Mitophagy, the selective removal of damaged mitochondria, is thought to be critical to maintain neuronal homeostasis. Mutations of proteins in the pathway cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration. In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage. Mitophagy-associated proteins were sequentially recruited to depolarized mitochondria followed by sequestration into autophagosomes. The localization of these mitophagy events had a robust somal bias. In basal and induced conditions, engulfed mitochondria remained in non-acidified organelles for hours to days, illustrating efficient autophagosome sequestration but delayed lysosomal fusion or acidification. Furthermore, expression of an ALS-linked mutation in the pathway disrupted mitochondrial network integrity and this effect was exacerbated by oxidative stress. Thus, age-related decline in neuronal health or expression of disease-associated mutations in the pathway may exacerbate the slow kinetics of neuronal mitophagy, leading to neurodegeneration. Mitochondrial damage or depolarization decreases function and signals maintenance mechanisms to repair or eliminate damaged organelles.The mitochondrial network is a dynamic interconnected system of organelles undergoing continuous renewal and rearrangement to support cellular needs. Given that neurons have high-energy demands at sites far from the soma, the targeted delivery and regulated removal of mitochondria are essential at these distal sites . These oDue to the unique characteristics of neurons, multiple quality control mechanisms likely function independently to maintain mitochondrial health . In one Mitophagy is primarily regulated by two proteins linked to familial PD, PTEN-Induced Putative Kinase 1 (PINK1) and Parkin . PINK1 rmito-QC mitophagy probe also did not reveal deficits in tissues of high metabolic demand in mice (mito-QC+/+Pink1-/-) or in dopaminergic neurons of PINK1 and Parkin mutant flies  mice display mild phenotypes with no dopaminergic neuron loss, a hallmark of PD, implying there are additional compensatory pathways contributing to neuronal mitophagy in vivo . In sharnt flies . Howevernt flies . Using tnt flies . Furthernt flies . Interesnt flies . Thus, tmito-QC mice illustrated mitophagic events in somatodendritic compartments, with no axonal events reported  deprivation for 1 or 6 hr, noted as AO-free . In paraTo quantitatively assess whether these mild oxidative stresses initiated mitochondrial damage, intracellular ROS was detected by a fluorogenic probe, CellROX. The signal intensity of CellROX was low in control neurons and increased significantly in both the AO-free and AA conditions . Since rIn neurons, we examined the endogenous expression levels of mitophagy-associated proteins, including Parkin, TBK1, OPTN, LC3, and LAMP1 . ProteinWe next determined whether our mitochondrial damage paradigm was sufficient to not only induce organelle damage, but to initiate neuronal mitophagy. In HeLa cells expressing exogenous Parkin, OPTN was efficiently recruited to fragmented mitochondria within 30 min following CCCP treatment . In contTo investigate the extent of OPTN association with damaged mitochondria, we quantified the percent of OPTN puncta on linear  versus rounded (i.e. damaged) mitochondria. OPTN association with rounded mitochondria significantly increased after mitophagy induction with AO-free treatment compared to control. In contrast, the percentage of OPTN puncta on linear mitochondria was similar in either condition . It shouIn non-neuronal cells, OPTN, NDP52, and TAX1BP1 are proposed to mediate the autophagic clearance of damaged mitochondria, while p62 clusters organelles without directly facilitating mitophagy . In neurWT significantly increased the percentage of cells with OPTN-positive mitochondria rings following 1 h AO-free treatment compared to KD alone  and was used to discriminate between axons and dendrites. A higher percentage of AO-free treated neurons displayed OPTN-positive mitochondria compared to control cells , it is possible that these experiments undersampled axonal events. To address this possibility, we searched specifically for mitophagic events in the axons of control and treated cells. We found a very low frequency of OPTN-positive mitochondrial fragments in axons that was not different between control and treated neurons .Next, we investigated the spatial dynamics of OPTN-mediated neuronal mitophagy by acquiring z-stacks of neuronal compartments  following mitophagy treatments . Cells wol cells . Strikinol cells ; this trTo follow up on this point, we used longer (6 h AO-free) and harsher (2 h AA) treatments to induce mitophagy and again observed similar levels of OPTN-positive mitochondria in the soma, but rarely in the axon . To testII-III was used to discriminate between compartments, so neurons were instead transfected with Mito-SNAP, Halo-OPTN, and mScarlet-LC3 , a fluorescent dye that marks acidic vesicular compartments, to monitor the organelle acidification that is required for mitochondrial degradation . In sharS211 across all treatments compared to control  to initiate complete turnover of the mitochondrial network. We found a similar time course of mitochondrial fragmentation and engulfment by LC3, complete within ~1–2 hr. However, the time course of mitophagosome acidification was markedly faster in HeLa cells as compared to primary neurons, with >80% of OPTN-positive mitochondrial fragments also positive for LysoT within 1 h after mitophagy induction . Thus, wWhile LAMP1 is routinely used as a marker of lysosomes, recent work has demonstrated that a portion of LAMP1-positive organelles did not colocalize with lysosomal hydrolases, implying not all LAMP1 is on degradation-competent vesicles but also on late endosomes . Under oTo test this idea, we examined mitochondrial flux using a tandem Cox8-EGFP-mCherry reporter. The fluorescence signal of EGFP, but not mCherry, quenches in acidic environments and thus the shift in fluorescence from yellow to red is commonly used to measure the efficiency of mitophagy . Under aSince we observed differences in the localization of LAMP1 and LysoT with mitophagosomes, we performed similar experiments using a tandem SEP-LAMP1-RFP reporter, where superecliptic pHluorin (SEP) is fused to the lumen of LAMP1-RFP . Thus, wAs previously mentioned, two types of OPTN localization were observed with damaged mitochondria, OPTN rings and puncta . OPTN puTo investigate long-term autolysosome sequestration hours after initial damage, we performed SNAP-tag pulse-chase experiments. Neurons were transfected with Mito-SNAP, Halo-OPTN, and in some experiments LAMP1-EGFP. The following day, mitophagy was initiated and mitochondria were labeled with the first SNAP ligand for 30 min. Subsequently, treated neurons were placed in control maintenance media, 2 h SNAP block was used to saturate the remaining SNAP-binding sites, and neurons were incubated overnight. Prior to imaging, cells were labeled with a second SNAP ligand  for 30 min and in some experiments labeled with LysoT . As a reTwenty-four hours post-treatment, we visualized spherical, single labeled ‘Old’ Mito-SNAP puncta that were engulfed by Halo-OPTN rings. Strikingly, these OPTN-positive mitochondria were negative for ‘Young’ Mito-SNAP and LAMP1-EGFP or LysoT , indicatE478G is a heterozygous missense mutation identified in both sporadic and familial cases of ALS  plays a role in mitochondrial quality control in neurons, where it functions as an additional E3 ubiquitin ligase in a parallel pathway to Parkin . Other sE478G was subcloned from HA-OPTNE479G  to a pHaloTag vector (Promega), YFP-Parkin and mCherry-Parkin , mutant mCherry-Parkin T240R was generated from site-directed mutagenesis, untagged-Parkin (mCherry-Parkin was removed), YFP-NavII-III (Addgene 26056), GFP-Ub (Addgene 11928), TBK1 (Addgene 23851) was recloned into a pSNAPf vector (New England Biolabs), LAMP1-EGFP was subcloned from Addgene 1817 where RFP was replaced with EGFP, LAMP2-EGFP , SEP-LAMP1-RFP  and SNAP-WIPI2B (subcloned from GFP-WIPI2B). All constructs were verified by DNA sequencing. ON-TARGETPlus Rat OPTN (246294) siRNA SMARTpool (L-097177-02-0005) and ON-TARGETPlus Rat Prkn (56816) siRNA SMARTpool (L-090709-02-0005) were purchased from Dharmacon. Reagents used include the following: TMRE , Antimycin A , Oligomycin A , LysoTracker Green DND-26  and Deep Red , MitoTracker Green FM (ThermoFisher M7514), CellMask Orange Plasma membrane Stain (ThermoFisher 10045), B-27 Supplement minus antioxidants , and CellROX Deep Red Reagent . HaloTag constructs were labeled with Janelia Fluor 646 HaloTag  and SNAP-Tag constructs were labeled with JF646-SNAP , SNAP-Cell TMR-Star , SNAP-Cell 430 , and SNAP-Cell Block . For a list of key reagents, see Constructs used include the following: Mito-DsRed , Mito-SNAP (recloned from Mito-DsRed into a pSNAPf [New England Biolabs]), Cox8-EGFP-mCherry was subcloned from Addgene 78520, pEGFP-LC3B , mScarlet-LC3B (EGFP was replaced with mScarlet from Addgene 85054), mCherry-EGFP-LC3 , pEGFP-OPTN , Halo-OPTN , Halo-OPTN2 incubator; AraC (5 μM) was added the day after plating to prevent glia cell proliferation. For transfections, neurons (5–8 DIV) were transfected with 0.8–1.2 μg of total plasmid DNA using Lipofectamine 2000 Transfection Reagent (ThermoFisher) and incubated for 18–24 hr; DNA and siRNAs (45 pmol) mixtures were incubated 36–48 hr.Sprague Dawley rat hippocampal neurons at embryonic day 18 were obtained from the Neurons R Us Culture Service Center at the University of Pennsylvania. Cells  were plated on 25 mm acid-washed glass coverslips (World Precision) or in a 35 mm glass-bottom dishes (MatTek) that were precoated with 0.5 mg/ml poly-L-lysine (Sigma Aldrich). Cells were initially plated in Attachment Media  which was replaced with Maintenance Media , 100 units/ml penicillin, 100 μg/ml streptomycin, and 2% B-27 [ThermoFisher]) after 5 hr. Neurons were maintained at 37 C in a 5% CO2 incubator. For transfection, cells were plated on uncoated 35 mm glass-bottom dishes (MatTek), transfected with 2 μg of DNA using FuGene 6 (Promega), and incubated for 18 hr. HeLa cells were routinely tested for mycoplasma using a MycoAlert detection kit . Cells were authenticated by STR profiling using GenePrint10  at the DNA Sequencing Facility at The University of Pennsylvania.HeLa-M  cells were maintained in DMEM (Corning) that was supplemented with 1% GlutaMAX and 10% FBS. Cells were maintained at 37 C in a 5% COMitochondrial damaged was induced via oxidative stress through antioxidant removal or by inhibiting complex III of the electron transport chain using Antimycin A . For treatments, neuronal media was fully replaced with control MM, AO-free MM , or AA MM (MM plus 3 nM AA) for a time course of 1, 2 or 6 hr. For SNAP pulse-chase experiments, neurons were maintained in control media following treatment.In HeLa cells, mitochondrial damage was induced by oxidative stress using a bath application of 10 μM AA and 10 μM Oligomycin A. Cells were treated for 90 min then maintained in control media. Recruitment was assessed 0,1, 2, 4, and 6 h after treatment. One hour prior to imaging, SNAP  and Halo-tag ligands (100 nM) were applied for 30 min, followed by two quick washes and a 30 min washout totaling 1 hr; 2.5 μM Halo-tag ligand was used for HeLa cells. For SNAP pulse-chase experiments, SNAP Block was applied at 1 μM for 2 hr, followed by two quick washes and a 30 min washout. To asses mitochondrial membrane potential, neurons were loaded with 2.5 nM TMRE for 30 min, which occurred during the SNAP or