Neural metabolism in vivo

Neural Metabolism In Vivo

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Series: Advances in neurobiology ; v. Subjects: Brain chemistry. Molecular neurobiology. Bosshard, Felicitas L. Princz-Kranz and David Ratering, et al. Evans, Angus M. Brown and Bruce R. Hamilton, Kellen Brunaldi, Richard P. Bazinet and Paul A. Swerdlow Part 4. Ross Part 5.

Nabuurs, Hermien E.

Introduction

Neil and Joseph J. Vlassenko and Mark A. Hasselbalch and Olaf B. Boas, Gaute T. Einevoll, Richard B. Surprisingly, even though neurons rely on oxidative phosphorylation to generate energy; the expression of TCA and mitochondrial genes is not increased. Thus, it appears that a need to avoid aerobic glycolysis is the major reason underlying neuronal reliance on oxidative phosphorylation.

The genes with decreased expression are dimmed. The width of the arrows indicates increased and decreased pyruvate and lactate utilization at different steps in NPCs and neurons. In addition to the conventional regulators of glycolysis, TCA and oxidative phosphorylation, the critical role of UCP2 uncoupling protein 2 in promoting aerobic glycolysis and inhibiting oxidative phosphorylation has been established from multiple experimental models Pecqueur et al. Distinct from UCP1, which uncouples ATP synthesis from the proton gradient by transporting protons into the mitochondrial matrix, UCP2 exports malate and oxaloacetate from mitochondria into the cytosol thus limiting the entry of pyruvate into the TCA cycle Vozza et al.

As demonstrated in human embryonic stem cells and hematopoietic stem cells, UCP2 is critical for maintaining stem cell glycolytic metabolism, and its level decreases during differentiation Zhang et al. Moreover, mitochondrial structure cristae organization and size , dynamics fusion and fission and calcium concentration in the mitochondrial matrix are all involved in mitochondrial energy metabolism Mishra and Chan, Possible changes in these processes need to be investigated to further understand how mitochondrial metabolism is reprogrammed during neuronal differentiation.

In addition to transcriptional regulation, protein degradation could also be used to downregulate key metabolic enzymes. We suspect there might be additional mechanisms of this sort at the protein level accounting for the extremely low levels of HK2 and LDHA protein in neurons. Even under conditions of energy shortage due to mitochondrial deficiency, neurons cannot turn on aerobic glycolysis genes, such as HK2 and LDHA. It appears that neurons cannot use aerobic glycolysis or mitochondrial biogenesis to compensate for energy shortage. To explore the importance of turning off aerobic glycolysis, we attempted to reactivate aerobic glycolysis by constitutive expression of HK2 and LDHA during neuronal differentiation.

Glucose transporter levels dramatically decrease in neurons, which limits glucose uptake and the production of glycolytic pyruvate. Reactivating the conversion of pyruvate to lactate by expression of LDHA would decrease the amount of pyruvate available for mitochondrial oxidation. Therefore, turning off aerobic glycolysis allows the efficient use of pyruvate for energy production.

Many lines of evidence support the conclusion that lactate secreted by glial cells is a critical energy source for neurons in vivo Pellerin and Magistretti, , and blocking neuronal oxidative utilization of lactate affects neuronal survival and even memory formation Suzuki et al. Obviously, downregulation of LDHA, an enzyme catalyzing the conversion of pyruvate to lactate, would favor the reverse reaction, which is catalyzed by a tetramer composed of LDHB to generate pyruvate from exogenous lactate. This phenotype could not be reversed by extra pyruvate in the medium.

Interestingly, it has been reported that exposure of NPCs to hypoxia, which boosts aerobic glycolysis, leads to more glial cells during neuronal differentiation Xie et al.

We confirmed this observation in wild-type NPCs data not shown. Aerobic glycolysis is tightly associated with cellular redox status Ochocki and Simon, We surmise that enhanced glycolysis may generate a cellular redox status that shifts the lineage choice toward glial cells during differentiation of NPCs.

