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    Reply to the reviewers

    __We thank the three reviewers for their helpful and valuable comments. We plan to address their criticisms in a revised manuscript and hope that our manuscript will then be significantly improved. __

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The authors have presented a very interesting and compelling set of data regarding the impact of conditional deletion of the only known pathway allowing the uptake of pyruvate into mitochondria. The paper comprises two interwoven stories that are both important. The first is the remarkable finding that the majority of excitatory neurons in the cortex (i.e. those under the influence of the CaMKII promoter) show remarkable metabolic flexibility as they tolerate elimination of pyruvate oxidation, considered the major supplier of ATP in neurons. The data on this seem clear although the authors did not delve into the potential mechanisms of metabolic compensation that likely occurs. Instead they examined whether there was some mal-adaptive compensation and they found clear evidence of this: in the absence of MPC activity the mice are much more prone to epileptic seizures, unveiled experimentally by relatively standard protocols (kindling). The authors present largely very convincing evidence that this mal-adaptive compensation in turn ends up decreasing the activity of KV7.2/7.3 channels whose job is normally to limit runaway repetitive firing by mediating an hyperpolarizing K+ efflux following an action potential. This channel, put on the map as it was one of the downstream targets modulated by cholinergic metabotropic activation, is also know known to be controlled by Calmodulin and therefore cytosolic Ca levels. Overall, I think at its core this manuscript is interesting and important. There however several weaknesses, I fear, will diminish the impact on the eventual readership. If these points can be addressed, it will strengthen the longevity of these findings:

    1. It is puzzling why the authors resorted to using shRNA-mediated KD of MPC1 for some of the in vitro studies when they have gone to the trouble of making a floxed CRE-dependent mouse. Primary cells (e.g. Fig 1) or organotypic cultures (Fig. 6) from these mice would have made a more consistent set of starting conditions to compare data across the manuscript. As there viruses expressing the CRE recombinase are widely available this could have been used on mice simply harboring the floxed gene it they are worried about waiting for the expression of the CaMKII promoter for in-vitro conditions.

    This is indeed a good point. Indeed initially, when we started these experiments, we tried to use viruses expressing the CRE recombinase in cultured neurons from mice harboring the floxed gene as proposed by the reviewer. However, for reasons that we do not fully understand, the use of AAVs or lentiviruses expressing the CRE was found to be deleterious for the cultured neurons. In view of this toxicity we tried using TAT-CRE recombinase, a recombinant cell-permeant fusion recombinase, which we added directly to the medium. However, this strategy proved to be poorly efficient. We finally used cultures of Cre-floxed neurons in which we tried to knockout MPC1 gene using 4-hydroxytamoxifen in the culture medium. However, we did not obtain satisfying results because, as previously reported, cortical neurons grow poorly in the presence of 4-hydroxytamoxifen (Nichols et al., Cell Death and Disease, 2018. https://doi.org/10.1038/s41419-018-0607-9). For these reasons we turned to the shRNA strategy and to the use of 3 small molecule inhibitors of the MPC each with different chemical structures. Both the RNA interference and the pharmacological approaches gave similar results, reinforcing our confidence in the specificity of the results, and the unlikelihood of off-target effects.

    1. The data in Figure 5 gets a little less convincing as using extracellular glutamate to drive Ca elevations is so non-physiological that the results might really be distorted by the participation of something irrelevant to the story, even though it supports the overall interpretation for a role of Ca/CaM in the control of the channel. Similarly, the use of RU360 should be done with caution. The drug, although a useful antagonist of MCU in purified mitochondria, is famously finicky with respect to its ability to cross membranes and could well have off target impact. A much cleaner experiment would be to suppress the expression of MCU via KD. Presumably in the MPC-deficient neurons, this would have minimal impact on Ca signals. Given the frequent ambiguity associated with interpreting pharmacological results, coupled to the central importance of this finding in interpreting the entire paper, I think carrying out experiments with molecular genetic manipulation of MCU is warranted.

    The main point of this figure is to study the capacity of MPC1 KO neurons to handle intracellular calcium increase and to regulate calcium homeostasis. To this end, we used strategies described to acutely increase cytosolic calcium, either through membrane depolarization with KCl (Rienecker et al., ASN Neuro. 2020. https://doi.org/10.1177/1759091420974807) or through activation of glutamate receptors using glutamate (For example see Wong, Vis Neurosci, 1995 : DOI: 10.1017/s0952523800009469). It is important to mention that the concentration of glutamate used in our experiments (10 microM for 2 min) is well below the concentration normally used to induce excitotoxicity (100-500 microM for 30min). The fact that both stimulations provided similar results and clearly indicated a defect in the clearance of cytosolic calcium in MPC-deficent neurons.

