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 ...

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.

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Selective degradation of the mutant protein responsible for most cystic fibrosis, F508del cystic fibrosis transmembrane conductance regulator (CFTR), is initiated by Hsp27, which associates with the small ubiquitin-like modifier (SUMO) E2, Ubc9. They modify F508del with SUMO-2/3, directing F508del to a SUMO-targeted ubiquitin ligase, RNF4. This work implicates SUMO and RNF4 in quality control of a cytosolic transmembrane protein.

Highlights d In somatosensory ganglia, Prdm12 is specific to the nociceptive lineage d Prdm12 is necessary for the survival of developing nociceptors d Prdm12 initiates and maintains the expression of TrkA in developing nociceptors d Prdm12 acts in conjunction with bHLH proteins Ngn1/2 to promote a nociceptor fate

Because retention of mutant ␣ 1 -antitrypsin (␣ 1 -AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in ␣ 1 -AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of Chem. 275, 25015-25022) found that degradation of ␣ 1 -ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of ␣ 1 -ATZ in nonhepatocytic cell types or in cell types with levels of ␣ 1 -AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of ␣ 1 -ATZ. In each of these cell lines degradation of ␣ 1 -ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin ␤-lactone. Using the inducible expression system to regulate the relative level of ␣ 1 -ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of ␣ 1 -ATZ at high and low levels of ␣ 1 -AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of ␣ 1 -ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes.The classical and most common form of ␣ 1 -antitrypsin (␣ 1 -AT) 1 deficiency is a relatively unique genetic disease in that it is associated with injury to one tissue, pulmonary emphysema, by a loss-of-function mechanism and injury to another tissue, chronic hepatitis/hepatocellular carcinoma, by a gain-of-function mechanism. Many studies have provided evidence that emphysema results from lack of the anti-elastase activity of ␣ 1 -AT in the lung (reviewed in Refs. 1 and 2). Liver disease is due to toxic effects of aggregated ␣ 1 -ATZ retained in the ER of liver parenchymal cells. The gain-of-function mechanism is most clearly demonstrated by experiments in mice transgenic for human ␣ 1 -ATZ. These mice develop liver injury and hepatocellular carcinoma despite the fact that they have their own endogenous anti-elastases (3-5).The mutant Z allele of ␣ 1 -AT is characterized by a single nucleotide substitution, which results in the replacement of glutamate 342 by a bulky lysine residue (1, 2). The studies of Carrell and Lomas (6, 7) have shown that this substitution renders the ␣ 1 -AT molecule more susceptible to polymerization and that highly ordered aggregates accumulate in the ER of liver cells.One interesting observation, arising from unbiased nationwide screening studies of ␣ 1 -AT deficiency in Sweden, indicates that only 10 -15% of deficient individuals develop clinically significant liver disease (8,9...

In alpha1-antitrypsin (alpha1-AT) deficiency, a mutant form of alpha1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant alpha1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The alpha1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which alpha1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant alpha1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of approximately 110-130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic alpha1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the alpha1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that approximately 85% of alpha1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and approximately 15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of alpha1-AT Z as well as on its residual secretion.

SummaryTools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.