Review ARTICLE

Although NPCs do not rely exclusively on oxidative phosphorylation to generate energy, mitochondria are used for generation of biosynthetic precursors in these proliferating cells, and mitochondria themselves have to be duplicated for daughter cells. Cell cycle transcription factors, such as MYC and E2F, have been shown to promote metabolic and mitochondrial gene expression.

E2F also upregulates mitochondrial genes, a function conserved from flies to mammals Ambrus et al. During neuronal differentiation, mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size. Interestingly in this regard, in a high-throughput screen to identify small molecules interfering with mitochondrial abundance, hundreds of compounds were found to be capable of changing the cellular mitochondrial content; the majority of them also change cell size accordingly Kitami et al. Our finding illustrates an example of this relationship in a normal developmental context.

NPCs and neurons were permeabilized at room temperature for 15 min in 0. Nuclear staining was done with Hoechst Invitrogen. Cell lysates were prepared with lysis buffer containing 20 mM Tris pH 7. Quick neuron nuclear extract preparation: the cells were rinsed with PBS once, and plates placed on ice and 1 ml of ice cold Buffer A 25 mM Hepes pH 7.

The plate was scraped and cells were transferred into an Eppendorf tube, which was centrifuged and rinsed once with Buffer A no NP The establishment of neural progenitor cells from iPSCs and neuron differentiation were performed as previously described Brennand et al. The cell clumps were then transferred to ultra-low attachment plates. This step is critical for the purity of NPCs; only colonies type 4 showing sufficient maturity as described in Figure 1—figure supplement 2 were picked. Medium was changed every third day up to 3 weeks. The protocol was adapted from the previous method Ladewig et al.

The real-time PCR primers were designed according to the work by Kim et al. For each time point, two experimental duplicates were used for each independent NPC line and its differentiated neurons. Lentivirus was concentrated from filtered culture media 0. Neurons were grown in a 6-well plate. After growth in fresh medium for 12 hr, cells were washed quickly 3 times with cold PBS, and 0. Culture plates were transferred to dry ice for 30 min.

After thawing on ice, the methanol extract was transferred to a microcentrifuge tube. Chloroform 0. After cooling, the derivatization mixture was transferred to an autosampler vial for analysis. More details including the parameters of machine settings can be found in the publication from the center Scott et al. Protein concentrations were determined by DC protein assay Bio-Rad.

The OCR values were normalized by protein mass. For measurement of lactate levels in medium, medium from cultures of iPSCs, NPCs and neurons was freshly changed and collected after 12 hr; cells were then frozen on the plate and lysed by two freeze-and-thaw cycles in dry ice.

Comparisons were done using Student's t-test. Statistical analyses were performed using GraphPad Prism. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation" for consideration by eLife. Your article has been reviewed by two peer reviewers, including Andrew Pieper Reviewer 1 , and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. This work sheds new light on alterations in metabolic gene expression during differentiation of neural progenitor cells NPCs to neurons.

Authors observe suppressed glycolytic gene expression in neurons, along with reduced expression of the glycolytic regulators c-MYC and N-MYC. Enforcing constitutive expression of glycolytic genes impairs differentiation of NPCs into viable neurons. While both reviewers found merit and interest in the work, both indicated that some translation to the in vivo situation is required.

Neural Metabolism In Vivo

Additionally, Reviewer 2 has very cogent and important suggestions that must be taken into account in a revised manuscript. With respect to demethylation in neurons, it would be powerful to show somehow that reversing promoter methylation allowed neurons to become glycolytic again. This would be particularly valuable in the context of hypoxia because of the relevance to stroke. One possibility would be to try to suppress methylation in the proliferating NPCs using 5 aza during the differentiation assay, since it is not possible to cause demethylation with this agent in non-dividing neurons.

Obviously this treatment would alter the methylation status of many genes, so the authors may find that neuronal differentiation is impaired to the point that the experiment cannot be performed.

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We think authors should give this experiment serious thought — it should not be difficult and could be very informative. Congratulations: we are very pleased to inform you that your article, "Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation", has been accepted for publication in eLife.

The Reviewing Editor for your submission was Gail Mandel. We include the re-review comments of Reviewer 2 below; unfortunately those comments were accidentally omitted from the original decision. If the authors have this data and can include it, it would make their case stronger.

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