Upregulated expression of ubiquitin ligase TRIM21 promotes PKM2 nuclear translocation and astrocyte activation in experimental autoimmune encephalomyelitis

Reviewer #1 (Public Review):

Summary:

Yang, Hu et al. examined the molecular mechanisms underlying astrocyte activation and its implications for multiple sclerosis. This study shows that the glycolytic enzyme PKM2 relocates to astrocyte nuclei upon activation in EAE mice. Inhibiting PKM2's nuclear import reduces astrocyte activation, as evidenced by decreased proliferation, glycolysis, and inflammatory cytokine release. Crucially, the study identifies TRIM21 as pivotal in regulating PKM2 nuclear import via ubiquitination. TRIM21 interacts with PKM2, promoting its nuclear translocation and enhancing its activity, affecting multiple signaling pathways. Confirmatory analyses using single-cell RNA sequencing and immunofluorescence demonstrate TRIM21 upregulation in EAE astrocytes. Modulating TRIM21 expression in primary astrocytes impacts PKM2-dependent glycolysis and proliferation. In vivo experiments targeting this mechanism effectively mitigate disease severity, CNS inflammation, and demyelination in EAE.

The authors supported their claims with various experimental approaches, however, some results should be supported with higher-quality images clearly depicting the conclusions and additional quantitative analyses of Western blots.

Strength:

This study presents a comprehensive investigation into the function and molecular mechanism of metabolic reprogramming in the activation of astrocytes, a critical aspect of various neurological diseases, especially multiple sclerosis. The study uses the EAE mouse model, which closely resembles MS. This makes the results relevant and potentially translational. The research clarifies how TRIM21 regulates the nuclear import of PKM2 through ubiquitination by integrating advanced techniques. Targeting this axis may have therapeutic benefits since lentiviral vector-mediated knockdown of TRIM21 in vivo significantly reduces disease severity, CNS inflammation, and demyelination in EAE animals.

Weaknesses:

The authors reported that PKM2 levels are elevated in the nucleus of astrocytes at different EAE phases compared to cytoplasmic localization. However, Figure 1 also shows elevated cytoplasmic expression of PKM2. The authors should clarify the nuclear localization of PKM2 by providing zoomed-in images. An explanation for the increased cytoplasmic PKM2 expression should provided. Similarly, while PKM2 translocation is inhibited by DASA-58, in addition to its nuclear localization, a decrease in the cytoplasmic localization of PKM2 is also observed. This situation brings to mind the possibility of a degradation mechanism being involved when its nuclear translocation of PKM2 is inhibited.

In Figure 3D, the authors claim that PKM2 expression causes nuclear retention of STAT3, p65, and p50, and inhibiting PKM2 localization with DASA-58 suppresses this retention. The western blot results for the MOG-stimulated group show high levels of STAT3, p50, and p65 in nuclear localization. However, in the MOG and DASA-58 treated group, one would expect high levels of p50, p65, and STAT3 proteins in the cytoplasm, while their levels decrease in the nucleus. These western blot results could be expanded. Additionally, intensity quantification for these results would be beneficial to see the statistical difference in their expressions, especially to observe the nuclear localization of PKM2.

The discrepancy between Figure 7A and its explaining text is confusing. The expectation from the knocking down of TRIM21 is the amelioration of activated astrocytes, leading to a decrease in inflammation and the disease state. The presented results support these expectations, while the images showing demyelination in EAE animals are not highly supportive. Clearly labeling demyelinated areas would enhance readers' understanding of the important impact of TRIM21 knockdown on reducing the disease severity.

Reviewer #2 (Public Review):

This study significantly advances our understanding of the metabolic reprogramming underlying astrocyte activation in neurological diseases such as multiple sclerosis. By employing an experimental autoimmune encephalomyelitis (EAE) mouse model, the authors discovered a notable nuclear translocation of PKM2, a key enzyme in glycolysis, within astrocytes.

Preventing this nuclear import via DASA 58 substantially attenuated primary astrocyte activation, characterized by reduced proliferation, glycolysis, and inflammatory cytokine secretion.
Moreover, the authors uncovered a novel regulatory mechanism involving the ubiquitin ligase TRIM21, which mediates PKM2 nuclear import. TRIM21 interaction with PKM2 facilitated its nuclear translocation, enhancing its activity in phosphorylating STAT3, NFκB, and c-myc. Single-cell RNA sequencing and immunofluorescence staining further supported the upregulation of TRIM21 expression in astrocytes during EAE.

Manipulating this pathway, either through TRIM21 overexpression in primary astrocytes or knockdown of TRIM21 in vivo, had profound effects on disease severity, CNS inflammation, and demyelination in EAE mice. This comprehensive study provides invaluable insights into the pathological role of nuclear PKM2 and the ubiquitination-mediated regulatory mechanism driving astrocyte activation.

The author's use of diverse techniques, including single-cell RNA sequencing, immunofluorescence staining, and lentiviral vector knockdown, underscores the robustness of their findings and interpretations. Ultimately, targeting this PKM2-TRIM21 axis emerges as a promising therapeutic strategy for neurological diseases involving astrocyte dysfunction.

While the strengths of this piece of work are undeniable, some concerns could be addressed to refine its impact and clarity further; as outlined in the recommendations for the authors.

Reviewer #3 (Public Review):

Summary:

Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme in glycolysis and its translocation to the nucleus in astrocytes in various nervous system pathologies has been associated with a metabolic switch to glycolysis which is a sign of reactive astrogliosis. The authors investigated whether this occurs in experimental autoimmune encephalomyelitis (EAA), an animal model of multiple sclerosis (MS). They show that in EAA, PKM2 is ubiquitinated by TRIM21 and transferred to the nucleus in astrocytes. Inhibition of TRIM21-PKM2 axis efficiently blocks reactive gliosis and partially alleviates symptoms of EAA. Authors conclude that this axis can be a potential new therapeutic target in the treatment of MS.

Strengths:

The study is well-designed, controls are appropriate and a comprehensive battery of experiments has been successfully performed. Results of in vitro assays, single-cell RNA sequencing, immunoprecipitation, RNA interference, molecular docking, and in vivo modeling etc. complement and support each other.

Weaknesses:

Though EAA is a valid model of MS, a proposed new therapeutic strategy based on this study needs to have support from human studies.

Reviewer #4 (Public Review):

Summary:

The authors report the role of the Pyruvate Kinase M2 (PKM2) enzyme nuclear translocation as fundamental in the activation of astrocytes in a model of autoimmune encephalitis (EAE). They show that astrocytes, activated through culturing in EAE splenocytes medium, increase their nuclear PKM2 with consequent activation of NFkB and STAT3 pathways. Prevention of PKM2 nuclear translocation decreases astrocyte counteracts this activation. The authors found that the E3 ubiquitin ligase TRIM21 interacts with PKM2 and promotes its nuclear translocation. In vivo, either silencing of TRIM21 or inhibition of PKM2 nuclear translocation ameliorates the severity of the disease in the EAE model.

Strengths:

This work contributes to the knowledge of the complex action of the PKM2 enzyme in the context of an autoimmune-neurological disease, highlighting its nuclear role and a novel partner, TRIM21, and thus adding a novel rationale for therapeutic targeting.

Weaknesses:

Despite the relevance of the work and its goals, some of the conclusions drawn would require more thorough proof:

I believe that the major weakness is the fact that TRIM21 is known to have per se many roles in autoimmune and immune pathways and some of the effects observed might be due to a PKM2-independent action. Some of the experiments to link the two proteins, besides their interaction, do not completely clarify the issue. On top of that, the in vivo experiments address the role of TRIM21 and the nuclear localisation of PKM2 independently, thus leaving the matter unsolved.

Some experimental settings are not described to a level that is necessary to fully understand the data, especially for a non-expert audience: e.g. the EAE model and MOG treatment; action and reference of the different nuclear import inhibitors; use of splenocyte culture medium and the possible effect of non-EAE splenocytes.

The statement that PKM2 is a substrate of TRIM21 ubiquitin ligase activity is an overinterpretation. There is no evidence that this interaction results in ubiquitin modification of PKM2; the ubiquitination experiment is minimal and is not performed in conditions that would allow us to see ubiquitination of PKM2 (e.g. denaturing conditions, reciprocal pull-down, catalytically inactive TRIM21, etc.).