Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons

  1. Akane Hatsuda
  2. Junko Kurisu
  3. Kazuto Fujishima
  4. Ayano Kawaguchi
  5. Nobuhiko Ohno
  6. Mineko Kengaku

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Abstract

Dendritic outgrowth in immature neurons is enhanced by neuronal activity and is considered one of the mechanisms of neural circuit optimization. It is known that calcium signals affect transcriptional regulation and cytoskeletal remodeling necessary for dendritic outgrowth. Here, we demonstrate that activity-dependent calcium signaling also controls mitochondrial homeostasis via AMP-activated protein kinase (AMPK) in growing dendrites of differentiating mouse hippocampal neurons. We found that the inhibition of neuronal activity induced dendritic hypotrophy with abnormally elongated mitochondria. In growing dendrites, AMPK is activated by neuronal activity and dynamically oscillates in synchrony with calcium spikes, and this AMPK oscillation was inhibited by CaMKK2 knockdown. AMPK activation led to phosphorylation of MFF and ULK1, which initiate mitochondrial fission and mitophagy, respectively. Dendritic mitochondria in AMPK-depleted neurons exhibited impaired fission and mitophagy and displayed multiple signs of dysfunction. Genetic inhibition of fission led to dendritic hypoplasia that was reminiscent of AMPK-deficient neurons. Thus, AMPK activity is finely tuned by the calcium-CaMKK2 pathway and regulates mitochondrial homeostasis by facilitating removal of damaged components of mitochondria in growing neurons during normal brain development.

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  1. Version published to 10.1242/dev.201930
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    Reply to the reviewers

    1. General Statements [optional]

    We thank the reviewers for their comments and very helpful suggestions to improve the manuscript. All the reviewers address that further confirmation of the causality of activity-induced AMPK activation and AMPK-induced mitochondrial fission and mitophagy regulating dendritic outgrowth in immature neurons would strengthen the significance of this study. We believe that this is the first study demonstrating that AMPK mediates activity-dependent dendritic outgrowth of immature neurons, and that regulation of mitophagy is critical for dendrite development.

    We can perform most of the experimentations and corrections requested by the reviewers. We have already made several revisions and are currently working on additional experiments. All experiments will be finished in several weeks and we expect to submit a full revision by the due date.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1-1.- MMP alone is not a good indicator of mitochondrial health. For instance, ATPase inhibitor causes increase in MMP and complex I inhibition diminish MMP and in both cases mitochondrial function is impaired. On the other hand, authors use increased flickering and mitochondrial ROS production as an indicator of enhanced respiration but they could also be used as indicators of mitochondrial dysfunction. Other assays, such as oxygen consumption, are needed to assess the mitochondrial function.”

    *Related comments by Reviewer #2-C. In figure 6 it is unclear what is the significance of the TMRM "flickering" parameter quantified and the difference between the control and knockdown condition is small on average. *

    Increase in TMRM flickering and mitochondrial ROS production, which we used as indicators of enhanced mitochondrial respiration, can certainly also be caused by mitochondrial dysfunction. We think it difficult to adopt an oxygen consumption assay in our system, as the transfection efficiency in the primary hippocampal culture is low (~10%). Instead, we plan to assess the mitochondrial function in control and AMPK deficient cells by using an ATP FRET sensor targeted to mitochondria (Mito-ATeam, Imamura et al., PNAS, 2009; Yoshida et al., Methods Mol Biol 2017). Mito-ATeam will be transfected in neurons to compare mitochondrial ATP synthesis in control and AMPK deficient neurons.

    *Reviewer #1-2.- It would be interesting to show a better characterization of the mitophagy flux and to test whether pharmacological or genetic stimulation of mitophagy could revert the effect of AMPK KD on dendritic outgrowth, ultimately linking AMPK, mitophagy and dendritic outgrowth. The latter experiments may be challenging but not impossible, for example see (PMID: 27760312). *

    We understand that it is important to demonstrate more strongly the correlation of the AMPK-induced peripheral fission and subsequent mitophagy of fragmented mitochondria with dendritic outgrowth. We will attempt the suggested experiment to see if induction of autophagy could revert the dendritic hypoplasia by AMPK KD. However, because AMPK deficiency generates elongated mitochondria defective in fission rather than fragmented mitochondria that are failed to undergo mitophagy, we doubt that activating mitophagy will properly remove damaged mitochondria.

    In parallel to the above experiments, we currently analyze if inhibition of mitochondrial fission or mitophagy would phenocopy the hypoplastic dendrites of AMPK-deficient neurons, and if the activation of fission would rescue the phenotypes of AMPK KD, to strengthen the causality of AMPK-dependent fission, autophagy and dendrite outgrowth. So far we have observed that inhibition of mitochondrial fission by MFF knockdown or inhibition of autophagy by bafilomycin treatment strongly suppress dendrite outgrowth. MFF knockdown also leads to the elongation of mitochondria with decreased association of p62-puncta, strikingly reminiscent of AMPK-deficient neurons. Please see attached figures. Completed analyses will be included in the full revision.

    *Reviewer #1-4.- Results clearly indicate that AMPK enhances mitochondrial fission, and that AMPK is necessary for proper dendritic outgrowth. However, as indicated, the role of AMPK-dependent mitochondrial fission in promoting dendritic growth is not well demonstrated. A possible, and not very difficult experiment, would be the expression of non-phosphorylable MFF S155/172 mutant (perhaps is also needed to knock down the endogenous MFF). Use of this mutant would abolish AMPK-dependent mitochondrial fission while preserving its other functions. *

    Related comments by Reviewer #3-3. The authors could further confirm the claim by examining how mutations in Mff and ULK2 which cannot be phosphorylated by AMPK can rescue defects in mitochondrial fission and spine density.

    We will examine if the expression of non-phosphorylable MFF S155/172 mutant would cause defective autophagy and dendritic arbor growth similarly to AMPK KD neurons. In addition, we will test whether MFF S155/172 mutant would inhibit activity-induced mitochondrial fission to strengthen the link between activity-AMPK-MFF-autophagy axis and dendritic outgrowth.

    *Reviewer #1-Minor 2.- It is intriguing that as shown in Fig. 2A, rather than an increase in pAMPK/AMPK at DIV5 seems there is less phosphorylation despite FRET analysis indicate more AMPK activation. On the other hand, most of the blots in Fig. 6 seem to be overexposed. *

    The exposure time of WB in Fig. 2A was adjusted so that all lanes can be compared. We will fix the exposure time.

    *Reviewer#2-A. Most of the evidence on the role of AMPKa2 relies on a shRNA-based strategy. The authors have performed this approach with the best practice, including selecting 2 shRNA plasmids for each gene, and performing a rescue experiment with shRNA -resistant cDNA. Yet, it is critical to provide stronger evidence with all the tools available to demonstrate the role of AMPKa2 in dendritic development. This is especially important because the effect reported by the authors is a transient effect: indeed, dendritic development appears abnormal in very young neurons (P5) but largely normal afterwards (P10). Hence one cannot discard a non specific effect on cell viability or sampling effect. The number of neurons counted is fairly low (about 30 neurons per condition) and it is not clear if they come from several independent cultures. It is known that plasmid preparation can impact cell viability and performing the experiment with only one batch of plasmid prep could explain why one plasmid would produce a short-lived effect on cell morphology. Two shRNA constructs are presented in figure S2A but only one is used for morphological experiments quantified in S2D-E with again a very low N number. The specific experiments I would recommend would be to increase the N: at least 25-30 neurons counted per culture, 3 independent cultures, and presenting the results of the two shRNA plasmids for both AMPKa1 and AMPKa2. Furthermore, the immunofluorescence validation of knockdown provided in figure S2B is not really convincing, a nuclear marker is lacking to determine where cells are (it seems that many cells are present in the image, maybe some of them with low AMPKa2 expression as well). A quantification should be provided as well as evidence for shRNA #1 and #2. **

    We thank the reviewer for valuable suggestions to improve our manuscript. All the knockdown analyses were done from three independent experiments using different mouse litters and multiple batches of plasmid prep. N number was low because of a low transfection efficiency in the primary culture. We will repeat experiments and increase the sample number. We will also present results of the two shRNA constructs. We will redo the immunofluorescence for validation of shRNA knockdown and replace Extended Data Fig. 2B which was pointed out as not being clear.

    *Reviewer #2-C. The observation, in vivo, that dendritic development is normal at P10 is intriguing but this reconciles the observation of altered dendritic development with previous studies demonstrating that AMPK knockout has little effect on brain development, as well as previous studies (Mairet-Coello et al. Neuron 2013, Lee et al. Nat commun 2022) targeting AMPKa2 in the hippocampus of AD mouse models by in utero electroporation. This is a critical aspect of the paper and as stated in the discussion, the previous studies only looked at the end product (neuronal morphology appears normal after development) but not the process of neuronal development and maturation. The in vitro experiment offer the possibility to study dendritic development over time in the same population of neurons, either through selected time points, or through time lapse imaging. This would strengthen one of the most original aspect of this work. *

    We thank the reviewer for an important suggestion. We will analyze if dendritic morphology and mitochondria would recover in later stages in culture. However, the dendritic growth defects in AMPK KD neurons are apparently more severe in culture and our preliminary results have shown that dendritic growth defects and mitochondrial elongation persist until 10DIV. We anticipate that AMPK deficiency is complemented by certain compensation mechanisms in vivo that are not present in culture, such as chemical signals or synaptic inputs from correct afferents. We will confirm the recovery of dendritic outgrowth in vivo using an AMPK alpha2 knockout mouse. We will include the results in vivo and in vitro in the revised manuscript.

    The authors use a FRET probe to witness AMPK activity, and this part raises a lot of questions. A lot of the signal matches the regularly spaced activity peaks suggesting that FRET response is a coincidence detector of calcium waves. Hence, is the FRET signal influenced by intracellular calcium concentration, or changes in pH? To address this question, the proper control would be to use a FRET biosensor with a mutated AMPK phosphorylation site and demonstrating the absence of response to calcium waves. *

    We think it unlikely that the FRET probe detects calcium concentration or pH change, as its kinetics and timing are different from calcium spikes. For confirmation, we will examine a FRET probe lacking phosphorylation sites to negate that calcium waves directly activate the FRET probe.

    Also, the parameter used for quantification is a so-called "number of FRET peaks over 3 minutes" for which the biological significance is unknown. On average there are 1-2 such "peaks" in control conditions (figure 4). These peaks have low amplitude, sometimes around 0.05-0.1 of the YFP/CFP ratio, which is about what is expected even in AMPKa2 knockdown cells (figure S4C). Are there changes in the baseline of FRET signal? *

    We monitor FRET at 3-5 sec intervals and is set to 3 minutes due to gradual photobleaching. Although the event frequency is 0-4 times per 3 minutes observation, it is nearly absent in AMPK KD (1 small peak in 3 cells out of 40 cells) or activity deprivation, which we consider a significant difference. We have replaced Figure 4B, 4D, 4I, 4J andExtended Data Fig.4E. The basal FRET signal is lowered in AMPK KD cells, but also varies depending on the expression level of the probe. For comparision of the results shown in Figure 4 and Extended Data Fig.4, we have changed the y-axis to the normalized FRET signal {FRET/FRETbaseline} and jRGECO signals (DF/F0) in Fig. 4F, Extended Data Fig.4C, 4D.

    *Finally, given that calcium peaks and AMPK activity peaks overlap, one key observation is the continued presence of calcium peaks upon AMPKa2 knockdown in figure S4D. Yet, the scale for jRGECO1 intensity in figure S4D differs from the scale in figure 4, making it difficult to interpret. It seems that on average the delta (peak-baseline) is 2000 in wild-type cells (figure 4), compared to 500 in AMPKa2 knockdown cells, which suggests a strong reduction in calcium signal amplitude upon knockdown of AMPK. This should be clarified to demonstrate that the FRET probe peaks are really due to AMPK activity. Also, the effect of STO-609 should be added to this figure. *

    We think that the presence of calcium transients in AMPK KD cells supports our conclusion that AMPK is downstream of calcium signaling. The amplitude of calcium spikes was actually lowered in AMPK KD cells. We think it is due to the reduction of the cell size and complexity in KD cells. To negate that AMPK inhibition affects calcium influx, we will examine if acute inhibition by an AMPK inhibitor will suppress only FRET signals but not calcium waves. In addition, we will monitor calcium waves and FRET signals in neurons treated with STO-609 or AICAR. STO-609 and AICAR should decrease and increase FRET signals without affecting calcium influx.

    • Other comments by Reviewer #2*
    • Similarly, the number of events in figure 5F-G is really low. Is a difference between 0.02 in the control group and 0.01 in the knockdown group physiologically relevant?*

    Since p62 puncta contact only a small mitochondrial region, the overlap area of mitochondria with p62 in the total mitochondrial area is small. We will analyze the number of p62 puncta associated with mitochondria per unit dendritic area.

      • Lines 339-350, the authors discuss about a putative regulatory loop involving AMPK dephosphorylation. Since this part of the discussion is based on the FRET signal, the authors should consider if an alternative explanation could be the kinetics of the biosensor dephosphorylation.* We will revise Discussion to argue about alternative possibilities of dynamic oscillation of the FRET signal when we get data from the above experiments.

    *In terms of significance, I would have two major criticisms. The first is that it appears that many of the findings by the authors are redundant with observations of the roles of CAMKK2-AMPK-MFF-ULK1 in AD model mice, see for example the work by Polleux (Mairet-Coello et al. Neuron 2013, Lee et al., Nat commun 2022). As said above, my opinion is that the paper should put more emphasis on the transient effect of AMPK, which would be a novel observation and, as the authors rightfully discuss, a phenotype potentially overlooked in previous studies of AMPK KO mice. The second is that many points in the discussion seem to be over reached and are not entirely supported by the data. As an example lines 298-299 "leading to mitochondrial dysfunction with low respiratory activity" (not addressed in this manuscript), lines 312-313 "multiple signatures of mitochondrial dysfunction such as reduced delta-Psi-m and ROS production" (biological significance of these parameters?), lines 332-334 "AMPK phosphorylation dynamically oscillates in dendrites, depending on Ca2+ influx and CAMKK2 activity, while it is independent of LKB1" (the authors don't study AMPK phosphorylation, and the experimental data has many limitations that need addressing), etc. *

    We thank reviewer’s guidance. We think this is the first study showing AMPK function in dendritic arbor growth in immature neurons before synaptogenesis. We will rewrite the manuscript to emphasize that neuronal activity in immature neurons regulates dendrite formation via AMPK in a short time window during brain development. Discussion will be revised according to the data of the ongoing additional experiments.

    Reviewer#3-1. All these studies are done in invitro neuronal culture modal with transfection of ShRNAs to Knockdown AMPK. An alternative possibility is that authors could use an AMPK Conditional Knockout mouse models Conditional deletion of (AMPKα1/α2 (AMPKα1−/−; AMPKα2F/F; Emx1-Cre) derived neurons for this study.

    We showed the effect of AMPK knockdown in hippocampal neurons in culture and in vivo (Fig. 2). For validation, we also examined CRISPR interference (Extended data Fig.2). We will examine in vivo phenotypes in pyramidal neurons in AMPK alpha2 knockout mice to further validate our observation.