Halo-tag ligand washout. Lysosome acidification was determined using Lysotracker (25–50 nM) that was incubated for 30 min during SNAP or Halo-tag ligand washout. For live-cell imaging, neurons were imaged in Imaging Media (HibernateE [Brain Bits] supplemented with 2% B27 and 33 mM D-glucose). Again, 2% B27, minus antioxidants replaced the standard B27 for AO-free conditions and 3 nM AA was added to the imaging media for AA conditions. HeLa cells were imaged in Leibovitz’s (1X) L-15 Medium (Gibco) supplemented with 10% FBS. For TMRE experiments, 2.5 nM TMRE was added to all Imaging Media. Mitochondrial and autophagosome motility was monitored in the mid-axon of 7–8 DIV neurons that were imaged at a rate of 4 timepoints/sec for 5 min. Neurons were imaged in an environmental chamber at 37 C on a Perkin Elmer UltraView Vox spinning disk confocal on a Nikon Eclipse Ti Microscope with a Plan Apochromat Lambda 60 × 1.40 NA and an Apochromat 100 × 1.49 NA oil-immersion objectives and a Hamamatsu EMCCD C9100-50 camera driven by Volocity . Z-stacks were acquired at 150 nm step-size.Hippocampal neurons  were plated in 96 well black plate with clear bottom (Corning) that was precoated with 0.5 mg/ml poly-L-lysine, grown for 7 DIV, and treated to induce mitochondrial damage. CellROX (5 μM) was added to each well 30 min prior to the end of treatment . Cells were washed three times with PBS and fluorescence was immediately analyzed using a plate reader. For fluorescent imaging of intracellular ROS, CellROX (5 μM) and MitoTracker (20 nM) were added 30 min prior to the end of treatment , cells were incubated with CellMask (5 μg/ml) for 5 min, and washed with media three times; neurons were immediately imaged.4, 150 mM NaCl, pH 7.4), permeabilized with ice cold methanol for 8 min at −20 C, and blocked for 1 h in blocking solution (5% goat serum and 1% BSA in PBS). Samples were incubated in primary antibodies diluted in blocking solution for 1 h and were washed three times with PBS; these steps was repeated with secondary antibodies. Following the three PBS washes after secondary antibodies, 0.1 mg/ml of Hoechst 33342 reagent  was added to samples and incubated for 10 min; one last PBS wash occurred after this step. Coverslips were mounted in ProLong Gold (Life Technologies) and images were acquired on a Leica DMI6000B inverted epifluorescence microscope with a 63 × 1.4 NA oil-immersion objective and a Hamamatsu ORCA-R2 charge-coupled device camera driven by LAS-AF (Leica Microsystems) or the previously mentioned Perkin Elmer spinning disk confocal.Neurons were fixed for 10 min using warm 4% PFA (HeLa cells) or 4%/4 %Sucrose . Cells were washed two times with PBS  for 30 min at 4 C. Samples were centrifuged at 4 C for 10 min at 15,800 x g, supernatant was collected, and a BCA assay was performed to determine total protein concentration. Supernatants (20–40 μg) were analyzed by SDS-PAGE and transferred onto PDVF Immobilon FL (Millipore). Membranes were dried for 1 hr, rehydrated in methanol, and stained for total protein . Following imaging of the total protein, membranes were destained, blocked for 1 h in Odyssey Blocking Buffer TBS (LI-COR), and incubated overnight at 4 C primary with antibodies diluted in Blocking Buffer with 0.2% Tween-20. Membranes were washed four times for 5 min in 1xTBS Washing Solution , incubated in secondary antibodies diluted in Odyssey Blocking Buffer TBS (LI-COR) with 0.2% Tween-20% and 0.01% SDS for 1 hr, and again washed four times for 5 min in the washing solution. Membranes were immediately imaged using an Odyssey CLx Infrared Imaging System (LI-COR).http://svi.nl) to remove background noise and increase resolution and signal-to noise. It should be noted that fluorescence intensity measurement for TMRE and CellRox experiments were quantified from unprocessed z-stacks. For deconvolution, up to 50 iterations of the Classic Maximum Likelihood Estimation (CMLE) algorithm with theoretical PSF was performed. Background was automatically corrected, the signal to noise ratio was 20–30, and all other settings were default. The number of iterations and signal to noise ratio varied based on the construct. Volume renderings of somal mitochondria and a representative OPTN-positive mitochondrion were generated using the normal shading mode in the volume function of Imaris Software (Bitplane); images were rotated to highlight the mitochondrial network.After live-cell confocal imaging of neurons, z-stack images were processed using Huygens Professional Deconvolution Software  was used to plot all graphs and determine statistical significance. Adobe Illustrator was used to prepare all figures and images.After image processing, mitochondrial fragments and protein ring or puncta formation in z-stack images were manually identified and counted using Fiji ; only clFor measurements of CellROX using a plate reader, the average CellROX fluorescence intensity from eight wells for each experimental treatment was quantified and normalized to the control. Values for each treatment were averaged across biological replicates. For fluorescence imaging measurements of CellROX, fluorescence intensities were quantified from unprocessed z-stack images. The mean gray value for each soma was determined by averaging the values of five individual areas (2.2 × 2.2 μm square) in the soma. For axons and dendrites, the mean gray value of a minimum of 40 mitochondria were manually measured per image and averaged across biological replicates. Fluorescence intensities were quantified from max projections of unprocessed z-stack images. In Fiji, the mean gray value for each cell was determined by averaging the values of five individual areas (2.2 × 2.2 μm square) in the soma. In the axon, the mean gray value of mitochondria was manually traced and measured for each cell.The somal and mitochondrial volume for each neuron was determined using the volume measurement function in Volocity Quantitation. Mitochondrial content was determined by dividing the mitochondrial volume by the somal volume.Bands were quantified using Image Studio Software (LI-COR). For each experimental trial, a total protein normalization factor was calculated based on the total protein from each lane. Band intensities for proteins of interest were quantified and divided by the total protein normalization factor corresponding to the same lane. Treatments were normalized relative to the control sample and presented as fold change over control.In Fiji, kymographs were generated for each axon using a three-pixel line width and the MultipleKymograph plugin. Kymographs were analyzed using MATLAB software (MathWorks). Axonal transport was quantified as described previously . The areIn Fiji, line scans were generated using a pixel line width to trace OPTN-positive mitochondria and a three-pixel line width to trace axons and dendrites. Intensity values were normalized by dividing the dataset by its maximum. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This paper describes a systematic analysis of recruitment of the autophagy cargo adaptor to damaged mitochondria in hippocampal neurons. Under mild oxidative stress, OPTN is recruited to damaged mitochondria and promotes recruitment of additional autophagy proteins and formation of an autophagosome, occurring robustly in the cell soma. OPTN- and LAMP1-positive structures remained in a non-acidic state for extended time periods, suggesting that acidification of lysosomes containing mitochondria is rate-limiting for degradation. This work is relevant to Parkinson's disease and ALS.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Mitophagosome acidification is rate-limiting in the maintenance of the somal mitochondrial network in neurons" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.This paper addresses the interesting question of how the autophagy cargo adaptor OPTN is employed in post-mitotic neurons to remove damaged mitochondria downstream of the Parkin ubiquitin ligase. Parkin is recruited to damaged mitochondria in response to PINK1 activation and catalyzes ubiquitylation of a number of proteins on the outer membrane of mitochondria. OPTN then binds to ubiquitin chains to promote recruitment of the autophagy machinery. The authors characterize this process in neurons in the presence of "endogenous" mitochondrial damage. Through a series of studies, they provide evidence that mitoautophagosomes acidify slowly, suggesting that acidification is a rate-limiting step in mitophagy.While the reviewers all feel that the paper is timely and interesting, there are several concerns that need to be addressed before the paper can be considered further. These concerns generally fall into 4 areas:1) OPTN overexpression. The paper extensively employs ectopic expression of tagged OPTN. Although this is very useful for detection of OPTN-coated mitochondria, it raises issues in the context of the model wherein mitolysosome acidification is rate-limiting in neurons. Depending upon the levels of overexpression, it is possible that autophagosome formation is accelerated in some way that makes acidification seem slow (rate-limiting). The paper also tries to make comparisons with HeLa cells in this context. Because the abundance of endogenous OPTN relative to the tagged OPTN isn't shown in either the case of neurons or HeLa cells, it is difficult to know the extent to which this may be an issue in the experiments that are shown. It is important to show the levels of overexpression either in the population, or preferably in individual cells using endogenous OPTN antibodies. This issue is also important in light of the fact that there are a substantial number of OPTN puncta that are not co-localized with mitochondria. Are these aggregates resulting from overexpression or are they something else? DO such OPTN puncta appear in neurons using endogenous antibodies? In terms of overexpression of OPTN accelerating early steps, it may be possible to visualize mitoautophagosomes using GFP-LC3, and since this is functioning at a step downstream of OPTN recruitment, it may allow an independent assessment of whether acidification is rate-limiting.2) Lysosome acidification as the rate-limiting step. All three reviewers had concerns about this strong conclusion, which is also featured in the Title of the paper. This reflects, in part, the OPTN overexpression as discussed above, as well as the approaches being used to examine acidification. The reviewer' feel that a more direct method, including measurement of mitophagic flux using Keima or mito-QC would strengthen the results. Is there evidence of mitochondria inside lysosomes that have not yet either activated the Keima red-shift or converted the yellow GFP-mCherry signal to mCherry only, which would indicate non-instantaneous acidification, and if so, what is the time constant for conversion to the acidic state? There is also some concern that the LAMP1 marker used for marking lysosomes may also, when overexpressed, mark autophagosomes, making the designation of mitolysosomes complicated. It may be possible to follow local diffusion  of lysotracker from a lysosome into an OPTN-positive autophagosome in order to measure the length of time from full OPTN recruitment to acidification. Previous studies have used this approach to examine the kinetics by live-cell imaging.3) A third issue brought up by the reviewers concerns the question of whether the pathway being examined is the Parkin-PINK1 pathway or not. This is the expectation given the fact that OPTN is recruited to the damaged mitochondria, but isn't formally demonstrated. Might it be possible to deplete PINK1 or Parkin by shRNA and examine the number of OPTN-positive mitochondria that appear over time in individual cells relative to cells transfected with control shRNA?4) The current work implies that the delay in acidification is specific to mitophagosomes. However, the acidification rate of autophagosomes in general, under the authors experimental conditions, was not tested. The authors could monitor the formation of OPTN -ve LC3 puncta using their mild depolarising media and determine if these equally take as long to become acidified.Reviewer #1:This paper describes an analysis of mitophagy in neuronal cells. Mitophagy is perhaps best understood in the context of PINK1 and PARK2-dependent mitochondrial ubiquitylation and targeting to the autophagosome via an OPTN-TBK1-dependent pathway . The majority of studies examining PARK2-dependent mitophagy have been done in cancer cell lines overexpressing PARK2 and there has been conflicting data for PARK2-dependent mitophagy in neuronal cell lineages. This paper tries to address some of these conflicting data and also presents a new model related to lysosomal acidification as being rate-limiting for mitophagy in neurons.In mito-QC or mito-Keima mice that lack PINK1 or PARK2, there is no discernable defect in mitophagic flux across all/most tissues, indicating that the majority of mitophagic flux is through a PARK2-independent pathway. However, this doesn't necessarily mean that there isn't a small amount of flux (not detectable by the methods used) that is still PARK2-dependent in specific classes of neurons. Additionally, previous studies have used various types of mitochondrial depolarizing agents to examine mitophagy in neurons in culture. In some experiments, mitophagy is initiated in the axon while in others, it occurs in the soma. While there are discrepancies in some of the conclusions, it's also probably fair to say that some of the conclusions are not really based on strong experiments. As such, there is still much that is unclear concerning when, where, and how mitochondria are degraded by autophagy in neurons.This study uses removal of anti-oxidants (AOF) from the media as a way to induce endogenous mitochondrial damage, with the idea that it will be possible to examine mitophagy mechanisms in a more physiological setting. The paper is primarily based on imaging.Major comments:Throughout the paper, the authors employ overexpression of Halo-OPTN. There is the distinct possibility that overexpression of OPTN can artificially accelerate steps in mitophagy that are downstream of PARK2-action on mitochondria. This complicates the interpretation of the data in terms of the rates of mitochondrial turnover and the conclusions related to lysosomal acidification being limiting. The recent finding that autophagy receptors (NDP52 in particular) can recruit ULK1-FIP200 raises the question as to whether having additional receptor in the cell could accelerate autophagosome formation around ubiquitylated mitochondira. Additionally, in Figure 1I, there appear to be many Halo-OPTN puncta that are not co-localizing with mitochondria. It is unclear what these puncta are. Do they reflect Halo-OPTN aggregates, for example, resulting from overexpression? Or are they OPTN localized on other autophagic cargo?In Figure 2 the authors examine localization of PARK2, TBK1 and OPTN with mitochondria and correlate it with TMRE signal. In some sense, it's a bit hard to judge because they are only showing AOF treated samples and so there isn't a systematic quantification of the number of OPTN-coated mitochondria in the absence of oxidative stress. There are a lot of OPTN-positive, TBK1-positive puncta whose identity are unknown. I also am unsure what the authors mean concerning Figure 2G, where the authors say that the number of OPTN on linear mitochondria are "low and similar in either conditions". The value is ~29%, which is much higher that the OPTN on fragmented mitochondria, so this doesn't seem to make sense. I am also not sure about the argument made concerning the aspect ratio with and without AOF in Figure 2H. The highest signal in the control sample is also the <1.5 ratio, and this only marginally increased with AOF. So, it would seem to be hard to conclude that this small mitochondria directly represents the ones that are subsequently tagged with OPTN.Figure 3 looks at the frequency of OPTN encircled mitochondria in axons, dendrites and soma, indicating that virtually no OPTN-positive mitochondria are seen in the axon. The frequency of OPTN-positive mitochondria relative to the total mitochondrial volume in the soma indicates that with AOF, there is a very small amount of damaged mitochondria relative to the total mitochondrial volume. Given this very low value, it would appear to take many axons in order to achieve a similar mitochondrial volume in order to directly compare the frequency of damage and capture by OPTN. So based on a stochastic damage argument, it may be very difficult to detect damaged mito in the axon but that doesn't necessarily mean it doesn't happen there. So, the conclusion in subsection “OPTN-mediated mitophagy occurs preferentially in the somatodendritic compartment of hippocampal neurons” might be too strong.The experiment in Figure 5 is a bit hard to interpret. Since LAMP1 is overexpressed, it is possible that it is marking organelles such as endosomes, in addition to lysosomes. In any event, the apparent number of LysoT positive puncta is far lower than the number of LAMP1 positive puncta, albeit not in the same cells. The major conclusion is that lysosomes are acidified more slowly in neurons, and results in HeLa cells overexpressing OPTN and LAMP1 are used to compare the rates of lysosome acidification. Interpretation of this experiment is difficult because there is no indication of the levels of expression of OPTN/cell. If OPTN overexpression can accelerate the assembly of a fully coated autophagosome around the damaged mitochondria, and therefore the rate of fusion with a lysosome since full closure is required, then the difference in the apparent rate of acidification could simply represent the levels of OPTN in each cell being examined. Since there are no measurements of OPTN levels/per cell and specifically in the cells that are being imaged, it makes it challenging to make the conclusion that rates of acidification are intrinsically slower in neurons. If much less overexpressed OPTN is present in neurons than HeLa, this could explain the difference seen. The authors examined cathepsin cleavage, and find only minor differences as measured in a western blot assay. Since cathepsin processing also requires low lysosomal pH, this a priori would suggest no issue with lysosomal acidification at a global level in the neurons, raising the question of precisely how acidification is regulated.With regards to the conclusions in Figure 5, an ultrastructure analysis in the cited Cheng et al., 2018 paper suggests that LAMP1 can be present on immature double membrane autophagosome-like structures. Is it possible that some of the LAMP1-positive signals lacking LysoT positivity are actually autophagosomes that are LAMP1 positive but not yet fused with a lysosome? Could LAMP1 overexpression affect the interpretation of the experiments?Subsection “Low-level induction of ROS leads to selective mitochondrial damage without compromising the overall mitochondrial network”. In the two color old/new mitochondrial experiment, it's hard to imagine how the age can be precisely controlled using the described protocol. The SNAP tag for mito localization contains a COXVIII MTS sequence. As such, it would seem like any mitochondria with a functional translocon could import the SNAP-tag protein, regardless of whether it is old or new, however the authors state that the two populations are "distinct". The interpretation that old mitochondria can import the newly synthesized snap tag is consistent with most of the mitochondria being double labeled . Because the mass of old mitochondria is much larger than the mass of new mitochondria that has been made since the new dye was added (dye added for 1 hour) and the number of those mitochondria not undergoing intermixing by fusion is small, the frequency with which such a "new" mitochondria would be identified as damaged and associated with OPTN would be particularly low, relative to the much more abundant old mito. So it seems possible, that this experiment doesn't really have the statistical power to rule out similar rates of old and new mitochondrial engulfment. The authors argue that the absence in green signal means that they are exclusively old, but it also may be that the signal to noise isn't equivalent. The labeling period for old was 24 hours while the labeling for new was only 1 hour. Therefore, all things being equal in terms of mitochondrial assembly/fission/fusion, there should be far less signal for new snap relative to old snap/per individual mito.In Figure 7, the authors examine an OPTN mutant found in ALS, and see general but small effects on mitochondrial size. This experiment is somewhat limited by the fact that OPTN deletion by siRNA is only ~60%, and it is possible that the overexpressed OPTN mutant (not sure how much it is overexpressed as they didn't show a western of the transfected cells) may also act as a dominant negative – binding TBK1 but not Ub chains. What isn't clear is whether the authors have missed an opportunity to statistically determine whether the OPTN mutant encircles damaged mito. The experiments are largely limited to mito morphology.An important weakness of the analysis is the absence of a direct demonstration of mitophagic flux.Reviewer #2:In their manuscript, Evans and Holzbaur present data arguing two main points regarding mitophagy in neurons: (1) that mitophagy occurs mainly in the soma and not in axons, contrary to some previous work by others, and (2) that neuronal mitophagy is significantly slower relative to other cell types, as result of slow acidification of mitoautophagosomes. Although the experiments are generally well-performed additional evidence is required in support of their main conclusions.Major comments:1) The implication throughout the paper is that the optineurin rings forming around mitochondria in response to mild oxidative stress are due to PINK1/Parkin dependent mitophagy. This should be demonstrated with knockdown or knockout of PINK1 and Parkin. This is particularly important given lack of clarity on the role of PINK1/Parkin mitophagy in neurons in response to more physiologically relevant stressors.2) A major finding of the manuscript is that mitophagy occurs primarily in the cell soma and not the axons, in contrast to the findings of Ashrafi et al. As a rationale for this discrepancy, they suggest that they are applying a milder stress  that does not lead to global bioenergetic collapse. This explanation doesn't seem satisfactory, however, as Ashrafi et al., also, used a variety of experimental setups to induce local damage mitochondria without affecting the overall bioenergetics of the neuron. An alternative explanation for the discrepancy might be that the particular exposure used by Evans and Holzbaur (withholding antioxidants) preferentially increases oxidative stress and mitochondrial damage in the cell soma as opposed to the axons. Does ROS increase equally in the axon and cell body with antioxidant withdrawal? Does the authors treatment result in TMRE negative mitochondria in axons that are not captured by Optineurin in contrast to the cell soma? When the authors treat with antimycin for 2 hours  do