the scope of the current work.

    Reviewer #2

    (Evidence, reproducibility and clarity (Required)):

    Hewitt et al. describe and characterize for the first time the ortholog of pdzd8 in Drosophila melanogaster. In accordance with pdzd8's previously described function as a member of mitochondrial-ER contact sites (MERCs) the authors show reduced MERCs upon RNAi mediated depletion of pdzd8 via TEM, SIM and a split-GFP based contact site sensor. Pdzd8 depletion results in the increased life span as well as improved locomotor activity in aging flies while increase of MERCs with a synthetic tether accelerates the age-related declines in survival and locomotion. Moreover, pdzd8 depleted flies are more resistant against mitochondrial toxins. The authors correlate these protective effects of pdzd8 knockdown with an increase in mitophagy using a mitophagy sensor and describe a rescue of locomotor defects in an Alzheimer disease fly model by pdzd8 depletion.

    **Major comments:**

    1.The authors quantify the number of MERCs in thin sections of TEM (Fig 1B and C). It would add to the paper if the authors would show a representative reconstruction of the quantified somata, as a 3D reconstruction would visualize ER-Mito contacts more reliable than thin sections.

    We agree that the 3D reconstruction of TEM images would provide a satisfying addition to the current analyses, however such advanced techniques are not readily available. The current samples used to collect these data cannot be used to generate 3D reconstructions. To counter this, we have used three independent methods to analyse the changes in MERCs, all of which show a decrease in MERCs in the flies with less pdzd8 supporting that these observations are reproducible and robust.

    2.The authors quantify MERCs in pdzd8 KD also by SIM (Fig1F, G). However, they quantify the number of MERCs in epidermal cells while they also show SIM images of larval neurons (Fig S1D). For consistency and to support their claim of MERC reduction in neurons, we ask the authors to include the quantification based on larval neurons especially as the authors show that pdzd8 is predominantly expressed in the CNS.

    Unfortunately, the soma of larval neurons have extremely limited cytosol (see fig. S1D) which creates very challenging conditions to discern the spatial separation of ER and mitochondria by light microscopy. While co-localisation of organelle markers in such cells has been reported in the literature, we are extremely concerned that the lack of space within the cytosol renders such analysis unreliable. However, we will attempt to quantify the extent of co-localisation of the ER and mitochondria in these cells. In contrast, epidermal cells are much larger providing greater spatial separation of ER and mitochondria. Notably, we complement the co-localisation analysis of epidermal cells with two additional approaches, TEM analysis and the SPLICS reporter construct, to demonstrate pdzd8-RNAi results in decreased MERCs specifically in neurons.

    3.The authors describe a decreased NMJ volume in Fig 4G. It would improve and complete the functional characterization of pdzd8 in flies if the authors can provide further data whether pdzd8 KD causes a general synaptic defect. Can the authors show morphological synaptic defects in the existing TEM data of the adult brain or provide additional ERG recordings, which would elucidate the functional consequences of pdzd8 depletion in the CNS?

    Our TEM data are not suitable for us to properly analyse defects in synaptic morphology as our images centered around the cell bodies where the organelle morphology was easiest to distinguish and there are very few synapses. While it is not surprising that the knockdown of pdzd8 has some detrimental effects, we chose to focus our efforts on trying to determine the cause of the protective effect on locomotor activity in aged flies rather than to exhaustively characterise the myriad phenomena which may be impacted as a knock-on effect of the disrupted cell biology that we have demonstrated. We hope to further explore the detrimental functional consequences of pdzd8 depletion on such phenomena as neurotransmission in future work.

    1. Hewitt et al. suggest a beneficial effect of increased turnover of mitochondria for healthy aging. To convince readers we would like to ask the following:

    a) This claim is based on their observation of increased mitophagy in pdzd8 depleted flies using one reporter (Fig 5). Can the authors support their data with an alternative method as this is one of the key claims of the manuscript?

    The mitoQC tool is well established in the field and we have found it to perform better but consistent with mito-Keima (Lee et al. 2018 JCB doi: 10.1083/jcb.201801044). We would be happy to consider other assays if the reviewer can suggest an unbiased and established alternative.

    b) An increased turnover of Mitochondria would also suggest that there are more "young" mitochondria present in the pdzd8 KD neurons. Can the authors experimentally address that?

    We understand the reviewer’s point here but due to the continual fission and fusion, as well as piecemeal turnover of mitochondria (see Vincow et al. 2019 Autophagy doi: 10.1080/15548627.2019.1586258), the concept of ‘young’ versus ‘old’ mitochondria is misplaced. The mitochondrial network essentially exists as a milieu of components which are produced and degraded at different rates.

    c)Furthermore, we would like to ask the authors to use also the MERC tether as control in the mitophagy assay. This would allow further conclusions about the role of the mitophagy, its protective effect during aging and the role of MERCs in this process.