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    *Reviewer #1-1.- Authors use mitochondrial membrane potential (MMP), MMP flickering and mitochondrial ROS production as indicators of mitochondrial function, but this is not convincing. To analyze MMP, authors use TMRM fluorescence normalized by mitochondria area. This is not correct, using this strategy would mean that a symmetric fission would instantly double MMP and fusion would half MMP. The analysis must be made by tracing ROIs of the same surface in different mitochondria and determining TMRM fluorescence in these ROIs. *

    We have reanalyzed TMRM fluorescence using the method indicated. As a result, TMRM fluorescence show a slight but significant decrease (p=0.0071) in AMPK KD cells. Extended Data Fig. 5C has been replaced accordingly. We thank the Reviewer for kind guidance!

    Reviewer #1-3.- The authors treat neurons with glutamate to support the view that synaptic activity activates AMPK and promotes mitochondrial fission. However, the concentration used (100 mM) may be excitotoxic. Synaptic activity can be induced by electric field stimulation, although this require equipment that may not be available in the authors' lab. Another alternative is network disinhibition with bicuculine or to use lower concentrations of glutamate. In any case, since neurons are immature and may respond differently from mature neurons, it would be worth to verify synaptic activity by analyzing Ca2+ transients.

    *Reviewer #1-Minor 3.- It is necessary more explanation about spontaneous Ca2+ transients in immature cultures. What percentage of neuros experience it? Is it synchronized? *

    *Related comments by Reviewer#2-D. It is well established and thus not surprising that AMPK activity increases in response to synaptic activity. It is more surprising to witness such an effect of activity in very immature neurons, where presumably synapses are sparse and not well developed. For example dendritic segments in Figure 1E and 3A don't have dendritic spines. Western-blot and/or immunofluorescence of synaptic markers with comparison to fully mature neurons would complete figure 1 and make the case whether the reported effects are marginal or a strong driver for dendritic development and AMPK regulation. *

    We thank the reviewers’ point that we failed to emphasize in the original manuscript.

    We focus on AMPK function during activity-dependent dendritic outgrowth in immature neurons before the onset of synaptogenesis. It has been shown that synaptogenesis occurs in dissociated hippocampal cultures between 7-12 DIV (eg, Renger et al., Neuron 2001) and that developing dendrites at 5 DIV are activated by ambient glutamate which is spontaneously released from nearby immature axon terminals and undergo spontaneous Ca2+ transients, and this non-synaptic activity is important for dendritic outgrowth (Andreae and Burrone, Cell Rep 2015). We have observed that Ca2+ transients in individual neurons are variable in frequency and magnitude and are not synchronized in consistent with previous studies. We have performed immunofluorescence with a synaptic marker PSD95 and confirmed that dendritic spines are not yet differentiated and PSD95 is sparsely distributed along the dendritic shaft in DIV5 hippocampal neurons. We describe the nature of Ca2+ transients in the Results more clearly and provide high magnified images and immunofluorescence with a synaptic marker PSD95 of the neurons at DIV5 and DIV13 as a new Fig. 1A. We believe that this is the first indication of AMPK function in non-synaptic neuronal activity during dendritogenesis.

    We have observed induction of mitochondrial fission in neurons treated with 1 µM glutamate. Extended Data Fig. 1E has been replaced accordingly. Since GABA is known to induce depolarization in immature neurons (Soriano et al., PNAS 2008), we would like to exclude bicuculine treatment from this analysis.

    *Reviewer #1-5.- The statistical analysis seems appropriate, but it is confusing that sometimes non-parametric and sometimes parametric tests are used. It is not indicated which test is used to determine normality since the methods section lacks a statistical analysis section.

    We have revised Methods and have described statistical analysis in detail.

    *Reviewer #1-Minor 1.- Authors should double check the analysis shown in Fig. 1A. As it is shown, Ca2+ transients are 2-3% higher than basal, when the video shown in video 1 seem to indicate much more. *

    Thank you for pointing this out. In the original version, the percentile change was erroneously measured across the entire visual field, including areas without neurons. We have replaced Fig. 1B (original Fig. 1A) with reanalyzed data in the proximal region of the apical dendrite.

    *Reviewer #1-Minor 4.- It is interesting that AMPK KD in vivo impairs dendritic architecture at P5, however at P10 the defect seem to be somehow compensated. This result apparently detracts from the relevance of the findings, however last year was published a paper in which in an animal model of Huntington's disease dendritic architecture is delayed during the first week but normalizes thereafter. Despite later normalization in dendritic architecture, this early defect in maturation has effects in adulthood as pharmacological restoration of arborization during the neonatal period suppresses some phenotypes observed in adulthood (PMID: 36137051). I believe that discussing this paper would help the reader to recognize the potential relevance of the findings. *

    *Related comments by Reviewer #2: Nonetheless let aside the technical concern, if their findings hold true, this is an intriguing mechanism. There are interesting parallels to be made with observations of altered morphology and excitability of neurons in Huntington's disease model mice during the first postnatal week. These changes spontaneously reverts and are undetectable in the second week (Braz et al. Science 2022). Thus, precedent suggests that indeed dendritic development can take a slow course, and this study also suggests that this is important later since normalization of abnormal excitability during the first week in HTT mice prevents some of the phenotypes later in life. Here again, an interesting parallel could be made with the known role of AMPK in synaptic loss in AD models. *

    We thank the reviewers for the supportive comments. We will refer this paper and discuss about potential significance of the transient defects in early dendrite morphology in AMPK deficient neurons.

    *Reviewer#2-B. The Crispr method lacks validation which should be provided somehow. The drug-based experiment relies on compound C, a notoriously non specific AMPK inhibitor (see for example Bain et al. Biochem J 2007, or Vogt et al. Cell Signal 2011). Data obtained with Compound C is hard to interpret given the number of kinases that are affected by the drug and should be removed from the manuscript. *

    We have added immunofluorescence images for validation of AMPK deletion by CRISPRi (Extended Data Fig. 2F).

    We think the results of Compound C treatment support our conclusion in combination with KD and CRISPRi, but will delete the results in accordance with this comment.

    • Other comments by Reviewer#2*
    • Figure 5A-C relies on the quantification of fission events that appear very rare (0.4 event per 20 minutes). The difference between the two groups is between 0.1 and 0.2 events on average. Since this was quantified on a fairly low number of cells (N=14), it is hard to appreciate exactly how many events have been observed and the actual physiological relevance. Furthermore individual datapoints should be added to the figure to estimate variability.*

    The number of fission events was counted in mitochondria in a unit length of dendrite of similar diameter, and normalized by the number of mitochondria. The values were thus small as they represent average number of events in one mitochondrion in 20 minutes. We have replaced the Fig. 1K, 3F, 5B and 5C to show the number of fission events in mitochondria included in a unit length of dendrites of similar diameter. Individual data points have been included.

    Reviewer #3-4. Authors showed activity-dependent calcium signaling controls mitochondrial homeostasis and dendritic outgrowth via AMP-activated protein kinase (AMPK) in developing hippocampal neurons do the cortical mitochondria respond the same way as the hippocampal neurons?

    Thank you for the comment. As pyramidal neurons in the cerebral cortex and hippocampus are basically the same origin, it is likely that they share the same signaling. We use hippocampal neurons in this study to perform quantitative analysis of dendritic morphology in the same type of neurons. Primary cultures of cortical neurons contain multiple different cell types, making it difficult to analyze the same cell type.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    *Reviewer #1: If activity is observed in only a portion of the neurons, taking advantage of the stablished long-term live imaging protocol in the authors' lab, it would be interesting to study in the same culture whether neurons that experience spontaneous activity develop more than those that do not. *

    We prefer not to carry out this analysis, as activity-dependent dendritic growth has already been well described in previous papers. It will take considerable time to observe the number of neurons for analysis of correlation between Ca2+ transients and dendrite morphology. We would like to focus our effort to demonstrate AMPK signaling during activity-dependent dendritic growth.

    Reviewer#3-2. Another technical issue here, most of the experiments are carried out on Neurobasal media, which has a lot of glucose plus substitution of glutamax might be not the perfect conditions for AMPK. Authors could not obtain evidence supporting the regulation of mitochondria biogenesis by PGC1α phosphorylation and expression. This surprise me, if you could reduce the glucose concentration if might change.

    We observed little or no changes in phosphorylation of PGC1alpha by enhancing or suppressing neuronal activity or AMPK activity. As mitochondrial biogenesis is very active in growing neurons, we surmise that PGC1alpha and mitochondrial biogenesis is regulated by multiple mechanisms during neuronal differentiation and AMPK activation/inhibition might not induce visible changes. We agree the reviewer that there is room to seek the conditions under which changes in PGC1alpha can be detected, but we do not see why Neurobasal plus glutamax is not suitable for this study. Multiple papers studying AMPK function in cultured neurons use similar culture media (Sample et al., Mol. Cell. Biol., 2015; Muraleedharan et al., Cell. Rep., 2020; Lee et al., Nat. Commun., 2022). We might see PGC1 phosphorylation by glucose deprivation, as it decreases glycolysis-derived ATP and thereby activates AMPK. Since we focus on AMPK activation by calcium signals, we are afraid that it would be difficult to distinguish AMPK activation by ATP deficiency or calcium signaling in glucose deficient condition. In addition, glucose deprivation would affect neuronal activity (which consumes large amount of ATP) and neuronal differentiation including dendritic outgrowth.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Hatsuda and colleagues builds on previous work from their group and others, to further investigate the activity-dependent dendritic arbor development and AMPK-dependent mitochondrial quality control. It recently been illustrated that over-activation of the CAMKK2-AMPK kinase dyad mediates synaptic loss through coordinated phosphorylation of MFF-dependent mitochondrial fission and ULK2-dependent mitophagy (Aβ42 oligomers trigger synaptic loss through CAMKK2-AMPK-dependent effectors coordinating mitochondrial fission and mitophagy). Though this is progress is modest, present study showed neuronal activity induces activation of two downstream effectors of AMPK, MFF and ULK1, which are the key regulators of mitochondrial fission and mitophagy.

    Major Concerns:

    1. All these studies are done in invitro neuronal culture modal with transfection of ShRNAs to Knockdown AMPK. An alternative possibility is that authors could use an AMPK Conditional Knockout mouse models Conditional deletion of (AMPKα1/α2 (AMPKα1−/−; AMPKα2F/F; Emx1-Cre) derived neurons for this study.
    2. Another technical issue here, most of the experiments are carried out on Neurobasal media, which has a lot of glucose plus substitution of glutamax might be not the perfect conditions for AMPK. Authors could not obtain evidence supporting the regulation of mitochondria biogenesis by PGC1α phosphorylation and expression. This surprise me, if you could reduce the glucose concentration if might change.
    3. The authors could further confirm the claim by examining how mutations in Mff and ULK2 which cannot be phosphorylated by AMPK can rescue defects in mitochondrial fission and spine density.
    4. Authors showed activity-dependent calcium signaling controls mitochondrial homeostasis and dendritic outgrowth via AMP-activated protein kinase (AMPK) in developing hippocampal neurons do the cortical mitochondria respond the same way as the hippocampal neurons?

    Significance

    It recently been illustrated that over-activation of the CAMKK2-AMPK kinase dyad mediates synaptic loss through coordinated phosphorylation of MFF-dependent mitochondrial fission and ULK2-dependent mitophagy (Aβ42 oligomers trigger synaptic loss through CAMKK2-AMPK-dependent effectors coordinating mitochondrial fission and mitophagy). Though this is progress is modest, present study showed neuronal activity induces activation of two downstream effectors of AMPK, MFF and ULK1, which are the key regulators of mitochondrial fission and mitophagy.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript 'Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons' by Hatsuda and collaborators aims at studying the interrelationship between neuronal activity, mitochondria dynamics (ie. fusion and fission mechanisms) and dendritic development. The authors provide evidence linking activity-dependent activation of a CAMKK2-AMPK pathway and the regulation of mitochondria fission and autophagy. Based on the literature, they focus on the roles of the mitochondria fission factor MFF and the autophagy regulator ULK1, both previously known targets of AMPK.

    This work parallels previous observations in the context of Alzheimer's disease and as such the discovery of a molecular link between CAMKK2-AMPK, MFF/ULK1 and the regulation of dendritic mitochondria is not surprising. The change of biological context raises interesting question although the relevance of these observations is not addressed in this manuscript.

    As a general comment, the work is well structured, reads easily. Iconography and figures organization are good. Major criticisms would concern the tools used to study AMPK and challenge some of the observations, as such I believe these are essential to address to validate the findings.

    Major comments

    • A. Most of the evidence on the role of AMPKa2 relies on a shRNA-based strategy. The authors have performed this approach with the best practice, including selecting 2 shRNA plasmids for each gene, and performing a rescue experiment with shRNA -resistant cDNA. Yet, it is critical to provide stronger evidence with all the tools available to demonstrate the role of AMPKa2 in dendritic development. This is especially important because the effect reported by the authors is a transient effect: indeed, dendritic development appears abnormal in very young neurons (P5) but largely normal afterwards (P10). Hence one cannot discard a non specific effect on cell viability or sampling effect. The number of neurons counted is fairly low (about 30 neurons per condition) and it is not clear if they come from several independent cultures. It is known that plasmid preparation can impact cell viability and performing the experiment with only one batch of plasmid prep could explain why one plasmid would produce a short-lived effect on cell morphology. Two shRNA constructs are presented in figure S2A but only one is used for morphological experiments quantified in S2D-E with again a very low N number. The specific experiments I would recommend would be to increase the N: at least 25-30 neurons counted per culture, 3 independent cultures, and presenting the results of the two shRNA plasmids for both AMPKa1 and AMPKa2. Furthermore, the immunofluorescence validation of knockdown provided in figure S2B is not really convincing, a nuclear marker is lacking to determine where cells are (it seems that many cells are present in the image, maybe some of them with low AMPKa2 expression as well). A quantification should be provided as well as evidence for shRNA #1 and #2.
    • B. To complete the shRNA-based experiments, the authors use a single cell Crispr approach, as well as a pharmacological approach. The Crispr method lacks validation which should be provided somehow. The drug-based experiment relies on compound C, a notoriously non specific AMPK inhibitor (see for example Bain et al. Biochem J 2007, or Vogt et al. Cell Signal 2011). Data obtained with Compound C is hard to interpret given the number of kinases that are affected by the drug and should be removed from the manuscript.
    • C. The observation, in vivo, that dendritic development is normal at P10 is intriguing but this reconciles the observation of altered dendritic development with previous studies demonstrating that AMPK knockout has little effect on brain development, as well as previous studies (Mairet-Coello et al. Neuron 2013, Lee et al. Nat commun 2022) targeting AMPKa2 in the hippocampus of AD mouse models by in utero electroporation. This is a critical aspect of the paper and as stated in the discussion, the previous studies only looked at the end product (neuronal morphology appears normal after development) but not the process of neuronal development and maturation. The in vitro experiment offer the possibility to study dendritic development over time in the same population of neurons, either through selected time points, or through time lapse imaging. This would strengthen one of the most original aspect of this work.
    • D. It is well established and thus not surprising that AMPK activity increases in response to synaptic activity. It is more surprising to witness such an effect of activity in very immature neurons, where presumably synapses are sparse and not well developed. For example dendritic segments in Figure 1E and 3A don't have dendritic spines. Western-blot and/or immunofluorescence of synaptic markers with comparison to fully mature neurons would complete figure 1 and make the case whether the reported effects are marginal or a strong driver for dendritic development and AMPK regulation. Furthermore the authors use a FRET probe to witness AMPK activity, and this part raises a lot of questions. A lot of the signal matches the regularly spaced activity peaks suggesting that FRET response is a coincidence detector of calcium waves. Hence, is the FRET signal influenced by intracellular calcium concentration, or changes in pH? To address this question, the proper control would be to use a FRET biosensor with a mutated AMPK phosphorylation site and demonstrating the absence of response to calcium waves. Also, the parameter used for quantification is a so-called "number of FRET peaks over 3 minutes" for which the biological significance is unknown. On average there are 1-2 such "peaks" in control conditions (figure 4). These peaks have low amplitude, sometimes around 0.05-0.1 of the YFP/CFP ratio, which is about what is expected even in AMPKa2 knockdown cells (figure S4C). Are there changes in the baseline of FRET signal? Finally, given that calcium peaks and AMPK activity peaks overlap, one key observation is the continued presence of calcium peaks upon AMPKa2 knockdown in figure S4D. Yet, the scale for jRGECO1 intensity in figure S4D differs from the scale in figure 4, making it difficult to interpret. It seems that on average the delta (peak-baseline) is 2000 in wild-type cells (figure 4), compared to 500 in AMPKa2 knockdown cells, which suggests a strong reduction in calcium signal amplitude upon knockdown of AMPK. This should be clarified to demonstrate that the FRET probe peaks are really due to AMPK activity. Also, the effect of STO-609 should be added to this figure.

