Molecular Genetics Of Heritable Human Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The hallmarks of GSD-Ia are impaired glucose homeostasis and long-term risk of hepatocellular adenoma and carcinoma (HCA/HCC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a G6Pase-alpha-expressing, recombinant adeno-associated virus (rAAV) vector that expressed 3-63% of normal hepatic G6Pase-alpha activity (AAV mice) maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-alpha activity required to prevent tumor formation remained unknown. We now constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-alpha and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase- expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-alpha activity maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-alpha activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-alpha activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing 3% of normal hepatic G6Pase-alpha activity. The results establish the threshold of hepatic G6Pase-alpha activity required to prevent HCA/HCC and show that GSD-Ia mice harboring less than 2% of normal hepatic G6Pase-alpha activity are at risk of tumor development. The predominant subtypes of HCA in GSD-Ia are inflammatory HCA (IHCA, 52%) and beta-catenin-mutated HCA (bHCA, 28%). We have shown that the non-tumor-bearing (NT), rAAV vector-treated GSD-Ia mice (AAV-NT mice) expressing a wide range (0.9-63%) of normal hepatic G6Pase-alpha activity maintain glucose homeostasis and display physiologic features mimicking animals living under calorie restriction (CR). We now show that in AAV-NT mice, the signaling pathways of the CR mediators, AMPK and SIRT1are activated. AMPK/ SIRT1 inhibit the activity of STAT3 and NFB, the pro-inflammatory and cancer-promoting transcription factors. SIRT1 also inhibits cancer metastasis via increasing the expression of E-cadherin, a tumor suppressor, and decreasing the expression of mesenchymal markers. Consistently, in AAV-NT mice, hepatic levels of active STAT3 and NFB-p65 were reduced as were expression of mesenchymal markers, STAT3 targets, NFB targets, and beta-catenin targets, all of which were consistent with the promotion of tumorigenesis. AAV-NT mice also expressed increased levels of E-cadherin and fibroblast growth factor 21 (FGF21), targets of SIRT1, and beta-klotho, which can acts as a tumor suppressor. Importantly, treating AAV-NT mice with a SIRT1 inhibitor markedly reversed many of the observed anti-inflammatory/anti-tumorigenic signaling pathways. In summary, activation of hepatic AMPK/SIRT1 and FGF21/beta-klotho signaling pathways combined with down-regulation of STAT3/NFB-mediated inflammatory and tumorigenic signaling pathways can explain the absence of hepatic tumors in AAV-NT mice. The most severe complications in GSD-Ia are the development of HCA and HCC of unknown etiology. The global knock-out G6pc-/- mice die early, well before HCA/HCC can develop, making mechanism studies of HCA/HCC difficult. We therefore generated the liver-specific G6pc knock-out mice (L-G6pc-/-) that survive to adulthood and develop HCA. A recent report showed that G6Pase-alpha deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the underlying mechanism is unclear. Studies have also shown that autophagy-deficient mice develop HCA, suggesting that autophgy deficiency contributes to HCA development in GSD-Ia. We first employed the L-G6pc-/- mice to investigate the molecular mechanism underlying defective autophagy. We show that in mice, liver-specific knockout of G6Pase-alpha leads to downregulation of SIRT1 signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and FoxO family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-alpha-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-alpha deficiency results in activation of ChREBP, a lipogenic transcription factor, increased expression of PPAR-gamma, a lipid regulator, and suppressed expression of PPAR-alpha, a master regulator of fatty acid beta-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-alpha deficiency. Importantly, a rAAV vector-mediated restoration of hepatic G6Pase-alpha expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-alpha deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia. We further show that impaired SIRT1 signaling mediated by hepatic G6Pase-alpha deficiency inhibits the activity of PGC-1alpha, a master regulator of mitochondrial biogenesis and function. Hepatic SIRT1 overexpression increases PGC-1alpha activity and corrects mitochondrial dysfunction. The dysfunctional mitochondria in G6Pase-alpha deficiency results in a time-dependent accumulation of damaged mitochondria, a potential source of reactive oxygen species (ROS) that contribute to tumorgenesis. Collectively, these data show that G6Pase-alpha deficiency downregulates SIRT1-PGC-1alpha signaling, leading to accumulation of damaged mitochondria and increased ROS generation, contributing to HCA/HCC development in GSD-Ia. GSD-Ib, deficient in the G6PT, is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of HCA. Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. We therefore investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt-/-) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt-/- mice with G-CSF corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt-/- mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the beta2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil migration and adhesion via aberrant expression of beta2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib. The G6pt-/- mice manifest both the metabolic and myeloid dysfunction characteristic of human GSD-Ib. When left untreated, the G6pt-/- mice rarely survive weaning, reflecting the juvenile lethality seen in human patients. Studies have shown that the choice of transgene promoter can impact targeting efficiency, tissue-specific expression, and the level of immune respo