Next-generation Cu(II) selective peptide shuttles prevent Cu(Aβ)-induced toxicity and microglial activation in organotypic hippocampal slices

  1. Michael Okafor
  2. David Schmitt
  3. Stéphane Gasman
  4. Laurent Raibaut
  5. Christelle Hureau
  6. Peter Faller
  7. Nicolas Vitale

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Abstract

Alzheimer’s disease (AD) remains the most prevalent neurodegenerative disease with hallmarks including the apparition, in specific areas of the brain, of intracellular neurofibrillary tangles and extracellular amyloid plaques. The latter result from an abnormal metabolism of Amyloid-β precursor protein (APP) leading to its accumulation in plaques. Ex vivo analysis of AD patients’ brains, show an abnormally elevated concentration of metals including Cu, Zn and Fe in these plaques. Some studies have also demonstrated altered Cu levels in the entire brain and more specifically in regions heavily affected in AD. These modifications are often accompanied by a decline in neuronal Cu levels and by an increase in the proportion of extracellular labile Cu, which in turn promotes reactive oxygen species formation. To correct this Cu dyshomeostasis, we designed and synthesized novel Cu(II)-selective peptide shuttles, capable of swiftly retrieving Cu from extracellular Aβ and subsequently transporting and releasing Cu inside cells. We demonstrate here the capacity of this new Cu-shuttles, DapHH-αR5W4 NBD and HDapH-αR5W4 NBD , to protect organotypic hippocampal slices (OHSCs) from Cu(Aβ)-induced insult and their capability to rescues Cu-induced microglial activation and proliferation.

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  1. Version published to 10.1101/2024.09.04.611242v2 on bioRxiv
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    Reply to the reviewers

    Reviewer #1:

    Major Comments:

    1. The data in the paper strongly suggests that the new copper shuttles are selective for copper and have faster binding kinetics (Fig 1) than the previous one. However, the data regarding the copper shuttling from the copper(Aβ) peptides is not very convincing. It appears to be due to the Cu effect alone (Fig.3), as the reduction in viability with Cu(II)+ AscH- is almost the same as the Cu(II)(Aβ)+AscH-. To convincingly show that the peptide shuttle can strip copper from (Aβ) peptides, the authors need to show that the copper is bound to the (Aβ) peptide before it is used in the experiment. Rightfully so, the effect of the toxicity of Cu(II)+ AscH- is similar to that of Cu(II)(Aβ16)+AscH-. This is due to the fact that Aβ16 is not toxic to the cells, so therefore there is no compounded effect of Cu and Aβ16 as seen for Cu(II)(Aβ40). As for the toxicity of Cu(II)+ AscH-, it is be similar to Cu(II)(Aβ)+AscH- because Cu(II) will be bound to a weaker ligand in the medium and such loosely bound Cu is also able to produce ROS with AscH- with similar rates as Cu-Ab.

    Data from our lab and others have shown that in HEPES solution at pH 7.4, Aβ forms a complex with Cu. The present work is also in line with Cu-binding to Ab, as in Figure 1C (GSH), the rate of Cu withdrawal by the shuttle can only be explained by Cu bound to Ab, as Cu in the buffer binds to the shuttle much faster. Also, the AscH- consumption rate measured in Fig S5D-E are congruent of Cu bound to Ab, unbound Cu has a much faster rate of AscH- consumption (Santoro et al. 2018, doi.org/10.1039/C8CC06040A).

    The concentrations of Aβ and Cu used in our experimental condition were determined with a UV-Vis spectrophotometer.

    Minor comments:

    1. The paper does not cite Figure 1A and some supplementary figures, especially Supp. Fig. 1-2. All the figures and supplementary figures should be cited. This has been rectified for all the concerned figures.

    The data presentation in Figures 3B and S8 is confusing."-" signs indicate no addition or the blank box means no addition. Also, the AKH-αR5W4 has no "-" sign in the first bar. For clarity, please indicate the -, +, or no sign means in the figure legends. Also, what does "Batch A" refer to in Figure 3B?

    The figures have been modified as suggested by the reviewer.

    Page 7, correct (Error! Referencesource not found.Figure 1C).

    This has been rectified.