    Regarding the concern with RU360, we are aware of the problems with plasma membrane permeability associated with this compound, and for this reason we included a membrane permeabilizer (0.02% pluronic acid) to facilitate its entry into the cell. This was indicated in the Material and Methods section (line 585) as well as in the figure legend (line 948). In order to clarify this methodology, we will add this information in the main text. It should be noted that this concern would not apply to the electrophysiogical experiments, since in this case the compound was injected directly into the cell. We would like to add that we chose to inhibit the MCU using a chemical inhibitor rather than a shRNA because of the well known difficulty in obtaining a complete loss of function of the MCU using RNA interference (Nichols et al., Cell Death and Disease, 2018. https://doi.org/10.1038/s41419-018-0607-9). Nevertheless, as recommended by the reviewer, we will attempt to downregulate the expression of MCU using shRNA.

    1. The authors have not really made clear in this paper whether the ability to suppress the phenotype of the MPC deficiency with ketones is really related to a providing TCA cycle support or instead a pharmacological impact on non-TCA related targets (such as the Kv7.2/7.3 channels). Presumably the use of other ketones might circumvent this. The action of ketone bodies has been a topic of considerable interest in neuroscience, given the clinical relevance for childhood epilepsies. Previous studies for example have argued for direct inhibition of the vesicular glutamate transporter (Juge et al. Neuron 2010). The use of other ketones (acetoacetate) would narrow down the interpretations of the data.

    Our results point to 2 two possible mechanisms of ketone bodies: i) providing acetyl-CoA to the Krebs cycle, thereby stimulating OXPHOS and ii) direct action of 3-beta hydroxybutyrate on the activity of Kv7/7.3 channels. The reviewer is asking whether, in addition to 3-beta hydroxybutyrate, other ketone bodies, acetone or acetoacetate, may display antiepileptic activity, which would probably indicate that providing substrates to the TCA cycle is sufficient to prevent neuron-intrinsic hyperactivity and seizures. We agree that this in an interesting question and we will now test the effect of acetoacetate on PTZ-induced seizures in MPC KO mice.

    **other**

    1. In vitro - scramble controls only serve to demonstrate there is no general effect of treating cells with shRNAs, but do not address if there is an off-target effect. The most convincing thing here would be to have an shRNA-insensitive variant that rescues.

    We have used 2 different shRNAs and 3 chemically unrelated inhibitors of the MPC and in all cases we obtained similar results. Therefore, we think that it is unlikely that the effects we observe are due to an off-target activity. The experiment proposed by the reviewer is interesting but extremely difficult. The idea would be to reintroduce a shRNA-insensitive MPC1 into MPC1-deficient neurons treated with shRNA. This is difficult as it is known that the expression level of MPC1 needs to be matched to that of MPC2, otherwise it leads to depolarization of the mitochondria. Obtaining the right level of MPC1 would be extremely difficult to achieve in practice.

    1. Does rescuing CaMK binding to KCNQ channels rescue the phenotypes?

    The question raised by the Reviewer implies that CaM is not constitutively bound to KCNQ channels, which is a matter of debate. As we pointed out in the discussion, ‘Intracellular calcium decreases CaM-mediated KCNQ channel activity (32, 36) by detaching CaM from the channel or by inducing changes in configuration of the calmodulin-KCNQ channel complex (36).’ The CaM-KCNQ tethering is also described in a review by Alaimo and Villaroel, 2018 (doi:10.3390/biom80300579): ‘[…] CaM was first defined as an integral subunit constitutively tethered to the C-terminal region of Kv7.2/3 channels since Kv7.2 mutants that were deficient in CaM binding were unable to generate measurable currents [5,21]. However, this model has been questioned since Kv7.2 channels, carrying a hB mutation [40] or Kv7.4 hA mutated channels [41] that do not bind CaM, can still reach the plasma membrane and are functional.’

    When considering to manipulate CaM binding to KCNQ, it should also be considered that previous studies on this matter have mainly worked with heterologous systems and through genetic manipulations of CaM (by expression of a dominant negative or by overexpression of CaM) or of the KCNQ binding motif.

    Based on both theoretical and practical issues, we, thus, believe that it is not feasible to implement a straightforward approach that would be compatible with our mouse model.

    An alternative, indirect approach, as indicated by Reviewer #3, would be to test the effect of Ca2+ chelators. Although this is likely to introduce confounding effects through the inhibition of other Ca2+-dependent channels, we propose to focus on trying this option and assess whether a XE991-sensitive component will be unmasked in MPC1 deficient cells.

    1. As the authors imply that BHB activates KCNQ channels, showing this directly in their prep would provide some convincing data. If this is true, why doesn't BHB increase firing rate of WT neurons?