    Other comments

    • A. Figure 5A-C relies on the quantification of fission events that appear very rare (0.4 event per 20 minutes). The difference between the two groups is between 0.1 and 0.2 events on average. Since this was quantified on a fairly low number of cells (N=14), it is hard to appreciate exactly how many events have been observed and the actual physiological relevance. Furthermore individual datapoints should be added to the figure to estimate variability.
    • B. Similarly, the number of events in figure 5F-G is really low. Is a difference between 0.02 in the control group and 0.01 in the knockdown group physiologically relevant?
    • C. In figure 6 it is unclear what is the significance of the TMRM "flickering" parameter quantified and the difference between the control and knockdown condition is small on average. Rather, the data presented in figure S5 would suggest that there is no difference in TMRM signal.
    • D. Lines 339-350, the authors discuss about a putative regulatory loop involving AMPK dephosphorylation. Since this part of the discussion is based on the FRET signal, the authors should consider if an alternative explanation could be the kinetics of the biosensor dephosphorylation.

    Significance

    The manuscript by Hatsuda and collaborator studies the roles of neuronal AMPK in the development of hippocampal neurons, specifically the authors describe a transient effect on dendritic development. To this reviewer's opinion, this is the major findings of this paper. Although the physiological implications of this finding are unknown, this is beyond the scope of this paper.

    Yet in terms of significance, I would have two major criticisms. The first is that it appears that many of the findings by the authors are redundant with observations of the roles of CAMKK2-AMPK-MFF-ULK1 in AD model mice, see for example the work by Polleux (Mairet-Coello et al. Neuron 2013, Lee et al., Nat commun 2022). As said above, my opinion is that the paper should put more emphasis on the transient effect of AMPK, which would be a novel observation and, as the authors rightfully discuss, a phenotype potentially overlooked in previous studies of AMPK KO mice. The second is that many points in the discussion seem to be over reached and are not entirely supported by the data. As an example lines 298-299 "leading to mitochondrial dysfunction with low respiratory activity" (not addressed in this manuscript), lines 312-313 "multiple signatures of mitochondrial dysfunction such as reduced delta-Psi-m and ROS production" (biological significance of these parameters?), lines 332-334 "AMPK phosphorylation dynamically oscillates in dendrites, depending on Ca2+ influx and CAMKK2 activity, while it is independent of LKB1" (the authors don't study AMPK phosphorylation, and the experimental data has many limitations that need addressing), etc.

    Nonetheless let aside the technical concern, if their findings hold true, this is an intriguing mechanism. There are interesting parallels to be made with observations of altered morphology and excitability of neurons in Huntington's disease model mice during the first postnatal week. These changes spontaneously reverts and are undetectable in the second week (Braz et al. Science 2022). Thus, precedent suggests that indeed dendritic development can take a slow course, and this study also suggests that this is important later since normalization of abnormal excitability during the first week in HTT mice prevents some of the phenotypes later in life. Here again, an interesting parallel could be made with the known role of AMPK in synaptic loss in AD models.

    Reviewer expertise: I have expertise in neuronal metabolism

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Hatsuda et al study the role of synaptic activity-dependent Ca2+ signals in activating AMPK and regulating mitochondrial homeostasis and dendritic outgrowth in developing neurons. Using cultures of immature primary hippocampal neurons, the authors found that Ca2+ transients activate AMPK, which regulates mitochondrial fission and dendritic growth. AMPK KD and pharmacological manipulation of AMPK activity confirmed a role of AMPK in mitochondrial morphology that correlated with impaired mitophagy and mitochondrial function. These findings led the authors to conclude that activity-dependent activation of AMPK promotes mitochondrial fission, which facilitates removal of dysfunctional mitochondria and thus contributes to maintaining a healthy mitochondrial pool that is necessary in the intense energetic effort that requires dendritic outgrowth.

    Major comments:

    Meanwhile the activation of AMPK and its role in mitochondrial fission and dendritic outgrowth is in general well demonstrated, the conclusion that AMPK is necessary for proper dendritic outgrowth because by promoting asymmetric mitochondrial fission facilitates mitophagy of dysfunctional mitochondria and thus ensures adequate generation of energy for dendritic outgrowth still seems preliminary.