    The Giantin staining in Figure 2B is making it hard to visualize ATP7A trafficking. If the Giantin image overlay is removed, it may be easier to see the movement of ATP7A from the perinuclear region to the vesicles.

    The images have been modified to better appreciate the ATP7A change in distribution upon the increase in intracellular Cu level. We have reduced the number of conditions for which images are provided and provided individual staining for clarity. Zoomed images are also provided. The remainder of the conditions are in Figure S7B

    In the introduction, the authors mention, "These molecules have, however, a major pitfall as is seen for Elesclemol, a candidate for Menkes disease treatments 32. The authors cite reference " Tsvetkov, P. et al. Copper induces cell death by targeting lipoylated TCA cycle proteins." The paper showing elesclomol as a candidate for Menkes disease treatments is Guthrie L et al., Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice. Science. 2020.

    We thank the reviewer for pointing this out, which was apparently not clearly explained. Our intention here was to show that a major pitfall of shuttles like Elesclomol, as seen in the study by Tsvetkov, P. et al. Science (2022), is cuprotoxicity. The sentence has been clarified and the work of Guthrie L et al is cited for Elesclomol as a candidate for Menkes disease.

    Reviewer #2 :

    Major issues:

    1. This reviewer is not convinced that the authors' experimental system is well suited for studies of glia activation and protective effects. With the exception of a couple of panels it is very hard to see differences. The authors should significantly improve the quality of images in Figure 5 to make this set of data convincing. We thank the reviewer for his/her detailed evaluation and for bringing to light the quality of the image in Figure 5. We have therefore improved the quality of the images by improving the signal to noise ratio to better show the differences between conditions.

    Similarly, the quality of giantin staining is low and needs to be improved and more experimental details are needed (see details below).

    As stated in our answer to reviewer 1, the images have been modified to better appreciate ATP7A redistribution upon increase of intracellular Cu levels. We have reduced the number of conditions for which images are provided and provided individual staining for clarity. Zoomed images are also provided. The remainder of the conditions are in Figure S7B.

    Given that shuttles are found within vesicles, the authors should discuss the mechanism through which Cu is released into the cytosol to trigger ATP7B trafficking.

    The mechanism of Cu escape from endosomes remains poorly understood. However, supported by our recent observations that Cu quickly (within 10 min) dissociates from the Cu-shuttle AKH-αR5W4NBD in endosomes (Okafor et al., 2024, /doi.org/10.3389/fmolb.2024.1355963), we discuss the potential involvement CTR1/2 and DMT1 (page 16).

    There are numerous small writing issues that make paper difficult to read. The authors are encouraged to carefully edit their manuscript.

    We thank the reviewer for pointing this out and several errors have been corrected whereas various sentences have been clarified.

    Minor issues

    * „A solution of monomerized Aβ complex in 10% DMEM (diluted with DMEM salt solution) was prepared in microcentrifuge tubes" - here and further the description of media composition is confusing What is the rest 90%?

    This has been rectified. The composition of the salt solution that makes up the 90% has been provided (page 4).

    * „Afterwards, AscH- was added to the tubes and vortexed, the mixture was then added to PC12 cells" - concentration of ascorbate is mentioned only once (later in the figure legend) where it can be barely found, also without explaining the choice of concentration. Additionally, ascorbate's product code is not listed. Please, correct.

    These points have been rectified.

    * Description of the cell (PC12 line) handling conditions is absent (growth medium, passage number used etc) and should be included.

    This information is now provided.

    * ATP7A delocalization assay. Details for the secondary antibodies are absent (full name (e.g. AlexaFluor 488), manufacturer, code) and should be added.

    Missing information has been added.

    * page 6: „Next, we investigated the capacity of the shuttles to withdraw Cu(II) from cell culture media, DMEM 10% and DMEM/F12 1:1 (D/F)." Here and further explanation is needed why the mixture of DMEM/F12 is needed (F12 is also not listed in the materials list).

    DMEM/F12 is a media that is commercially available used for some cell types, and it has been added to the materials list (page 4).

    * Page 7. Legend to the figure 1B: „Conditions: Cu(II)=AKH-αR5W4NBD=DapHH-αR5W4NBD=HDapH-αR5W4NBD= 5 μM, DMEM 10%, D/F 100%, 25{degree sign}C, n=3." - „DMEM/F12" ratio equals to „100%" is confusing, please clarify

    This has been clarified.

