PREreview of GLUD1 dictates muscle stem cell differentiation by controlling mitochondrial glutamate levels

This review reflects comments and contributions from Marina Schernthanner and Pablo Ranea-Robles. Review synthesized by Pablo Ranea-Robles.

In this study, the authors studied the role of GLUD1 in satellite cells in the muscle. These cells are responsible for dynamic changes in muscular cells upon different signals, such as exercise. It is well known that metabolic changes play an important role in the fate decision of these cells toward proliferation or differentiation. Here, they found that GLUD1 and glutamine anaplerosis are decreased in differentiation, in contrast to what happens during proliferation. Using inducible KO cells and mice, they show how GLUD1 deficiency induces differentiation to myotubes and imbalance fusion of fibers. This phenotype was associated with an accumulation of glutamate only in mitochondria together with decreased levels of mitochondrial aspartate. In consequence, the malate-aspartate shuttle was inhibited, disturbing the NAD/NADH ratio between the cytosolic and mitochondrial compartments. In conclusion, they establish the role of GLUD1 in muscle satellite cells as a brake on differentiation, allowing proper proliferation using glutamine to feed the TCA cycle. Overall, we found this an excellent study, highly relevant, easy to read, and with multiple techniques and models to sustain their conclusions. Therefore, we would like to congratulate the authors on such a high-impact study. Below, a few comments we think could improve the manuscript and its understanding by the broad scientific community.

Major comments:

• We had no major comments

Minor comments:

• Could authors expand on how they know/tested that the custom media they used worked fine on the protocols of differentiation/proliferation? Was it possible to control for the amount of FBS in proliferation vs differentiation media (there appears to be a big difference between 30% and 0.2% FBS) without affecting the maintenance/differentiation of cells? FBS per se contains a number of growth factors, which could influence metabolomic analyses?

• I would recommend adding some implications/impact of these results more on a big picture over muscular function.

• The graphical abstract is excellent. I would just recommend to make the changes in NAD/NADH more clear, and indicate better the status of WT cell (quiescent vs prolif)

• Are there more number of cells after 48h in fig 2?

• What is the ratio Glu/alpha-kg in the cytosol?

• Metabolic changes were done with proliferation media, as I understand it, why did the authors not perform metabolomic analysis with the differentiation media?

• Figure 5D - can the authors show the metabolite levels of the cytosolic fraction as well (maybe in a supplementary figure)? Given that few differences were observed in whole cell lysates, as shown in B, one would expect that mitochondrial and cytosolic metabolite levels display opposite trends that balance each other out, correct?

• Figure 4 - aren't these minimal differences in transcription upon loss of Glud1 surprising, given the strong phenotypic difference in figure 1? What mediates precocious differentiation if not transcriptional changes? Could it be that the authors are dealing with somewhat heterogeneous populations here and thus relative enrichment of MuSC vs differentiated cell populations might not be readily picked up by bulk RNA-seq?

• Figure 3C-D - couldn't a reduction in GFP+ cells in theory also be due to increased cell death of GFP+ PAX7+ MuSC? Have the authors excluded this possibility by f.e. showing that there is no difference in TUNEL+(general cell death, DNA damage marker) and Caspase-3+ (apoptotic) cells between KO and WT MuSCs? For D) addition of nuclear signals to indicate cell fusion as expected in muscle fibers/myotubes would be helpful.

Suggestions for future studies:

•  It would be extremely interesting to evaluate a possible rescue of the phenotype in vivo. Perhaps with alanine supplementation?

Competing interests

The author declares that they have no competing interests.