they see the same preferential increase in mitophagy in the soma or are mitochondria in the axons also targeted? Ashrafi saw axonal events as soon as 45 minutes, do the authors see axonal events at these early timepoints? Ashafri noted increased axonal events with the use of lysosomal inhibitors. If these are used by the authors, do they then see axonal events?3) In Figure 5 the authors compare the speed of mitophagosome maturation in neurons and HeLa cells. They conclude that the difference in speed reflects a difference in processes downstream of mitochondrial ubiquitination, but the level mitochondrial ubiquitination is likely very different in neurons with endogenous Parkin subjected to mild oxidative stress and HeLa cells over-expressing Parkin and fully depolarized with OA. Could this account for the difference in maturation rate? Do the authors still see a difference in the speed of mitophagosome maturation when the two cell types are subjected to more equal treatment – e.g., in neurons and HeLa cells that are both overexpressing Parkin and are subjected to the same oxidative stress ? Additionally, the authors note that acidification of mitoauthophagosomes is delayed in neurons? How long is it delayed? What do they see at time points longer than one day?Reviewer #3:In the manuscript by Evans and Holzbaur, the authors examine the dynamics of OPTN recruitment to mitochondria and subsequent delivery to lysosomes following mild oxidative stress in primary rat hippocampal neurons. The authors mainly use transfected neurons and live-cell microscopy and find that OPTN is rapidly recruited to a subset of mitochondria, primarily in the soma, following anti-oxidant removal from the cell media. This then correlated with subsequent recruitment of mitophagy and lysosomal markers. Surprisingly, and the main take home message from the manuscript, was that the OPTN puncta on mitochondria do not become efficiently acidified , from which the authors conclude that this is a rate-limiting step during neuronal mitophagy.This is an intriguing manuscript with many interesting observations. The identification of "mild" depolarising conditions that trigger a mitophagy response will be useful to the field and certainly does appear more physiological that the use of CCCP or oligomycin/antimycin. The microscopy images are impressive and convincing in what they show. However, I think more work is needed to determine whether this phenomenon is specific to mitophagosomes and neurons in general.Major comments:1) I think more robust analyses of lysosome acidification are needed. It is surprising and very interesting that there is a large delay in acidification of mitolysosomes. Is this a general phenomenon, i.e. are acidified lysosomes scarce in these cells? What is the percentage of LAMP1 structures  that are LysoTracker +ve? If it is low, could this mean that most of the LAMP1-GFP is not in lysosomes? Could the authors use another lysosomal marker to confirm?2) Related to the above point, the authors only use LysoTracker to estimate lysosomal acidification. Is there another way the authors could estimate this – for example using ratiometric fluorophores, such as Keima or mCherry-GFP?3) Is the delay in acidification specific to mitolysosomes? What about autophagosomes in general? The authors could monitor the formation of OPTN -ve LC3 puncta under their conditions and determine if these equally take as long to become acidified. The authors may wish to discuss Ralph Nixon's work  that argues autophagosome fusion with lysosomes is very efficient in neurons.4) Are the authors sure it is mitophagosome acidification that is rate-limiting and not an earlier step in the pathway? For example, is it possible that the autophagosome is not fully sealed (despite appearing circular by fluorescence microscopy). The authors could look at early autophagosome markers such as ULK1, ATG5 or WIPI that are not present on mature autophagosomes. Or mature autophagosome markers such as STX17.5) The authors imply that this delay is specific to neurons, yet they only look in hippocampal neurons and compare this to HeLa cells overexpressing Parkin. I think the authors need to provide some more data in relevant cell lines for a better comparison. What about cortical neurons, as well and comparing with primary fibroblasts ?eLife. Your revised article has been evaluated by Suzanne Pfeffer (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Based on the comments of reviewer 3, a couple of changes in the manuscript are still needed. First, some data that are described in the rebuttal don't appear in the paper, so Figure 4 may need to be updated to include data that were discussed in the rebuttal. In addition, there are a couple of suggested changes to the text that will need to be addressed.Reviewer #1 and 2:Overall, the authors have done a good job of addressing the previous comments. I have no further concerns and feel that the paper should be accepted.Reviewer #3:This is a re-review of the manuscript by Evans et al., describing the slow maturation/impaired acidification of mitophagosomes in the soma of neurons. I think the authors have done a great job in strengthening their manuscript – my main concern was with the lysosomal acidification studies and the use of the dual reporters means the data are much more convincing. I just have a few small points remaining:1) The LAMP2 data, mentioned in subsection” Acidification of OPTN-positive mitochondria is a rate-limiting step in neuronal mitophagy” and in the comments to reviewers does not seem to be in the manuscript. The authors mention Figure 4E, but these are only LC3-related. Please include the data.2) Likewise, the authors show data in the reviewers comments that the OPTN/LC3 structures are WIPI -ve. I do not see these data in the manuscript. This shows that the structures are not early autophagosomal in nature and thus they will be of interest to readers (and not just this reviewer) and so should be included . I'm assuming WIPI2B is expressed in these cells and does indeed form puncta with LC3 when autophagy is induced?3) I also take on the point that the authors have made in response to my concern raised in point 5 – i.e. that HeLa cells overexpressing Parkin are a suitable system to compare to the neurons. The HeLa cell has been instrumental in the field in terms of building up the mechanism of PINK/Parkin mitophagy. But this is not a "normal" cell. Normally, it does not undergo this pathway as it does not express Parkin! I think to avoid doing more experiments, the authors can be more accurate in what they state and say that this delay in acidification does not occur in HeLa cells expressing Parkin. For example in subsection “Acidification of OPTN-positive mitochondria is a rate-limiting step in neuronal mitophagy”: "…..much slower than the corresponding time course in non-neuronal cells." Should be replaced with "…..much slower than the corresponding time course in HeLa cells." This paper addresses the interesting question of how the autophagy cargo adaptor OPTN is employed in post-mitotic neurons to remove damaged mitochondria downstream of the Parkin ubiquitin ligase. Parkin is recruited to damaged mitochondria in response to PINK1 activation and catalyzes ubiquitylation of a number of proteins on the outer membrane of mitochondria. OPTN then binds to ubiquitin chains to promote recruitment of the autophagy machinery. The authors characterize this process in neurons in the presence of "endogenous" mitochondrial damage. Through a series of studies, they provide evidence that mitoautophagosomes acidify slowly, suggesting that acidification is a rate-limiting step in mitophagy.While the reviewers all feel that the paper is timely and interesting, there are several concerns that need to be addressed before the paper can be considered further. These concerns generally fall into 4 areas:1) OPTN overexpression. The paper extensively employs ectopic expression of tagged OPTN. Although this is very useful for detection of OPTN-coated mitochondria, it raises issues in the context of the model wherein mitolysosome acidification is rate-limiting in neurons. Depending upon the levels of overexpression, it is possible that autophagosome formation is accelerated in some way that makes acidification seem slow (rate-limiting). The paper also tries to make comparisons with HeLa cells in this context. Because the abundance of endogenous OPTN relative to the tagged OPTN isn't shown in either the case of neurons or HeLa cells, it is difficult to know the extent to which this may be an issue in the experiments that are shown. It is important to show the levels of overexpression either in the population, or preferably in individual cells using endogenous OPTN antibodies. This issue is also important in light of the fact that there are a substantial number of OPTN puncta that are not co-localized with mitochondria. Are these aggregates resulting from overexpression or are they something else? DO such OPTN puncta appear in neurons using endogenous antibodies? In terms of overexpression of OPTN accelerating early steps, it may be possible to visualize mitoautophagosomes using GFP-LC3, and since this is functioning at a step downstream of OPTN recruitment, it may allow an independent assessment of whether acidification is rate-limiting.We thank the reviewers for their comments and agree that it is important to consider the expression level of OPTN when discussing the rates of neuronal mitophagy. As requested, we compared OPTN expression in non-transfected and Halo-OPTN transfected cells . We also confirmed these findings using two independent aOPTN antibodies . To further avoid over-expression artifacts, we select neurons within a narrow fluorescence range in our imaging experiments.We determined that ~60-70% of OPTN puncta are not associated with mitochondria . OPTN has been implicated in other processes, such as aggrephagy . As a result, these Halo-OPTN puncta may be localized to other autophagic cargos. Consistent with this, we found that roughly 50% of nonmitochondrial associated OPTN puncta colocalized with LAMP1-EGFP, as shown in Finally, we transfected neurons with a mitochondrial marker (Mito-SNAP) and a tandem mCherry-GFP-LC3 to look at acidification in the absence of overexpressed OPTN. We continued to find consistent evidence of spherical mitochondria that colocalized with both GFP and mCherry, further corroborating our findings that while these mitochondria are engulfed within autophagosomes, they are not yet acidified . A representative image is provided in 2) Lysosome acidification as the rate-limiting step. All three reviewers had concerns about this strong conclusion, which is also featured in the Title of the paper. This reflects, in part, the OPTN overexpression as discussed above, as well as the approaches being used to examine acidification. The reviewer' feel that a more direct method, including measurement of mitophagic flux using Keima or mito-QC would strengthen the results. Is there evidence of mitochondria inside lysosomes that have not yet either activated the Keima red-shift or converted the yellow GFP-mCherry signal to mCherry only, which would indicate non-instantaneous acidification, and if so, what is the time constant for conversion to the acidic state? There is also some concern that the LAMP1 marker used for marking lysosomes may also, when overexpressed, mark autophagosomes, making the designation of mitolysosomes complicated. It may be possible to follow local diffusion  of lysotracker from a lysosome into an OPTN-positive autophagosome in order