    * Page 8-9. Legend to the Figure 2A. „Similar observations were obtained with 5 different cell cultures." Same remark goes to the legend to supplementary figure 7 ("Similar observations were obtained with at least 3 different cell cultures"). Do the authors mean independent experiments or different cell lines? Please clarify. If different cell lines, consider including these data into the supplement.

    Indeed we meant independent experimentations. This has been clarified.

    * Page 8-9, figure 2B. Giantin is a cis-golgi marker, which should localize perinuclearly. In the cells shown the signal is diffuse and appears non-specific. Please improve the quality.

    We have reduced the number of conditions for which images are provides and are providing individual staining for clarity. Zoomed images are also provided allowing visualization of the typical cis-Golgi distribution of Giantin.

    * Page 8-9, figure 2B. ATP7A is shown in green. The authors did not specify the secondary antibody has been used for it. If the secondary antibody used for labeling of ATP7A has green fluorescence then how does one distinguish between the transporter signal and signal of the green fluorescent shuttle? Please provide more details.

    We thank the reviewer for pointing this point as we missed to mention this technical issue in the original manuscript. The Cu-shuttles labeled with NBD indeed emit in the green signal, but they are not fixable under our conditions and are washed out during ICC procedure. Accordingly, they do generate any background signal and do not interfere with the ICC as shown by the controls and test conditions (Figure S7B and Figure 2B). This is now mentioned (page 11).

    * Page 9 and Figure 2B. Why did authors use Cu(II)EDTA for the experiment? What was the concentration? Please, add this information as well as Cu(II)GTSM treatment conditions to the experiment description in materials and methods.

    EDTA is a strong chelator of Cu(II), however due to its negative charge it cannot penetrate the plasma membrane thus importing Cu. It is therefore used as a negative control, to eliminate the speculation of Cu non-specifically crossing the plasma membrane or through a channel.

    * Figure 2 and supplementary figure 7. It would be beneficial to have higher magnification images. Please, add them, if possible.

    These higher magnification images have been provided.

    * Page 11. „In conclusion, the novel Cu(II)-selective peptide shuttles .... capable of instantly preventing ... toxicity on PC12 cells, whereas ... instantly rescue Cu(II)Aβ1-42 toxicity". Authors should be more careful with terminology. According to the materials and methods, the survival assay was carried out after 24h of cells' treatment with the reagents. Effect visible after 24h and „instant rescue" is not the same, Please clarify or modify the wording

    In principle, the peptides cannot reverse the production of ROS, however they prevent ROS production. Therefore, for the peptides to have an effect, they have to instantly halt ROS production. This is justified by the novel shuttles being more effective than AKH-αR5W4NBD in preventing toxicity, given we modified just the Cu binding sequence. We have however restricted the use of the term instantly to ROS production.

    * Page 13, figure 5, panels C and D. In both quantitations Cu(II) was used as one of the control conditions. Why in panel D the percentage of activated microglial cells (second graphs from right) is several fold higher (appr. 150% vs >500%)?

    This variability was observed throughout our set of experiments and could be linked to the quality of the hippocampal slices used. Slight variations in the age of the animals or in the traces of metals in the mediums are likely explanations. However, the different groups that are compared represent experiments performed simultaneously.

    * Supplementary Figure S3B. The lowest solid line does not correspond to any color in the legend (please, check and correct). However, by the method of exclusion, one may conclude that it refers to Cu(II)+HDapH-shuttle. What could be a potential explanation for stronger quenching of this shuttle by binding Cu(II) directly from the spiked media comparing to when it is pre-complexed with copper (also supported by the panel D)?

    The stronger quenching of this shuttle by binding Cu(II) directly from the spiked media comparing to when it is pre-complexed with copper is not significant.

    * In discussion the authors mention that the designed shuttles are prone to degradation in 48 hours. In the viability assays, they treat cells for 24 hours, in the fluorescent and confocal microscopy experiments for one hour or less. What is the lifetime of these shuttle peptides in the cells?

