In the classical form of alpha1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant alpha1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain-of-toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.

In the classical form of ␣ 1 -antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant ␣ 1 -antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble ␣ 1 -antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic ␣ 1 -antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with ␣ 1 -antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble ␣ 1 -antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in ␣ 1 -antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.The histological hallmark of the classical form of ␣ 1 -antitrypsin (AT) 2 deficiency is liver cells containing periodic acidSchiffϩ/diastase-resistant globules. From many years of research on the disease, we now know that these globules represent rough endoplasmic reticulum (ER) distended by accumulation of the mutant ATZ molecule (where ATZ is the Z variant of ␣ 1 -antitrypsin). The wild type AT is a classical liverderived secretory glycoprotein that is delivered by the circulating blood to tissues to subserve its predominant function of inhibiting the neutrophil serine proteases neutrophil elastase, cathepsin G, and proteinase 3. The point mutation that characterizes the ATZ variant converts glutamate 342 to lysine and is sufficient to result in selective retention of the glycoprotein in the ER (reviewed in Refs. 1, 2). Thus, AT deficiency could be considered a prototype, naturally occurring "ER stress" state. Characterization of the structure of AT and its functional correlates led to the remarkable observation that the substitution of lysine for glutamate 342 conferred on the ATZ molecule a tendency to polymerize and aggregate (3, 4). Although it is still not clear whether the tendency to polymerize is the cause, or an effect, of ER retention, there is clear-cut evidence that polymers and aggregates of this molecule are formed in the ER, and there is growing evidence that these polymers and aggregates play a role in how liver cells respond and wh...

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models.