    Activation of KCNQ channels is expected to reduce (not increase) neuronal firing. When exposed to BHB, we indeed found that WT cells also show a trend towards decreased excitability (p=0.08). We will report this trend in the revised figure 5F. Given that KCNQ channels are already available to be recruited upon repetitive firing in WT cells (to a larger extent as compared to KO, as indicated by our data with XE991) it is conceivable that a further potentiating effect of BHB at the concentration used for ex vivo recordings (2 mM) will be limited.

    1. How does the anti-epileptic effects of ketones in this study relate to previous reports of regulation of KATP channels? One of main concerns is that ketones might have a parallel anti-epileptic effect in the MPC1 KO mice that is unrelated to the mechanism proposed here.

    The ketogenic diet is likely to exert several effects including disruption of glutamatergic synaptic transmission, inhibition of glycolysis, and activation of ATP-sensitive potassium channels as pointed out by the reviewer. We do not exclude that inhibition of the MPC could also have an impact on the KATP channels and we are currently exploring this possibility. However, such work to dissect the potential implication of the KATP channels would go well beyond the scope of the present paper. Nevertheless, we will plan to certainly raise this important possibility in the discussion.

    **Minor comments:**

    1- What is the MPC1 KO efficiency in CaMK neurons? The western blot in 2c is from the whole cortex and therefore does not show that.

    This is indeed a good comment, however, please note that the estimation of MPC1 KO efficiency has also been evaluated in synaptosomes isolated from MPC1 KO cortices. These structures are mainly isolated from neurons (Carlin et al., JCB, 1980. 10.1083/jcb.86.3.831). As shown in figure 2C, these synaptosomes are massively enriched for CamKII and contain less astrocytic marker GFAP in comparison to the whole cortex. The amount of MPC1 in the synaptosomes prepared from the KO animals is strongly decreased. Nevertheless, as recommended by the reviewer, we plan to quantify the efficiency of the KO by performing a double immunostaining for MPC1 and a specific marker for neurons.

    2- Mitochondrial Ca2+ levels are not measured directly, for which there are many tools. This is needed to demonstrate definitively that there is a defect in Ca2+ handling."

    The reviewer raised an important point and we plan to monitor the levels of mitochondrial calcium in MPC-deficient neurons using the mito-Aequorin, a luminescent quantitative probe targeted to mitochondria (Granatiero et al., Cold Spring Harb. Protoc. 2014. 10.1101/pdb.top066118)

    Reviewer #1 (Significance (Required)):

    see above.

    **Referee Cross-commenting**

    It seems we are in reasonable agreement about the pros & cons of the manuscript. I agree that alternative approaches to RU360 are warranted.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    De la Rossa and colleagues examined the consequences of conditionally knocking out MPC1,a subunit of the mitochondrial pyruvate carrier. They found that despite decreased levels of oxidative phosphorylation in excitatory neurons, phenotypically these conditional knockout mice were normal at rest. However, when challenged by inhibition of GABA neurotransmission, these animals developed severe seizure activity and expired. These authors then showed that neurons with an absence of MPC1 were hyperexcitable in part through abnormal calcium homeostasis, which was associated with a reduction in M-type inhibitory potassium channel activity. Intriguingly, the ketogenic diet and the major ketone body beta-hydroxybutyrate were able to reverse these changes.

    This is a carefully conducted research study that reveals cell type-specific alterations of MPC1 deletion and functional consequences. The study design was logical and involved an exhaustive array of methodologies. The manuscript was generally well written and organized, and there are no major concerns. This study shows a direct causal relationship between impaired bioenergetics at the level of mitochondrial, and subsequent behavioral seizures, and is perhaps the most direct demonstration to date that an intrinsic disturbance of metabolic function can result in seizure activity (through changes in calcium regulation and impairment of ion channel activity). This will be an important contribution to the scientific literature.

    **MINOR:**

    1. Page 4, line 86: Would recommend changing "paroxystic" to "paroxysmal" (the latter which is a more recognized term). We will make the change.

    Page 5, line 124: recommend including the concentration of beta-hydroxybutyrate used when first mentioned. In general, concentration and dose information were difficult to find, as well as route of administration (for kainate, page 7, line 175). This type of information was not conveniently presented.

    We will follow the reviewer’s recommendation.

    Page 5, line 128: "both overcomed" is awkward. Would recommend using "both reversed".

    We fully agree and will make the change in the revised manuscript.

    Page 8, line 193: the authors probably meant "astro-MPC1-WT mice", not "neuro-MPC1-WT mice".

    Thank you for the acurate look. This will be changed.

    Page 12, lines 280-282: the authors might want to mention that chronic exposure of BHB might reduce the hyperexcitability of neuro-MPC1-KO mice.

    This point could indeed be discussed.

    Please review entire manuscript and use consistent tense. For example, on page 13, line 309, to maintain the past tense, it should read "We first assessed whether..."

    Thanks for the recommendation.

    Page 13, line 318: the authors used 10 mM BHB when examining calcium levels, but they earlier used 2 mM. They need to explain why they used a different concentration; and 2 vs 10 mM are quite different.

    The reviewer makes a valid point. When we performed the in vitro experiments, we used 10 mM BHB, which is slightly higher than the amount of ketone bodies measured in the blood of mice fed on a ketogenic diet for 2 days (Supplementary figure 4). This concentration of BHB has also been used by others (see for example: Izumi et al., JCI 1998, 101:1121-1132). Later on, when electrophysiology experiments were performed, the person in charge of these experiments followed a previously published protocol by Yellen and colleagues, in which the authors had used 2 mM BHB (Ma et al., J. Neurosci 2007,27: 3618-3625). This explains the differences between the concentrations used in vitro and in vivo.

    Page 13, line 323: it is not necessary to say "...interesting study published during the preparation of this manuscript." This phrase should be deleted, and the relevant reference simply cited.

    We will follow the reviewer’s recommendation.

    The authors need to explain more clearly in the beginning what exactly is meant by "paradoxical" hyperactivity. They provide greater meaning later in the manuscript, but this should be clarified at the outset.

    We will explain why we used this adjective in the beginning as recommended by the reviewer.

    Reviewer #2 (Significance (Required)):

    This is a very important study to show how primary defects in metabolism (i.e., disruption of the mitochondrial pyruvate carrier) can lead to epilepsy. Moreover, it details a primary mechanism that connects cellular bioenergetics to membrane excitability (through changes in calcium homeostasis and M-current function).

    This is a well-conducted study that utilizes a multiplicity of experimental tools to link biochemistry with seizure activity. This type of study is not so readily done, and strengthens the notion that primary defects in metabolism can result in epileptic seizures.

    This study is unique and attempts successfully to be more than just correlational. Hence it is a valuable contribution to the field.

    The audience will likely consist of metabolic geneticists, neurologists/epileptologists, and neuroscientists. This is a beautiful study that runs the translational spectrum from biochemistry to behavior.

    My expertise is in the field of translational epilepsy research, with a focus on mitochondria, metabolism, the ketogenic diet and ketone bodies. Thus, I am qualified to critically evaluate the entire manuscript.

    **Referee Cross-commenting**

    After reading comments and reviewing the manuscript again, would agree with Reviewer #1, and would change recommendation to MAJOR REVISION.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This manuscript tests the genetic requirement of the mitochondrial pyruvate carrier (MPC) in regulation of neuronal excitability. The authors find that MPC deficiency in glutamatergic neurons is associated with aerobic glycolysis, inhibition of the M-type K channels, and neuronal hyperexcitability that manifests in increased sensitivity to chemical pro-convulsants without changes in resting conditions. Alterations in Ca homeostasis in MPC-deficient neurons is consistent with reduced mitochondrial membrane potential and attendant diminution of mitochondrial calcium buffering capacity. The authors further show that the effect of MPC deficiency can be phenocopied by treatment of wild type neurons with a chemical inhibitor of the mitochondrial Ca uniporter (MCU). Based on these data, it is proposed that reduced mitochondrial Ca uptake causes neuronal hyperexcitability in the absence of MPC. Overall, the manuscript presents detailed electrophysiology and in vivo seizure studies. However, there is significant disconnect between the actual data in Fig. 6 and the authors' conclusions/proposed mechanism. In particular, the evidence for the role of Ca in the hyperexcitability due to MPC deficiency is the weak link in the authors' argument.

    1. The studies linking reduced mitochondrial Ca uptake to hyperexcitability in MPC-deficient neurons (Fig. 6) have several limitations that significantly weaken the paper: 1a. The Ca measurements in cortical neurons (Fig. 6A-F) are performed under conditions (glutamate/KCl) that are fundamentally different from those used in electrophysiology of CA1 pyramidal neurons (Fig. 6G-N). The electrophysiological excitation is much briefer and less extreme than the chemical stimulation, and it is not clear that the Ca dysregulation occurs at the earliest times (see Fig. 6A).

    This point was also raised by reviewer 1. Please see our response to point 2.

    1b. The conclusion that MCU is functionally responsible for MPC's effect on neuronal excitability is singularly based on the use of RU360 as a chemical inhibitor of MCU but the specificity of this reagent is questionable. Evidence for a cause and effect relationship that directly implicates altered MCU/mitochondrial Ca buffering has not been provided.

    This accurate point was also raised by the reviewer 1. Please see our response to point 2 for a complete response. We will downregulate expression of MCU using shRNAs. We will also measure the mitochondrial calcium level in the hope of better understanding whether the phenotype of the MPC-deficient mice is due to impaired mitochondrial calcium uptake.