    The lifetime of the shuttle peptide in the cells is currently unknown. However, after 24h incubation of PC12 cells with the AKH-αR5W4NBD, DapHH-αR5W4NBD and HDapH-αR5W4NBD, the Cu shuttles lose their punctate distribution and appear diffuse inside the cells. We have recently shown that AKH-αR5W4NBD cycles through different endosomal compartments and eventually reaches the lysosomes where it could be degraded (Okafor et al., 2024, /doi.org/10.3389/fmolb.2024.1355963). Therefore, the diffuse distribution of the fluorescence signal could suggest degradation of the Cu-shuttles.

    * From the microscopy observations, the mechanism of entry of apo-shuttles (with no Cu(II) in the complex) and in complex with Cu(II) looks quite different. Namely, in figure S7 the fluorescent signal is very strong in the plasma membrane with significantly less vesicular pattern when compared to figure 2A. It is especially apparent for DapHH shuttle at 15 minutes of incubation. Can authors hypothesize/discuss the reason for these differences?

    The difference of the shuttle’s signal in the presence or absence of Cu binding, is due to fluorescence quenching by Cu bound and was at the heart of the design of these shuttles. Hence a strong signal at the plasma membrane is seen in the absence of Cu as these CPP-based shuttles interact strongly with the plasma membrane. However in presence of Cu, they become less visible due to quenching by Cu. Interestingly however, is that when Cu dissociates from the shuttle inside the cells (likely in acid endosomes), this quenching is suppressed and the fluorescence reappears. This is now better explained (page 10).

    * Please, show the figures in the supplementary file in the same order as you refer to them.

    This has been rectified.

    * Introduction. Description of the shuttle peptides: „(3) a cell penetrating peptide (CPP), αR5W4, with sequence RRWWRRRWWR, for cell entry35" - one R is the middle is extra.

    This has been rectified.

    *Kd units are missing (pages 2, 3 and 15) and should be added.

    This has been added.

    * Figure 1A is either not referred at all or mislabeled.

    * Page 7, Figure 1B: x axis on the second panel (+Mn+) misses a label.

    * Page 8. „Upon addition of DapHH-αR5W4NBD or HDapH-αR5W4NBD, an immediate slow-down in ROS production was observed (Figure 1D and S1E), ..." - mislabeled supplementary figure, please, correct.

    * Page 11. „...but not in the presence of AKH-αR5W4NBD which required pre-incubation to prevent toxicity (Figure 3AFigure)." Please, correct the reference to the figure.

    * Page 11. „This is in line with the faster retrieval ... previously demonstrated in vitro (Figure 1)" - please, specify the panel.

    * Supplementary materials and methods, subsection „Retrieval of Cu by peptide shuttles from Aβ", page 2: „The same was done for 10 μM Cu(II)...to give the estimated 100% saturated emission level." - check the spelling of the shuttle species.

    * Supplementary Figure S4. By the behavior of AKH-shuttle in the presence of copper and other metals, it looks that panels are shuffled, i.e. panel C looks corresponding to the panel B with DMEM/F12 conditions, whish is also supported by the values in the Table S1. Please, check and correct, if needed.

    * Supplementary figure S9, panel A. Apparently, mislabeled images with Abeta1-42 and Cu(II)Abeta1-42. Please, correct.

    We apologize for the different issues in referencing figures. This has been rectified.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Minor Concerns

    I think that authors can add some concepts of general interest on AD, as follows

    evidence showed that AD top-line disease-modifying drugs employing monoclonal antibodies (donanemab, lecanemab, and aducanumab) that tag Aβ, based on the 'Amyloid cascade hypothesis', are able to rid the brain of Aβ plaques, but the drug benefits consist in a reduction of 35% of cognitive decline. The remaining disease burden (more than 65%) has no disease-modifying therapeutic options, at the moment. Furthermore, monoclonal antibodies against Aβ have strong side- events (ARIA). On this basis, it could be suggested that removing Aβ plaque might not be sufficient to slow the 100% percentage of clinical decline in AD. This is why the Cu(II) shuttle invention presented by the candidate may represent a valid and concrete means to fight AD, since also meta-analyses demonstrate that Cu and more specifically non-Cp Cu is increased in AD (PMID: 34219710). The authors can add some of these clinical considerations in the Discussion.