to measure the length of time from full OPTN recruitment to acidification. Previous studies have used this approach to examine the kinetics by live-cell imaging.mito-QC previously described . We identified OPTN-positive mitochondria and looked for the presence or absence of EGFP fluorescence coincident with mCherry fluorescence to identify non-acidified and acidified organelles. Using this assay, we found OPTN-positive mitochondria that were dual labeled with the tandem reporter, suggesting they were in non-acidified compartments. This data is now included in the revised manuscript .We agree that additional methods were needed to further strengthen our conclusion that lysosome acidification is rate-limiting in neuronal mitophagy. To address this point, we utilized the mitochondrial matrix directed tandem fluorescent reporter Cox8-EGFP-mCherry to look at mitophagic flux ; this construct is similar to the To use LAMP1 as a second, independent reporter for acidified degradation-competent organelles, we utilized a dual labeled SEP-LAMP1-RFP construct where superecliptic pHluorin (SEP) is fused to the lumen of LAMP1-RFP . The fluorescent signal of the SEP will quench in acidic environments ). We felt that a dual reporter system would provide more reliable measurements than the local diffusion of lysotracker from a lysosome. As a control experiment, we used this construct to determine the number of acidified lysosomes per cell  and found it to be consistent with other methods used. We then identified OPTN-positive mitochondria and quantified the relative percentage that were dual or single labeled with the tandem LAMP1 probe. We determined the majority of sequestered mitochondria were dual positive for green and red fluorescence with the SEP-LAMP1-RFP tandem probe, again suggesting that this represents a substantial population of non-acidified organelles .3) A third issue brought up by the reviewers concerns the question of whether the pathway being examined is the Parkin-PINK1 pathway or not. This is the expectation given the fact that OPTN is recruited to the damaged mitochondria, but isn't formally demonstrated. Might it be possible to deplete PINK1 or Parkin by shRNA and examine the number of OPTN-positive mitochondria that appear over time in individual cells relative to cells transfected with control shRNA?To formally demonstrate whether this pathway is PINK1/Parkin-dependent, we performed Parkin knock-down and rescue experiments. The percent of neurons with OPTN-positive mitochondria was determined in neurons following antioxidant removal for 1 hour, see Figure 2—figure supplement 2 of the revised manuscript. We observed an overall 30% reduction in Parkin expression by immunoblot analysis of cell lysates, but it is important to note that this measurement is an underestimate of the reduction in Parkin expression in the imaged neurons, as transfection rates are low in hippocampal neurons and the immunoblot measurement is from an entire plate of cells and so includes neurons that did not take up siRNA. When imaging neurons, we selected cells that were transfected and therefore have also taken up siRNA. Importantly, this level of knock-down was sufficient to significantly inhibit the number of cells with OPTN-positive rings, from ~35% down to less than 10%. This deficit was rescued by reexpression of WT Parkin. These data along with our recruitment data indicate that the PINK1-Parkin pathway is involved, although we cannot rule out the involvement of other possible ROS-induced E3 ligases in addition to Parkin. This point is directly addressed in the Discussion section.4) The current work implies that the delay in acidification is specific to mitophagosomes. However, the acidification rate of autophagosomes in general, under the authors experimental conditions, was not tested. The authors could monitor the formation of OPTN -ve LC3 puncta using their mild depolarising media and determine if these equally take as long to become acidified.We agree that it is important to determine whether our mitophagy induction paradigm alters autophagosome acidification in general or if it is specific to mitophagosomes. As requested, we identified OPTN-negative LC3 puncta and monitored acidification using the tandem mCherry-GFP-LC3 reporter. We determined that 75% of autophagosomes in a cell were acidified and that there was no significant difference in acidification between control or AO-free treated conditions at 1 or 6 hours. Additionally, we monitored lysosome acidification and observed similar results. See Figure 5—figure supplement 1 of the revised manuscript.Reviewer #1:This paper describes an analysis of mitophagy in neuronal cells. Mitophagy is perhaps best understood in the context of PINK1 and PARK2-dependent mitochondrial ubiquitylation and targeting to the autophagosome via an OPTN-TBK1-dependent pathway . The majority of studies examining PARK2-dependent mitophagy have been done in cancer cell lines overexpressing PARK2 and there has been conflicting data for PARK2-dependent mitophagy in neuronal cell lineages. This paper tries to address some of these conflicting data and also presents a new model related to lysosomal acidification as being rate-limiting for mitophagy in neurons.In mito-QC or mito-Keima mice that lack PINK1 or PARK2, there is no discernable defect in mitophagic flux across all/most tissues, indicating that the majority of mitophagic flux is through a PARK2-independent pathway. However, this doesn't necessarily mean that there isn't a small amount of flux (not detectable by the methods used) that is still PARK2-dependent in specific classes of neurons. Additionally, previous studies have used various types of mitochondrial depolarizing agents to examine mitophagy in neurons in culture. In some experiments, mitophagy is initiated in the axon while in others, it occurs in the soma. While there are discrepancies in some of the conclusions, it's also probably fair to say that some of the conclusions are not really based on strong experiments. As such, there is still much that is unclear concerning when, where, and how mitochondria are degraded by autophagy in neurons.This study uses removal of anti-oxidants (AOF) from the media as a way to induce endogenous mitochondrial damage, with the idea that it will be possible to examine mitophagy mechanisms in a more physiological setting. The paper is primarily based on imaging.Major comments:1) Throughout the paper, the authors employ overexpression of Halo-OPTN. There is the distinct possibility that overexpression of OPTN can artificially accelerate steps in mitophagy that are downstream of PARK2-action on mitochondria. This complicates the interpretation of the data in terms of the rates of mitochondrial turnover and the conclusions related to lysosomal acidification being limiting. The recent finding that autophagy receptors (NDP52 in particular) can recruit ULK1-FIP200 raises the question as to whether having additional receptor in the cell could accelerate autophagosome formation around ubiquitylated mitochondira. Additionally, in Figure 1I, there appear to be many Halo-OPTN puncta that are not co-localizing with mitochondria. It is unclear what these puncta are. Do they reflect Halo-OPTN aggregates, for example, resulting from overexpression? Or are they OPTN localized on other autophagic cargo?Halo-OPTN does form puncta that do not colocalize with mitochondria. However, we observed a similar distribution of endogenous OPTN in the soma of neurons and in HeLa cells, please see Figure 2—figure supplement 1 and additional images above. These observations are consistent with previous work demonstrating the punctate appearance of endogenous OPTN .It is possible that some of these puncta localize with autophagic cargos. OPTN has been suggested to function in aggrephagy and some of these Halo-OPTN puncta could localize with aggregated proteins . Consistent with this, we observed that many of the Halo-OPTN puncta colocalize with EGFP-LC3 and LAMP1-EGFP (see above for representative images). However, it is difficult to determine whether these puncta represent OPTN-mediated clearing events or overexpressed protein that is being cleared from the cell, as both are destined for autophagosome engulfment and degradation via lysosomal fusion.2) In Figure 2 the authors examine localization of PARK2, TBK1 and OPTN with mitochondria and correlate it with TMRE signal. In some sense, it's a bit hard to judge because they are only showing AOF treated samples and so there isn't a systematic quantification of the number of OPTN-coated mitochondria in the absence of oxidative stress. There are a lot of OPTN-positive, TBK1-positive puncta whose identity are unknown. I also am unsure what the authors mean concerning Figure 2G, where the authors say that the number of OPTN on linear mitochondria are "low and similar in either conditions". The value is ~29%, which is much higher that the OPTN on fragmented mitochondria, so this doesn't seem to make sense. I am also not sure about the argument made concerning the aspect ratio with and without AOF in Figure 2H. The highest signal in the control sample is also the <1.5 ratio, and this only marginally increased with AOF. So, it would seem to be hard to conclude that this small mitochondria directly represents the ones that are subsequently tagged with OPTN.On average ~95% of OPTN-positive mitochondria were TMRE negative in both control and AO-free conditions. Since mitophagy can occur at a basal level, OPTN-positive mitochondria were found in both control and treated conditions, but the number of events was higher in AO-free treated conditions compared to control . In Figure 2D-F, we observed two types of localization that occurred using upstream mitophagy-associated proteins, the formation of rings around damaged mitochondria and puncta that colocalized with isolated mitochondrial fragments. We highlighted examples of each using arrows, but every event was not highlighted.As shown in Figure 1—figure supplement 1D, ~30-40% of total OPTN puncta were found on mitochondria. Of this population, most were localized to linear mitochondria under both control and AO-free conditions. Although, the percent associated with rounded mitochondria was observed to increase significantly upon mild stress in the AO-free condition. We have now clarified this description in the revised manuscript.We measured the mitochondrial aspect ratio to determine whether we could quantify minor changes in the mitochondrial network. Overall, we observed a small but significant decrease in the mitochondrial aspect ratio. The binned data illustrate that the fraction of mitochondria with an aspect ratio of ≤1.5 was greater in neurons treated with AO-free media compared to control . Additionally, we measured the diameter of OPTN-positive mitochondrial rings and found they were on average ~1.2 µm (data not shown). Together these data suggest that some of the small mitochondria represent the ones that are engulfed by OPTN.Figure 3 looks at the frequency of OPTN encircled mitochondria in axons, dendrites and soma, indicating that virtually no OPTN-positive mitochondria are seen in the axon. The frequency of OPTN-positive mitochondria relative to the total mitochondrial volume in the soma indicates that with AOF, there is a very small amount of damaged mitochondria relative to the total mitochondrial volume. Given this very low value, it would appear to take many axons in order to achieve a similar mitochondrial volume in order to directly compare the frequency of damage and capture by OPTN. So based on a stochastic damage