    1c. There is a large variation in the effect of 10 uM RU360 on firing frequency, comparing Fig. 6H and N (blue traces), including the shape of the traces and values at ramp number 6. This calls into question the reliability of the comparisons in each separate figure.

    Data presented in each single graph in the main Figures were obtained from groups of littermates through recordings conducted in consecutive days. Some caution is warranted when comparing data between different figures (i.e. between different experimental series), as several factors may contribute to inter-experiment variability, including variability between different batches of animals. However, the difference pointed out by the reviewer regarding the values of cell firing reported in Fig. 6H and N is only apparent. When applying depolarizations with ramps of 5s, a fair amount of WT cells infused with RU-360 show high instantaneous firing frequency, especially for the last ramps that steeply reach high current levels. This leads to accommodation/inactivation of the action potential towards the end of the ramps, as shown in the example trace in Fig 6G. As a result, the current-frequency plot deviates from linearity, as it is the case in Fig 6H (blue trace) and, even more evidently, in Fig 6N. We have now reanalyzed the same recordings from WT cells infused with 10 µM RU-360 and measured the firing frequency in response to a square depolarizing step (250 pA) of 0.5 or 1 second. No difference was found between the firing frequencies of the cells from Fig 6H and Fig. 6N (group 1 and group 2, respectively, in the figure below). Although the ramps may lead to some distortion for higher stimulation levels, we have decided to show results from ramps consistently throughout the main figures because this protocol with continuously increasing currents allows us to measure more precisely the rheobase and the firing threshold (as opposed to the stepwise increments of a square stimulation).

    1d. The calcium > PIP2 > M-type K+ channel axis is well established but has not been fully explored in the context of MPC deficiency. The use of a calcium chelator will likely be informative in this context, and would be better evidence for a role of Ca in the MPC effects.

    Although the use of a Ca2+ chelator such as BAPTA is likely to introduce confounding effects through the inhibition of other Ca2+-dependent channels, we will try this option and assess whether a XE991-sensitive component will be unmasked in MPC deficient cells.

    1e. The ability of BHB to rescue various parameters in this and other figures in the paper is interesting but does not directly speak to the specific mechanism as to how MPC deficiency affects neuronal excitability. BHB's effect is consistent with the metabolic flexibility of neurons when the TCA cycle cannot be fueled by glucose/pyruvate (as in GLUT1 or MPC deficiency).

    The mechanism we propose to explain the hyperexcitability of MPC-deficient neurons relies on the low mitochondrial membrane potential and their decreased capacity to buffer calcium. Based on our data, we propose that calcium accumulation in the cytosol disrupts the CaM-KCNQ interaction leading to hyperexcitability. Indeed, BHB could act in two possible (and parallel) ways. 1: directly on the M-type channels, 2. on mitochondria by providing acetylCoA to the TCA cycle. The use of an alternative ketone body will be informative in disentangling these two possibilities.

    The manuscript (and the field) will benefit from a more scholarly discussion and integration of published literature:

    2a. The published studies on the outcome of pharmacologic MPC inhibition in neurons (Ref 18, Divakaruni et al.) are not only consistent with the bioenergetic effect in Fig. 1, but more importantly, show that interference with MPC does not lead to broad deficiencies in energy metabolism but rather remodel fuel utilization patterns to alternative substrates that feed the TCA cycle (BHB, leucine, etc). For this reason, terms such as "mitochondrial dysfunction" and "OXPHOS deficiency" used throughout the manuscript to describe MPC deficiency are vague and imprecise. In addition, this metabolic flexibility may explain lack of defects under resting conditions. In light of these considerations, the argument as to whether aerobic glycolysis in MPC-deficient neurons explains the lack of phenotype in resting conditions (p 17) seems one-sided. Overall, the studies in ref 18 are relevant to the current manuscript and should be better integrated in the discussion.

    We fully agree with the possibility that the rewiring of cell metabolism in MPC-deficient neurons in the presence of leucine, BHB and other metabolites could explain the lack of phenotype in resting conditions. We thank the reviewer for this highly relevant comment which we will include in the revised discussion.

    2b. Several references are cited to describe the role of OXPHOS vis-à-vis aerobic glycolysis in neuronal function. At times, however, the authors' statements are not consistent with what these papers actually show (or do not show). For example, see the use of refs 6 and 44 on p17 of the discussion, where the authors state that aerobic glycolysis uncoupled from OXPHOS is sufficient to provide ATP for normal neurotransmission, but this does not mean OXPHOS is not needed.

    We agree that these references are not appropriate here and they will be removed.

    2c. Although the XE991 experiments support an important role for the M-type channels in the altered excitability with deficiency, it is not clear that the proposed mechanism can explain all of the electrophysiological differences, particularly those resting properties that are measured without a Ca challenge to the neurons. It would be good to discuss other possible mechanisms that could affect neuronal excitability.