    There is only a very brief description of the scenario of evidence of the involvement of copper in Alzheimer's, especially from a clinical point of view, I mean the scenario resulting from clinical studies carried out on AD patients. This would have highlighted the unmet medical need to which these new compounds (the Cu shuttles) can provide an answer. At least for a subpopulation of Alzheimer's patients, and we know that there are different subtypes of Alzheimer's disease (for example 10.1016/j.neurobiolaging.2004.04.001, but authors can find others), these Cu(II) selective shuttles could provide beneficial effects. Literature reports about a percentage of AD patients with increased levels of Cu (some papers on this topic e can be easily retrieved,), who may primarily benefit from these compounds. These can be easily identified as it is also characterized by a different biochemical, cognitive, and genetic profile. The current study is timely since AD patients with high Cu can be easily identified since they are characterized by a different biochemical, cognitive, and genetic profile as per recent findings (PMID: 37047347). This information can improve the quality of the manuscript by providing information about the unmet clinical need that this study can answer

    We thank the reviewer for his very positive evaluation and for his suggestion that gives more perspective to our work. Accordingly, we have added these parts to the introduction and discussion sections.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: The paper addresses the design and synthesis of novel copper (Cu)-selective peptide transporters to prevent Cu(Aβ)-induced toxicity and microglial activation in organotypic hippocampal slices.This is a very interesting study. I would define the study as pioneering and I hope that it is a seminal study, as it could be a study that opens the doors to future studies in the sector but above all applications in the clinical field. The methods are very complex and demonstrate an excellent knowledge of the biochemistry of beta-amyloid and copper. Methods are also clear and provide information for reproducibility

    Minor Concerns

    I think that authors can add some concepts of general interest on AD, as follows evidence showed that AD top-line disease-modifying drugs employing monoclonal antibodies (donanemab, lecanemab, and aducanumab) that tag Aβ, based on the 'Amyloid cascade hypothesis', are able to rid the brain of Aβ plaques, but the drug benefits consist in a reduction of 35% of cognitive decline. The remaining disease burden (more than 65%) has no disease-modifying therapeutic options, at the moment. Furthermore, monoclonal antibodies against Aβ have strong side- events (ARIA). On this basis, it could be suggested that removing Aβ plaque might not be sufficient to slow the 100% percentage of clinical decline in AD. This is why the Cu(II) shuttle invention presented by the candidate may represent a valid and concrete means to fight AD, since also meta-analyses demonstrate that Cu and more specifically non-Cp Cu is increased in AD (PMID: 34219710). The authors can add some of these clinical considerations in the Discussion

    there is only a very brief description of the scenario of evidence of the involvement of copper in Alzheimer's, especially from a clinical point of view, I mean the scenario resulting from clinical studies carried out on AD patients. This would have highlighted the unmet medical need to which these new compounds (the Cu shuttles) can provide an answer. At least for a subpopulation of Alzheimer's patients, and we know that there are different subtypes of Alzheimer's disease (for example 10.1016/j.neurobiolaging.2004.04.001, but authors can find others), these Cu(II) selective shuttles could provide beneficial effects. Literature reports about a percentage of AD patients with increased levels of Cu (some papers on this topic e can be easily retrieved,), who may primarily benefit from these compounds. These can be easily identified as it is also characterized by a different biochemical, cognitive, and genetic profile. The current study is timely since AD patients with high Cu can be easily identified since they are characterized by a different biochemical, cognitive, and genetic profile as per recent findings (PMID: 37047347). This information can improve the quality of the manuscript by providing information about the unmet clinical need that this study can answer

    Significance

    The significance of the study relies on that the Cu(II) selective shuttles can import Cu into cells and also release Cu once inside the cells, which was shown to be bioavailable, as revealed by the delocalization of ATP7A from the TGN to the sub-plasmalemma zone in PC12 cells. The novelty is well expressed by the implementation of Cu(II) selective shuttles that can release Cu inside the cells. Thus, they can restore Cu physiological levels in conditions of brain Cu deficiency that typify the neuronal cells in AD. Furthermore, this Cu trafficking can be finely tuned, thus preventing potential adverse drug reactions when transferred into human clinical phase I and II studies. This application may represent a step forward concerning previous copper attenuating compounds/Cu(II) ionophores such as Clioquinol and GTSM which mediated non-physiological Cu import into the cells that have likely contributed to neurotoxicity processes in previous unsuccessful phase II clinical trials.

