Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism

Homozygous loss-of-function variants in Sucla2 result in developmental lethality.

“homozygous loss-of-function variants in Sucla2 resulting in developmental lethality.7,14”

• cites [7] Gut, 2020, SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease, Nat. Commun. sourceSupported
• cites [14] Donti, 2014, Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion, Dis. Model. Mech. sourceSupported

The only reported mouse model of constitutive Sucla2 deficiency exhibits embryonic lethality.

“Currently, the only reported mouse model of constitutive Sucla2 deficiency exhibits embryonic lethality, demonstrating a continued need for an adult in vivo model of SCS deficiency.14”

• cites [14] Donti, 2014, Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion, Dis. Model. Mech. sourcePartly supported

  The Results section reports that homozygous Sucla2 mutant embryos died late in gestation, with no live-born pups. The cited paper establishes embryonic lethality in its model, but the passages do not establish that it was the only reported mouse model of constitutive Sucla2 deficiency. No page number is available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The succinyllysine antibody used in this study exhibits little to no cross-reactivity to other acyl-species.

“the succinyllysine antibody used within this study has been shown to exhibit little to no cross-reactivity to these other acyl-species41”

• cites [41] Head, 2022, Aberrant methylmalonylation underlies methylmalonic acidemia and is attenuated by an engineered sirtuin, Sci. Transl. Med. sourceCould not verify

  The Discussion describes weak or absent cross-reactivity of a methylmalonyllysine antibody with other acylated lysines. The Results describe an anti-malonyllysine antibody that does not cross-react with succinyllysine. Neither passage establishes the cross-reactivity of a succinyllysine antibody; searches for that antibody and its specificity found no such statement in our copy. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

2,176 peptides from 640 proteins have a 2-fold or greater abundance in the SCS-deficient cortex.

“Of the hypersuccinylated proteome, 2,176 peptides from 640 proteins had a 2-fold or greater abundance in the SCS-deficient cortex (Figure 3E, red).”

Nearly all enriched pathways from the less-active TF-TG relationships were related to neuronal functions, including neuronal signaling, neurotransmitter release, and nervous system development.

“Nearly all enriched pathways were related to neuronal functions, including neuronal signaling, neurotransmitter release, and nervous system development”

Not all hypersuccinylated metabolic enzymes demonstrate functional changes in activity.

“In addition, not all hypersuccinylated metabolic enzymes demonstrate functional changes in activity.”

Forebrain-specific KO of Sucla2 results in complete survival of the KO mice.

“The spatial restriction of Sucla2 deficiency resulted in complete survival of the KO mice (Table S1)”

There is a positive correlation between mutant-specific changes in accessibility of chromatin promoter regions and differentially expressed genes.

“a positive correlation (p < 0.0001) between mutant-specific changes in accessibility of chromatin promoter regions and differentially expressed genes is observed (Figures 5G and 5H).”

Succinyl-CoA levels are increased by 50% in mutant cerebral cortex compared with controls.

“results indicate a 50% increase in mutant cellular concentrations of succinyl-CoA compared with controls (Figure 2D; Data S1).”

Of the 20 most affected succinylation marks, 19 are in mitochondrial proteins.

“Of the 20 most affected succinylation marks (with highest KO:WT fold change [FC]), 19 are in mitochondrial proteins”

On a proteomic level, 121 of the 145 differentially expressed mitochondrial proteins are upregulated in the mutant cortex.

“On a proteomic level, 121 of the 145 differentially expressed mitochondrial proteins are upregulated in the mutant cortex (Data S3).”

Mitochondrial complex I activity is reduced 40% on average in the mutant cortex.

“mitochondrial complex I activity is reduced 40% on average (Figure 4E).”

ATAC-seq revealed genotype-specific clustering for changes in the chromatin landscape of both enhancer and promoter regions.

“Multidimensional scaling of both datasets revealed stark genotype-specific clustering for changes in the chromatin landscape of both enhancer and promoter regions (Figures 5A and 5C).”

mtDNA depletion is not observed in the Sucla2-deficient mouse forebrain.

“mtDNA depletion is not observed in the Sucla2-deficient mouse forebrain.”

The distributions of subcellular localizations are the same for all succinylated proteins identified regardless of enrichment.