    1. Authors use mitochondrial membrane potential (MMP), MMP flickering and mitochondrial ROS production as indicators of mitochondrial function, but this is not convincing. To analyze MMP, authors use TMRM fluorescence normalized by mitochondria area. This is not correct, using this strategy would mean that a symmetric fission would instantly double MMP and fusion would half MMP. The analysis must be made by tracing ROIs of the same surface in different mitochondria and determining TMRM fluorescence in these ROIs. But even in the case that there were changes in MMP, that it does not seem to be the case, MMP alone is not a good indicator of mitochondrial health. For instance, ATPase inhibitor causes increase in MMP and complex I inhibition diminish MMP and in both cases mitochondrial function is impaired. On the other hand, authors use increased flickering and mitochondrial ROS production as an indicator of enhanced respiration but they could also be used as indicators of mitochondrial dysfunction. Other assays, such as oxygen consumption, are needed to assess the mitochondrial function.
    2. It would be interesting to show a better characterization of the mitophagy flux and to test whether pharmacological or genetic stimulation of mitophagy could revert the effect of AMPK KD on dendritic outgrowth, ultimately linking AMPK, mitophagy and dendritic outgrowth. The latter experiments may be challenging but not impossible, for example see (PMID: 27760312).
    3. The authors treat neurons with glutamate to support the view that synaptic activity activates AMPK and promotes mitochondrial fission. However, the concentration used (100 M) may be excitotoxic. Synaptic activity can be induced by electric field stimulation, although this require equipment that may not be available in the authors' lab. Another alternative is network disinhibition with bicuculine or to use lower concentrations of glutamate. In any case, since neurons are immature and may respond differently from mature neurons, it would be worth to verify synaptic activity by analyzing Ca2+ transients.
    4. Results clearly indicate that AMPK enhances mitochondrial fission, as previously reported, and that AMPK is necessary for proper dendritic outgrowth. However, as indicated, the role of AMPK-dependent mitochondrial fission in promoting dendritic growth is not well demonstrated. For example, AMPK could regulate dendritic outgrowth through its role on cytoskeletal dynamics. A possible, and not very difficult experiment, would be the expression of non-phosphorylable MFF S155/172 mutant (perhaps is also needed to knock down the endogenous MFF). Use of this mutant would abolish AMPK-dependent mitochondrial fission while preserving its other functions.
    5. The statistical analysis seems appropriate, but it is confusing that sometimes non-parametric and sometimes parametric tests are used. It is not indicated which test is used to determine normality since the methods section lacks a statistical analysis section.

    Minor comments:

    1. Authors should double check the analysis shown in Fig. 1A. As it is shown, Ca2+ transients are 2-3% higher than basal, when the video shown in video 1 seem to indicate much more.
    2. It is intriguing that as shown in Fig. 2A, rather than an increase in pAMPK/AMPK at DIV5 seems there is less phosphorylation despite FRET analysis indicate more AMPK activation. On the other hand, most of the blots in Fig. 6 seem to be overexposed.
    3. It is necessary more explanation about spontaneous Ca2+ transients in immature cultures. What percentage of neuros experience it? Is it synchronized? If activity is observed in only a portion of the neurons, taking advantage of the stablished long-term live imaging protocol in the authors' lab, it would be interesting to study in the same culture whether neurons that experience spontaneous activity develop more than those that do not.
    4. It is interesting that AMPK KD in vivo impairs dendritic architecture at P5, however at P10 the defect seem to be somehow compensated. This result apparently detracts from the relevance of the findings, however last year was published a paper in which in an animal model of Huntington's disease dendritic architecture is delayed during the first week but normalizes thereafter. Despite later normalization in dendritic architecture, this early defect in maturation has effects in adulthood as pharmacological restoration of arborization during the neonatal period suppresses some phenotypes observed in adulthood (PMID: 36137051). I believe that discussing this paper, and others with similar message (if they exist, I do not know), would help the reader to recognize the potential relevance of the findings.

    I believe that all the proposed experiments would strongly help to support the claims of the paper and are perfectly feasible during the time given for a revision and economically affordable.

    Significance

    As authors already cite in their work, several groups have shown that mitochondrial fission is necessary for proper dendritic growth. Here, the authors have proposed that synaptic activity in immature neurons induce mitochondrial fission via AMPK activation and subsequent MFF phosphorylation. Many of the findings here are confirmations of previous work, for instance activity-dependent activation of AMPK (PMID: 25698741, PMID: 27012879) and induction of mitochondrial fission by AMPK via MFF phosphorylation (PMID: 26816379). The novelty of the work is in studying these processes in immature neurons and how this affects dendritic growth, which is of interest for cellular neuroscientist, but I do not think it represents a conceptual breakthrough. A more detailed understanding of the mechanism proposed, i.e. mitochondrial fission facilities removal of dysfunctional mitochondria to maintain the high energy demands of growing dendrites, would greatly enhance the significance of the study.

    This reviewer specializes in neuronal cell biology, specifically the study of the mechanisms by which mitochondrial function regulates aspects of neuronal physiology, including neuritic outgrowth.

  10. Version published to 10.1101/2022.12.27.521995v2 on bioRxiv
  11. Version published to 10.1101/2022.12.27.521995v1 on bioRxiv