argument, it may be very difficult to detect damaged mito in the axon but that doesn't necessarily mean it doesn't happen there. So, the conclusion in subsection “OPTN-mediated mitophagy occurs preferentially in the somatodendritic compartment of hippocampal neurons” might be too strong.3/µm3  and the axonal mitochondrial content to be ~0.02 µm3/µm3 (data not shown). Based on these values and the number of somas analyzed in Figure 3, 210-280 axons would need to be analyzed. Since this is not the ideal system for such analysis, we cannot definitively rule out the possibility that an event occurred that we did not image. As a result, we have changed the statement to say “with limited events in dendrites and axons.” Additionally, we discuss this caveat and provide more axonal analysis in the Results section.We thank the reviewer for the insightful comment and agree that it may be difficult to detect damaged mitochondria in the axon based on the stochastic damage argument. We determined the somal mitochondrial content was ~0.2 µmThe experiment in Figure 5 is a bit hard to interpret. Since LAMP1 is overexpressed, it is possible that it is marking organelles such as endosomes, in addition to lysosomes. In any event, the apparent number of LysoT positive puncta is far lower than the number of LAMP1 positive puncta, albeit not in the same cells. The major conclusion is that lysosomes are acidified more slowly in neurons, and results in HeLa cells overexpressing OPTN and LAMP1 are used to compare the rates of lysosome acidification. Interpretation of this experiment is difficult because there is no indication of the levels of expression of OPTN/cell. If OPTN overexpression can accelerate the assembly of a fully coated autophagosome around the damaged mitochondria, and therefore the rate of fusion with a lysosome since full closure is required, then the difference in the apparent rate of acidification could simply represent the levels of OPTN in each cell being examined. Since there are no measurements of OPTN levels/per cell and specifically in the cells that are being imaged, it makes it challenging to make the conclusion that rates of acidification are intrinsically slower in neurons. If much less overexpressed OPTN is present in neurons than HeLa, this could explain the difference seen. The authors examined cathepsin cleavage, and find only minor differences as measured in a western blot assay. Since cathepsin processing also requires low lysosomal pH, this a priori would suggest no issue with lysosomal acidification at a global level in the neurons, raising the question of precisely how acidification is regulated.We quantified the number of LAMP-positive organelles that are LysoT positive in cells overexpressing LAMP1 or LAMP2, and determined that ~89% of LAMP1 and ~88% of LAMP2 organelles colocalized with LysoT, suggesting no overall defects with lysosomal acidification. Additionally, we confirmed this finding using the SEP-LAMP1-RFP construct. We have further clarified this important issue in the revised manuscript . To address the question of OPTN expression levels, as discussed above in our general response, we now provide images of non-transfected and Halo-OPTN overexpressed cells to illustrate representative levels of endogenous and overexpressed OPTN in the cells we chose for analysis . Thus, it would appear that only mitophagolysosome acidification is slow. We plan to follow up on this interesting observation in the future.With regards to the conclusions in Figure 5, an ultrastructure analysis in the cited Cheng et al., 2018 paper suggests that LAMP1 can be present on immature double membrane autophagosome-like structures. Is it possible that some of the LAMP1-positive signals lacking LysoT positivity are actually autophagosomes that are LAMP1 positive but not yet fused with a lysosome? Could LAMP1 overexpression affect the interpretation of the experiments?Cheng et al., (2018) showed that there is a gradient in the number of LAMP1 organelles that contain Cathepsin D, with soma being higher than the axons and dendrites. In the Results for Figure 5, we argue that the observed discrepancy between LAMP1+/total and LysoT+/total may be in part due to the possibility that some of LAMP1 signal is on ‘late’ mitophagosomes (LC3LAMP1-positive) that have yet to fuse with degradative-competent organelles.To address this issue more directly, we utilized a tandem SEP-LAMP1-RFP reporter where we could monitor acidified degradative-competent organelles. Using this method, we determined that the vast majority of OPTN rings around damaged mitochondria were dual labeled, indicating non-acidified compartments .Subsection “Low-level induction of ROS leads to selective mitochondrial damage without compromising the overall mitochondrial network”. In the two color old/new mitochondrial experiment, it's hard to imagine how the age can be precisely controlled using the described protocol. The SNAP tag for mito localization contains a COXVIII MTS sequence. As such, it would seem like any mitochondria with a functional translocon could import the SNAP-tag protein, regardless of whether it is old or new, however the authors state that the two populations are "distinct". The interpretation that old mitochondria can import the newly synthesized snap tag is consistent with most of the mitochondria being double labeled . Because the mass of old mitochondria is much larger than the mass of new mitochondria that has been made since the new dye was added (dye added for 1 hour) and the number of those mitochondria not undergoing intermixing by fusion is small, the frequency with which such a "new" mitochondria would be identified as damaged and associated with OPTN would be particularly low, relative to the much more abundant old mito. So it seems possible, that this experiment doesn't really have the statistical power to rule out similar rates of old and new mitochondrial engulfment. The authors argue that the absence in green signal means that they are exclusively old, but it also may be that the signal to noise isn't equivalent. The labeling period for old was 24 hours while the labeling for new was only 1 hour. Therefore, all things being equal in terms of mitochondrial assembly/fission/fusion, there should be far less signal for new snap relative to old snap/per individual mito.In Figure 7, the labeling period is the same for both ‘first’ and ‘second’ SNAP ligands . The first SNAP ligand labeled expressed Mito-SNAP from the time of transfection until the SNAP Block labeling, ~24 hours; this is termed the ‘old’ mito population. The second SNAP ligand labeled Mito-SNAP that was expressed from the time after SNAP Block until imaging, ~22 hours; this is termed the ‘young’ mito population. The dark SNAP Block was added to saturate any remaining binding sites after the first SNAP labeling .It is possible that mitochondria with a functional translocon could import the SNAP-tag protein and ligand, but we would argued that the damaged OPTN-positive mitochondria would not have the capacity to import new protein or be labeled by the new ligand because it is sequestered. Thus, these rounded structures would indicate that they were labeled and engulfed before the addition of the new SNAP-ligand. In the revised manuscript, we more clearly describe the labeling protocol in the text and figure legends and have updated the labeling schematic in the revised Figure 7.In Figure 7, the authors examine an OPTN mutant found in ALS, and see general but small effects on mitochondrial size. This experiment is somewhat limited by the fact that OPTN deletion by siRNA is only ~60%, and it is possible that the overexpressed OPTN mutant (not sure how much it is overexpressed as they didn't show a western of the transfected cells) may also act as a dominant negative – binding TBK1 but not Ub chains. What isn't clear is whether the authors have missed an opportunity to statistically determine whether the OPTN mutant encircles damaged mito. The experiments are largely limited to mito morphology.Please see the revised Figure 8—figure supplement 1C for a western blot showing relative equal levels of overexpression of Halo-OPTN WT and E478G. However, since neuronal transfection efficiency is low, it is not surprising that a 60% reduction in OPTN was observed. This reduction in OPTN expression is from an entire plate of cells, which includes cells that did not take up the siRNA. When imaging neurons, we selected cells that are transfected and therefore were more likely to also have taken up the siRNA.E478G fails to engulf damaged mitochondria , we sought to understand the functional significance of expression of this mutant on mitochondrial and neuronal health. Using our mitophagy-inducing paradigm, we think these experiments provide new insights into how expression of this mutant could lead to neurodegeneration.Since it was previously shown that OPTNAn important weakness of the analysis is the absence of a direct demonstration of mitophagic flux.We now include mitophagic (Cox8-EGFP-mCherry) flux assays and complementary autophagic (mCherry-GFP-LC3) and lysosomal (SEP-LAMP1-RFP) flux experiments, please see the new Figure 6, Figure 6—figure supplement 1, and Figure 5—figure supplement 1.Reviewer #2:In their manuscript, Evans and Holzbaur present data arguing two main points regarding mitophagy in neurons: (1) that mitophagy occurs mainly in the soma and not in axons, contrary to some previous work by others, and (2) that neuronal mitophagy is significantly slower relative to other cell types, as result of slow acidification of mitoautophagosomes. Although the experiments are generally well-performed additional evidence is required in support of their main conclusions.Major comments:1) The implication throughout the paper is that the optineurin rings forming around mitochondria in response to mild oxidative stress are due to PINK1/Parkin dependent mitophagy. This should be demonstrated with knockdown or knockout of PINK1 and Parkin. This is particularly important given lack of clarity on the role of PINK1/Parkin mitophagy in neurons in response to more physiologically relevant stressors.As previously stated, we performed Parkin knock-down and rescue experiments. We observed a 30% reduction in Parkin expression by immunoblot analysis. It is important to note that this measurement is an underestimate, as lysates were generated from an entire plate of hippocampal neurons, which transfect at a low efficiency and therefore includes many cells that did not take up the siRNA. When imaging neurons, we selected cells that were transfected and therefore did take up the Parkin siRNA. This level of knock-down was sufficient to significantly inhibit the number of cells with OPTN-positive rings, from ~35% down to less than 10%, which was rescued by re-expression of WT Parkin. This data has been included in the revised manuscript, please see Figure 2—figure supplement 2.2) A major finding of the manuscript is that mitophagy occurs primarily in the cell soma and not the axons, in contrast to the findings of Ashrafi et al. As a rationale for this discrepancy, they suggest that they are applying a milder stress  that does not lead to global bioenergetic collapse. This explanation doesn't seem satisfactory, however, as Ashrafi et al., also, used a variety of experimental setups to induce local damage mitochondria without affecting the overall bioenergetics of the neuron. An alternative explanation for the discrepancy might be that the particular exposure used by Evans and Holzbaur (withholding antioxidants) preferentially increases oxidative stress and