Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses
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eLife assessment
This study examines an interplay between synaptic mitochondria and glutamate receptor exocytosis in C. elegans. Collectively, the solid results support the idea that mitochondrial function influences receptor dynamics at postsynaptic sites. This is important because tight control of synaptic function likely integrates several mitochondrial functions: energy production, calcium buffering, and (here) reactive oxygen species signaling.
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Abstract
Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca 2+ ) via the mitochondrial Ca 2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion.
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Author Response
Reviewer #2 (Public Review):
In this paper, the authors discover that postsynaptic mitochondria in C. elegans govern glutamate receptor trafficking dynamics. The core results are two-fold. For one, they find that loss or inhibition of mcu-1 - the C. elegans mitochondrial calcium uniporter - increases GLR-1 glutamate receptor accumulation at the postsynaptic dendritic sites and enhances its trafficking dynamics. The authors hypothesize that this effect on glutamate receptors may have something to do with mitochondrial ROS production. This is because ROS is a by-product of normal oxidative phosphorylation, downstream of calcium import. Indeed, the generation of artificially high amounts of mitochondrial ROS has the opposite effect of mcu-1 loss: decreased glutamate receptor subunit accumulation. Collectively, the …
Author Response
Reviewer #2 (Public Review):
In this paper, the authors discover that postsynaptic mitochondria in C. elegans govern glutamate receptor trafficking dynamics. The core results are two-fold. For one, they find that loss or inhibition of mcu-1 - the C. elegans mitochondrial calcium uniporter - increases GLR-1 glutamate receptor accumulation at the postsynaptic dendritic sites and enhances its trafficking dynamics. The authors hypothesize that this effect on glutamate receptors may have something to do with mitochondrial ROS production. This is because ROS is a by-product of normal oxidative phosphorylation, downstream of calcium import. Indeed, the generation of artificially high amounts of mitochondrial ROS has the opposite effect of mcu-1 loss: decreased glutamate receptor subunit accumulation. Collectively, the results support the idea that mitochondrial function can control receptor dynamics at synaptic sites. This is interesting because tight control of synaptic function likely combines several mitochondrial functions: energy production, calcium buffering, and (here) ROS signaling.
STRENGTHS
• The C. elegans genetic model is a strength because the authors are able to make refined conclusions by classical loss-of-function mutants (e.g., mcu-1) along with an impressive cytological toolkit to examine GLR-1 dynamics.
• The use of pharmacology as a second means to test those genetic conclusions is a strength.
• The authors' careful reagent verification of reporters (Ca2+, ROS, etc.) is a strength.
• The ability to link fundamental mitochondrial processes to GLR-1 exocytosis will expand how the field thinks about mitochondrial synapse function.
WEAKNESSES
For the most part, the data in the paper support the conclusions, and the authors were careful to try experiments in multiple ways. But please see below:
• (Main Point) The data are good, but they fall short of mechanism (e.g., Line 322). Figure 6 is accurate as drawn. But calcium and ROS are not abstract signals. They are likely exerting affirmative actions on specific targets. The Discussion does acknowledge this in terms of ROS and it speculates on possible targets.
We thank the reviewer for their analytical review of our manuscript. We agree that all molecular players involved in the proposed mechanism were not identified by the data presented, so we modified the text to remove overstatements. We also agree that Ca2+ and ROS signaling is not abstract. Rather, there are specific and diverse targets of both Ca2+ and ROS signaling. Follow-up experiments are underway to identify and provide evidence for the necessity of potential ROS/Ca2+ targets in this proposed mechanism. For the current manuscript, we have modified our verbiage in an attempt to not mislead or overstate what our results suggest (e.g., changes/additions to the beginning of the ‘Discussion’, lines 365-377 and 385-388) and updated the illustration of the proposed model to include dashed lines that, as mentioned in the figure legend, indicate indirect action by ROS and Ca2+ (see revised Figure 7).
The general idea seems to be that mitochondria import calcium through MCU-1 (and interacting factors). As a result, oxidative phosphorylation successfully occurs and mitochondrial ROS is a signaling by-product that signals glutamate receptors not to undergo exocytosis. But there are other interpretations of what might happen in between. In fact, if OXPHOS is disrupted, it is known that this can generate a lot more mitochondrial ROS than the normal by-product levels.