    Furthermore, the originality of the current study relies on the fact that these shuttles can be tracked in real-time, once in the cell, since they employ a fluorophore moiety sensitive to Cu binding. Furthermore, DapHH-αR5W4NBD and HDapH-αR5W4NBD, can import bioavailable Cu(II) and can prevent ROS production by Cu(II)Aβ instantly, due to the much faster Cu-binding. Importantly, DapHH-αR5W4NBD and HDapH-αR5W4NBD shuttles have been also capable of preventing OHSC slices from Cu-induced neurotoxicity, microglial proliferation, and activation. Another important feature of the Cu shuttles is that they can be designed to control their site of cell delivery. In fact, previous ionophores had the tendency to accumulate in the mitochondria, and, in doing so, excess Cu in the mitochondria might have competed with other transitional metals (mainly Fe) and triggered mitochondrial dysfunction as well as cuproptosis. These are the main strengths of the study.

    The audience of this article is currently that of expert biochemists, but by adding aspects regarding the unmet clinical need relating to the large population of AD patients with high copper in the introduction and discussion, the article can capture the attention of clinicians.

    I am a neuroscientist working on biochemical pathways and metals in Alzheimer's disease.

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    Referee #2

    Evidence, reproducibility and clarity

    This is an interesting work characterizing a new generation of copper shuttles with an improved ability to retrieve copper intracellularly from amyloid beta (Ab). In the in-vitro experiments, the authors demonstrate that both DapHH-αR5W4NBD and HDapH-αR5W4NBD have faster Cu(II) retrieval kinetic than the previously characterized shuttle. The authors show the ability of on Cu(II)-DapHH-αR5W4NBD and Cu(II)-HDapH-αR5W4NBD to release copper intracellularly by monitoring changes in the intracellular pattern of the copper transporter ATP7A. Using PC12 cells, the author found that one of the shuttles, DapHH-αR5W4NBD can rescue Cu(II)Aβ1-42 toxicity, and this and other shuttles, show some protective effects in organotypic slices. Overall, the chemical and biochemical data are clear and the ability of new shuttles to deliver Cu to vesicles is convincingly demonstrated. Similarly, the protective effects on plasma membrane permeability in hippocampal staining are also apparent.

    Major issues:

    1. This reviewer is not convinced that the authors' experimental system is well suited for studies of glia activation and protective effects. With the exception of a couple of panels it is very hard to see differences. The authors should significantly improve the quality of images in Figure 5 to make this set of data convincing.
    2. Similarly, the quality of giantin staining is low and needs to be improved and more experimental details are needed (see details below)
    3. Given that shuttles are found within vesicles, the authors should discuss the mechanism through which Cu is released into the cytosol to trigger ATP7B trafficking.
    4. There are numerous small writing issues that make paper difficult to read. The authors are encouraged to carefully edit their manuscript

