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Referee #1
Evidence, reproducibility and clarity
Summary:
In the manuscript entitled "Nfe2l1-mediated proteasome function controls muscle energy metabolism in obesity", Lemmer and colleagues observe elevated 20S proteasome activity along with increased expression of the transcription factor Nfe2l1, a known stimulator of proteasome subunit biogenesis, in muscle tissue of diet-induced obese mice. To understand the role of Nfe2l1 in regulating skeletal muscle proteostasis, they use siRNA to knockdown Nfe2l1 in cultured C2C12 muscle cells and cross Nfe2l1 floxed mice with and Acta1-Cre line to KO Nfe2l1 specifically in muscle fibers. Nfe2l1mKO mice show reduced body and muscle size, impaired enzymatic activity of some proteasome subunits, an accumulation of ubiquitylated proteins, a fast-to-slow shift in muscle fiber phenotype and metabolic abnormalities, including impaired mitochondrial function and mild increases in relative energy expenditure. Multi-omics analysis at transcript, protein, ubiquitinated protein and metabolite levels indicate a strong influence of Nfe2l1 loss on muscle homeostasis. These affects appear to predominately affect fast-type (i.e. Gastrocnemius) rather than slow-type (i.e. soleus) muscles. Finally, Nfe2l1mKO mice fail to gain weight on a high fat diet and are therefore spared the typical metabolic alterations associated with obesity.
Major comments:
- The authors push the idea that the UPS, via Nfe2l1, plays an 'adaptive' role in regulating muscle proteostasis, however, all signaling experiments investigating the effect of muscle fiber Nfe2l1 KO are performed under basal conditions.
- Due to the use of ACTA1-Cre to conditionally KO Nfe2l1 in muscle fibers, Nfe2l1 is also absent during development. It is therefore difficult to distinguish the acute effects of Nfe2l1 on muscle proteostasis from those that may result from developmental impairment. It is conceivable that remodeling of muscle architecture would be more active during development than in mature muscle and therefore perhaps more sensitive to impairments in proteasome biogenesis. Use of an inducible Cre system (e.g. the tamoxifen inducible HSA-MerCreMer model) and/or the addition of acute Nfe2l1 overexpression experiments would be needed to dissociate acute, primary effects of Nfe2l1 from the secondary features of long term Nfe2l1 KO and disruption of proteostasis.
- It is unclear what the HFD experiments reported in Figure 7 add to our understanding of the role of Nfe2l1 in skeletal muscle. I could understand if an inducible Nfe2l1mKO system was used to test the role of Nfe2l1 in already obese mice... but a failure of a mouse displaying myopathic features to put on weight is not the same as an alteration that improves metabolic health. As Nfe2l1mKO mice do not become obese, the authors are unable to directly test what role the upregulation of muscle Nfe2l1 plays in maintaining proteostasis in obesity. On the other hand, I find it hard to conclude that the absence of muscle Nfe2l1 is beneficial for metabolic health if fed a HFD, especially given the reported increase in p62 and LC3B (indicative of autophagy impairment) and the impaired muscle mitochondrial function. Further investigations in older mice would be required to determine the long-term impact of muscle Nfe2l1 KO on whole-body health under both normal and high-fat diet feeding conditions.
- The authors note several interesting muscle phenotypes, including a fast-to-slow fiber type transition and an increased expression of neonatal myosin heavy chain isoforms (Myh3 & 8). The representative images seem to indicate that IIA (green) fibers are larger in Nfe2l1mKO mice. I would recommend quantifying fiber type-specific cross sectional area in Gastrocnemius muscle sections from these mice, as well as confirming the increased 'regeneration' phenotype by quantifying the prevalence of centralized nuclei.
- Figure 3D: Why would Nfe2l1 KD lead to a larger increase in ubiquitylated proteins after proteasome inhibition? If Nfe2l1 KD reduces proteasome subunit gene expression (presumably also protein content) and proteasome activity (although this effect is rather mild), then blocking a proteasome with lower activity should lead to a lower accumulation of ubiquitylated proteins, despite an accumulation of ubiquitylated proteins under basal conditions.
- Fig3C&G: The finding that Nfe2l1 KD/KO mildly reduces chymotrypsin-like and caspase-like (in mice) activity, but strongly increases trypsin-like activity is surprising. As these activities are mediated by different Beta subunits within the 20S core particle, it would be important to also test whether protein levels of PSMB5 (Chymotrypsin), PSMB6 (Caspase) and PSMB7 (trypsin) are altered by Nfe2l1 in accordance with differences in their measured activity.
- The authors have a tendency to use vague terms to describe changes in proteostasis resulting from Nfe2l1 KO, for example: 'recalibration', 'adaptive', 'fine-tuning', 'remolding', 'remodeling', 'rewiring'. While it is understandable that Nfe2l1 and the UPS will have different roles under different conditions, the use of vague language makes it difficult to understand whether they are referring to reduced or increased proteasome activity. Please be clear and precise as the direction of the changes observed. The same goes for the extension of conclusions made on measures of proteasome activity to mean activity of the UPS / protein breakdown. Specific examples are described within the minor comments section.