We do agree that an alternative explanation could be that genetic or pharmacological inhibition of mitochondrial Ca2+ uptake disrupts oxidative phosphorylation, and as a result, inefficiencies or uncoupling in the electron transport chain would lead to an even greater increase in mitochondrial ROS production. Although oxidative phosphorylation was not directly measured, one of our post hoc analyses of GLR-1 transport suggests ATP levels are comparable between controls, mcu-1 mutants, and with Ru360 treatment: the velocity of GLR-1 transport is unchanged between these experimental groups. The processivity of molecular motors (which dictates transport velocity) is highly sensitive to relative ATP abundance. Thus, if ATP levels were dramatically decreased in mcu-1 mutants or following Ru360 treatment, then one would expect a detectable change in GLR-1 transport velocities, but we observed no change (see revised Figure S2E and related discussion at lines 183-190). Although these results do not directly indicate whether ATP production is altered with loss or inhibition of MCU-1, it does suggest that basal ATP levels remain sufficient to support the metabolic demands of GLR-1 transport.
This reviewer wonders if excess ROS would cause an extreme response. Or alternatively, if scavenging ROS via pharmacological scavengers or SOD expression would reverse the effects.
These are good points, and we have previously published experiments that address each of them. First, we have seen that globally increasing ROS with various concentrations of H2O2 within the physiological range (<100 nM) decreased GLR-1 transport to a similar extent (PMID: 32847966) indicating that there is not a dose-dependent decrease in GLR-1 transport. We have also assessed GLR-1 transport after treatment with concentrations of H2O2 well above the physiological range (e.g., 500 nM), but these high concentrations obliterated all GLR-1 transport. Contrary to what one may expect, we showed that decreasing ROS via pharmacological or genetic means (probably below physiological range) decreased GLR-1 transport (PMID: 35622512) via a Ca2+ independent mechanism. In other words, ROS scavenging did not have the opposite effect on GLR-1 transport, but we have not combined ROS scavenging with optical induction of ROS production (e.g., via KillerRed) nor have we assessed the potential influence of ROS scavenging on synaptic recruitment. Although we agree that these are important follow-up experiments, they will require a more sensitive ROS indicator because current genetically encoded in vivo ROS sensors cannot detect decreases in ROS levels below the physiological range (< 10 nM) (PMID: 31586057).
Small Points
• 33.3 mHz - just making sure, do the authors mean once every 30 seconds? That would be more straightforward.
Yes, we do mean a 1-second pulse of light every 30 seconds. We have clarified this in the manuscript text (line 115).
• Figure 2 is confusing. The text says that the mcu-1 mutants have a GLR-1::GFP FRAP rate that is comparable to controls (Lines 165-167). But Figure 2E suggests that it is markedly less, which is the opposite result of the slight increase in rate resulting from Ru360 treatment. And is the explanation why the GLR-1::GFP results differ from the SEP::GLR-1 results a difference between total GFP vs. surface GFP?
The confusion is due to an incorrect statement in the results text. We have corrected this error and appreciate the reviewer for bringing it to our attention (lines 173-174).
• I could not watch Video 2 (not sure if it is the file or just the copy I downloaded).
We thank the reviewer for bringing this to our attention and we believe we have remedied the issue.
• It is good that the authors tried both optical stimulation and mechanical stimulation (dropping culture plates to stimulate the worms, Figure 3). Why was the mechanical stimulation set aside for further tests in the paper?
Mechanical stimulation consisted of dropping culture plates containing 2-3 C. elegans onto a lab bench every 30 seconds for 5 or 10 minutes. This mechanical stimulation paradigm was technically cumbersome and was less effective at inducing changes in mito-roGFP fluorescence that optical stimulation. This is likely due to habituation to the mechanical stimulus which has been well-characterized in C. elegans. The optical stimulation was therefore used as it is a more reliable and repeatable method for stimulating the AVA neuron.
• Does this process affect all kinds of transport, or is it just the glutamate receptors? Was anything else examined?
Transport of other proteins has not been examined in the context of mitoROS signaling. Our attempts at visualizing and quantifying the transport, synaptic delivery and exocytosis of other synaptic proteins in vivo has proven to be more technically challenging likely due to relatively lower expression in the C. elegans neurons suitable for transport analysis.
