Homozygous, PIZZ alpha(1)-antitrypsin (alpha(1)-AT) deficiency is associated with chronic liver disease and hepatocellular carcinoma resulting from the toxic effects of mutant alpha(1)-anti-trypsin Z (alpha(1)-ATZ) protein retained in the endoplasmic reticulum (ER) of hepatocytes. However, the exact mechanism(s) by which retention of this aggregated mutant protein leads to cellular injury are still unknown. Previous studies have shown that retention of mutant alpha(1)-ATZ in the ER induces an intense autophagic response in hepatocytes. In this study, we present evidence that the autophagic response induced by ER retention of alpha(1)-ATZ also involves the mitochondria, with specific patterns of both mitochondrial autophagy and mitochondrial injury seen in cell culture models of alpha(1)-AT deficiency, in PiZ transgenic mouse liver, and in liver from alpha(1)-AT-deficient patients. Evidence for a unique pattern of caspase activation was also detected. Administration of cyclosporin A, an inhibitor of mitochondrial permeability transition, to PiZ mice was associated with a reduction in mitochondrial autophagy and injury and reduced mortality during experimental stress. These results provide evidence for the novel concept that mitochondrial damage and caspase activation play a role in the mechanism of liver cell injury in alpha(1)-AT deficiency and suggest the possibility of mechanism-based therapeutic interventions.

Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2,000 Americans. The Z mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes and chronic liver injury. Autophagy is one of several proteolytic mechanisms activated to cope with this hepatocellular protein burden, and is likely important in disposal of the unique polymerized conformation of the a1AT mutant Z protein, which is thought to be especially injurious to the cell. Recent data indicates that rapamycin may more efficiently upregulate autophagy when given in weekly dose pulses, as compared to a daily regimen. Therefore, we evaluated the effect of rapamycin on PiZ mice, a well characterized model which recapitulates human a1AT liver disease. Daily dosing had no effect on autophagy, on accumulation of a1AT mutant Z protein, or on liver injury. Weekly dosing of rapamycin did increase autophagic activity, as shown by increased numbers of autophagic vacuoles. This was associated with reduction in the intrahepatic accumulation of a1AT mutant Z protein in the polymerized conformation. Markers of hepatocellular injury, including cleavage of caspase 12 and hepatic fibrosis, were also decreased. In conclusion, this is the first report of a successful in vivo method for reduction of intrahepatic a1AT mutant Z polymerized protein. Application of this finding may be therapeutic in patients with a1AT deficiency by reducing the intracellular burden of the polymerized, mutant Z protein and by reducing the progression of liver injury.

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2000 births. The Z mutation confers an abnormal conformation on the protein, resulting in an accumulation within the endoplasmic reticulum of hepatocytes rather than appropriate secretion. The accumulation of the mutant protein is strikingly heterogeneous within the liver. Homozygous ZZ children and adults have an increased risk of chronic liver disease, which is thought to result from this variable intracellular accumulation of the a1AT mutant Z protein. Previous reports have suggested that autophagy, mitochondrial injury, apoptosis, and other pathways may be involved in the mechanism of hepatocyte injury, although the interplay of these mechanisms in vivo is unclear. In this study, we examine a well-characterized in vivo model of a1AT mutant Z liver injury, the PiZ mouse, to better understand the pathways involved in this disease. The results show an increase in the stimulation of the apoptotic cascade in hepatocytes, the magnitude of which strongly correlates to the absolute amount of the a1AT mutant Z protein accumulated within the individual cell. Increases in apoptotic regulatory proteins are also detected. Conclusion: These data, combined with previous work, permit for the first time the construction of a hypothetical hepatocellular injury cascade for this disease involving mitochondrial injury, caspase activation, and apoptosis, which takes into account the heterogeneous nature of the mutant Z protein accumulation within the liver. Further development of this hypothetical cascade will focus future research on this and other metabolic liver diseases. T he genetic disease alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2000 births. 1 The Z mutation confers an abnormal conformation on the nascent polypeptide, resulting in an accumulation of the mutant protein within the endoplasmic reticulum (ER) of hepatocytes rather than the appropriate, highly efficient secretion of the wild-type (WT) protein. Homozygous, ZZ individuals have an increased risk of chronic liver disease and hepatocellular carcinoma resulting from this intracellular accumulation of the a1AT mutant Z protein.Studies of the a1AT mutant Z protein molecule have shown that the nascent polypeptide has a tendency to form unique protein homopolymers. 1,2 Although this loop-sheet insertion mechanism of a1AT mutant Z protein polymerization, in which the reactive site loop of one molecule inserts into a surface groove in a neighboring molecule, does not involve the formation of covalent bonds, physical-chemical studies of these polymers suggest that this conformation is highly favored. It is proposed and supported by some published data that the liver injury in humans with a1AT deficiency is directly related to the hepatic accumulation of the polymerized a1AT mutant Z protein. [3][4][5] Our laboratory and others have reported a series of studies that have begun to examine the mechani...

