Variants in autophagy genes MTMR12 and FAM134A are putative modifiers of the hepatic phenotype in α1-antitrypsin deficiency.

BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

Multiple Genes Core to ERAD, Macroautophagy and Lysosomal Degradation Pathways Participate in the Proteostasis Response in α1-Antitrypsin Deficiency.

BACKGROUND & AIMS: In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD. METHODS: We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems. RESULTS: Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria. CONCLUSIONS: Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype.

A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy.

Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans.