Marquez J et al. (2025), Polyamine metabolism is dysregulated in COXFA4-...

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HGG Adv 2025 Feb 10;62:100418. doi: 10.1016/j.xhgg.2025.100418.

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Polyamine metabolism is dysregulated in COXFA4-related mitochondrial disease.

Marquez J , Viviano S , Beckman E , Thies J , Friedland-Little J , Lam CT , Deniz E , Shelkowitz E .

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Most of the chemical energy that organisms rely on to support cellular function is generated through oxidative phosphorylation, a metabolic pathway in which electron donors NADH and FADH are oxidized through a series of successive steps to generate adenosine triphosphate. These redox reactions are orchestrated by a series of five protein complexes that sit within the mitochondrial membrane. Deficiency of cytochrome c oxidase, the fourth of these complexes, is a recognized cause of mitochondrial disease. COXFA4 encodes one of the protein subunits of cytochrome c oxidase, and variants in COXFA4 have recently been reported in individuals with a range of symptoms. These symptoms can include feeding difficulties, poor growth, cardiomyopathy, Leigh or Leigh-like disease, and neurodevelopmental delay, although these symptoms vary widely between individuals. However, a mechanistic understanding of the connection between COXFA4 loss and these varied disease manifestations is lacking. Using animal modeling in Xenopus, we explored the ramifications of coxfa4 loss of function on the early developing heart. We then conducted a hypothesis naive analysis of cellular gene expression in the context of COXFA4 deletion and discovered a downstream deficiency in the ornithine decarboxylase pathway. Small-molecule-based modulation of the ornithine decarboxylase pathway in our model modified the extent of disease, including improvement of cardiac function. Our findings point to a mechanism by which COXFA4 dysfunction leads to tissue-specific disease.

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Species referenced: Xenopus tropicalis
Genes referenced: azin1 cox6a2 ndufa4 oaz1 odc1

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Phenotypes: Xtr Wt + ndufa4 CRISPR (Fig 2. BC) [+]