    Our results point to M-type channels as important players in the phenotype of the MPC-deficient mice. Previous reports indicate that inhibition of this channel by XE991 can modulate input resistance, membrane potential and firing threshold of pyramidal cells (e.g. Shah et al, 2018, doi/10.1073/pnas.0802805105; Hu et al. 2007, DOI:10.1523/JNEUROSCI.4463-06.2007; Petrovic et al., 2012, doi:10.1371/journal.pone.0030402). We also found that XE991 induced a shift towards more negative potentials in the firing threshold of WT cells, but not in MPC1 deficient cells (-3.3±0.6 vs. -0.4±1.0, n=9, 8, p=0.027). However, we agree with the reviewer that the phenotype is probably highly complex and that additional mechanisms may contribute to modulate the intrinsic excitability of MPC-deficient neurons. One such mechanism could be closure of KATP channels, which we are currently investigating. This will be discussed.

    Reviewer #3 (Significance (Required)):

    The significance of the advance: The studies provide genetic evidence for the role of MPC in neuronal excitability.

    The work in the context of existing literature: Please see specific comments above under point 2 regarding the need for a scholarly discussion and integration of existing literature.

    Audience that might be interested: mitochondrial bioenergetics and metabolism and metabolic control of neuronal excitation.

    Keywords describing expertise: metabolism, mitochondria and electrophysiology.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript tests the genetic requirement of the mitochondrial pyruvate carrier (MPC) in regulation of neuronal excitability. The authors find that MPC deficiency in glutamatergic neurons is associated with aerobic glycolysis, inhibition of the M-type K channels, and neuronal hyperexcitability that manifests in increased sensitivity to chemical pro-convulsants without changes in resting conditions. Alterations in Ca homeostasis in MPC-deficient neurons is consistent with reduced mitochondrial membrane potential and attendant diminution of mitochondrial calcium buffering capacity. The authors further show that the effect of MPC deficiency can be phenocopied by treatment of wild type neurons with a chemical inhibitor of the mitochondrial Ca uniporter (MCU). Based on these data, it is proposed that reduced mitochondrial Ca uptake causes neuronal hyperexcitability in the absence of MPC. Overall, the manuscript presents detailed electrophysiology and in vivo seizure studies. However, there is significant disconnect between the actual data in Fig. 6 and the authors' conclusions/proposed mechanism. In particular, the evidence for the role of Ca in the hyperexcitability due to MPC deficiency is the weak link in the authors' argument.

    1. The studies linking reduced mitochondrial Ca uptake to hyperexcitability in MPC-deficient neurons (Fig. 6) have several limitations that significantly weaken the paper:

    1a. The Ca measurements in cortical neurons (Fig. 6A-F) are performed under conditions (glutamate/KCl) that are fundamentally different from those used in electrophysiology of CA1 pyramidal neurons (Fig. 6G-N). The electrophysiological excitation is much briefer and less extreme than the chemical stimulation, and it is not clear that the Ca dysregulation occurs at the earliest times (see Fig. 6A).

    1b. The conclusion that MCU is functionally responsible for MPC's effect on neuronal excitability is singularly based on the use of RU360 as a chemical inhibitor of MCU but the specificity of this reagent is questionable. Evidence for a cause and effect relationship that directly implicates altered MCU/mitochondrial Ca buffering has not been provided.

    1c. There is a large variation in the effect of 10 uM RU360 on firing frequency, comparing Fig. 6H and N (blue traces), including the shape of the traces and values at ramp number 6. This calls into question the reliability of the comparisons in each separate figure.

    1d. The calcium > PIP2 > M-type K+ channel axis is well established but has not been fully explored in the context of MPC deficiency. The use of a calcium chelator will likely be informative in this context, and would be better evidence for a role of Ca in the MPC effects.

    1e. The ability of BHB to rescue various parameters in this and other figures in the paper is interesting but does not directly speak to the specific mechanism as to how MPC deficiency affects neuronal excitability. BHB's effect is consistent with the metabolic flexibility of neurons when the TCA cycle cannot be fueled by glucose/pyruvate (as in GLUT1 or MPC deficiency).

    1. The manuscript (and the field) will benefit from a more scholarly discussion and integration of published literature:

    2a. The published studies on the outcome of pharmacologic MPC inhibition in neurons (Ref 18, Divakaruni et al.) are not only consistent with the bioenergetic effect in Fig. 1, but more importantly, show that interference with MPC does not lead to broad deficiencies in energy metabolism but rather remodel fuel utilization patterns to alternative substrates that feed the TCA cycle (BHB, leucine, etc). For this reason, terms such as "mitochondrial dysfunction" and "OXPHOS deficiency" used throughout the manuscript to describe MPC deficiency are vague and imprecise. In addition, this metabolic flexibility may explain lack of defects under resting conditions. In light of these considerations, the argument as to whether aerobic glycolysis in MPC-deficient neurons explains the lack of phenotype in resting conditions (p 17) seems one-sided. Overall, the studies in ref 18 are relevant to the current manuscript and should be better integrated in the discussion.

    2b. Several references are cited to describe the role of OXPHOS vis-à-vis aerobic glycolysis in neuronal function. At times, however, the authors' statements are not consistent with what these papers actually show (or do not show). For example, see the use of refs 6 and 44 on p17 of the discussion, where the authors state that aerobic glycolysis uncoupled from OXPHOS is sufficient to provide ATP for normal neurotransmission, but this does not mean OXPHOS is not needed.

    2c. Although the XE991 experiments support an important role for the M-type channels in the altered excitability with deficiency, it is not clear that the proposed mechanism can explain all of the electrophysiological differences, particularly those resting properties that are measured without a Ca challenge to the neurons. It would be good to discuss other possible mechanisms that could affect neuronal excitability.

    Significance

    The significance of the advance: The studies provide genetic evidence for the role of MPC in neuronal excitability.

    The work in the context of existing literature: Please see specific comments above under point 2 regarding the need for a scholarly discussion and integration of existing literature. Audience that might be interested: mitochondrial bioenergetics and metabolism and metabolic control of neuronal excitation.

    Keywords describing expertise: metabolism, mitochondria and electrophysiology.

    Read the original source
    Was this evaluation helpful?
  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    De la Rossa and colleagues examined the consequences of conditionally knocking out MPC1,a subunit of the mitochondrial pyruvate carrier. They found that despite decreased levels of oxidative phosphorylation in excitatory neurons, phenotypically these conditional knockout mice were normal at rest. However, when challenged by inhibition of GABA neurotransmission, these animals developed severe seizure activity and expired. These authors then showed that neurons with an absence of MPC1 were hyperexcitable in part through abnormal calcium homeostasis, which was associated with a reduction in M-type inhibitory potassium channel activity. Intriguingly, the ketogenic diet and the major ketone body beta-hydroxybutyrate were able to reverse these changes.

    This is a carefully conducted research study that reveals cell type-specific alterations of MPC1 deletion and functional consequences. The study design was logical and involved an exhaustive array of methodologies. The manuscript was generally well written and organized, and there are no major concerns. This study shows a direct causal relationship between impaired bioenergetics at the level of mitochondrial, and subsequent behavioral seizures, and is perhaps the most direct demonstration to date that an intrinsic disturbance of metabolic function can result in seizure activity (through changes in calcium regulation and impairment of ion channel activity). This will be an important contribution to the scientific literature.

    MINOR:

    1. Page 4, line 86: Would recommend changing "paroxystic" to "paroxysmal" (the latter which is a more recognized term).
    2. Page 5, line 124: recommend including the concentration of beta-hydroxybutyrate used when first mentioned. In general, concentration and dose information were difficult to find, as well as route of administration (for kainate, page 7, line 175). This type of information was not conveniently presented.
    3. Page 5, line 128: "both overcomed" is awkward. Would recommend using "both reversed".
    4. Page 8, line 193: the authors probably meant "astro-MPC1-WT mice", not "neuro-MPC1-WT mice".
    5. Page 12, lines 280-282: the authors might want to mention that chronic exposure of BHB might reduce the hyperexcitability of neuro-MPC1-KO mice.
    6. Please review entire manuscript and use consistent tense. For example, on page 13, line 309, to maintain the past tense, it should read "We first assessed whether..."
    7. Page 13, line 318: the authors used 10 mM BHB when examining calcium levels, but they earlier used 2 mM. They need to explain why they used a different concentration; and 2 vs 10 mM are quite different.
    8. Page 13, line 323: it is not necessary to say "...interesting study published during the preparation of this manuscript." This phrase should be deleted, and the relevant reference simply cited.
    9. The authors need to explain more clearly in the beginning what exactly is meant by "paradoxical" hyperactivity. They provide greater meaning later in the manuscript, but this should be clarified at the outset.

    Significance

    This is a very important study to show how primary defects in metabolism (i.e., disruption of the mitochondrial pyruvate carrier) can lead to epilepsy. Moreover, it details a primary mechanism that connects cellular bioenergetics to membrane excitability (through changes in calcium homeostasis and M-current function).

    This is a well-conducted study that utilizes a multiplicity of experimental tools to link biochemistry with seizure activity. This type of study is not so readily done, and strengthens the notion that primary defects in metabolism can result in epileptic seizures.

    This study is unique and attempts successfully to be more than just correlational. Hence it is a valuable contribution to the field.

    The audience will likely consist of metabolic geneticists, neurologists/epileptologists, and neuroscientists. This is a beautiful study that runs the translational spectrum from biochemistry to behavior.

