Knockdown of Alpha-1 Antitrypsin with antisense oligonucleotide does not exacerbate smoke induced lung injury.

Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein's primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

Development and validation of an ambulatory piglet model for short bowel syndrome with ileo-colonic anastomosis.

IMPACT STATEMENT: Short bowel syndrome is associated with significant comorbidities and mortality. This study is important as unlike current systems, it provides a validated piglet model which mirrors anatomical, histological, and serological characteristics observed in human SBS. This model can be used to advance knowledge into mechanistic pathways and therapeutic modalities to improve outcomes for SBS patients. This study is novel in that in addition to significant reduction in the remnant bowel and noted liver disease, we also developed a method to emulate ileocecal valve resection and described gut adaptive responses which has important clinical implications in humans.

Mechanisms of Parenteral Nutrition-Associated Liver and Gut Injury.

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.

Developing a Novel Ambulatory Total Parenteral Nutrition-Dependent Short Bowel Syndrome Animal Model.

BACKGROUND: Short bowel syndrome (SBS) results from extensive bowel resection. Patients with SBS require total parenteral nutrition (TPN) for survival. Understanding mechanisms contributing to TPN-associated liver injury and gut atrophy are critical in developing SBS therapies. Existing SBS models using tethered animals have significant limitations and are unlike ambulatory human SBS patients. We hypothesized that we could induce SBS in piglets and develop an ambulatory TPN-SBS model. MATERIAL AND METHODS: Eighteen neonatal pigs received duodenal and jugular catheters. They were fitted with a jacket holding TPN and a miniaturized pump. Six piglets had 90% small bowel resection and catheter placement (SBS group). Non-SBS piglets were randomized into enteral nutrition (EN) or TPN. RESULTS: Bowel resection was successfully accomplished in SBS animals. Weight gain was similar in all groups. SBS animals had increased serum bilirubin compared to EN. Mean conjugated bilirubin ± SD was 0.045 ± 0.01 for EN, (P = 0.03 EN versus TPN and P = 0.03 SBS versus EN) and 1.09 ± 1.25 for TPN, (P = 0.62 TPN versus SBS). Gut density was reduced in the TPN group compared to EN and SBS groups. Mean gut density ± SD was 0.11 ± 0.04 for TPN (P = 0.0004 TPN versus SBS and P = 0.00007 TPN versus EN) and not statistically different for EN versus SBS (P = 0.32). CONCLUSIONS: We created a novel, ambulatory TPN-SBS model using piglets, mimicking long-term TPN delivery in human SBS patients. Our model demonstrated TPN-related conjugated hyperbilirubinemia and compensatory gut hypertrophy, as noted in humans with SBS. This model holds great potential for future research.

Role of the Gut⁻Liver Axis in Driving Parenteral Nutrition-Associated Injury.

For decades, parenteral nutrition (PN) has been a successful method for intravenous delivery of nutrition and remains an essential therapy for individuals with intolerance of enteral feedings or impaired gut function. Although the benefits of PN are evident, its use does not come without a significant risk of complications. For instance, parenteral nutrition-associated liver disease (PNALD)-a well-described cholestatic liver injury-and atrophic changes in the gut have both been described in patients receiving PN. Although several mechanisms for these changes have been postulated, data have revealed that the introduction of enteral nutrition may mitigate this injury. This observation has led to the hypothesis that gut-derived signals, originating in response to the presence of luminal contents, may contribute to a decrease in damage to the liver and gut. This review seeks to present the current knowledge regarding the modulation of what is known as the "gut⁻liver axis" and the gut-derived signals which play a role in PN-associated injury.

NorUDCA promotes degradation of α1-antitrypsin mutant Z protein by inducing autophagy through AMPK/ULK1 pathway.

Alpha-1 Antitrypsin (α1AT) Deficiency is a genetic disease in which accumulation of α1AT mutant Z (α1ATZ) protein in the ER of hepatocytes causes chronic liver injury, liver fibrosis, and hepatocellular carcinoma. No effective medical therapy is currently available for the disease. We previously found that norUDCA improves the α1AT deficiency associated liver disease by promoting autophagic degradation of α1ATZ protein in liver in a mouse model of the disease. The current study unravels the novel underlying cellular mechanism by which norUDCA modulates autophagy. HTOZ cells, modified from HeLa Tet-Off cells by transfection with the resulting pTRE1-ATZ plasmid and expressing mutant Z proteins, were studied in these experiments. The role of norUDCA in inducing autophagy, autophagy-mediated degradation of α1ATZ and the role of AMPK in norUDCA-induced autophagy were examined in the current report. NorUDCA promoted disposal of α1ATZ via autophagy-mediated degradation of α1ATZ in HTOZ cells. Activation of AMPK was required for norUDCA-induced autophagy and α1ATZ degradation. Moreover, mTOR/ULK1 was involved in norUDCA-induced AMPK activation and autophagy in HTOZ cells. Our results provide novel mechanistic insights into the therapeutic action of norUDCA in promoting the clearance of α1ATZ in vitro and suggest a novel therapeutic approach for the treatment of α1ATZ deficiency disease and its associated liver diseases.