    We remind the reviewer that this MERC tether is constructed from an RFP with N- and C-terminal tethering peptides. The presence of this RFP prevents the proper analysis of the mitoQC mCherry signal. However, given the dramatic phenotypes we think that it is unlikely that a decrease in mitophagy alone can explain the detrimental effects of increased tethering.

    1. In Fig6 A,B the authors should include also the pdzd8 KD to support their claim that the rescue of climbing defects correlates with an reduction of MERCs.

    We thank the reviewer for this suggestion and we will perform this experiment.

    Moreover, it would be beneficial for their final conclusion, if the authors could show that increases mitophagy in the background of Ab42 expressing flies.

    We thank the reviewer for this suggestion and we will perform this experiment.

    **Minor comments:**

    1.Can the authors add to the figure legend of Fig 1F how the ER and Mitochondria were labeled?

    We have added the constructs to the figure legend (full genotypes for all figures are given in Table S2).

    2.Error bars should be added in the quantification of MERCs in Fig1C.

    The MERCs are quantified in three brains per genotype but as there were variable numbers of sections suitable for imaging from each brain the total values are combined to give a single percentage.

    3.A reference to Supplementary Fig S1D is missing in the main text.

    This figure is referenced in line 135

    4.Can the authors label the individual genotypes in Fig S3C and 4F?

    Figure labels and legends have been modified to clarify this.

    5.Can the author specify which brain region they imaged in Fig 5C?

    The regions imaged and quantified were chosen for their clear organelle morphology rather than targeting a specific brain region. All images were from the protocerebrum and the methods and figure legends have been updated to note this.

    6.Are the ATP levels normalized to ADP in Fig S3D? Can the authors specify in the figure and figure legend to what ATP was normalized?

    Figure labels and legends have been modified to clarify the ATP levels are normalised to total protein quantification of the samples.

    7.Please sort the supplementary figures in accordance to their reference order in the text.

    We thank the reviewer for checking this. This figure order will be rechecked in the final version as addressing reviewer comments is likely to lead to further changes.

    Reviewer #2 (Significance (Required)):

    The authors present here novel insights about the functional role of a new member of the MERCs, pdzd8, using RNAi mediated depletion and Drosophila melanogaster as a model system. As MERCs receive more attention especially in the context of their potential role in neurological diseases, the author's manuscript will be of high interest to the scientific community. The in vivo model combined with multiple different technical approaches add to the significance of the paper. There are some controls and additional experiments that are required to support the author's main claims and complete the functional characterization of pdzd8 (see major comments).

    Field of expertise: neuroscience, fly genetics, neurodegeneration.

    Reviewer #3

    (Evidence, reproducibility and clarity (Required)):

    This manuscript entitled "Decreasing pdzd8-mediated mitochondrial-ER contacts in neurons improves fitness by increasing mitophagy" by Hewitt and collaborators describes the role of the Drosophila ortholog of PDZD8 in ER-mitochondria contacts in neurons and the physiological consequence of pdzd8 loss. The authors show that ER-mitochondria contacts are reduced in fly neurons expressing a pdzd8-RNAi construct. Decreasing pdzd8 expression in neurons was accompanied by a slowed age-associated decline in locomotor activity, and an increased lifespan. In presence of mitochondrial toxins, neurons deficient for pdzd8 were protected. Finally, the authors showed that pdzd8 silencing increased mitophagy in aged neurons, and protected against neurodegeneration in a model of Alzheimer's disease.

    **Major points:**

    1)There are important controls that are missing. RNAi expression often affects off-target genes which could unfortunately modify the observed phenotypes. The authors should verify that a) the phenotypes observed by RNAi-mediated pdzd8 silencing can be rescued by the expression of an RNAi-insensitive pdzd8 construct (the authors should verify the rescue of the most crucial phenotypes described in the manuscript); b) the RNAi-LacZ-line that they use as control in the paper does not behave differently from a WT line, which could be induced by an off-target effect of the RNAi-LacZ (again with the most crucial phenotypes).

    While the Drosophila community is fortunate to have a plethora of readily available tools for interrogating the function of nearly all genes in the genome – tools which form the foundation of most work in Drosophila labs worldwide – the availability is not limitless. In this instance, the transgenic RNAi line generated as a resource for the community comprises a 500 bp hairpin, computed to be the most selective target for that gene. Being a 500 bp sequence it is unrealistic to be able to establish an RNAi-resistant variant that still faithfully functions as normal. Nevertheless, although imperfect we show in Figure S3B that pdzd8-RNAi rescues the climbing defect produced by overexpressing pdzd8, providing evidence the construct is specifically acting on this sequence.

