mTOR-dependent loss of PON1 secretion and antiphospholipid autoantibody production underlie autoimmunity-mediated cirrhosis in transaldolase deficiency

Abstract

Transaldolase deficiency predisposes to chronic liver disease progressing from cirrhosis to hepatocellular carcinoma (HCC). Transition from cirrhosis to hepatocarcinogenesis depends on mitochondrial oxidative stress, as controlled by cytosolic aldose metabolism through the pentose phosphate pathway (PPP). Progression to HCC is critically dependent on NADPH depletion and polyol buildup by aldose reductase (AR), while this enzyme protects from carbon trapping in the PPP and growth restriction in TAL deficiency. Although AR inactivation blocked susceptibility to hepatocarcinogenesis, it enhanced growth restriction, carbon trapping in the non-oxidative branch of the PPP and failed to reverse the depletion of glucose 6-phosphate (G6P) and liver cirrhosis. Here, we show that inactivation of the TAL-AR axis results in metabolic stress characterized by reduced mitophagy, enhanced overall autophagy, activation of the mechanistic target of rapamycin (mTOR), diminished glycosylation and secretion of paraoxonase 1 (PON1), production of antiphospholipid autoantibodies (aPL), loss of CD161⁺ NK cells, and expansion of CD38⁺ Ito cells, which are responsive to treatment with rapamycin in vivo. The present study thus identifies glycosylation and secretion of PON1 and aPL production as mTOR-dependent regulatory checkpoints of autoimmunity underlying liver cirrhosis in TAL deficiency.

Keywords: Aldose reductase; Autophagy; Cirrhosis; Glucose 6-phosphate; Hepatocarcinogenesis; Ito cell; Mechanistic target of rapamycin; Mitochondrial oxidative stress; Mitophagy; PON1; Paraoxonase 1; Pentose phosphate pathway; Rapamycin; Transaldolase; Tricarboxylic acid cycle; UDP-GlcNAc.

Figure 1.. Cirrhosis in TAL deficiency is resistant to inactivation of AR.

A, Detection of fibrosis in Gömöri-trichrome-stained liver tissues from TALKO and DKO mice. Pro-fibrotic Ito cells or fat-storing hepatic stellate cells are indicated with arrows in areas of higher magnification. B, Formation of vascular reticulum in livers of TALKO and DKO mice upon in situ perfusion via the inferior vena cava. C, Assessment of CD38⁺ in WT, TALKO, ARKO, and DKO mice by flow cytometry of non-hepatocyte fraction isolated after in situ liver perfusion. NAD levels were assessed in liver extracts in WT, TALKO, ARKO, and DKO mice by LC- MS/MS. D, Assessment of NK and NKT cells in WT, TALKO, ARKO, and DKO mice by flow cytometry of non-hepatocyte fraction isolated after in situ liver perfusion. E, Effect of heparin administration on hepatocyte yield and viability after in situ liver perfusion. Mice were injected with or without 5 U heparin per g of body weight in 100 μl of PBS prior to perfusion (61).

Figure 2.. The TALAR axis regulates expression of genes involved cirrhosis, mitochondrial metabolism and mTOR activation in the livers.

Five age-matched mice were used for each of four genotypes: WT, TALKO, ARKO, and DKO.. A, Concordant changes in expression of 193 genes by RNAseq analysis in TALKO and DKO livers relative to WT controls at false discovery rate (FDR) p value < 0.05. B, Chromosomal proximity of H19, Igf2, TALDO1, and ZNF143 in synthenic genomic loci in humans and mice. Upper panel, schematic mapping of H19, Igf2, TALDO1, and ZNF143 along human chromosome 11 and mouse chromosome 7. Lower panel, nucleotide positions of H19, Igf2, TALDO1, and ZNF143 along human chromosome 11 and mouse chromosome 7. C, Western blot detection of ZNF143, IGF2, FKBP2, Deptor, NDUFB8, SDHB, SLC25A1, and Drp1. Representative blots and bar charts of cumulative analysis of five mice per genotype are shown for each gene. *, two-tailed t-test p < 0.05.