No Gut No Gain! Enteral Bile Acid Treatment Preserves Gut Growth but Not Parenteral Nutrition-Associated Liver Injury in a Novel Extensive Short Bowel Animal Model.

BACKGROUND: Parenteral nutrition (PN) provides nutrition intravenously; however, this life-saving therapy is associated with significant liver disease. Recent evidence indicates improvement in PN-associated injury in animals with intact gut treated with enteral bile acid (BA), chenodeoxycholic acid (CDCA), and a gut farnesoid X receptor (FXR) agonist, which drives the gut-liver cross talk (GLCT). We hypothesized that similar improvement could be translated in animals with short bowel syndrome (SBS). METHODS: Using piglets, we developed a novel 90% gut-resected SBS model. Fifteen SBS piglets receiving PN were given CDCA or control (vehicle control) for 2 weeks. Tissue and serum were analyzed posteuthanasia. RESULTS: CDCA increased gut FXR (quantitative polymerase chain reaction; P = .008), but not downstream FXR targets. No difference in gut fibroblast growth factor 19 (FGF19; P = .28) or hepatic FXR (P = .75), FGF19 (P = .86), FGFR4 (P = .53), or Cholesterol 7 α-hydroxylase (P = .61) was noted. PN resulted in cholestasis; however, no improvement was noted with CDCA. Hepatic fibrosis or immunostaining for Ki67, CD3, or Cytokeratin 7 was not different with CDCA. PN resulted in gut atrophy. CDCA preserved (P = .04 vs control) gut mass and villous/crypt ratio. The median (interquartile range) for gut mass for control was 0.28 (0.17-0.34) and for CDCA was 0.33 (0.26-0.46). CONCLUSIONS: We note that, unlike in animals with intact gut, in an SBS animal model there is inadequate CDCA-induced activation of gut-derived signaling to cause liver improvement. Thus, it appears that activation of GLCT is critically dependent on the presence of adequate gut. This is clinically relevant because it suggests that BA therapy may not be as effective for patients with SBS.

Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.

Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.

Pathophysiology of Alpha-1 Antitrypsin Deficiency Liver Disease.

Classical alpha-1 antitrypsin (a1AT) deficiency is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults (Teckman and Jain, Curr Gastroenterol Rep 16(1):367, 2014). The vast majority of the liver disease is associated with homozygosity for the Z mutant allele, the so-called PIZZ. These homozygous individuals synthesize large quantities of a1AT mutant Z protein in the liver, but the mutant protein folds improperly during biogenesis and approximately 85% of the molecules are retained within the hepatocytes rather than appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein molecules retained within hepatocytes are directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time. Some of these molecules adopt an unusual aggregated or "polymerized" conformation (Duvoix et al., Rev Mal Respir 31(10):992-1002, 2014). It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end-organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (Lindblad et al., Hepatology 46(4):1228-1235, 2007). The hepatocytes with the largest accumulations of mutant Z polymers undergo apoptotic death and possibly other death mechanisms. This intracellular death cascade appears to involve ER stress, mitochondrial depolarization, and caspase cleavage, and is possibly linked to autophagy and redox injury. Cells with lesser burdens of mutant Z protein proliferate to maintain the liver cell mass. This chronic cycle of cell death and regeneration activates hepatic stellate cells and initiates the process of hepatic fibrosis. Cirrhosis and hepatocellular carcinoma then result in some patients. Since not all patients with the same homozygous PIZZ genotype develop end-stage disease, it is hypothesized that there is likely to be a strong influence of genetic and environmental modifiers of the injury cascade and of the fibrotic response.

Semiquantitation of Monomer and Polymer Alpha-1 Antitrypsin by Centrifugal Separation and Assay by Western Blot of Soluble and Insoluble Components.

Alpha-1 antitrypsin (a1AT) deficiency, in its classical form, is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults. The vast majority of liver disease is associated with homozygosity for the Z mutant allele, also called PiZZ. This homozygous allele synthesizes large quantities of a1AT mutant Z protein in the liver, but the mutant protein also folds improperly during biogenesis. As a result, approximately 85% of the molecules are retained within the hepatocytes instead of being appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein retained within hepatocytes is directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time and others adopt an unusual aggregated or "polymerized" conformation. It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. It is widely accepted that the disease causing factor in mutant Z-alpha-1 antitrypsin deficiency (AATD-Z) is the toxic build-up of the mutant Z protein. Since misfolding of some but not all of the Z protein during its maturation leads to homopolymerization, an assay to assess the amount of normally folded ATZ and accumulated polymeric ATZ would be very useful. Here we describe a method to semiquantitatively assess these two fractions in a tissue or cell culture source.