Minor comments:
Introduction: Nfe2l1 does not restore proteasome activity per se, but stimulates proteasome subunit biogenesis and thereby increases proteasome content. This would not necessarily influence activity, which also relies on the presence of substrate/ubiquitination.
Introduction: 'we investigate remodeling of the muscle UPS in obesity and define the role of Nfe2l1 as a new regulator of muscle biology'. This statement is an overreach, particularly seeing as a role for Nfe2l1 has already been described in skeletal muscle, albeit under a different context (ref. 29).
Results: "Of note, leptin levels in chow-fed animals were indifferent". I guess this is a typo? Should be 'different' not 'indifferent'.
Results: "These global changes are in line with the notion that UPS is activity is rewired and metabolism impacted by HFD feeding." Please use specific language to describe the changes you see.
Results: "The data supported the hypothesis that Nfe2l1 stimulates protein degradation via the proteasome, as the dominant lysine-linkage was the proteasome-targeting linkage K48, accounting for more than 86 % and being significantly higher in muscle of mKO mice compared to tissue of WT controls (Fig. 4G)." While it is clear that depleting a protein contributing to proteasome biogenesis would impair proteasome function, this would not be sufficient to say that Nfe2l1 promotes protein degradation via the proteasome. So far, there is no evidence that increasing Nfe2l1 increases protein degradation.
Figure 1L: What is the unit of measurement for gene expression?
Figure 2G: There appears to be significant freeze damage in H&E and SDH sections from Nfe2l1mKO mice. Perhaps you can find better representative images.
Results: "In summary, these results establish Nfe2l1 as an adaptive regulator of proteasomal activity and ubiquitylation in cultured myocytes". Why do these results establish Nfe2l1 as an 'adaptive' regulator? These are steady state conditions. Results so far would only indicate that Nfe2l1 controls proteasome subunit biogenesis in myocytes, which is well known in other cell types and has also been shown in skeletal muscle tissue.
Results: "The proteome showed many significantly regulated proteins and in general a higher protein load in the mKO condition (Fig. 4A), potentially caused by impaired proteasomal protein degradation." What is meant by a 'higher protein load'
Discussion: "Here, we show that proteasomal activity and management of ubiquitin levels in muscle is a regulated and critical process in obesity, as proteasome levels and function are increased in obesity." This is actually not shown. As Nfe2l1 KO mice do not become obese, it is unclear what role this increase plays under the conditions of obesity.
Discussion: "Interestingly, at the same time, total ubiquitin levels are largely unchanged, which suggests a dynamic recalibration of the rates of protein synthesis and degradation, including the processes necessary for ubiquitylation and its targets". The authors seem to be interpreting ex vivo proteasome activity assays as a readout of protein breakdown rates in vivo. These Proteasome activity assays are only a readout of proteasome content, not activity, since substrate entry into the 26S proteasome is tightly controlled by its cap structure. Ex vivo, substrates able to independently access the inside of the 20S proteasome (and hence the active protease sites) are provided in abundance.
Discussion: "However, overall proteasomal activity was lower and ubiquitin levels higher, indicating the predominant role of Nfe2l1 determining rates of UPS in myocytes." The reduction in activity was not so strong that it could be considered predominant. Furthermore, proof is only provided for Nfe2l1 regulating proteasome content... not rates of UPS breakdown, which also relies on the ubiquitination part of the system.
Discussion: "There seems to be profound crosstalk between proteostatic mechanisms in muscle, as we found in the proteome of Nfe2l1 mKO muscle that autophagy pathways are markedly upregulated, including p62 and LC3B levels (Extended Data Fig. 1B-C)". This should be first introduced into the results section.
Discussion: "Uncoupling of mitochondria and loss of mitochondrial membrane potential in myocytes are associated with the induction of FGF21 (33), a myokine that is implicated in regulating energy metabolism. We find that FGF21 and GDF15 expression were higher in muscle of mKO mice compared to WT controls, and for GDF15 also plasma levels were elevated (Extended Data Fig. 2A-D)." This should be included in results section.
Significance
General assessment: after identifying increased proteasome activity and an associated increase in Nfe2l1 expression in the muscle of obese mice, this work provides strong evidence that muscle fiber Nfe2l1 expression is necessary for muscle fiber development / homeostasis, with wide ranging effects of muscle fiber Nfe2l1 KO, including on body and muscle size, fiber type composition and mitochondrial content and function. On the other hand, muscle fiber Nfe2l1 KO mice fail to become obese, making it hard to draw conclusions on the role of increased Nfe2l1 in the muscle of obese mice.
Advance: This study complements recent work showing a role for increased Nfe2l1 expression in maintaining proteostasis under a different proteostatic challenge, and suggests a role for muscle Nfe2l1 in response to obesity.
Audience: This study is likely to be of interest to readers interested in proteostasis, the UPS and muscle biology.
Expertise: Muscle proteostasis and aging.
