The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells
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eLife Assessment
This report describes evidence that the main driving force for stimulation of glycolysis in dentate granule cell neurons in acute hippocampal slices from mouse by electrical activity comes from influx of Na+ including Na+ exchanging into the cell for Ca2+. The findings are presented very clearly and the authors' interpretations seem reasonable. This is important and impactful because it identifies the major energy demand in excited neurons that stimulates glycolysis to supply more ATP.
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Abstract
Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca 2+ influx, but is not activated by feedforward Ca 2+ signaling (Díaz-García et al., 2021a). Since ATP-powered Na + and Ca 2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na + , while intracellular Ca 2+ could only increase NADH production if both forward Na + /Ca 2+ exchange and Na + /K + pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na + ] increases were reduced >50% by blocking Ca 2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na + /K + pump, which is strongly potentiated by Na + influx through the Na + /Ca 2+ exchanger during extrusion of Ca 2+ following stimulation.
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Author Response
Reviewer #1 (Public Review):
This report describes evidence that the main driving force for stimulation of glycolysis in cultured DGC neurons by electrical activity comes from influx of Na+ including Na+ exchanging into the cell for Ca2+. The findings are presented very clearly and the authors' interpretations seem reasonable. This is important and impactful because it identifies the major energy demand in excited neurons that stimulates glycolysis to supply more ATP.
Strengths are the highly rigorous use of fluorescent probes to directly monitor the concentrations of NADH/NAD+, Ca2+ and Na+. The strategies directly test the roles of Na+ and Ca2+.
A weakness is an ambiguity about the effects of ouabain to inhibit the Na+/K+ ATPase directly and the absence of biochemical controls to validate the interpretation of …
Author Response
Reviewer #1 (Public Review):
This report describes evidence that the main driving force for stimulation of glycolysis in cultured DGC neurons by electrical activity comes from influx of Na+ including Na+ exchanging into the cell for Ca2+. The findings are presented very clearly and the authors' interpretations seem reasonable. This is important and impactful because it identifies the major energy demand in excited neurons that stimulates glycolysis to supply more ATP.
Strengths are the highly rigorous use of fluorescent probes to directly monitor the concentrations of NADH/NAD+, Ca2+ and Na+. The strategies directly test the roles of Na+ and Ca2+.
A weakness is an ambiguity about the effects of ouabain to inhibit the Na+/K+ ATPase directly and the absence of biochemical controls to validate the interpretation of the ouabain experiment.
We appreciate the reviewer's comments about the work. While we can not rule out non-specific effects of ouabain at the concentrations needed to block Na+/K+ ATPase in these experiments, we do think that we can rely on the prior biochemical work characterizing the multiple components of ouabain binding in fresh mouse brain tissue, which is a close match to the acute mouse brain slice tissue used here.
Reviewer #2 (Public Review):
This study seeks to determine how neuronal glycolysis is coupled to electrical activity. Previous studies had found that glycolytic enzymes cluster within nerve terminals (in C. elegans) during activity. Furthermore, the glucose transporter GLUT4 is recruited to synaptic surface during activity. The authors previously showed that Ca2+ does not stimulate glycolysis in active neurons. Here, the authors show that the cytosolic Na+, not Ca2+, and the activity of the Na+/K+ pump drive glycolysis. However, it is important to note that in this study, glycolysis was examined in the soma, not nerve terminals, where some of the previous studies were conducted. A few other caveats in the interpretation of the findings are listed below:
- The NADH/NAD+ ratio is used throughout as the only measurement reflecting glycolytic flux.
In this and previous work, we have validated that increased cytosolic NADH production (whose major sources are related to glycolysis), rather than altered NADH reoxidation, produces the changes in NADH/NAD+ ratio.
- It has been hypothesized that the close association of glycolytic enzymes with ion transporters (such as the Na+/K+ pump) is meant to provide localized ATP to power these pumps. How does bulk glycolysis (monitored with NADH/NAD+ ratio) relate to localized/compartmentalized glycolysis?