    Minor issues

    • „A solution of monomerized Aβ complex in 10% DMEM (diluted with DMEM salt solution) was prepared in microcentrifuge tubes" - here and further the description of media composition is confusing What is the rest 90%?
    • „Afterwards, AscH- was added to the tubes and vortexed, the mixture was then added to PC12 cells" - concentration of ascorbate is mentioned only once (later in the figure legend) where it can be barely found, also without explaining the choice of concentration. Additionally, ascorbate's product code is not listed. Please, correct.
    • Description of the cell (PC12 line) handling conditions is absent (growth medium, passage number used etc) and should be included.
    • ATP7A delocalization assay. Details for the secondary antibodies are absent (full name (e.g. AlexaFluor 488), manufacturer, code) and should be added
    • page 6: „Next, we investigated the capacity of the shuttles to withdraw Cu(II) from cell culture media, DMEM 10% and DMEM/F12 1:1 (D/F)." Here and further explanation is needed why the mixture of DMEM/F12 is needed (F12 is also not listed in the materials list).
    • Page 7. Legend to the figure 1B: „Conditions: Cu(II)=AKH-αR5W4NBD=DapHH-αR5W4NBD=HDapH-αR5W4NBD= 5 μM, DMEM 10%, D/F 100%, 25{degree sign}C, n=3." - „DMEM/F12" ratio equals to „100%" is confusing, please clarify
    • Page 8-9. Legend to the Figure 2A. „Similar observations were obtained with 5 different cell cultures." Same remark goes to the legend to supplementary figure 7 ("Similar observations were obtained with at least 3 different cell cultures"). Do the authors mean independent experiments or different cell lines? Please clarify. If different cell lines, consider including these data into the supplement
    • Page 8-9, figure 2B. Giantin is a cis-golgi marker, which should localize perinuclearly. In the cells shown the signal is diffuse and appears non-specific. Please improve the quality
    • Page 8-9, figure 2B. ATP7A is shown in green. The authors did not specify the secondary antibody has been used for it. If the secondary antibody used for labeling of ATP7A has green fluorescence then how does one distinguish between the transporter signal and signal of the green fluorescent shuttle? Please provide more details
    • Page 9 and Figure 2B. Why did authors use Cu(II)EDTA for the experiment? What was the concentration? Please, add this information as well as Cu(II)GTSM treatment conditions to the experiment description in materials and methods.
    • Figure 2 and supplementary fugure 7. It would be beneficial to have higher magnification images. Please, add them, if possible.
    • Page 11. „In conclusion, the novel Cu(II)-selective peptide shuttles .... capable of instantly preventing ... toxicity on PC12 cells, whereas ... instantly rescue Cu(II)Aβ1-42 toxicity". Authors should be more careful with terminology. According to the materials and methods, the survival assay was carried out after 24h of cells' treatment with the reagents. Effect visible after 24h and „instant rescue" is not the same, Please clarify or modify the wording
    • Page 13, figure 5, panels C and D. In both quantitations Cu(II) was used as one of the control conditions. Why in panel D the percentage of activated microglial cells (second graphs from right) is several fold higher (appr. 150% vs >500%)?
    • Supplementary Figure S3B. The lowest solid line does not correspond to any color in the legend (please, check and correct). However, by the method of exclusion, one may conclude that it refers to Cu(II)+HDapH-shuttle. What could be a potential explanation for stronger quenching of this shuttle by binding Cu(II) directly from the spiked media comparing to when it is pre-complexed with copper (also supported by the panel D)?
    • In discussion the authors mention that the designed shuttles are prone to degradation in 48 hours. In the viability assays, they treat cells for 24 hours, in the fluorescent and confocal microscopy experiments for one hour or less. What is the lifetime of these shuttle peptides in the cells?
    • From the microscopy observations, the mechanism of entry of apo-shuttles (with no Cu(II) in the complex) and in complex with Cu(II) looks quite different. Namely, in figure S7 the fluorescent signal is very strong in the plasma membrane with significantly less vesicular pattern when compared to figure 2A. It is especially apparent for DapHH shuttle at 15 minutes of incubation. Can authors hypothesize/discuss the reason for these differences?
    • Please, show the figures in the supplementary file in the same order as you refer to them.
    • Introduction. Description of the shuttle peptides: „(3) a cell penetrating peptide (CPP), αR5W4, with sequence RRWWRRRWWR, for cell entry35" - one R is the middle is extra.
    • Kd units are missing (pages 2, 3 and 15) and should be added
    • Figure 1A is either not referred at all or mislabeled
    • Page 7, Figure 1B: x axis on the second panel (+Mn+) misses a label
    • Page 8. „Upon addition of DapHH-αR5W4NBD or HDapH-αR5W4NBD, an immediate slow-down in ROS production was observed (Figure 1D and S1E), ..." - mislabeled supplementary figure, please, correct.
    • Page 11. „...but not in the presence of AKH-αR5W4NBD which required pre-incubation to prevent toxicity (Figure 3AFigure)." Please, correct the reference to the figure.
    • Page 11. „This is in line with the faster retrieval ... previously demonstrated in vitro (Figure 1)" - please, specify the panel.
    • Supplementary materials and methods, subsection „Retrieval of Cu by peptide shuttles from Aβ", page 2: „The same was done for 10 μM Cu(II)...to give the estimated 100% saturated emission level." - check the spelling of the shuttle species
    • Supplementary Figure S4. By the behavior of AKH-shuttle in the presence of copper and other metals, it looks that panels are shuffled, i.e. panel C looks corresponding to the panel B with DMEM/F12 conditions, whish is also supported by the values in the Table S1. Please, check and correct, if needed.
    • Supplementary figure S9, panel A. Apparently, mislabeled images with Abeta1-42 and Cu(II)Abeta1-42. Please, correct.