Preserved Gut Microbial Diversity Accompanies Upregulation of TGR5 and Hepatobiliary Transporters in Bile Acid-Treated Animals Receiving Parenteral Nutrition.

BACKGROUND: Parenteral nutrition (PN) is a lifesaving therapy but is associated with gut atrophy and cholestasis. While bile acids (BAs) can modulate intestinal growth via gut receptors, the gut microbiome likely influences gut proliferation and inflammation. BAs also regulate the bile salt export pump (BSEP) involved in cholestasis. We hypothesized that the BA receptor agonist oleanolic acid (OA) regulates gut TGR5 receptor and modulates gut microbiota to prevent PN-associated injury. MATERIALS AND METHODS: Neonatal piglets were randomized to approximately 2 weeks of isocaloric enteral nutrition (EN), PN, or PN + enteral OA. Serum alanine aminotransferase, bilirubin, BAs, hepatic BSEP, gut TGR5, gut, liver morphology, and fecal microbiome utilizing 16S rRNA sequencing were evaluated. Kruskal-Wallis test, pairwise Mann-Whitney U test, and multilevel logistic regression analysis were performed. RESULTS: PN support resulted in gut atrophy substantially prevented by OA. The median (interquartile range) for villous/crypt ratio was as follows: EN, 3.37 (2.82-3.80); PN, 1.73 (1.54-2.27); and OA, 2.89 (2.17-3.34; P = .006). Pairwise comparisons yielded P = .002 (EN vs PN), P = .180 (EN vs OA), P = .026 (PN vs OA). OA upregulated TGR5 and BSEP without significant improvement in serum bilirubin ( P = .095). A decreased microbial diversity and shift toward proinflammatory phylum Bacteroidetes were seen with PN, which was prevented by OA. CONCLUSIONS: OA prevented PN-associated gut mucosal injury, Bacterioides expansion, and the decreased microbial diversity noted with PN. This study demonstrates a novel relationship among PN-associated gut dysfunction, BA treatment, and gut microbial changes.

Autophagy induced by exogenous bile acids is therapeutic in a model of α-1-AT deficiency liver disease.

The bile acid nor-ursodeoxycholic acid (norUDCA) has many biological actions, including antiapoptotic effects. Homozygous PIZZ α-1-antitrypsin (A1AT)-deficient humans are known to be at risk for liver disease, cirrhosis, and liver cancer as a result of the accumulation of the toxic, A1AT mutant Z protein within hepatocytes. This accumulation triggers cell death in the hepatocytes with the largest mutant Z-protein burdens, followed by compensatory proliferation. Proteolysis pathways within the hepatocyte, including autophagy, act to reduce the intracellular burden of A1AT Z protein. We hypothesized that norUDCA would reduce liver cell death and injury in A1AT deficiency. We treated groups of PiZ transgenic mice and wild-type mice with norUDCA or vehicle, orally, and examined the effects on the liver. The PiZ mouse is the best model of A1AT liver injury and recapitulates many features of the human liver disease. Mice treated with norUDCA demonstrated reduced hepatocellular death by compensatory hepatocellular proliferation as determined by bromodeoxyuridine incorporation (3.8% control, 0.88% treated, P < 0.04). Ki-67 staining as a marker for hepatocellular senescence and death was also reduced (P < 0.02). Reduced apoptotic signaling was associated with norUDCA, including reduced cleavage of caspases-3, -7, and -8 (all P < 0.05). We determined that norUDCA was associated with a >70% reduction in intrahepatic mutant Z protein (P < 0.01). A 32% increase in hepatic autophagy associated with norUDCA was the likely mechanism. norUDCA administration is associated with increased autophagy, reduced A1AT protein accumulation, and reduced liver injury in a model of A1AT deficiency.

Oleanolic Acid Improves Gut Atrophy Induced by Parenteral Nutrition.