Figure 3.. Regulation of mitochondrial homeostasis by the TAL-AR axis.

A, Immunofluorescence microscopy of mitochondria. Left panel, representative images colored by size and shape. Right panel, cumulative analyses represent mean ± SEM of 5 mice per genotype. *, p < 0.05 relative to WT. B, Electron microscopy of liver mitochondria. Left panel, formation of autophagosomes from mitochondrial membranes, also termed phagophores (autophagosomes/mitochondrion), are indicated by white arrows. Right panel, cumulative analysis of mitochondrial autophagosome formation. C, Western blot analysis of LC3-I and LC3-II isoforms and mTOR in purified mitochondrial and cytosol fractions of liver from WT, TALKO, ARKO, and DKO mice. Left panel, representative blots. Right panel, cumulative analysis of LC3-I, LC3-II, LC3-II/LC3-I, and p-mTOR^S2448 in 4–5 mice per genotype. *, p < 0.05 relative to WT.

Figure 4.. Diminished glycosylation of PON1 in TAL deficiency.

A, Western blot analysis of PON1 and PLTP expression in liver of WT, TALKO, ARKO, and DKO mice using five animals per genotype. Left panel, representative western blots. Each lysate was validated by expression of TAL and AR, using β-actin as loading control. Right panel, Bar charts show expression of top two isoforms and lowest molecular weight isoform of PON1 and PLTP relative to β-actin. *, p < 0.05 relative to WT. B, Western blot analysis of PON1 expression and paraoxonase activity in the serum of WT, TALKO, ARKO, and DKO mice. C, Detection of glycosylation substrates in the liver of WT, TALKO, ARKO, and DKO mice. *, p < 0.05 relative to WT; differences at p < 0.05 between other mouse strains are indicated by brackets. D, Schematic diagram of UDP- GlcNAc biosynthesis required for glycosylation of PON1. E, Mapping of N-glycosylation at position N253 in PON1 peptide “HANWTLTPLK” in WT liver extracts. High-energy collisional dissociation tandem mass spectroscopy (HCD-MS2) predominantly detected Man8GlcNAc2 with lesser amounts of Man9 and Man7 N-glycans. F, Treatment of hepatocyte lysates in vitro with peptide-N-glycosidase F, PNGase F. Western blot represents five experiments. Black arrows indicate glycosylated isoforms, while red arrow indicates non-glycosylated isoform.

Figure 5.. Rapamycin restrains autophagy and oxidative stress and reduces aPL production and Ito cell expansion in TALKO mice.

WT and TALKO mice were with rapamycin (3 mg/kg sc 3 times weekly) from 35 weeks of age for 10 weeks. A, Western blot analysis of LC3, p62 and NDUFS3 expression, and mTORC1 pathway activation in livers of WT, TALKO, ARKO and DKO mice. Representative western blots (left panels) and cumulative analysis of LC3-I, p62, NDUFS3, phosphorylated (p-mTOR) and total mTOR, and p4E-BP1 protein levels were been determined relative to β-actin loading controls (right panels) in age-matched WT, TALKO, ARKO, and DKO mice and compared to WT and TALKO mice treated with rapamycin. *, p < 0.05 relative to WT based on two-tailed t-test. B, Effect of in vivo rapamycin treatment on PON1 protein levels in the liver. C, Effect of in vivo rapamycin treatment on PON1 protein levels in the serum. D, Effect of in vivo rapamycin treatment on serum aPL, ACLA and anti-Apo-H, antibody levels and Ito cell counts in the liver. Analyses were performed in age-matched untreated control WT, TALKO, ARKO and DKO female mice and rapamycin-treated WT and TALKO female mice. *, p < 0.05 relative to WT based on two-tailed t-test.