Reviewer #3 (Public Review):
Reactive oxygen species (ROS) have been previously shown to regulate glutamate receptor phosphorylation, long-distance transport, and delivery of glutamate receptors to synapses, however, the source of ROS is unclear. In this study, the authors test if mitochondria act as a signaling hub and produce ROS in response to neuronal activity in order to regulate glutamate receptor trafficking. The authors use a variety of optogenetic tools including the calcium reporter mitoGCaMP and the ROS reporter mito-roGFP to monitor changes in calcium and ROS, respectively, in mitochondria after activating neurons with ChRimson in the genetic model organism C. elegans. Repeated stimulation of interneurons called AVA with ChRimson leads to increased calcium uptake into mitochondria in dendrites and increased mitochondrial ROS production. The mitochondrial calcium uniporter mcu-1 is required for these effects because mcu-1 genetic loss of function or treatment with Ru360, a drug that inhibits mcu-1, inhibits the uptake of calcium into mitochondria and ROS production after neuronal activation. Mcu-1 genetic loss of function is correlated with an increase in exocytosis of glutamate receptors but a decrease in glutamate receptor transport and delivery to dendrites. This study suggests that mitochondria monitor neuronal activity by taking up calcium and downregulating glutamate receptor trafficking via ROS, as a means to negatively regulate excitatory synapse function.
Strengths
-The use of multiple optogenetic tools and approaches to monitor mitochondrial calcium, reactive oxygen species, and glutamate receptor trafficking in live organisms.
-Identifying a novel signaling role for dendritic mitochondria which is to monitor neuronal activity (via calcium uptake into mitochondria) and generate a signal (reactive oxygen species) that regulates glutamate receptors at synapses.
Weaknesses
-Although the use of KillerRed to generate ROS downstream of mcu-1 is a clever approach, the fact that activation of KillerRed results in reduced GLR-1 exocytosis, delivery, and transport raises the concern that KillerRed is generating a high level or ROS that might be toxic to cellular processes. Experiments showing that other cellular processes are not affected by KillerRed activation and testing if reduced ROS production mimics the effects of blocking mcu-1 would strengthen the conclusions in this study.
We thank the reviewer for their careful analyses of our findings. It is plausible that KillerRed could cause toxic levels of ROS, in fact, it was originally used to instigate oxidative stress-induced apoptosis to achieve cell-specific ablation. These cell ablation protocols required 20+ minutes of KillerRed activation with substantially higher levels of irradiation (e.g., 3.8 mW/mm [PMID: 24209746] vs. our light dosage of 25 µW/mm2). Additionally, our transgenic C. elegans strains expressing KillerRed were designed to have a relatively low KillerRed expression and were screened for low expression based on KillerRed’s fluorescence. Using these strains, we were able to minimally activate KillerRed in the AVA neuron resulting in ROS elevations at mitochondria that were comparable to neuronal activity-induced increases in mitochondrial ROS as measured by mito-roGFP. Specifically, we found that 10 minutes of mechano-stimulation and 5 minutes of ChRimson stimulation increased the fluorescence ratio (Fratio) of mito-roGFP nearly two-fold (Figure 4A-B and 4C-E). A 15-second pulse of light focused on a small region activating mitoKR in the AVA neurite also caused similar two-fold increase in the mito-roGFP Fratio (Figure 4C-E) comparable to what neuronal activity induced. Our 5-minute global KillerRed activation less effectively increased the mito-roGFP Fratio at mitochondria in the AVA neurite compared to neuronal activity (revised Figure 4B and 4H) but was sufficient in decreasing GLR-1 transport (revised Figure 5G-H). So, we decided to do all experiments with 5 minutes of global KillerRed activation since lower activation levels of KillerRed were more likely to achieve non-toxic, signaling levels of ROS. Since we strongly agree that this data is important for tool validation, we have reorganized the manuscript such that these data are now a primary figure (see revised Figure 4 and new results sub-section starting at line 252).
Additionally, we added supplemental transport velocity data. This data shows that local photoactivation as well as whole-cell activation of KillerRed does not alter transport velocity of GLR-1 vesicles within the neurite (revised Figure S4A and S4B and lines 272-276 and 287-289), which would be the case if ATP, microtubules, or actin dynamics were affected. This supports that our local and whole-cell activation protocol does not cause toxic levels of ROS production.
Lastly, the reviewer questions whether decreasing ROS alters GLR-1 transport, synaptic delivery and exocytosis in a similar fashion to loss or inhibition of mcu-1, and if so, would further support the proposed mechanism. We have decreased ROS via genetic (catalase overexpression) and pharmacological (using the mitochondria-targeted antioxidant MitoTEMPO) means and seen that diminished ROS levels decrease GLR-1 transport albeit to a lesser degree than that caused by loss/inhibition of mcu-1 (PMID: 35622512). To determine if decreased GLR-1 transport during diminished ROS levels involves mcu-1, we would need to assess GLR-1 transport in mcu-1 mutants while ROS is decreased (e.g., using MitoTEMPO treatment) to see if their combined effect phenocopies the effect of mcu-1(lf) or decreased ROS alone. However, as mentioned previously, we are unable to measure ROS levels below the sensitivity of roGFP but within physiological range so we cannot currently calibrate or validate our methods for scavenging ROS in vivo. This is why we have not yet analyzed synaptic delivery or exocytosis rates of GLR-1 in the context of decreased ROS, but these would be interesting follow-up experiments that may further support our model once more sensitive ROS sensors are available.
Reviewer #4 (Public Review):
Using optogenetic stimulation, the authors presented compelling evidence that neuronal activity increases mitochondrial calcium levels, facilitated by the mitochondrial uniporter MCU-1. Through ratiometric measurements, they showed that mitochondrial ROS levels also increase due to neuronal activity via MCU-1. Subsequent FRAP studies were employed to investigate the trafficking of the AMPA receptor, GLR-1. By integrating genetic and pharmacological methodologies, the recovery rate of GLR-1 was assessed. The authors concluded that increased mitochondrial ROS due to neuronal activity reduces the trafficking and exocytosis of AMPA receptors. They proposed that mitochondrial ROS serves as a homeostatic mechanism regulating AMPA receptor trafficking and abundance, thus maintaining synaptic strength. This research is crucial as it provides a direct link between mitochondrial signaling and AMPA receptor trafficking.
However, there are several significant concerns regarding the methodologies and quantifications employed in this manuscript. The authors utilized GLR-SEP to label surface AMPA receptors and relied on the "FRAP rate" as an indicator of the exocytosis rate. The absence of direct visualization of exocytosis using GLR-SEP, and the lack of direct measurements of exocytosis events, casts doubt on the conclusions about ROS's impact on AMPA receptor exocytosis. Furthermore, the "FRAP rate" determined in this study is a combination of recovery rates (incorporating both endosomal trafficking and diffusion) with the mobile fractions of AMPA receptors, potentially weakened interpretations of the findings. A more comprehensive discussion addressing the conflicting effects of MCU-1 and ROS on GLR-GFP FRAP recovery and dendritic trafficking would enable readers to grasp the intricate roles of mitochondrial calcium and ROS in modulating synaptic receptors.
We appreciate the reviewer’s attention to detail while reviewing our article. Their major concern about directly visualizing exocytosis events is valid since changes in exocytosis and endocytosis would dictate the amount of SEP::GLR-1 at the synaptic membrane. However, streaming imaging of SEP in vivo is technically difficult showing only few exocytosis events and provides short “snapshots” (1-2 minutes, longer streaming imaging causes photobleaching and photo-toxicity) which must be extrapolated to longer time frames. Our 16-minute SEP::GLR-1 FRAP protocol allows us to capture all plasma membrane recruitment and quantify the relative balance between exo- and endocytosis. It also allows for longer observational periods during which we can detect changes in GLR-1 recruitment to and retention at the synaptic membrane in genetic mutants and with drug treatments. In addition, our photobleaching approach involves photobleaching a ~40-60 µm region proximally and distally to the imaging region which limits the influence of receptor diffusion on the FRAP rate. The reviewer makes a valid point that receptor endocytosis rates would also influence the SEP::GLR-1 FRAP rate. We have now changed the text in the results and discussion to include this information (lines 155-161, and changing “exocytosis” to “synaptic recruitment” throughout the manuscript when discussing SEP::GLR-1 FRAP results [e.g, at lines 169, 208, and 321]).
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eLife assessment
This study examines an interplay between synaptic mitochondria and glutamate receptor exocytosis in C. elegans. Collectively, the solid results support the idea that mitochondrial function influences receptor dynamics at postsynaptic sites. This is important because tight control of synaptic function likely integrates several mitochondrial functions: energy production, calcium buffering, and (here) reactive oxygen species signaling.