“the distributions of subcellular localizations are the same for all succinylated proteins identified regardless of enrichment (Figure 3G)”

Increased succinylation of mitochondrial complex I correlates with functionally significant deficiency of the enzymatic complex without global loss in protein abundance.

“increased succinylation of mitochondrial complex I correlates with functionally significant deficiency of the enzymatic complex without global loss in protein abundance (Figures 4E, 4F, and S4).”

• Also stated in: § Limitations of the study · boosted

  “while the data presented clearly associate hypersuccinylation of mitochondrial complex I with functionally reduced enzymatic function”

A large proportion of highly hypersuccinylated proteins with multiple succinylation sites are mitochondrial localized.

“Furthermore, a large proportion of highly hypersuccinylated proteins with multiple succinylation sites are mitochondrial localized (Figure 3H)”

Increased mutant abundance of at least one succinylation mark was detected on each of the five major histone proteins (H1, H2A, H2B, H3, and H4) in the mutant cortex.

“Increased mutant abundance of at least one succinylation mark was detected on each of the five major histone proteins (H1, H2A, H2B, H3, and H4) in the mutant cortex (Figure 5I).”

Targeted metabolomic analysis demonstrates altered amino acid and intermediate energy metabolism in the model.

“Targeted metabolomic analysis demonstrating altered amino acid and intermediate energy metabolism provide additional evidence of altered metabolism in the model (Figures 2C–2F).”

Nearly half of the identified SCS-dependent changes in the succinylome are localized to mitochondrial proteins.

“with nearly half of the identified SCS-dependent changes in the succinylome localized to mitochondrial proteins”

Sucla2 gene expression is reduced by 68% in the mutant cerebral cortex compared with wild-type controls.

“molecular validation via qPCR confirmed a 68% reduction in Sucla2 gene expression in the mutant cerebral cortex when compared with wild-type (WT) controls (Figure 1B).”

There are marked elevations in methylmalonic, succinyl (C4-DC), and propionyl (C3) acylcarnitines in the mutant cortex.

“marked elevations in methylmalonic and succinyl (C4-dicarboxylic of C4-DC) and propionyl (C3) acylcarnitines were observed (Figure 2F).”

Hypersuccinylation of complex V and citrate synthase is not associated with changes in enzyme activities.

“hypersuccinylation of complex V (CV) and TCA cycle enzyme citrate synthase (CS) are not associated with changes in enzyme activities (Figures S3E and S3F).”

There is a 14-fold increase in methylmalonic acid concentration in Sucla2-deficient cortex compared with WT controls.

“there is a 14-fold increase in the concentration of methylmalonic acid (MMA) measured in whole-cell lysates of Sucla2-deficient cortex compared with WT controls (Figure 2C; Data S1).”

The amino acids with significant changes in steady-state concentrations are directly involved in TCA cycle metabolism.

“Interestingly, the amino acids with significant changes in steady-state concentrations are directly involved in TCA cycle metabolism.”

92% of quantified succinylated peptides are significantly hypersuccinylated in the mutant cortex.

“92% of the quantified succinylated peptides were significantly (p < 0.05) hypersuccinylated in the mutant cortex (Figure 3E, blue and red).”

Forebrain-specific KO of Sucla2 results in decreased protein expression of both SUCLG1 and SUCLG2 at levels comparable to SUCLA2 loss.

“western blot analysis indicates that forebrain-specific KO of Sucla2 results in decreased protein expression of both SUCLG1 and SUCLG2 at levels comparable to SUCLA2 loss (Figures 1C and 1D).”

Four of the 13 hypersuccinylated lysines in NDUFV1 are among the most significantly enriched succinylation sites within the Sucla2-deficient cortex.

“4 of the 13 hypersuccinylated lysines in NDUFV1 are among the most significantly enriched succinylation sites within the Sucla2-deficient cortex (Figure 4A).”

Succinylated lysine is increased 100-fold in Sucla2 mutant cortex by Western blot analysis.

“This hypothesis was confirmed via Western blot analysis showing a 100-fold increase of succinylated lysine in Sucla2 mutant cortex (Figures 3A, 3B, and S2).”

The present research presents a viable adult mouse model of forebrain-specific homozygous Sucla2 deficiency.

“the present research presents a viable adult mouse model of forebrain-specific homozygous Sucla2 deficiency.”

RNA-seq differential expression analysis shows a significant change in expression for 13% of the 24,421 total mapped genes.

“The DE analysis shows a significant (FDR < 0.05) change in expression for 13% of the 24,421 total mapped genes, clearly distinguishing mutant cortex from controls (Data S6; Figures 5E and 5F).”

Succinylated proteins are enriched for mitochondrial localization.

“Succinylated proteins are enriched for mitochondrial localization (p < 0.0001, Figure S2C)”

There is an 80%-90% reduction of the entire SCS complex in the Sucla2−/− cerebral cortex.

“These data demonstrate significant post-transcriptional downregulation leading to 80%–90% reduction of the entire SCS complex in the Sucla2−/− cerebral cortex.”

Total SCS activity is reduced by 58% in the mutant cortex.

“providing further evidence of significant reduction (58%) of total SCS activity.”

Both mitochondrial and nuclear localized sirtuins with desuccinylase activity, SIRT5 and SIRT7, are upregulated.

“This idea is further evidenced by upregulation of both the mitochondrial and nuclear localized sirtuins with desuccinylase activity, SIRT5 and SIRT7 (Table S4).”

About 60% of the genes encoding subunits of ETC complexes are significantly upregulated in the mutant cortex.

“we see about 60% of the genes encoding subunits of ETC complexes are significantly (FDR < 0.05) upregulated in the mutant cortex (Figure S4)”

The transcription factors with the largest reduction in TF-TG regulatory activities in the Sucla2 mutant cortex include members of the EGR and POU3 TF families.

“the TFs that had the largest reduction in TF-TG regulatory activities in the Sucla2 mutant cortex include members of the EGR and POU3 TF families”

Pathway analysis of genes with reduced expression revealed that neuronal pathways including nervous system development are downregulated in the SCS-deficient mice.

“pathway analysis of genes with reduced expression revealed that neuronal pathways including nervous system development are downregulated in the SCS-deficient mice”

The skewing toward more closed chromatin is accompanied by computational evidence for an overall loss in neuronal transcriptional networks in the mutant forebrain.

“the skewing toward more closed chromatin is accompanied by computational evidence for an overall loss in neuronal transcriptional networks in the mutant forebrain (Figure 5L).”

mtDNA depletion is not present within the SUCLA2-deficient cortex.

“mtDNA depletion is not present within the SUCLA2-deficient cortex”

GO enrichment analysis of upregulated genes within the Sucla2 mutant cortex revealed increased metabolic pathways.

“while the GO enrichment analysis of upregulated genes within the Sucla2 mutant cortex revealed increased metabolic pathways”

The functional deficit in complex I activity provides further evidence that hypersuccinylation is associated with ETC dysfunction in the context of SCS deficiency.

“Therefore, the functional deficit in complex I activity provides further evidence that hypersuccinylation is associated with ETC dysfunction in the context of SCS deficiency.”

The mechanisms for SCS-related changes in the succinylome occur across the cell.

“suggesting that the mechanisms for SCS-related changes in the succinylome occur across the cell.”

Hypersuccinylation has a significant effect on mitochondrial and metabolic processes.

“suggesting that hypersuccinylation has a significant effect on mitochondrial and metabolic processes.”

The conditional KO model allows for in vivo investigations of metabolic and molecular perturbations of Sucla2 deficiency without the confounding influence of OXPHOS defects caused by mtDNA depletion.

“this conditional KO model allows for in vivo investigations of metabolic and molecular perturbations of Sucla2 deficiency without the confounding influence of OXPHOS defects caused by mtDNA depletion.”

PECA analysis suggests an overall loss of transcriptional regulation in the Sucla2 KO model.

“suggesting an overall loss of transcriptional regulation in the Sucla2 KO model, which coincides with a skewing toward less-accessible promoter regions (Figure 5D).”

Accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex.

“Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex.”

Respiratory complex dysfunction is associated with severe hypersuccinylation.

“These results demonstrate a respiratory complex dysfunction associated with severe hypersuccinylation.”

Increased succinylation within this model is likely reliant on perturbations in the steady-state levels of succinyl-CoA generated by the TCA cycle.

“the increased succinylation within this model is likely reliant on perturbations in the steady-state levels of succinyl-CoA generated by the TCA cycle.”

