The specific AMPK activator A-769662 ameliorates pathological phenotypes following mitochondrial DNA depletion

  1. Gustavo Carvalho
  2. Bruno Repolês
  3. Tran V.H. Nguyen
  4. Josefin M.E. Forslund
  5. Farahnaz Ranjbarian
  6. Isabela C. Mendes
  7. Micol Falabella
  8. Mara Doimo
  9. Sjoerd Wanrooij
  10. Robert D.S. Pitceathly
  11. Anders Hofer
  12. Paulina H. Wanrooij

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Abstract

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis that also plays a role in preserving mitochondrial function and integrity. Upon a disturbance in the cellular energy state that increases AMP levels, AMPK activity promotes a switch from anabolic to catabolic metabolism to restore energy homeostasis. However, it is currently unclear how severe of a mitochondrial dysfunction is required to trigger AMPK activation, and whether stimulation of AMPK using specific agonists can improve the cellular phenotype following mitochondrial dysfunction. Using a cell model of mitochondrial disease characterized by progressive mitochondrial DNA (mtDNA) depletion and deteriorating mitochondrial metabolism, we show that mitochondria-associated AMPK becomes activated early in the course of the advancing mitochondrial dysfunction, before any quantifiable decrease in the ATP/(AMP+ADP) ratio or respiratory chain activity. Moreover, stimulation of AMPK activity using the specific small-molecule agonist A-769662 alleviated the mitochondrial phenotypes caused by the mtDNA depletion and restored normal mitochondrial membrane potential. Notably, the agonist treatment was able to partially restore mtDNA levels in cells with severe mtDNA depletion, while it had no impact on mtDNA levels of control cells. The beneficial impact of the agonist was also observed in cells from patients suffering from mtDNA depletion. However, the positive effects of A-769662 in the two experimental cell models appeared to involve at least partially different mechanisms. These findings improve our understanding of the effects of specific small-molecule activators of AMPK on mitochondrial and cellular function, and suggest a potential utility for these compounds in disease states involving mtDNA depletion.

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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors investigate the role of the AMP-activated protein kinase (AMPK) in mitochondrial dysfunction. Using HEK293T cells as model system they induce expression of either the wild-type or a dominant-negative variant of the mitochondrial DNA polymerase, which results in depletion of mtDNA and a decreased mitochondrial membrane potential. Using different time points of Pol induction they correlate the mitochondrial defects with activation of AMPK and make the interesting observation that only the mitochondrial associated fraction of AMPK becomes activated at an early stage of mitochondrial dysfunction. The authors then apply a known AMP activator (A-769662) and assess its impact on mtDNA levels and respiratory chain subunit steady state levels. Finally, they compare the findings using the HEK cells with patient derived fibroblasts, which show the same response to the activator.

    Regarding the so far provided data I have the following concerns:

    • I do not agree with the statement that the mechanisms in the HEK and patient cells are different. First of all, there is no analysis of the mechanism in the HEK cells nor the patient fibroblasts. Secondly, the control cell line (Pol WT overexpression) is also showing a decrease in mitochondrial membrane potential, but no change in mtDNA - which is fully reflecting the observation of the patient cells.
    • Figure 1H-J: the authors claim that CIV activity is decreasing. However, CIV is virtually absent in these samples and therefore the statement that CIV activity is decreased is not correct.
    • It is not clear why the authors used the dominant negative D1135A variant in the HEK system and not the most common dominant patient mutations (of which they in the end use the patient fibroblasts).
    • Supplemental Figure S1A/B: Which time point of induction is shown here?
    • The rho zero cell line and the control UV treatment are not described in the materials and methods section.

    Significance

    The observation that mitochondrial-associated AMPK reacts much earlier than the global AMPK pool to the mitochondrial dysfunction is interesting. The other observations described in the manuscript were rather to be expected given previous publications. Overall, the study primarily provides descriptive findings, which, in my view, seem preliminary at this stage and requires significant revisions for it to be truly valuable to the scientific community.