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	Figure 1. Homozygous deletion of COXFA4 leads to Mitochondrial Disease. 3 siblings from one family presented with either hypertrophic cardiomyopathy or developmental delays. (A) Pedigree showing 3 affected siblings with * indicating individuals with sequencing completed as part of a trio exome. (B) Images of individual IV-3 at age 7 and 13 years and individual IV-6 at age 4 months and 9 years. (C) MRI imaging of individual IV-3 demonstrating thin corpus collosum (white arrow) and hyperintensities near the grey-white matter junction (white arrowheads). (D) Echocardiogram images from short axis view of individual IV-6 heart demonstrating early cardiac hypertrophy improving over time
	Figure 2. Phenotypic assessment of coxfa4 loss of function in Xenopus reveals craniofacial and cardiac dysmorphology. 1-cellstage embryos were injected with either Cas9, Cas9 along with a guide RNA targeting coxfa4, or Cas9 along with a guide RNA targeting coxfa4 along with COXFA4 mRNA and phenotypically assessed at stage 45. (A) Schematic of injection at the 1-cell stage and phenotyping at Stage 45. (B) Schematic of fractional shortening measurements and measurements of 3 replicates of stage 45 Cas9 injected controls (n = 66), coxfa4 crispant (n = 111) and COXFA4 rescue embryos (n = 15) cardiac function imaged with OCT. (C) Cartilage schematic along with representative images and measurements of 3 replicates of stage 45 Cas9 injected controls (n = 15), coxfa4 crispant (n = 15) and COXFA4 rescue embryo Alcian blue stained craniofacial cartilage. (D) In-gel activity of complex IV and complex I for control and coxfa4 crispant embryos with densitometry measurements across 3 replicates. * indicates p < 0.05 by two-tailed T-test. Error bars indicate standard deviation. A: anterior, CR: CRISPR L: left, P: posterior, R: right.
	Figure 3. Multimodal expression analysis in COXFA4 deficient cells and Xenopus uncovers ODC deficits. RNA sequencing in iPSCs and RT-qPCR in Xenopus demonstrated decreased expression of multiple components of the ODC pathway. (A) Plot of mean change in expression levels from 3 biological replicates of bulk RNA sequencing in iPSCs deficient in COXFA4 compared to controls with top 20 hits by Manhattan distance delineated. (B) Reactome pathway overrepresentation analysis showing all overrepresented pathways with false discovery rate less than 0.05 in downregulated genes based on RNA sequencing. Count corresponds to number of encoded proteins identified for each pathway. (C) Plot of mean change in expression levels from 3 biological replicates of bulk RNA sequencing in iPSCs deficient in COXFA4 compared to controls with a subset of ODC related hits delineated. (D) Plot of relative expression of odc1, azin1, and oaz1 in Cas9 injected controls (n = 30), coxfa4 crispant (n = 30) and COXFA4 rescue embryos (n = 30) demonstrating decreased expression of these ODC components in coxfa4 crispant embryos. (E) Plot of relative polyamine abundance based on fluorometric probe binding in Cas9 injected controls (n = 40), coxfa4 crispant (n = 40) and COXFA4 rescue embryos (n = 40) demonstrating decreased abundance in coxfa4 crisspant embryos, with shapes indicating individual replicate values. * indicates p < 0.05 by two-tailed Ttest. Error bars indicate standard deviation.
	Figure 4. Depletion of ODC pathway components may lead to depletion of polyamines. Imbalance of polyamine synthesis may play a role in pathogenesis of disease stemming from COXFA4 loss of function. (A) ODC pathway depicting typical role in polyamine production. (B) Hypothesized imbalance in ODC pathway due to depleted pathway components as suggested by RNA sequencing and expression in Xenopus models. The ODC pathway is disrupted by 1) decreased ODC and AZIN relative to OAZ resulting in increased proteasomal degradation of ODC and 2) Increased OAZ inhibition of polyamine transport due to excess OAZ relative to ODC and AZIN. AZIN: antizyme inhibitor, OAZ: ornithine carboxylase antizyme, ODC: ornithine decarboxylase, SPDS: spermidine synthase, SPMS: spermine synthase
	Figure 5. Modifying the ODC pathway influences cardiomyopathy severity. Supplementation of polyamines or expression of COXFA4L2 improves cardiomyopathy while inhibition of ODC exacerbates cardiomyopathy. (A) Measurements of 3 replicates of stage 45 coxfa4 crispant embryos raised with polyamine supplementation (n = 49) and coxfa4 crispant embryos injected with COXFA4L2 mRNA (n=15). Data for Cas9 injected controls (n = 66) and coxfa4 crispant (n = 111) embryos presented for comparison. (B) Kaplan-Meier survival analysis of 3 replicates Cas9 injected embryos (n = 150), coxfa4 crispant embryos (n = 150), and coxfa4 crispant embryos raised with polyamine supplementation (n = 150). Line indicates average survival over 3 replicates and shaded region indicates standard deviation. (C) In-gel activity of complex IV in coxfa4 crispant embryo hearts, coxfa4 crispant embryo hearts with polyamine supplementation, coxfa4 crispant embryo hearts exposed to allicin, coxfa4 crispant embryo hearts injected with COXFA4L2, and embryo hearts injected with Cas9 only as a control with densitometry measurements across 3 replicates. ANOVA P value was calculated as a comparison of coxfa4 CRISPR injected embryos to all other groups. Post hoc Tukey test of multiple comparisons was carried out with * indicating p < 0.05. Error bars indicate standard deviation. (C) Protein alignment of COXFA4 and COXFA4L2 demonstrating similarities at the amino acid level in red boxes. (D) Comparison of protein structures of COXFA4 and COXFA4L2 and expression patterns for Coxfa4 and Coxfa4l2 during murine development.
	Figure 6. Single cell expression data support variable tissue specific function of Coxfa4 and Coxfa4l2. Expression of Coxfa4l2 is more spatiotemporally restricted in cardiomyocytes than in hepatocytes when compared to expression of Coxfa4 in these same tissues. (A) 3- dimensional uniform manifold approximation and projection of murine hepatocytes from E8.75 to E16 with corresponding plots of Coxfa4 and Coxfa4l2 expression. (B) 3-dimensional uniform manifold approximation and projection of murine cardiomyocytes from E8.5 to E16 with corresponding plots of Coxfa4 and Coxfa4l2 expression.