Even if glycolysis is indeed localized to the plasma membrane (an interesting and difficult-to-address hypothesis), we believe that because the mitochondrial shuttles are the main pathway for NADH re-oxidation, and most mitochondria are not localized to the plasma membrane, changes in glycolytic NADH production are likely to be reflected in changes of the bulk cytosolic NADH/NAD+.
- Related to point 2, most of the Peredox measurements in the paper have been made at baseline, in the absence of electrical activity. Therefore, it is not clear how the findings relate to activity-driven glycolysis.
The ion exchange experiments and even the faster Ca2+ puff experiments can mimic but indeed cannot match the speed of activity-driven changes in ion concentrations. Unfortunately, it is impossible to induce normal electrical activity in neurons in the absence of extracellular Na+. We believe that the complete inability of Ca2+ elevation alone (without Na+-Ca2+ exchange) to stimulate glycolysis, combined with the substantial Ca2+ contribution to activity-driven glycolysis, makes a good argument that Ca2+ entering during activity is likely to stimulate glycolysis via Na+ entry and the Na+/K+ ATPase.
- The finding that inhibition of SERCA during stimulation actually elevates cytosolic NADH level argues against Na+ being the only ion that regulates glycolysis.
The ability of SERCA inhibition to produce a small increase in activity-driven glycolysis is consistent with the simple argument that reduced SERCA-driven uptake of Ca2+ into ER results in additional Ca+ removal via Na+/Ca2+ exchange (which can then affect glycolysis via Na+ levels).
- The finding that "SBFI ΔF/F transients were longer in duration than the RCaMP LT transient" does not necessarily mean that Na+ elevation lasts longer than Ca2+ in the cell. This could be an artefact of the SBFI on/off rate relative to RCaMP. In fact, prolonged elevation of cytosolic Na+ would make neurons refractive to depolarization in AP trains.
The rates of Na+ binding and unbinding to SBFI are likely to occur on the microsecond timescale (based on the known properties of crown ether molecules), much faster than the observed transient duration of approximately one minute. Prolonged elevation of cytosolic Na+ alone (to the levels seen here) should not cause neurons to be refractory to firing; refractoriness typically occurs in the setting of prolonged depolarization and consequent inactivation of NaV channels.
Reviewer #3 (Public Review):
Meyer et al have studied the mechanisms of glycolysis activation in the hippocampus during neuronal activity. The study is logically laid out, uses sophisticated fluorescence lifetime imaging technology and smart experimental designs. The support for intracellular [Na+] vs [Ca2+] rise driving glycolysis is strong. The evidence for the direct involvement of the Na+/K+ pump is based only on pharmacology using ouabain but the Na+/K+ pump is admittedly not an easy subject for specific perturbations. I still think that the Authors should strengthen the support for the pathway.
We are happy that the reviewer feels that the evidence for Na+ rather than Ca2+ as the effector of glycolysis is strong. The tools for investigating the role of the Na+/K+ pump (NKA) are indeed limited to pharmacology, because (as the reviewer says) there are not many other options. The requirement for Na+ elevation (which stimulates NKA activity) to trigger glycolysis and the ability of ouabain, a specific NKA inhibitor, to prevent this seem like strong implication of NKA in the mechanism of glycolysis activation. Genetic manipulation of the NKA may be unable to change the level of pump activity, because of compensation by altered expression of other subunits (PMID 17234593); it also is unclear how any chronic manipulation would shed light on the role of NKA in triggering glycolysis. But perhaps future studies of knock-in mice in which the α1 isoform of NKA has made more sensitive to ouabain (PMIDs 15485817; 34129092) might allow the identification of the NKA as the target of ouabain in this situation to be made even more secure.
Also, there is a long list of publications on the connection between the Na+/K+ pump and glycolysis. It might be useful to highlight the role of the NCX- Na+/K+ pump coupling in the activation of glycolysis in the title.