Succinylation can lead to functional changes in enzyme activity in an enzyme-specific manner.

“Therefore, succinylation can lead to functional changes in enzyme activity. The changes occur in an enzyme-specific manner, however, indicating that the effect of succinylation on protein function is complex”

Knockdown of Sucla2 by neuron-specific cre-recombination leads to SCS loss within the neurons of the forebrain.

“These data suggest that the knockdown of Sucla2 by neuron-specific cre-recombination16 leads to SCS loss within the neurons of the forebrain.”

Protein succinylation plays a significant role in the pathogenesis of SCS deficiency.

“Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.”

Inhibition of the N-module may be a likely mechanism of complex I deficiency.

“These data suggest that inhibition of the N-module may be a likely mechanism of complex I deficiency; however, identifying the exact mechanism requires further investigation.”

The metabolic profile of the SCS-deficient model mirrors the molecular hallmarks of human SCS deficiency, including methylmalonic aciduria and elevations of C3 and C4-DC acyl-carnitines.

“the metabolic profile of the SCS-deficient model (Figures 2C and 2F) mirrors the molecular hallmarks of human SCS deficiency, including methylmalonic aciduria and elevations of C3 and C4-DC acyl-carnitines.13,20,34”

• cites [13] Carrozzo, 2007, SUCLA2 mutations are associated with mild methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness, Brain sourceNot checked — no full text of the source
• cites [20] Ostergaard, 2007, Mitochondrial encephalomyopathy with elevated methylmalonic acid is caused by SUCLA2 mutations, Brain sourceNot checked — no full text of the source
• cites [34] Jaberi, 2013, The novel mutation p.Asp251Asn in the beta-subunit of succinate-CoA ligase causes encephalomyopathy and elevated succinylcarnitine, J. Hum. Genet. sourceNot checked — no full text of the source

The data suggest the possibility of increased mitochondrial biogenesis within the Sucla2 mutant cortex.

“These data, along with the increased mtDNA content (Figure 2B), suggest the possibility of increased mitochondrial biogenesis within the Sucla2 mutant cortex.”

Changes in the transcriptome resulting from SCS deficiency appear to result from a complex and dynamic interaction of multiple regulatory mechanisms.

“changes in the transcriptome resulting from SCS deficiency appear to result from a complex and dynamic interaction of multiple regulatory mechanisms that require further study.”

Evidence suggests that increased succinylation is involved in the pathogenesis of SCS deficiency.

“Evidence suggests that increased succinylation is involved in the pathogenesis of SCS deficiency.”

The data presented in this study were collected within the whole cortex, so the contribution of and consequences to other cell types within the Sucla2 mutant forebrain cannot be specifically assessed with the datasets described.

“this study is limited in that the data presented herein were collected within the whole cortex. Therefore, the contribution of and consequences to other cell types within the Sucla2 mutant forebrain cannot be specifically assessed with the datasets described.”

In this model, hypersuccinylation is associated, at least in part, to perturbations in cellular energy metabolism.

“In this model, hypersuccinylation is associated, at least in part, to perturbations in cellular energy metabolism.”

Loss of SCS has significant consequences on neuronal cell health.

“these results suggest that loss of SCS has significant consequences on neuronal cell health.”

This mouse model presents forebrain-specific SUCLA2 deficiency with metabolic, biochemical, and functional hallmarks reflective of clinical phenotypes in humans.

“Ultimately, this mouse model presents forebrain-specific SUCLA2 deficiency with metabolic, biochemical, and functional hallmarks reflective of clinical phenotypes in humans”

• AssumptionRests on an assumption: Pathogenic variants in SCS cause a severe mitochondrial disorder for which the pathomechanisms are incompletely understood.

SCS loss may also affect neuronal pathways directly involved in encephalopathic pathogenesis.

“suggesting that, in addition to metabolic dysfunction, SCS loss may also affect neuronal pathways directly involved in encephalopathic pathogenesis (Data S4).”

Remodeling of the chromatin landscape and an altered transcriptomic profile links Sucla2 mutations to loss of neuronal transcriptional regulation.

“remodeling of the chromatin landscape and an altered transcriptomic profile links Sucla2 mutations to loss of neuronal transcriptional regulation, revealing another mechanism for potential neuronal dysfunction in SCS deficiency.”