    Significance

    Delivering copper to various cells and tissue to improve cells function or removal excess copper to decrease pathology is an important and timely goal. This work describe new membrane-permeable reagents, "shuttles" with improved intracellular copper release and protective effects in PC12 cells. While, the results are overall interesting, the quality of writing and data presentation needs to be improved.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript titled "Next-generation Cu(II) selective peptide shuttles prevent Cu(Aβ)-induced toxicity and microglial activation in organotypic hippocampal slices" the authors have designed and synthesized two novel peptide shuttles that specifically bind to copper in the extracellular medium and transport them into the cells where copper is released and used for the copper-dependent function. The new copper shuttles are based on the previously published copper shuttle reported by the same group. Compared to the older peptide shuttle, which required pre-incubation for an hour in cellular media before adding AscH- to prevent copper(Aβ)-induced toxicity, the new copper shuttles reported in this article do not require pre-incubation. Overall, the manuscript is well written, experiments are controlled, and data are clear. The authors need to clarify some of the issues mentioned below:

    Major Comments:

    1. The data in the paper strongly suggests that the new copper shuttles are selective for copper and have faster binding kinetics (Fig 1) than the previous one. However, the data regarding the copper shuttling from the copper(Aβ) peptides is not very convincing. It appears to be due to the Cu effect alone (Fig.3), as the reduction in viability with Cu(II)+ AscH- is almost the same as the Cu(II)(Aβ)+AscH-. To convincingly show that the peptide shuttle can strip copper from (Aβ) peptides, the authors need to show that the copper is bound to the (Aβ) peptide before it is used in the experiment.

    Minor comments:

    1. The paper does not cite Figure 1A and some supplementary figures, especially Supp. Fig. 1, 2. All the figures and supplementary figures should be cited.
    2. The data presentation in Figures 3B and S8 is confusing."-" signs indicate no addition or the blank box means no addition. Also, the AKH-αR5W4 has no "-" sign in the first bar. For clarity, please indicate the -, +, or no sign means in the figure legends. Also, what does "Batch A" refer to in Figure 3B?
    3. Page 7, correct (Error! Referencesource not found.Figure 1C).
    4. The Giantin staining in Figure 2B is making it hard to visualize ATP7A trafficking. If the Giantin image overlay is removed, it may be easier to see the movement of ATP7A from the perinuclear region to the vesicles.
    5. In the introduction, the authors mention, "These molecules have, however, a major pitfall as is seen for Elesclemol, a candidate for Menkes disease treatments 32. The authors cite reference " Tsvetkov, P. et al. Copper induces cell death by targeting lipoylated TCA cycle proteins." The paper showing elesclomol as a candidate for Menkes disease treatments is Guthrie L et al., Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice. Science. 2020.

    Significance

    General Assessment: This well-written manuscript reports two novel peptide shuttles that specifically bind to copper in the extracellular medium and transport them into the cells where copper is released and available for the copper-dependent function. However, more convincing data is needed to show that the new peptide shuttles can pick copper from the copper bound to the (Aβ) peptides. In addition to their high specificity to copper, these copper shuttles can be tracked in real-time, making them well-suited for mechanistic studies to follow copper importation in cells, providing valuable new research tools to the copper community.

    Advance: The new copper shuttles in this manuscript are based on the previously published copper shuttle reported by the same group. Compared to the older peptide shuttle, which required pre-incubation for an hour in cellular media before adding AscH- to prevent copper(Aβ)-induced toxicity, the new copper shuttles reported in this article do not require pre-incubation and hence have faster binding kinetics.

    Audience: It will attract a broad audience, as the copper shuttles reported in this paper are promising drugs for Alzheimer's disease.

    My expertise: Mitochondria copper biology

  6. Version published to 10.1101/2024.09.04.611242v1 on bioRxiv