    Similarly, the availability of ‘control’ RNAi reagents is generous but still limited. This LacZ-RNAi line is one of a few well-established controls that has provided a cornerstone reference for a wealth of studies. Nevertheless, we will provide experimental data that aged climbing of nSyb>LacZ-RNAi is highly comparable to several other well-established control genotypes.

    1. Did the author analyzed their EM data in a blinded-way to minimize subjective bias? This type of analysis is complicated by the manual annotation of ultrastructures, which is by nature subjective. For instance, this reviewer would have annotated the two mitochondria in the middle of Fig 1B, right as "Mitochondria with ER contact", as there is a membrane tube present at the interface of these two organelles.

    The EM data were analysed blinded to the genotypes. This is noted in the methods section.

    1. There is a controversy in the field on the role of PDZD8: some papers show its involvement in ER-mitochondria contacts, others in ER-lysosome contacts. The authors should discuss this point in more details. Moreover, the authors should localize the protein in Drosophila neurons; is the protein associated with mitochondria or endo/lysosomes?

    We recognize that there is some debate in the field over the localization and role of PDZD8. However, since there is currently no antibody against the Drosophila protein and the sequence is sufficiently divergent such that antibodies against the mammalian protein will not recognize the fly protein, we are not well-positioned to determine the localization of Drosophila pdzd8. Consequently, we will expand our discussion to reflect the differing views.

    We can offer instead to quantify the localization of mouse PDZD8 in our newly generated NIH-3T3 Pdzd8-Halo knock in line to help resolve the controversy regarding the location(s) and function(s) of mammalian Pdzd8.

    1. The authors should specify in more details how the different quantifications were performed. For instance Fig 1G: how many samples were quantified (i.e. how many flies, and how many neurons); what is compared? Fields-of-view, neurons, flies...?

    Further details have been added to the figure legends 1G (now H), 4I, 5 and Fig S2.

    **Minor point:**

    1)Could the authors show the SIM images Fig1F together with the binarized images.

    These images have been added to Figure 1 and the legend and text updated accordingly.

    1. It is surprising to see that data otherwise similar are represented with so many different types of graph (For instance Fig 5, bar graph, box-plot, violin plot). Why individual data points are not always present on the graphs?

    The graphs will be redrawn using more consistent representations once the data for the revisions has been gathered.

    1. The way that data are presented is sometimes odd: for instance, line 101, the authors wrote "To establish that MERCs were decreased...". This would imply that they knew the result before performing the experiment. And later, line 103 "Accordingly...".

    These sentences have been rephrased “To determine whether MERCs were decreased..” and “These results showed the…”

    Reviewer #3 (Significance (Required)):

    This study about the role of pdzd8 is timely. The functional description of inter-organelle contacts is a hot topic in cell biology. There are several recent reports describing the identification of pdzd8 role in inter-organelle contact formation. This manuscript provides data on the role of pdzd8 in a whole organism and expands our understanding of this protein.

    My expertise: inter-organelle contacts (human cells)

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    This manuscript entitled "Decreasing pdzd8-mediated mitochondrial-ER contacts in neurons improves fitness by increasing mitophagy" by Hewitt and collaborators describes the role of the Drosophila ortholog of PDZD8 in ER-mitochondria contacts in neurons and the physiological consequence of pdzd8 loss. The authors show that ER-mitochondria contacts are reduced in fly neurons expressing a pdzd8-RNAi construct. Decreasing pdzd8 expression in neurons was accompanied by a slowed age-associated decline in locomotor activity, and an increased lifespan. In presence of mitochondrial toxins, neurons deficient for pdzd8 were protected. Finally, the authors showed that pdzd8 silencing increased mitophagy in aged neurons, and protected against neurodegeneration in a model of Alzheimer's disease.

    Major points:

    1)There are important controls that are missing. RNAi expression often affects off-target genes which could unfortunately modify the observed phenotypes. The authors should verify that a) the phenotypes observed by RNAi-mediated pdzd8 silencing can be rescued by the expression of an RNAi-insensitive pdzd8 construct (the authors should verify the rescue of the most crucial phenotypes described in the manuscript); b) the RNAi-LacZ-line that they use as control in the paper does not behave differently from a WT line, which could be induced by an off-target effect of the RNAi-LacZ (again with the most crucial phenotypes).