    My expertise is in the field of translational epilepsy research, with a focus on mitochondria, metabolism, the ketogenic diet and ketone bodies. Thus, I am qualified to critically evaluate the entire manuscript.

    Referee Cross-commenting

    After reading comments and reviewing the manuscript again, would agree with Reviewer #1, and would change recommendation to MAJOR REVISION.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors have presented a very interesting and compelling set of data regarding the impact of conditional deletion of the only known pathway allowing the uptake of pyruvate into mitochondria. The paper comprises two interwoven stories that are both important. The first is the remarkable finding that the majority of excitatory neurons in the cortex (i.e. those under the influence of the CaMKII promoter)show remarkable metabolic flexibility as they tolerate elimination of pyruvate oxidation, considered the major supplier of ATP in neurons. The data on this seem clear although the authors did not delve into the potential mechanisms of metabolic compensation that likely occurs. Instead they examined whether there was some mal-adaptive compensation and they found clear evidence of this: in the absence of MPC activity the mice are much more prone to epileptic seizures, unveiled experimentally by relatively standard protocols (kindling). The authors present largely very convincing evidence that this mal-adaptive compensation in turn ends up decreasing the activity of KV7.2/7.3 channels whose job is normally to limit runaway repetitive firing by mediating an hyperpolarizing K+ efflux following an action potential. This channel, put on the map as it was one of the downstream targets modulated by cholinergic metabotropic activation, is also know known to be controlled by Calmodulin and therefore cytosolic Ca levels. Overall, I think at its core this manuscript is interesting and important. There however several weaknesses, I fear, will diminish the impact on the eventual readership. If these points can be addressed, it will strengthen the longevity of these findings:

    1. It is puzzling why the authors resorted to using shRNA-mediated KD of MPC1 for some of the in vitro studies when they have gone to the trouble of making a floxed CRE-dependent mouse. Primary cells (e.g. Fig 1) or organotypic cultures (Fig. 6) from these mice would have made a more consistent set of starting conditions to compare data across the manuscript. As there viruses expressing the CRE recombinase are widely available this could have been used on mice simply harboring the floxed gene it they are worried about waiting for the expression of the CaMKII promoter for in-vitro conditions.

    2. The data in Figure 5 gets a little less convincing as using extracellular glutamate to drive Ca elevations is so non-physiological that the results might really be distorted by the participation of something irrelevant to the story, even though it supports the overall interpretation for a role of Ca/CaM in the control of the channel. Similarly, the use of RU360 should be done with caution. The drug, although a useful antagonist of MCU in purified mitochondria, is famously finicky with respect to its ability to cross membranes and could well have off target impact. A much cleaner experiment would be to suppress the expression of MCU via KD. Presumably in the MPC-deficient neurons, this would have minimal impact on Ca signals. Given the frequent ambiguity associated with interpreting pharmacological results, coupled to the central importance of this finding in interpreting the entire paper, I think carrying out experiments with molecular genetic manipulation of MCU is warranted.

    3. The authors have not really made clear in this paper whether the ability to suppress the phenotype of the MPC deficiency with ketones is really related to a providing TCA cycle support or instead a pharmacological impact on non-TCA related targets (such as the Kv7.2/7.3 channels). Presumably the use of other ketones might circumvent this. The action of ketone bodies has been a topic of considerable interest in neuroscience, given the clinical relevance for childhood epilepsies. Previous studies for example have argued for direct inhibition of the vesicular glutamate transporter (Juge et al. Neuron 2010). The use of other ketones (acetoacetate) would narrow down the interpretations of the data.

    other

    1. In vitro - scramble controls only serve to demonstrate there is no general effect of treating cells with shRNAs, but do not address if there is an off-target effect. The most convincing thing here would be to have an shRNA-insensitive variant that rescues.

    2. Does rescuing CaMK binding to KCNQ channels rescue the phenotypes?

    3. As the authors imply that BHB activates KCNQ channels, showing this directly in their prep would provide some convincing data. If this is true, why doesn't BHB increase firing rate of WT neurons?

    4. How does the anti-epileptic effects of ketones in this study relate to previous reports of regulation of KATP channels? One of main concerns is that ketones might have a parallel anti-epileptic effect in the MPC1 KO mice that is unrelated to the mechanism proposed here.

    Minor comments:

    1- What is the MPC1 KO efficiency in CaMK neurons? The western blot in 2c is from the whole cortex and therefore does not show that. 2- Mitochondrial Ca2+ levels are not measured directly, for which there are many tools. This is needed to demonstrate definitively that there is a defect in Ca2+ handling."

    Significance

    see above.

    Referee Cross-commenting

    It seems we are in reasonable agreement about the pros & cons of the manuscript. I agree that alternative approaches to RU360 are warranted.

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