-
Reviewer #2 (Public Review):
In this paper, the authors discover that postsynaptic mitochondria in C. elegans govern glutamate receptor trafficking dynamics. The core results are two-fold. For one, they find that loss or inhibition of mcu-1 - the C. elegans mitochondrial calcium uniporter - increases GLR-1 glutamate receptor accumulation at the postsynaptic dendritic sites and enhances its trafficking dynamics. The authors hypothesize that this effect on glutamate receptors may have something to do with mitochondrial ROS production. This is because ROS is a by-product of normal oxidative phosphorylation, downstream of calcium import. Indeed, the generation of artificially high amounts of mitochondrial ROS has the opposite effect of mcu-1 loss: decreased glutamate receptor subunit accumulation. Collectively, the results support the idea …
Reviewer #2 (Public Review):
In this paper, the authors discover that postsynaptic mitochondria in C. elegans govern glutamate receptor trafficking dynamics. The core results are two-fold. For one, they find that loss or inhibition of mcu-1 - the C. elegans mitochondrial calcium uniporter - increases GLR-1 glutamate receptor accumulation at the postsynaptic dendritic sites and enhances its trafficking dynamics. The authors hypothesize that this effect on glutamate receptors may have something to do with mitochondrial ROS production. This is because ROS is a by-product of normal oxidative phosphorylation, downstream of calcium import. Indeed, the generation of artificially high amounts of mitochondrial ROS has the opposite effect of mcu-1 loss: decreased glutamate receptor subunit accumulation. Collectively, the results support the idea that mitochondrial function can control receptor dynamics at synaptic sites. This is interesting because tight control of synaptic function likely combines several mitochondrial functions: energy production, calcium buffering, and (here) ROS signaling.
STRENGTHS
• The C. elegans genetic model is a strength because the authors are able to make refined conclusions by classical loss-of-function mutants (e.g., mcu-1) along with an impressive cytological toolkit to examine GLR-1 dynamics.
• The use of pharmacology as a second means to test those genetic conclusions is a strength.
• The authors' careful reagent verification of reporters (Ca2+, ROS, etc.) is a strength.
• The ability to link fundamental mitochondrial processes to GLR-1 exocytosis will expand how the field thinks about mitochondrial synapse function.
WEAKNESSES
For the most part, the data in the paper support the conclusions, and the authors were careful to try experiments in multiple ways. But please see below:
• (Main Point) The data are good, but they fall short of mechanism (e.g., Line 322). Figure 6 is accurate as drawn. But calcium and ROS are not abstract signals. They are likely exerting affirmative actions on specific targets. The Discussion does acknowledge this in terms of ROS and it speculates on possible targets.
The general idea seems to be that mitochondria import calcium through MCU-1 (and interacting factors). As a result, oxidative phosphorylation successfully occurs and mitochondrial ROS is a signaling by-product that signals glutamate receptors not to undergo exocytosis. But there are other interpretations of what might happen in between. In fact, if OXPHOS is disrupted, it is known that this can generate a lot more mitochondrial ROS than the normal by-product levels.
This reviewer wonders if excess ROS would cause an extreme response. Or alternatively, if scavenging ROS via pharmacological scavengers or SOD expression would reverse the effects.
Small Points
• 33.3 mHz - just making sure, do the authors mean once every 30 seconds? That would be more straightforward.
• Figure 2 is confusing. The text says that the mcu-1 mutants have a GLR-1::GFP FRAP rate that is comparable to controls (Lines 165-167). But Figure 2E suggests that it is markedly less, which is the opposite result of the slight increase in rate resulting from Ru360 treatment. And is the explanation why the GLR-1::GFP results differ from the SEP::GLR-1 results a difference between total GFP vs. surface GFP?
• I could not watch Video 2 (not sure if it is the file or just the copy I downloaded).
• It is good that the authors tried both optical stimulation and mechanical stimulation (dropping culture plates to stimulate the worms, Figure 3). Why was the mechanical stimulation set aside for further tests in the paper?
• Does this process affect all kinds of transport, or is it just the glutamate receptors? Was anything else examined?