mitochondrial damage in the cell soma as opposed to the axons. Does ROS increase equally in the axon and cell body with antioxidant withdrawal? Does the authors treatment result in TMRE negative mitochondria in axons that are not captured by Optineurin in contrast to the cell soma? When the authors treat with antimycin for 2 hours  do they see the same preferential increase in mitophagy in the soma or are mitochondria in the axons also targeted? Ashrafi saw axonal events as soon as 45 minutes, do the authors see axonal events at these early timepoints? Ashafri noted increased axonal events with the use of lysosomal inhibitors. If these are used by the authors, do they then see axonal events?Ashrafi et al., (2014) induced mitochondrial damage using much higher doses of AA (20 or 40 µM) or locally via mito-KillerRed. In the mito-KillerRed experiments, imaging was initially performed in HBSS buffer instead of Hibernate E to enhance the effects of ROS produced by mito-KillerRed. We argue that this approach may affect the overall bioenergetics of the neuron.We performed experiments to observe the ROS production of axonal mitochondria in neurons treated with antioxidant removal or by a much lower concentration of AA. We measured CellROX fluorescence intensity in the soma and processes of hippocampal neurons using fluorescence microscopy . In these experiments, we observed significant increases in ROS production, measured by increases in CellROX intensity, in both the soma and processes of hippocampal neurons following 6 hours AO-free or 2 hours of 3 nM AA treatment , suggesting that our global treatment targets all mitochondria. Further, since we observed shorter axonal mitochondria after 2 hours AA treatment, this timepoint should be sufficient to identify axonal events.To follow up on this point, we measured axonal mitochondrial health using TMRE and saw no differences in TMRE intensity across these conditions . However, this is not surprising as we did not observe differences in the somal mitochondrial population, where we routinely saw mitophagic events . These observations are fully consistent with our approach of inducing low level damage without grossly perturbing the overall mitochondrial network.We treated cells for 2 hours with AA and looked for colocalization of OPTN with fragmented mitochondria in the axon. Of the 21 neurons analyzed, we did not observe OPTN-positive mitochondria in the axon. Yet, similar treatments resulted in 35% of neurons having OPTN-positive mitochondria rings in the soma , suggesting preferential increases in the somal compartment. Additional experiments were performed where AA treatments were reduced to 1 hour. Again, we observed mitophagic events were rare in the axon , but routinely found in soma . However, we think it likely that if we did use a 10-20-fold higher dose of AA, we might see more evidence of axonal mitophagy as previously described . It is possible that neurons are in a survival state, where axonal mitochondria undergo mitophagy locally to preserve the health of the neuron. We clarify this point in the revised manuscript. In general, however, under the paradigm described here axonal mitophagic events were rare across all conditions and did not increase with mild oxidative stress relative to control or with longer incubation times (1 vs 6 hours).3) In Figure 5 the authors compare the speed of mitophagosome maturation in neurons and HeLa cells. They conclude that the difference in speed reflects a difference in processes downstream of mitochondrial ubiquitination, but the level mitochondrial ubiquitination is likely very different in neurons with endogenous Parkin subjected to mild oxidative stress and HeLa cells over-expressing Parkin and fully depolarized with OA. Could this account for the difference in maturation rate? Do the authors still see a difference in the speed of mitophagosome maturation when the two cell types are subjected to more equal treatment – e.g., in neurons and HeLa cells that are both overexpressing Parkin and are subjected to the same oxidative stress ? Additionally, the authors note that acidification of mitoauthophagosomes is delayed in neurons? How long is it delayed? What do they see at time points longer than one day?While we appreciate the point that we could use alternate paradigms to induce mitochondrial stress or attempt to equalize levels of Parkin expression to make HeLa cells a closer model of neuronal mitophagy, we thought that the more important question was to determine whether there is a universal deficit in autophagosome/lysosome acidification in hippocampal neurons subjected to mild oxidative stress, or whether there is a specific deficit in the acidification and eventual degradation of mitophagolysosomes. We therefore focused on this point. New data now included in the manuscript reveal that there is no overall perturbation in the extent of either autophagosomal or lysosomal acidification in neurons incubated in AO-free media as compared to control conditions . Thus, our data suggest that delayed acidification of mitophagosomes or ‘late’ mitophagosomes is specific in neurons. Using our experimental conditions, it would be difficult to continuously image cells for days. However, in Figure 7 we include data demonstrating the persistence of pulse-labeled OPTN-positive mitochondria that had yet to undergo autophagosome or lysosome engulfment 24 hours after mitophagy induction. Thus, the delay in acidification of mitophagolysosomes appears to be on the order of hours to days.Reviewer #3:In the manuscript by Evans and Holzbaur, the authors examine the dynamics of OPTN recruitment to mitochondria and subsequent delivery to lysosomes following mild oxidative stress in primary rat hippocampal neurons. The authors mainly use transfected neurons and live-cell microscopy and find that OPTN is rapidly recruited to a subset of mitochondria, primarily in the soma, following anti-oxidant removal from the cell media. This then correlated with subsequent recruitment of mitophagy and lysosomal markers. Surprisingly, and the main take home message from the manuscript, was that the OPTN puncta on mitochondria do not become efficiently acidified , from which the authors conclude that this is a rate-limiting step during neuronal mitophagy.This is an intriguing manuscript with many interesting observations. The identification of "mild" depolarising conditions that trigger a mitophagy response will be useful to the field and certainly does appear more physiological that the use of CCCP or oligomycin/antimycin. The microscopy images are impressive and convincing in what they show. However, I think more work is needed to determine whether this phenomenon is specific to mitophagosomes and neurons in general.Major comments:1) I think more robust analyses of lysosome acidification are needed. It is surprising and very interesting that there is a large delay in acidification of mitolysosomes. Is this a general phenomenon, i.e are acidified lysosomes scarce in these cells? What is the percentage of LAMP1 structures  that are LysoTracker +ve? If it is low, could this mean that most of the LAMP1-GFP is not in lysosomes? Could the authors use another lysosomal marker to confirm?To investigate the acidification of lysosomes, we measured the percentage of LAMP1-EGFP structures that were positive for LysoT. We determined that ~89% of LAMP1 structures were also positive for LysoT (red arrows). In addition, we repeated these experiments with LAMP2GFP and observed ~88% of LAMP2 structures were also LysoT-positive. Thus, in our hands the majority of lysosomes in the soma are acidified in hippocampal neurons; representative images are shown in 2) Related to the above point, the authors only use LysoTracker to estimate lysosomal acidification. Is there another way the authors could estimate this – for example using ratiometric fluorophores, such as Keima or mCherry-GFP?As described above, we utilized SEP-LAMP1-RFP, a superecliptic pHluorin that is fused to the lumen domain of LAMP1-RFP  to measure lysosome acidification. In the soma, ~96% of LAMP1 organelles in control conditions were positive for RFP but negative for SEP, indicating that the majority of lysosomes are acidified organelles. Similar results were observed in 1 hour AO-free conditions. These data are now included in the revised manuscript, please see Figure 5—figure supplement 1.3) Is the delay in acidification specific to mitolysosomes? What about autophagosomes in general? The authors could monitor the formation of OPTN -ve LC3 puncta under their conditions and determine if these equally take as long to become acidified. The authors may wish to discuss Ralph Nixon's work  that argues autophagosome fusion with lysosomes is very efficient in neurons.We performed the requested experiment and determined that 71-85% of autophagosomes in the soma are acidified. Additionally, autophagosome acidification was not affected by antioxidant removal, with similar results found with either 1 or 6 h treatments . Furthermore, we observed a considerable amount of LC3-II via western blot across all conditions . Collectively, these results suggest autophagosome fusion and acidification is efficient in neurons and support the findings of Boland et al., (2008).4) Are the authors sure it is mitophagosome acidification that is rate-limiting and not an earlier step in the pathway? For example, is it possible that the autophagosome is not fully sealed (despite appearing circular by fluorescence microscopy). The authors could look at early autophagosome markers such as ULK1, ATG5 or WIPI that are not present on mature autophagosomes. Or mature autophagosome markers such as STX17.To determine whether earlier steps in the pathway were rate-limiting, we observed WIPI2B, an early autophagosome marker, colocalization with mitophagosomes. In the soma, we observed WIPI2B puncta that colocalized with LC3, indicating these are immature autophagosomes. However, in both control and AO-free conditions OPTN-LC3-positive mitochondria were negative for WIPI2B, suggesting that these autophagosomes are fully formed.5) The authors imply that this delay is specific to neurons, yet they only look in hippocampal neurons and compare this to HeLa cells overexpressing Parkin. I think the authors need to provide some more data in relevant cell lines for a better comparison. What about cortical neurons, as well and comparing with primary fibroblasts ?We thank the reviewer for their comment. The use of HeLa cells provides an alternative cell line to investigate mitophagy. Since HeLa cells are readily used to characterize mitophagy and we observed similar temporal dynamics for OPTN and LC3 translocation, we felt that it was a suitable system to compare for rates of mitophagosome acidification. Previous work has looked at the time course of mitophagy in cortical neurons where the authors suggested that mitophagy was slower and compartmentally restricted compared to non-neuronal cells , consistent with our findings – we further clarify this point in the revised manuscript.