    2)Did the author analyzed their EM data in a blinded-way to minimize subjective bias? This type of analysis is complicated by the manual annotation of ultrastructures, which is by nature subjective. For instance, this reviewer would have annotated the two mitochondria in the middle of Fig 1B, right as "Mitochondria with ER contact", as there is a membrane tube present at the interface of these two organelles.

    3)There is a controversy in the field on the role of PDZD8: some papers show its involvement in ER-mitochondria contacts, others in ER-lysosome contacts. The authors should discuss this point in more details. Moreover, the authors should localize the protein in Drosophila neurons; is the protein associated with mitochondria or endo/lysosomes?

    4)The authors should specify in more details how the different quantifications were performed. For instance Fig 1G: how many samples were quantified (i.e. how many flies, and how many neurons); what is compared? Fields-of-view, neurons, flies...?

    Minor point:

    1)Could the authors show the SIM images Fig1F together with the binarized images.

    2)It is surprising to see that data otherwise similar are represented with so many different types of graph (For instance Fig 5, bar graph, box-plot, violin plot). Why individual data points are not always present on the graphs?

    3)The way that data are presented is sometimes odd: for instance, line 101, the authors wrote "To establish that MERCs were decreased...". This would imply that they knew the result before performing the experiment. And later, line 103 "Accordingly...".

    Significance

    This study about the role of pdzd8 is timely. The functional description of inter-organelle contacts is a hot topic in cell biology. There are several recent reports describing the identification of pdzd8 role in inter-organelle contact formation. This manuscript provides data on the role of pdzd8 in a whole organism and expands our understanding of this protein.

    My expertise: inter-organelle contacts (human cells)

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Hewitt et al. describe and characterize for the first time the ortholog of pdzd8 in Drosophila melanogaster. In accordance with pdzd8's previously described function as a member of mitochondrial-ER contact sites (MERCs) the authors show reduced MERCs upon RNAi mediated depletion of pdzd8 via TEM, SIM and a split-GFP based contact site sensor. Pdzd8 depletion results in the increased life span as well as improved locomotor activity in aging flies while increase of MERCs with a synthetic tether accelerates the age-related declines in survival and locomotion. Moreover, pdzd8 depleted flies are more resistant against mitochondrial toxins. The authors correlate these protective effects of pdzd8 knockdown with an increase in mitophagy using a mitophagy sensor and describe a rescue of locomotor defects in an Alzheimer disease fly model by pdzd8 depletion.

    Major comments:

    1.The authors quantify the number of MERCs in thin sections of TEM (Fig 1B and C). It would add to the paper if the authors would show a representative reconstruction of the quantified somata, as a 3D reconstruction would visualize ER-Mito contacts more reliable than thin sections.

    2.The authors quantify MERCs in pdzd8 KD also by SIM (Fig1F, G). However, they quantify the number of MERCs in epidermal cells while they also show SIM images of larval neurons (Fig S1D). For consistency and to support their claim of MERC reduction in neurons, we ask the authors to include the quantification based on larval neurons especially as the authors show that pdzd8 is predominantly expressed in the CNS.

    3.The authors describe a decreased NMJ volume in Fig 4G. It would improve and complete the functional characterization of pdzd8 in flies if the authors can provide further data whether pdzd8 KD causes a general synaptic defect. Can the authors show morphological synaptic defects in the existing TEM data of the adult brain or provide additional ERG recordings, which would elucidate the functional consequences of pdzd8 depletion in the CNS?

    4.Hewitt et al. suggest a beneficial effect of increased turnover of mitochondria for healthy aging. To convince readers we would like to ask the following:

    a)This claim is based on their observation of increased mitophagy in pdzd8 depleted flies using one reporter (Fig 5). Can the authors support their data with an alternative method as this is one of the key claims of the manuscript?

    b)An increased turnover of Mitochondria would also suggest that there are more "young" mitochondria present in the pdzd8 KD neurons. Can the authors experimentally address that?

    c)Furthermore, we would like to ask the authors to use also the MERC tether as control in the mitophagy assay. This would allow further conclusions about the role of the mitophagy, its protective effect during aging and the role of MERCs in this process.

    5.In Fig6 A,B the authors should include also the pdzd8 KD to support their claim that the rescue of climbing defects correlates with an reduction of MERCs. Moreover, it would be beneficial for their final conclusion, if the authors could show that increases mitophagy in the background of Ab42 expressing flies.

    Minor comments:

    1.Can the authors add to the figure legend of Fig 1F how the ER and Mitochondria were labeled?

    2.Error bars should be added in the quantification of MERCs in Fig1C.

