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.

Mutant ␣ 1 -antitrypsin Z (␣ 1 -ATZ) protein, which has a tendency to form aggregated polymers as it accumulates within the endoplasmic reticulum of the liver cells, is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary ␣ 1 -antitrypsin (␣ 1 -AT) deficiency. Previous studies have suggested that efficient intracellular degradation of ␣ 1 -ATZ is correlated with protection from liver disease in ␣ 1 -AT deficiency and that the ubiquitin-proteasome system accounts for a major route, but not the sole route, of ␣ 1 -ATZ disposal. Yet another intracellular degradation system, autophagy, has also been implicated in the pathophysiology of ␣ 1 -AT deficiency. To provide genetic evidence for autophagy-mediated disposal of ␣ 1 -ATZ, here we used cell lines deleted for the Atg5 gene that is necessary for initiation of autophagy. In the absence of autophagy, the degradation of ␣ 1 -ATZ was retarded, and the characteristic cellular inclusions of ␣ 1 -ATZ accumulated. In wild-type cells, colocalization of the autophagosomal membrane marker GFP-LC3 and ␣ 1 -ATZ was observed, and this colocalization was enhanced when clearance of autophagosomes was prevented by inhibiting fusion between autophagosome and lysosome. By using a transgenic mouse with liver-specific inducible expression of ␣ 1 -ATZ mated to the GFP-LC3 mouse, we also found that expression of ␣ 1 -ATZ in the liver in vivo is sufficient to induce autophagy. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for ␣ 1 -ATZ and suggest that autophagic degradation plays a fundamental role in preventing toxic accumulation of ␣ 1 -ATZ.3 a monomeric 394-amino acid glycoprotein, is synthesized and secreted primarily by liver cells. It is a prototypic member of serine protease inhibitor (serpin) superfamily proteins and the most abundant of the circulating serpins. The principal role of ␣ 1 -AT in serum is to protect lung tissues from destructive proteases (elastase, cathepsin G, and proteinase 3) released by neutrophils during inflammation. Some genetic alterations in ␣ 1 -AT are responsible for defective secretion and thus cause serum ␣ 1 -AT deficiency (1-3). The most common causal mutation found in Caucasian populations is the replacement of Glu-342 by Lys that characterizes the Z mutant of ␣ 1 -AT (␣ 1 -ATZ). This substitution is sufficient to cause an abnormality in folding early in the secretory pathway with retention of the mutant ␣ 1 -ATZ molecule in the ER of liver cells. Homozygotes for the ␣ 1 -ATZ mutation (PIZZ) are characterized by serum levels of ␣ 1 -AT that are ϳ10 -15% of those in the general population and are susceptible to two major target organ injuries. Destructive lung disease/emphysema in adults is due to a loss-of-function mechanism. Chronic liver disease often first discovered in childhood, but also affecting adults, is due to a gain-of-toxic-function mechanism in which liver cell injury results from the hepatotoxic effects of retained ␣ 1 -ATZ...

In ␣ 1 -antitrypsin (␣1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein ␣1AT, ␣1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant ␣1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant ␣1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant ␣1AT. The results show that ER retention of polymerogenic mutant ␣1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of ␣1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic ␣1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant ␣1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic ␣1AT mutants.In the classical form of ␣1AT 2 deficiency, the mutant ␣1ATZ molecule is retained in the endoplasmic reticulum (ER) of liver cells rather than secreted. There is an 85-90% reduction in ␣1AT levels in the blood and body fluids. This deficiency affects ϳ1 in 1800 live births and results in the premature development of pulmonary emphysema in adult life. Chronic liver disease develops in a subgroup of homozygotes, usually becoming evident during childhood. There is also an increased incidence of hepatocellular carcinoma later in life. Emphysema is caused by a loss-of-function mechanism whereby lack of ␣1AT in the lung allows proteolytic destruction of the connective tissue matrix. In contrast, liver injury appears to involve a gain-of-toxic-function mechanism whereby the accumulation of mutant ␣1ATZ in the ER damages liver cells (1).Nevertheless, relatively little is known about the factors that predispose the "susceptible" subpopulation of PIZZ individuals to liver disease and/or protect the remainder of the PIZZ population from liver disease. By using skin fibroblast cell lines from PIZZ individuals with or without liver disease engineered for expression of ␣1ATZ, we have shown previously that there is a lag in ER degradation of mutant ␣1ATZ in cells from PIZZ individuals with liver disease (2). These results provided evidence that the response of cells to the accumulation of this mutant protein in the ER, particularly the degradative machinery, could play a role in determining the susceptibility to liver disease among ...