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Based on the comments of reviewer 3, a couple of changes in the manuscript are still needed. First, some data that are described in the rebuttal don't appear in the paper, so Figure 4 may need to be updated to include data that were discussed in the rebuttal. In addition, there are a couple of suggested changes to the text that will need to be addressed.Reviewer #3:This is a re-review of the manuscript by Evans et al., describing the slow maturation/impaired acidification of mitophagosomes in the soma of neurons. I think the authors have done a great job in strengthening their manuscript – my main concern was with the lysosomal acidification studies and the use of the dual reporters means the data are much more convincing. I just have a few small points remaining:1) The LAMP2 data, mentioned in subsection” Acidification of OPTN-positive mitochondria is a rate-limiting step in neuronal mitophagy” and in the comments to reviewers does not seem to be in the manuscript. The authors mention Figure 4E, but these are only LC3-related. Please include the data.In addition to providing the percent of LAMP1 and LAMP2 structures that were Lyso-T positive in the text, we now include images in the revised manuscript .2) Likewise, the authors show data in the reviewers comments that the OPTN/LC3 structures are WIPI -ve. I do not see these data in the manuscript. This shows that the structures are not early autophagosomal in nature and thus they will be of interest to readers (and not just this reviewer) and so should be included . I'm assuming WIPI2B is expressed in these cells and does indeed form puncta with LC3 when autophagy is induced?At the reviewer’s request, we now include the WIPI2B data in Figure 5—figure supplement 1. WIPI2B, an early autophagosome marker, has been shown to be critical in the autophagy pathway, as depletion of WIPI2 in DRG neurons significantly decreased autophagosome biogenesis . In hippocampal neurons, we observed WIPI2B puncta that colocalized with LC3 in the soma, indicating immature autophagosomes (examples are highlighted by green arrows).3) I also take on the point that the authors have made in response to my concern raised in point 5 – i.e. that HeLa cells overexpressing Parkin are a suitable system to compare to the neurons. The HeLa cell has been instrumental in the field in terms of building up the mechanism of PINK/Parkin mitophagy. But this is not a "normal" cell. Normally, it does not undergo this pathway as it does not express Parkin! I think to avoid doing more experiments, the authors can be more accurate in what they state and say that this delay in acidification does not occur in HeLa cells expressing Parkin. For example in subsection “Acidification of OPTN-positive mitochondria is a rate-limiting step in neuronal mitophagy”: "…..much slower than the corresponding time course in non-neuronal cells." Should be replaced with "…..much slower than the corresponding time course in HeLa cells."The statement has been changed to “much slower than the corresponding time course in HeLa cells expressing exogenous Parkin.”


FMR1 gene results in transcriptional silencing and the absence of the FMR1 encoded protein, FMRP. FMRP is an RNA-binding protein that regulates the transport and translation of a variety of brain mRNAs in an activity-dependent manner. The loss of FMRP causes dysregulation of many neuronal pathways and results in an intellectual disability disorder, fragile X syndrome (FXS). Currently, there is no effective treatment for FXS. In this review, we discuss reactivation of the FMR1 gene as a potential approach for FXS treatment with an emphasis on the use of small molecules to inhibit the pathways important for gene silencing.More than ~200 CGG repeats in the 5′ untranslated region of the  FMR1) gene. An unstable CGG repeat tract is present in the 5’ untranslated region (UTR) of the FMR1 gene. In the general population, the repeat tract has less than 45 repeats -6H-pyridocarbazol-2-ium) is a small molecule that stabilizes the hairpins formed by the CGG repeats in the RNA (rCGG) and disrupts protein-binding [FMR1 mRNA with PRC2 [FMR1-specific small molecules that are also able to block PRC2 recruitment may be desirable.While it has been reported that some inhibitors of epigenetic modifying enzymes do not have the global effects on gene expression that one might expect ,125, in -binding ,129. Tre-binding . Howeverith PRC2 . Since Cith PRC2 , and sucFMR1 gene in FXS cells, HTS can help identify new biologically active small molecules against known targets, for example, DNA methyltransferases, histone deacetylases, and histone methyltransferases, as well as identify additional targets that might provide new insights into the gene silencing mechanism. This is particularly important as many of the compounds that have been shown to reactivate the silenced FMR1 gene so far are toxic and may not be suitable for long-term use in humans.High-throughput screening (HTS) is an approach to accelerate drug discovery that involves testing a large number of potential biological modulators and effectors against a specific target. With respect to identifying compounds that can reactivate the The success of an HTS depends on a robust readout that can be measured using an assay that is reliable and economical. In addition to the detection assay, the cells used in the screen are also important. Most FXS fibroblasts are mosaic for PM and FM alleles as well as their methylation status. Hence, it is difficult to assess if the increase in FMRP levels is due to an increase in translation from transcriptionally active alleles or reactivation of the silenced alleles. Furthermore, primary fibroblasts have a finite life span making it difficult to generate the large number of cells required for HTS. These characteristics make primary human fibroblast cells less desirable for HTS. In contrast, FXS iPSC-derived neural stem cells (NSCs) or NPCs are suitable for HTS because they can be used to rapidly generate a large number of cells. Furthermore, given that FM alleles in FXS iPSCs remain methylated ,132, it FMR1 transcription have relied on both the detection of endogenous FMRP in FXS NSCs or NPCs and the use of reporter cell lines. In the following sections we review the library screens that have been done thus far to identify compounds that reactivate the FMR1 gene.In this respect, HTS designed for identifying drugs that can increase FMR1 gene [1280 compound library, and two hits were identified, protoporphyrin IX and SB216763. These hit compounds were further validated by the dose response in TR-FRET assay and in a quantitative reverse transcription (qRT)-PCR assay for FMR1 mRNA. The authors then screened a ~4000 compound FDA-approved library. Four additional compounds  were identified from this HTS. With the exception of sodium decanehydroxamate, which is known to have HDAC activity, the mode of action of these compounds is unknown. While the compounds identified in this HTS were effective at very high concentrations and thus not likely to be biologically useful, it provides proof of principle of the approach and suggests that better lead compounds might be identified using larger compound libraries. Two different HTS have been reported for identifying molecules that are able to reactivate the MR1 gene ,134. ForFMR1 allele with ~480 CGG repeats. The authors also used 5-aza-dC as a positive control compound for the HTS. A total of 2099 compounds were identified that induced a small FMRP increase, and 790 of those were further tested in dose response assays. Only one compound was identified that had a previously known mode of action—a hydroxamate-based HDAC inhibitor, similar to the one identified in the HTS by Kumari and Swaroop et al. [In another screen, Kaufmann et al.  used higp et al. . The ideFMR1 gene locus in FXS iPSCs and control H1 ESCs using CRISPR/Cas9 gene editing. The authors then generated NPCs from the H1 control and FXS FMR1-Nluc reporter lines and optimized the HTS in 384-well and 1536-well plates. Both 5-aza-C and 5-aza-dC were used as positive control compounds to screen a 128 epigenetic compound library and ~1134 FDA-approved drug library using NPCs differentiated from the FXS-FMR1-Nluc reporter iPSCs. While no new compounds were identified in these screens, the sensitivity and cost-effectiveness of these reporter lines will make it feasible to screen very large compound libraries.While HTS based on assays using antibodies to detect FMRP were successful at identifying a number of hit compounds, the cost of antibodies is a limiting factor. To eliminate the requirement for antibodies, Li et al.  insertedFMR1 mRNA levels. However, currently, RNA detection assays are not economical, and the generation of new reporter cell lines that allow the effect of compounds on transcription or translation to be distinguished would thus be highly desirable for large library screens.The FMRP-based screening assay and the Nano-luc reporter lines share a common drawback—namely, the negative effect of CGG repeats on translation, which limits the amount of protein that can be detected. Thus, these assays are less sensitive than assays that detect FMR1 gene is a potentially useful treatment option for FXS. However, many challenges to this approach remain. More work is needed to identify additional proteins important for gene silencing that can be targeted for gene reactivation. Conducting genome-wide CRISPR/siRNA screens for gene reactivation is one way to identify new targets. In addition, better model systems to understand the initiating events leading to gene silencing may allow the identification of novel proteins for pharmacological targeting. Early work in humans had led to the suggestion that FMR1 gene silencing occurs relatively late in embryonic development at around 10 weeks of gestation [FMR1 gene is actively transcribed in FXS ESCs and undergoes differentiation-induced silencing [per se is not required for gene silencing [FMR1 gene was not reactivated in iPSCs derived from FXS patients [FMR1 gene silencing occurs at an earlier stage of embryonic development. It is therefore possible that naïve FXS iPSCs or ESCs could provide a better cell model for understanding the very earliest events in FMR1 gene silencing [Restoring FMRP expression in FXS cells by reactivating the endogenous estation . If so, ilencing ,82,136. ilencing ,138. Morpatients ,132. Botilencing . FMR1 mRNA [FMR1 gene reactivation required. This would also reduce the risk of developing pathology resulting from rCGG expression. Furthermore, small molecules like Compound 1a that prevent gene re-silencing in a relatively gene-specific way while also reducing the deleterious effects of rCGG may be particularly useful [Additional challenges include the necessity for overcoming the negative effect of the CGG repeats on the translation efficiency of MR1 mRNA ,142,143 MR1 mRNA ,147,148.MR1 mRNA ,150,151.MR1 mRNA  and FM fMR1 mRNA ,154. Simy useful ,129. Fmr1 KO mice to suggest that increasing FMRP production during postnatal life may still have some clinical benefit [Finally, the challenge of if, when, and how a gene reactivation approach could be deployed needs to be considered. For families with a known history of FXS, preimplantation genetic diagnosis might be the preferred option . Most ne benefit ,158. WheFMR1 gene and suggested approaches for gene reactivation that are most likely to be effective. However, we still have a long way to go before this approach is therapeutically useful. A number of new tools are needed, including an animal model that recapitulates the repeat-mediated FMR1 gene silencing seen in FXS and human cell-based/organoid models that can be used to verify the compounds and approaches that work in the animal models. In addition, the identification of molecular biomarkers, a focus area for FXS, will be useful not only to test the efficacy of treatment strategies based on restoring FMRP expression but also for those aimed at compensating for its loss.Preliminary studies using cell-based models for FXS have shown that it is possible to reactivate the silenced