    At least some molecular insight into the regulation of the different cellular AMPK pools or detailed analysis on how the activator impacts on mtDNA or the general mechanism that results in stabilization of the mitochondrial membrane potential are necessary to provide sufficient novel findings for publication. This additional analysis would be necessary to strengthen the study's conclusions and broaden its relevance to a larger readership.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The authors aimed to investigate the functional consequences of AMPK agonist, A-769662, in improving the cellular energetic response to mitochondrial DNA depletion. AMP kinase plays a crucial role in switching the metabolic programming in the cells upon energetic stresses. It drives the activation of transcription factors of the mitochondrial genome as well as nuclear genes important for mitochondrial biogenesis (Herzig and Shaw., 2018, Bonekamp et al., 2021). Previous studies have shown that triggering the AMPK cascade has positive outcomes in mitochondrial disease models (Peralta et a., 2016, Moore et al., 2020). However, the mechanistic basis of their impact on mitochondrial function, especially mtDNA is not known.

    Brief Summary:

    In this study, the authors characterize the dynamics of mitochondrial dysfunction in response to severe mtDNA depletion, using a cell model and report that A-769662, a non-AMP mimetic AMPK agonist maintains cellular energy homeostasis by stimulating the AMPK cascade to restore membrane potential in mtDNA depleted cells. The positive effect was observed only in patient-derived, mtDNA-depleted cells and absent in control cells, suggesting that A-769662 mediates mitochondrial activity and cellular function via partially different mechanisms.

    Major Results:

    • The authors used a previously established inducible cell line to transiently express a dominant negative mutant of the mitochondrial DNA polymerase, Poly, as a model system for mtDNA depletion. Compared to the wildtype cells, mtDNA copy number, membrane potential, assembly and levels of respiratory chain complexes I, III and IV were severely reduced in the PolyD1135A cells (Figure 1A-K).
    • The authors checked the relative ratio of AMP/ADP and ATP in the PolyD1135A cells and found that ADP levels were elevated, as expected (Figure 2A). Consequently, the levels of phosphorylated AMPK in/at (?) the mitochondria were also increased, in good correlation with the depletion of mtDNA levels and respiratory complexes after 3 days of induction (Figure 2B-E). Activation of the pAMPK pathway was further confirmed by the increased levels of AMPK substrate ACC. However, metabolite levels and cell cycle profiles are mildly altered in the PolyD1135A cells, suggesting that the activation of mitochondrial AMPK is an early response to mtDNA dysfunction.
    • Chemical activation of AMPK by agonist A-769662 had a sustained positive effect on the membrane potential in both induced and uninduced cells. This was specific to AMPK signalling as evidenced by no change in membrane potential in cells transfected with AMPK siRNA (Figure 3A-C). However, the A-769662 treatment was insufficient to rescue the proliferation defects in the PolyD1135A mutant cells (Figure 3D), suggesting that the growth defect is independent of AMPK activation.
    • A-769662 treatment for 48 or 72h in PolyD1135A improved mtDNA copy number and respiratory complex subunit expression, while having no effect on control cells (Figure 4A-G). Upon A-769662 treatment, patient-derived mutant cell lines showed no change in mtDNA levels (Figure 4H). Interestingly, membrane potential was enhanced in both control and patient cell lines (Figure 4I), suggesting an overall activation of mitochondrial function and not a specific response to restoring mtDNA.

    Taken together the manuscript by Carvalho et al., proposes a stimulatory effect of AMP agonist, A-769662 on mtDNA depletion. However, since the model is not consistent in different model systems, the authors should provide stronger evidence for the utility of A-769662 as a therapeutic possibility for mtDNA disorders. Moreover, some mechanistical molecular insights into these largely descriptive results must be presented in a revised version. What drives AMPK localization to mitochondria? Is this kinase imported? Is it just attached? What regulates the distribution of AMPK between their different locations?

    Significance

    Major points:

    • The effect of A-769662 on mtDNA levels in the FlipIn-TRex cell line, harboring a severe mutation and clinically-relevant patient-derived cell lines are not comparable (Figure4A and 4H), suggesting that the amelioration of mitochondrial defect is probably dependent on the extent of mtDNA damage. The FlipIn system shows a loss of almost 85% of mtDNA on day 3 of induction (Figure 1A) whereas the patient-derived cells retain almost 60% of the mtDNA (Figure 4H). The authors argue that the two systems are not comparable. A good control would be to check an inducible FlipIn-TRex cell line with the same patient-derived mutations or alter the induction system, with a shorter induction time or reduced concentration of doxycycline, to have comparable levels of mtDNA depletion.
    • A more thorough investigation of A-769662 in different cell models of mtDNA dysfunction, possibly different disease-specific mutations in Poly, or cell types which contain AMPK complexes with -subunits (irresponsive to A-769662 stimulation) will be needed to claim its therapeutic merit.
    • To substantiate the claim that the downstream effect of A-769662 treatment is dependent on the metabolic context of the cell, it would be necessary to test the levels of crucial metabolites like mtDNA transcripts, ATP, NADH, in addition to dNTPs tested in the study. It would also help to compare the levels of these metabolites in of PolyD1135A cells, grown in galactose medium.
    • In Figure 2C, TFAM is nearly absent in the mitochondrial fraction of PolyD1135A cells, since Poly dysfunction triggers a reduced expression of the transcription machinery. Considering the mtDNA level upregulation is mild after if A-769662 treatment (about 8%, Figure 4A), it would be worthwhile to check if A-769662 could alter transcript levels of mtDNA and/or expression of mitochondrial transcription factors TFAM and TF2B.

    Minor points:

    • The data from Figure 1 conclusively show defects in mtDNA. It would be necessary to compare OCR, ROS and cellular ATP levels to demonstrate the extent of mitochondrial dysfunction in this model due to mtDNA depletion.
    • To further delineate the differences in mitochondrial bioenergetics in PolyD1135A , cells should be grown in galactose media and probed for respiratory fitness.
    • The study uses 100uM of A-769662 in the cell assays (Figures 3 and 4) and 200uM to test the activation of AMPK substrate ACC (Figure S2A-C). The authors should explain if a dose-dependent study was performed and how the concentration of A-769662 to be used was determined.

    A-769662 is a known AMPK activator with possible therapeutic effects in metabolic disorders, type II diabetes (Cool et al., 2006, Görransson et al., 2007): However, due to the wide range of effects it may have, it would be necessary to get to the molecular basis of how A-769662 targets mtDNA depletion. This study is a nice starting point to further probe into the benefits of A-769662, however it is not (yet) conclusive and definitely needs the clarification of the underlying molecular mechanisms.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Carvalho and colleagues addresses how an increase in AMPK signaling, triggered by the agonist A769662, contributes to ameliorate cellular phenotypes caused by mtDNA depletion. The authors build on previously established cellular models of mtDNA depletion, and use subcellular fractions in an attempt to distinguish pools of AMPK associated with different organelles. The data is very clearly presented, and the western blot data shown is of high quality. The authors also use cells from two patients with mtDNA depletion, in which they stimulate AMPK using A769662. The overall conclusion is that AMPK stimulation in the cell models with mtDNA depletion is advantageous to minimize the disease-related phenotypes.

    There is one fundamental weakness: there are several intracellular AMPK pools described, the major ones being in the cytoplasm, or associated with mitochondria, or with lysosomes, or in the nucleus. However, and importantly, while the authors convincingly show that there is no cytoplasm in their mitochondrial fractions, they do not control for the presence of lysosomal proteins. For the conclusions to be valid, it is absolutely essential to distinguish the effects of mitochondria-associated AMPK from lysosome-associated AMPK, because they may have different effectors and because they are activated by different mechanisms. Furthermore, the authors do not show what happens to AMPK in the patient cells, and this would be very informative. Finally, it would be important to put these findings in the context of other studies on AMPK signaling in response to other mitochondrial perturbations and that find AMPK to be down-regulated in chronic mitochondrial respiratory chain deficiency, as well as to more carefully reference the different intracellular AMPK pools. These studies might help to strengthen the discussion, given that the authors find AMPK signaling to be increased but show that increasing it pharmacologically has benefits - is A769662 activating different pools of AMPK?

    Significance

    The manuscript addresses an important question: how does the metabolic hub AMPK contribute to the phenotypes observed in chronic mitochondrial malfunction. Most of the studies on AMPK and mitochondrial malfunction focus on acute effects, which are not a model for mitochondrial diseases. Therefore, focusing on chronic effects is important to understand the long term consequences on AMPK signaling and its downstream signaling. Furthermore, if AMPK reactivation is beneficial (as this study proposes and other studies have also shown in other models of chronic mitochondrial malfunction), then this can become a new important therapeutic strategy.

  5. Version published to 10.1101/2024.03.14.584413 on bioRxiv