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eLife Assessment
This report describes evidence that the main driving force for stimulation of glycolysis in dentate granule cell neurons in acute hippocampal slices from mouse by electrical activity comes from influx of Na+ including Na+ exchanging into the cell for Ca2+. The findings are presented very clearly and the authors' interpretations seem reasonable. This is important and impactful because it identifies the major energy demand in excited neurons that stimulates glycolysis to supply more ATP.
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Reviewer #1 (Public Review):
This report describes evidence that the main driving force for stimulation of glycolysis in DGC neurons by electrical activity comes from influx of Na+ including Na+ exchanging into the cell for Ca2+. The findings are presented very clearly and the authors' interpretations seem reasonable. This is important and impactful because it identifies the major energy demand in excited neurons that stimulates glycolysis to supply more ATP.
Strengths are the highly rigorous use of fluorescent probes to directly monitor the concentrations of NADH/NAD, Ca2+ and Na+. The strategies directly test the roles of Na+ and Ca2+.
-
Reviewer #2 (Public Review):
This study seeks to determine how neuronal glycolysis is coupled to electrical activity. Previous studies had found that glycolytic enzymes cluster within nerve terminals (in C. elegans) during activity. Furthermore, the glucose transporter GLUT4 is recruited to synaptic surface during activity. The authors previously showed that Ca2+ does not stimulate glycolysis in active neurons. Here, the authors show that the cytosolic Na+, not Ca2+, and the activity of the Na/K pump drive glycolysis. However, it is important to note that in this study, glycolysis was examined in the soma, not nerve terminals, where some of the previous studies were conducted. A few other caveats in the interpretation of the findings are listed below:
1. The NADH/NAD ratio is used throughout as the only measurement reflecting glycolytic …
Reviewer #2 (Public Review):
This study seeks to determine how neuronal glycolysis is coupled to electrical activity. Previous studies had found that glycolytic enzymes cluster within nerve terminals (in C. elegans) during activity. Furthermore, the glucose transporter GLUT4 is recruited to synaptic surface during activity. The authors previously showed that Ca2+ does not stimulate glycolysis in active neurons. Here, the authors show that the cytosolic Na+, not Ca2+, and the activity of the Na/K pump drive glycolysis. However, it is important to note that in this study, glycolysis was examined in the soma, not nerve terminals, where some of the previous studies were conducted. A few other caveats in the interpretation of the findings are listed below:
1. The NADH/NAD ratio is used throughout as the only measurement reflecting glycolytic flux.
2. It has been hypothesized that the close association of glycolytic enzymes with ion transporters (such as the Na+/K+ pump) is meant to provide localized ATP to power these pumps. How does bulk glycolysis (monitored with NADH/NAD ratio) relate to localized/compartmentalized glycolysis?
3. Related to point 2, most of the peredox measurements in the paper have been made at baseline, in the absence of electrical activity. Therefore, it is not clear how the findings relate to activity-driven glycolysis.
4. The finding that inhibition of SERCA during stimulation actually elevates cytosolic NADH level argues against Na+ being the only ion that regulates glycolysis.
5. The finding that "SBFI ΔF/F transients were longer in duration than the RCaMP LT transient" does not necessarily mean that Na+ elevation lasts longer than Ca2+ in the cell. This could be an artefact of the SBFI on/off rate relative to RCaMP. In fact, prolonged elevation of cytosolic Na+ would make neurons refractive to depolarization in AP trains. -
Reviewer #3 (Public Review):
Meyer et al have studied the mechanisms of glycolysis activation in the hippocampus during neuronal activity. The study is logically laid out, uses sophisticated fluorescence lifetime imaging technology and smart experimental designs. The support for intracellular [Na+] vs [Ca2+] rise driving glycolysis is strong. The evidence for the direct involvement of the Na+/K+ pump is based only on pharmacology using ouabain but the Na+/K+ pump is admittedly not an easy subject for specific perturbations. I still think that the Authors should strengthen the support for the pathway.
Also, there is a long list of publications on the connection between the Na+/K+ pump and glycolysis. It might be useful to highlight the role of the NCX- Na+/K+ pump coupling in the activation of glycolysis in the title.
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