Synaptic Activity Regulates Mitochondrial Iron Metabolism to Enhance Neuronal Bioenergetics

Round 1

Reviewer 1 Report (Previous Reviewer 1)

I thank the Authors addressing my concerns.

I am satisfied with the revised manuscript.

Author Response

We thank the referee for the comments and positive feedback

Reviewer 2 Report (Previous Reviewer 2)

The article can be accepted

Author Response

We thank the referee for the comments and positive feedback

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In this manuscript, the Authors study the effect of “synaptic activity” on iron uptake and mitochondrial metabolism, indicating that activity increases the mitochondrial iron uptake improving long-term mitochondrial ability to perform oxidative phosphorylation.

Their experimental model is based on exposing primary cortical neurons to a 24h dis-inhibition protocol by applying bicuculline (GABA-A receptor antagonist) and 4AP (K+ channel antagonist). After this treatment, they assess several parameters:

-       Mitochondrial membrane potential measured with TMRM fluorescence is increased

-       Basal and maximal OCR is increased

-       Mitochondrial mass (estimated based on western blot quantification of two mitochondrial markers) is not changed, suggesting increased mitochondrial metabolic activity

-       Intracellular and mitochondrial iron levels are increased in stimulated neurons, based on a colorimetric assay and a fluorescent sensor

-       The iron chelator bipy prevents the increase in OCR in stimulated neurons, suggesting that iron is mediating it

-       Blocking protein synthesis with CHX prevents the increase in intracellular iron upon stimulation

-       Stimulation induces expression of Mfrn1 (that mediates mitochondrial iron import) and Fpn (that mediates iron export from the cells)

-       KD of Mfrn1 reduces mitochondrial iron levels and prevents increase in OCR upon stimulation

-       Forskolin, which activates adenyl cyclase, is sufficient to induce Mfrn1 expression, suggesting the transcription factor CREB mediates the effect

-       Expressing a dominant-negative CREB (A-CREB) prevents the induction of Mfrn1 and the increase in mitochondrial iron uptake upon stimulation

I believe that this is a very exciting subject, and the main findings are quite interesting, but the presentation of the data needs substantial improvement and possibly repeating or expanding some experiments.

Major points:

1.     The stimulation protocol is not properly characterized, and it cannot be simplified as “synaptic activity”, “synaptic stimulation” or “an episode of synaptic activity”, as it is suggested in the title and throughout the manuscript. The current protocol will trigger activity in the cultures neurons that will release neurotransmitters that in turns could activate a variety of receptors, including metabotropic receptors (some directly linked to induction of adenylate cyclase activity). Some pharmacology experiments using specific receptors antagonists would be extremely informative. In present form, this a general very broad stimulation protocol, and it is not clear if it leads to a persistent Ca2+ increase (for example) in neurons or if at some point desensitization occurs. The paper would greatly benefit from a proper characterization of the event occurring during the stimulation protocol. Moreover, considering that the authors use as a standard protocol the 24hs stimulation but observe transcriptional changes already at 4 hours, it would be even more interesting seeing what happens at shorter time points. In the present form, I think it is only fair to describe this as “prolonged disinhibition”.

2.     The data is very interesting, but it is suffering from very small sample sizes and a too minimalistic presentation. It would be informative to see the traces/time lapses of representative experiments (when applicable - see TMRM experiments or OCR measurements) to fully appreciate the described effects. Moreover, with sample sizes so small it is not appropriate to use parametric statistics. Also on the topic of small sample sizes, I appreciate that the authors disclaim that the same control samples are used across different figures, but this doesn’t seem an appropriate practice and would require statistical corrections.

Minor points:

1.     Is the stimulation protocol associated with induction of cell death/apoptosis or cell loss? This should be verified to make sure that the changes observed are not due to the selection of neurons that are more resistant because they are already richer in iron/more bioenergetically efficient and can survive the stress of the stimulation.

2.     The assays are based on the whole cells (soma, dendrites and axons), and at least from the images shown, most optical measurements are based on somatic regions. If the phenomenon is synaptic and not a general stimulation of the cells, it should be even more evident when considering post-synaptic regions. While this is obviously very challenging for assays performed on a whole plate/well, some of the optical/fluorescent assays could be performed more specifically in regions of high synaptic density.

3.     Manuscript should be edited more accurately. There are still visible sections that have been cut/pasted with no format adaptation.

Reviewer 2 Report

The manuscript shows that during synaptic activity, rat cortical neurons increase mitochondrial bioenergetics by transcriptionally inducing the expression of iron metabolism genes, accompanied by increased uptake of cellular and mitochondrial iron content. Further, the authors demonstrated for the first time the involvement of CREB in regulating mitoferrin 1 expression.

This is an interesting, convincing and well-organized work. It is well known that iron is involved in mitochondrial energy metabolism while little is known about its involvement in synaptic activity, strictly linked to energetic metabolism of neurons. The authors elegantly show that synaptic activity enhances mitochondrial iron uptake and bioenergetics, while iron chelation or mitoferrin 1 silencing impair mitochondrial bioenergetics. I have only minor requests:

Figures 2A and 2E: FerroOrange and MitoGreen fluorescence images are not of good quality. I suggest to upload sharper images

Discussion: I ask to deepen the discussion regarding the (probably unexpected) upregulation of ferroportin during synaptic activity, considering the simultaneous upregulation of genes that are involved in cellular iron entry. Please also deepen the suggested link with ferroptosis.