BACKGROUND: Nutrition support with parenteral nutrition (PN) is associated with gut atrophy. Prior studies have shown improvement with enteral chenodeoxycholic acid, a dual agonist for the farnesoid X receptor (FXR) and bile acid receptor TGR5. We hypothesized that gut growth is induced by TGR5 activation, and gut atrophy during PN administration could be prevented with the TGR5-specific agonist oleanolic acid (OA). METHODS: Neonatal pigs were implanted with duodenal and jugular vein catheters. Animals were provided equi-nutritious PN or enteral swine milk. A PN subgroup received enteral OA at 50 mg/kg/d. RESULTS: PN caused marked gut atrophy compared with enterally fed (EN) control animals. OA treatment led to preservation of gut mass demonstrated grossly and histologically. The mean ± SD gut weight as a percentage of body weight was 4.30 ± 0.26 for EN, 1.92 ± 0.06 for PN (P < .05, EN vs PN), and 3.39 ± 0.79 for PN+OA (P < .05, PN+OA vs PN). Mean ± SD gut density (g/cm) was 0.31 ± 0.03 for EN, 0.18 ± 0.03 for PN (P < .05 EN vs PN), and 0.27 ± 0.01 for PN+OA (P < .05 PN+OA vs PN). Histologically, a markedly decreased villous to crypt ratio was noted with PN, and OA significantly prevented this decrease. The mean ± SD v/c ratio was 3.51 ± 0.59 for EN, 1.69 ± 0.10 for PN (P < .05, EN vs PN), and 2.90 ± 0.23 for PN+OA (P < .05, PN+OA vs PN). Gut TGR5 messenger RNA expression was significantly elevated with OA treatment compared with both PN and EN. CONCLUSION: The bile acid-activated G protein-coupled receptor TGR5 agonist OA prevented gut atrophy associated with PN.

Validating hyperbilirubinemia and gut mucosal atrophy with a novel ultramobile ambulatory total parenteral nutrition piglet model.

Total parenteral nutrition (TPN) provides all nutrition intravenously. Although TPN therapy has grown enormously, it causes significant complications, including gut and hepatic dysfunction. Current models use animal tethering which is unlike ambulatory human TPN delivery and is cost prohibitive. We hypothesize that using ultramobile infusion pumps, TPN can be delivered cost-effectively, resulting in classical gut and hepatic injury, and we thus aim to establish a new model system. Neonatal pigs (n=8) were implanted with jugular vein and duodenal catheters. Animals were fitted in dual-pocket jackets. An ultramobile ambulatory pump was placed in one pocket and connected to the jugular vein or duodenal catheter. Isocaloric TPN or swine formula was placed in the other pocket. Rigorous Wifi-based video and scheduled monitoring was performed. After 14days, the animals were euthanized. The mean (±SD) daily weight gain (in grams) for enteral-fed control (EN) vs TPN animals was 102.4±10.8 and 91.03±12.1 respectively (P<.05). Total parenteral nutrition resulted in significant conjugated bilirubin elevation and hepatomegaly. Mean (±SD) serum conjugated bilirubin (in µmol/L) was 1.5±0.7 for EN and 6.3±2.8 for TPN (P<.05). Marked gut atrophy was noted with TPN. The mean (±SD) gut weight as a percent of body weight was 4.30±0.26 for EN and 2.62±0.48 for TPN (P<.05). Surgical sites healed well. All animals remained completely mobile. We thus established that TPN can be successfully delivered using ultramobile pumps and believe that this remains the first such description of an ambulatory piglet TPN model system. In addition to cholestasis and gut atrophy, classical TPN-induced injury was documented.

PiZ mouse liver accumulates polyubiquitin conjugates that associate with catalytically active 26S proteasomes.

Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.

Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin-related liver disease in mice.

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.

Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency.

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.

Oxidative stress contributes to liver damage in a murine model of alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency is a genetic disorder resulting in the expression of misfolded mutant protein that can polymerize and accumulate in hepatocytes, leading to liver disease in some individuals. Transgenic PiZ mice are a well-characterized model, which express human alpha-1-antitrypsin mutant Z protein (ATZ protein) and faithfully recapitulate the human liver disease. Liver tissue expressing alpha-1-antitrypsin mutant Z protein exhibits inflammation, injury and replacement of damaged cells. Fibrosis and hepatocellular carcinoma (HCC) develop in aging PiZ mice. In this study, microarray analysis was performed comparing young PiZ (ZY) mice to wild-type (WY), and indicated that there were alterations in gene expression levels that could influence a number of pathways leading to liver disease. Redox-regulating genes were up-regulated in ZY tissue, including carbonyl reductase 3 (CBR3), glutathione S-transferase alpha 1 + 2 (GSTA(1 + 2)) and glutathione S-transferase mu 3 (GSTM3). We hypothesized that oxidative stress could develop in Z mouse liver, contributing to tissue damage and disease progression with age. The results of biochemical analysis of PiZ mouse liver revealed that higher levels of reactive oxygen species (ROS) and a more oxidized, cellular redox state occurred in liver tissue from ZY mice than WY. ZY mice showed little evidence of oxidative cellular damage as assessed by protein carbonylation levels, malondialdehyde levels and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) staining. Aging liver tissue from PiZ older mice (ZO) had elevated ROS, generally lower levels of antioxidant enzymes than younger mice and evidence of cellular damage. These data indicate that oxidative stress is a contributing factor in the development of liver disease in this model of alpha-1-antitrypsin deficiency.

Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

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.