-
Reviewer #3 (Public Review):
Reactive oxygen species (ROS) have been previously shown to regulate glutamate receptor phosphorylation, long-distance transport, and delivery of glutamate receptors to synapses, however, the source of ROS is unclear. In this study, the authors test if mitochondria act as a signaling hub and produce ROS in response to neuronal activity in order to regulate glutamate receptor trafficking. The authors use a variety of optogenetic tools including the calcium reporter mitoGCaMP and the ROS reporter mito-roGFP to monitor changes in calcium and ROS, respectively, in mitochondria after activating neurons with ChRimson in the genetic model organism C. elegans. Repeated stimulation of interneurons called AVA with ChRimson leads to increased calcium uptake into mitochondria in dendrites and increased mitochondrial ROS …
Reviewer #3 (Public Review):
Reactive oxygen species (ROS) have been previously shown to regulate glutamate receptor phosphorylation, long-distance transport, and delivery of glutamate receptors to synapses, however, the source of ROS is unclear. In this study, the authors test if mitochondria act as a signaling hub and produce ROS in response to neuronal activity in order to regulate glutamate receptor trafficking. The authors use a variety of optogenetic tools including the calcium reporter mitoGCaMP and the ROS reporter mito-roGFP to monitor changes in calcium and ROS, respectively, in mitochondria after activating neurons with ChRimson in the genetic model organism C. elegans. Repeated stimulation of interneurons called AVA with ChRimson leads to increased calcium uptake into mitochondria in dendrites and increased mitochondrial ROS production. The mitochondrial calcium uniporter mcu-1 is required for these effects because mcu-1 genetic loss of function or treatment with Ru360, a drug that inhibits mcu-1, inhibits the uptake of calcium into mitochondria and ROS production after neuronal activation. Mcu-1 genetic loss of function is correlated with an increase in exocytosis of glutamate receptors but a decrease in glutamate receptor transport and delivery to dendrites. This study suggests that mitochondria monitor neuronal activity by taking up calcium and downregulating glutamate receptor trafficking via ROS, as a means to negatively regulate excitatory synapse function.
Strengths
-The use of multiple optogenetic tools and approaches to monitor mitochondrial calcium, reactive oxygen species, and glutamate receptor trafficking in live organisms.
-Identifying a novel signaling role for dendritic mitochondria which is to monitor neuronal activity (via calcium uptake into mitochondria) and generate a signal (reactive oxygen species) that regulates glutamate receptors at synapses.Weaknesses
-Although the use of KillerRed to generate ROS downstream of mcu-1 is a clever approach, the fact that activation of KillerRed results in reduced GLR-1 exocytosis, delivery, and transport raises the concern that KillerRed is generating a high level or ROS that might be toxic to cellular processes. Experiments showing that other cellular processes are not affected by KillerRed activation and testing if reduced ROS production mimics the effects of blocking mcu-1 would strengthen the conclusions in this study. -
Reviewer #4 (Public Review):
Using optogenetic stimulation, the authors presented compelling evidence that neuronal activity increases mitochondrial calcium levels, facilitated by the mitochondrial uniporter MCU-1. Through ratiometric measurements, they showed that mitochondrial ROS levels also increase due to neuronal activity via MCU-1. Subsequent FRAP studies were employed to investigate the trafficking of the AMPA receptor, GLR-1. By integrating genetic and pharmacological methodologies, the recovery rate of GLR-1 was assessed. The authors concluded that increased mitochondrial ROS due to neuronal activity reduces the trafficking and exocytosis of AMPA receptors. They proposed that mitochondrial ROS serves as a homeostatic mechanism regulating AMPA receptor trafficking and abundance, thus maintaining synaptic strength. This research …
Reviewer #4 (Public Review):
Using optogenetic stimulation, the authors presented compelling evidence that neuronal activity increases mitochondrial calcium levels, facilitated by the mitochondrial uniporter MCU-1. Through ratiometric measurements, they showed that mitochondrial ROS levels also increase due to neuronal activity via MCU-1. Subsequent FRAP studies were employed to investigate the trafficking of the AMPA receptor, GLR-1. By integrating genetic and pharmacological methodologies, the recovery rate of GLR-1 was assessed. The authors concluded that increased mitochondrial ROS due to neuronal activity reduces the trafficking and exocytosis of AMPA receptors. They proposed that mitochondrial ROS serves as a homeostatic mechanism regulating AMPA receptor trafficking and abundance, thus maintaining synaptic strength. This research is crucial as it provides a direct link between mitochondrial signaling and AMPA receptor trafficking.
However, there are several significant concerns regarding the methodologies and quantifications employed in this manuscript. The authors utilized GLR-SEP to label surface AMPA receptors and relied on the "FRAP rate" as an indicator of the exocytosis rate. The absence of direct visualization of exocytosis using GLR-SEP, and the lack of direct measurements of exocytosis events, casts doubt on the conclusions about ROS's impact on AMPA receptor exocytosis. Furthermore, the "FRAP rate" determined in this study is a combination of recovery rates (incorporating both endosomal trafficking and diffusion) with the mobile fractions of AMPA receptors, potentially weakened interpretations of the findings. A more comprehensive discussion addressing the conflicting effects of MCU-1 and ROS on GLR-GFP FRAP recovery and dendritic trafficking would enable readers to grasp the intricate roles of mitochondrial calcium and ROS in modulating synaptic receptors.
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