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

Alpha-1-antitrypsin (␣1AT) deficiency in its most common form is caused by homozygosity for the ␣1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant ␣1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative ␣1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff-positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized ␣1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of ␣1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic ␣1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of ␣1ATZ T he most common form of alpha-1-antitrypsin (␣1AT) deficiency is caused by homozygosity for the ␣1AT mutant Z gene. [1][2][3][4][5][6][7][8][9][10][11] The Z mutation confers an abnormal conformation on the nascent polypeptide, which is synthesized in the liver, resulting in accumulation within the endoplasmic reticulum (ER) of hepatocytes. ZZ individuals have an increased risk of chronic liver disease and hepatocellular carcinoma resulting from this intracellular accumulation of mutant ␣1ATZ protein.Studies of the mutant ␣1ATZ protein molecule have shown that the nascent polypeptide has the tendency to form unique protein homopolymers. 2,[12][13][14][15][16] Although the "loop sheet" insertion mechanism of ␣1ATZ polymerization-in which the reactive site loop of one molecule inserts into a surface groove in a neighboring moleculedoes not involve the formation of covalent bonds, physical/chemical studies of these polymers suggest that this conformation is highly favored. It has been widely hypothesized that liver injury in humans with ␣1AT deficiency is directly related to the hepatic accumulation of the polymerized ␣1ATZ protein, rather than to the monomeric form. Furthermore, in vitro studies of ␣1ATZ molecules show that the equilibrium favoring formation of polymers increases as temperature increases. 13 These data have been used to propose that progressive liver disease in ZZ humans is directly related to increased ␣1ATZ polymers formed during episodes of physiological fever. 1,8 Our laboratory has reported a series of studies that have begun to examine the mechanism of liver cell injury in ␣1AT deficie...

Parenteral nutrition (PN) has revolutionized the care of patients with intestinal failure by providing nutrition intravenously. Worldwide, PN remains a standard tool of nutrition delivery in neonatal, pediatric, and adult patients. Though the benefits are evident, patients receiving PN can suffer serious cholestasis due to lack of enteral feeding and sometimes have fatal complications from liver injury and gut atrophy, including PN-associated liver disease or intestinal failure-associated liver disease. Recent studies into gut-systemic cross talk via the bile acid-regulated farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) axis, gut microbial control of the TGR5-glucagon-like peptide (GLP) axis, sepsis, and role of prematurity of hepatobiliary receptors are greatly broadening our understanding of PN-associated injury. It has also been shown that the composition of ω-6/ω-3 polyunsaturated fatty acids given parenterally as lipid emulsions can variably drive damage to hepatocytes and cell integrity. This manuscript reviews the mechanisms for the multifactorial pathogenesis of liver disease and gut injury with PN and discusses novel ameliorative strategies. (Nutr Clin Pract. 2020;35:63-71)

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