    3.A reference to Supplementary Fig S1D is missing in the main text.

    4.Can the authors label the individual genotypes in Fig S3C and 4F?

    5.Can the author specify which brain region they imaged in Fig 5C?

    6.Are the ATP levels normalized to ADP in Fig S3D? Can the authors specify in the figure and figure legend to what ATP was normalized?

    7.Please sort the supplementary figures in accordance to their reference order in the text.

    Significance

    The authors present here novel insights about the functional role of a new member of the MERCs, pdzd8, using RNAi mediated depletion and Drosophila melanogaster as a model system. As MERCs receive more attention especially in the context of their potential role in neurological diseases, the author's manuscript will be of high interest to the scientific community. The in vivo model combined with multiple different technical approaches add to the significance of the paper. There are some controls and additional experiments that are required to support the author's main claims and complete the functional characterization of pdzd8 (see major comments).

    Field of expertise: neuroscience, fly genetics, neurodegeneration.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2. The authors present an in vivo analysis of pdzd8 (CG10362) and a synthetic ER-mitochondria tether in the regulation of locomotor activity, lifespan, and mitochondrial turnover of Drosophila melanogaster, using basic bioinformatics, RNAi, SPLICS, imaging and microscopies observations (i. e. TEM, SIM), fly lines, and a representative AD fly disease model, etc. The research methodologies were detailed in good order. The model system employed was suitable to address the research topic. The manuscript was written in a clear language and statistical analysis were correctly applied.

    Major comments:

    -Are the key conclusions convincing?

    Yes. The results/conclusions are logical and provide an overview of Pdzd8 in the regulation of mitochondrial quality control and neuronal homeostasis.

    -Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    No. Experiments were generally well performed, and all the data support the conclusions.

    -Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    No suggested experiments needed.

    -Are the data and the methods presented in such a way that they can be reproduced?

    Yes. The authors have followed proper experimental design and methods have been described in sufficient detail.

    -Are the experiments adequately replicated and statistical analysis adequate?

    Yes, they are.

    Minor comments:

    -Specific experimental issues that are easily addressable.

    No comment.

    -Are prior studies referenced appropriately?

    Yes. The relevant literatures have been cited appropriately.

    -Are the text and figures clear and accurate?

    1.Please pay attention to the correct spelling of the described protein name (Pdzd8) and gene name (should be in 'italic') throughout the manuscript, i. e. line 36, 98, and 556, etc.

    2.In figure 1C and its figure legend, please state what the numbers "201" and "195" stand for.

    3.Your data needs to be converted the lowercase letter "x" to math symbol "×" when representing times sign, i. e. line 523, 5x, etc.

    -Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    No comment.

    Significance

    -Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    Discoveries from this study include 1) characterization of the tethering protein Pdzd8 in Drosophila melanogaster, and 2) shed light on a possible way on how to enhance mitochondrial quality control and to help promote healthy aging of neurons by manipulating MERCs.

    -Place the work in the context of the existing literature (provide references, where appropriate).

    With this manuscript, the authors present a straightforward but sound piece of scientific research, with the intent to illustrate the consequences of neuronal depletion of pdzd8 in Drosophila melanogaster. Since Pdzd8 plays specific functions in ER-mitochondrial tethering complexes and dysregulations of MERCs are damaging to neurons, this protein represents a good potential target. In this context the characterization of Pdzd8 should represent an interesting starting point. To this purpose, the gene was knockdown and the tether construct was recombinantly produced. The fly lines were then subjected to analysis both at the organismal and at the cellular level.

    -State what audience might be interested in and influenced by the reported findings. Audience might include those who are in the field of neuroscience and pharmaceutical, and benefit from an awareness of this research.

    -Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Key words in my field of expertise: Ageing, neurodegenerative diseases, Alzheimer's disease, mitophagy, NAD+, neuroprotection. My group is investigating the molecular mechanisms of ageing and age-related neurodegeneration (especially AD) using cross-species model systems, ranging from human brain samples, iPSCs, C. elegans, Drosophila melanogaster, and mice, therefore I have sufficient expertise to evaluate this paper.

    Referees Cross-commenting

    To this reviewer the key novelty of this paper was the study of the regulation of the mitochondrial-ER contact sites (MERCs) in life and health. The data indicate that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial homeostasis, neuronal function, and lifespan. In a transitional perspective, this reviewer would ask to check whether this mechanism conserves in rodents or not (e.g. to to memory in the AD mice and to run lifespan in mitochondrial toxin condition). This may be to much. But will depend on the standard of the journal.

  9. Version published to 10.1101/2020.11.14.382861 on bioRxiv