	Figure 1. Homozygous deletion of COXFA4 leads to mitochondrial diseaseThree siblings from 1 family presented with either hypertrophic cardiomyopathy or developmental delays.(A) Pedigree showing 3 affected siblings, with asterisks indicating individuals with sequencing completed as part of a trio exome.(B) Images of individual IV-3 at ages 7 and 13 years and individual IV-6 at ages 4 months and 9 years.(C) MRI imaging of individual IV-3 demonstrating thin corpus callosum (arrow) and hyperintensities near the gray-white matter junction (arrowheads).(D) Echocardiogram images from short axis view of the heart of individual IV-6 demonstrating early cardiac hypertrophy improving over time.
	Figure 2. Phenotypic assessment of coxfa4 loss of function in Xenopus reveals craniofacial and cardiac dysmorphologyOne-cell-stage embryos were injected with either Cas9, Cas9 along with a guide RNA targeting coxfa4, or Cas9 along with a guide RNA targeting coxfa4 with COXFA4 mRNA and phenotypically assessed at stage 45.(A) Schematic of injection at the 1-cell stage and phenotyping at stage 45.(B) Schematic of fractional shortening measurements and measurements of 3 replicates of the cardiac function of stage 45 Cas9-injected controls (n = 66), coxfa4 crispant (n = 111), and COXFA4 rescue embryos (n = 15) imaged with OCT.(C) Cartilage schematic along with representative images and measurements of 3 replicates of stage 45 Cas9-injected controls (n = 15), coxfa4 crispant (n = 15), and COXFA4 rescue embryo Alcian blue-stained craniofacial cartilage.(D) In-gel activity of complex IV and complex I for control and coxfa4 crispant embryos with densitometry measurements across 3 replicates. Asterisk indicates p < 0.05 by 2-tailed t test.Error bars indicate SD. A, anterior; CR, CRISPR; L, left; ns, not significant; P, posterior; R, right.
	Figure 3. Multimodal expression analysis in COXFA4-deficient cells and Xenopus reveals ODC deficitsRNA sequencing (RNA-seq) in iPSCs and RT-qPCR in Xenopus demonstrated decreased expression of multiple components of the ODC pathway.(A) Plot of mean change in expression levels from 3 biological replicates of bulk RNA-seq in iPSCs deficient in COXFA4 compared to controls with top 20 hits by Manhattan distance delineated.(B) Reactome pathway overrepresentation analysis showing all overrepresented pathways with false discovery rate less than 0.05 in downregulated genes based on RNA-seq. Count corresponds to number of encoded proteins identified for each pathway.(C) Plot of mean change in expression levels from 3 biological replicates of bulk RNA-seq in iPSCs deficient in COXFA4 compared to controls with a subset of ODC-related hits delineated.(D) Plot of relative expression of odc1, azin1, and oaz1 in Cas9-injected controls (n = 30), coxfa4 crispant (n = 30), and COXFA4 rescue embryos (n = 30) demonstrating decreased expression of these ODC components in coxfa4 crispant embryos.(E) Plot of relative polyamine abundance based on fluorometric probe binding in Cas9-injected controls (n = 40), coxfa4 crispant (n = 40), and COXFA4 rescue embryos (n = 40) demonstrating decreased abundance in coxfa4 crispant embryos, with shapes indicating individual replicate values. Asterisk indicates p < 0.05 by 2-tailed t test.Error bars indicate SD.
	Figure 4. Depletion of ODC pathway components may lead to depletion of polyaminesImbalance of polyamine synthesis may play a role in pathogenesis of disease stemming from COXFA4 loss of function.(A) ODC pathway depicting typical role in polyamine production.(B) Hypothesized imbalance in ODC pathway due to depleted pathway components as suggested by RNA-seq and expression in Xenopus models. The ODC pathway is disrupted by (1) decreased ODC and AZIN relative to OAZ, resulting in increased proteasomal degradation of ODC and (2) increased OAZ inhibition of polyamine transport due to excess OAZ relative to ODC and AZIN.AZIN, antizyme inhibitor; OAZ, ornithine carboxylase antizyme; ODC, ornithine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase.
	Figure 5. Modifying the ODC pathway influences cardiomyopathy severitySupplementation of polyamines or expression of COXFA4L2 improves cardiomyopathy while inhibition of ODC exacerbates cardiomyopathy.(A) Measurements of 3 replicates of stage 45 coxfa4 crispant embryos raised with polyamine supplementation (n = 49) and coxfa4 crispant embryos injected with COXFA4L2 mRNA (n = 15). Data for Cas9-injected controls (n = 66) and coxfa4 crispant (n = 111) embryos presented for comparison.(B) Kaplan-Meier survival analysis of 3 replicates of Cas9-injected embryos (n = 150), coxfa4 crispant embryos (n = 150), and coxfa4 crispant embryos raised with polyamine supplementation (n = 150). Line indicates average survival over 3 replicates and shaded region indicates SD.(C) In-gel activity of complex IV in coxfa4 crispant embryo hearts, coxfa4 crispant embryo hearts with polyamine supplementation, coxfa4 crispant embryo hearts exposed to allicin, coxfa4 crispant embryo hearts injected with COXFA4L2, and embryo hearts injected with Cas9 only as a control with densitometry measurements across 3 replicates. ANOVA p value was calculated as a comparison of coxfa4 CRISPR injected embryos to all other groups. Post hoc Tukey test of multiple comparisons was carried out, with asterisk indicating p < 0.05. Error bars indicate SD.(D) Protein alignment of COXFA4 and COXFA4L2 demonstrating similarities at the amino acid level in red boxes.(E) Comparison of protein structures of COXFA4 and COXFA4L2 and expression patterns for Coxfa4 and Coxfa4l2 during murine development.
	Figure 6. Single-cell expression data support variable tissue-specific function of Coxfa4 and Coxfa4l2Expression of Coxfa4l2 is more spatiotemporally restricted in cardiomyocytes than in hepatocytes when compared to expression of Coxfa4 in the same tissues.(A) Three-dimensional uniform manifold approximation and projection of murine hepatocytes from embryonic day (E) 8.75 to E16, with corresponding plots of Coxfa4 and Coxfa4l2 expression.(B) Three-dimensional uniform manifold approximation and projection of murine cardiomyocytes from E8.5 to E16, with corresponding plots of Coxfa4 and Coxfa4l2 expression.