Mitochondrial autophagy and injury in the liver in {alpha}1-antitrypsin deficiency

Jeffrey H. Teckman,1 Jae-Koo An,1 Keith Blomenkamp,1 Bela Schmidt,2 and David Perlmutter2,3

1Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, Missouri 63110; and 2Departments of Pediatrics, 3Cell Biology and Physiology, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213

Submitted 16 April 2003 ; accepted in final form 12 December 2003


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

{alpha}1-antitrypsin; autophagy; mitochondria; quality control; cyclosporin A


THE CLASSIC FORM of {alpha}1-antitrypsin ({alpha}1-AT) deficiency, homozygous PIZZ {alpha}1-AT deficiency, is caused by a point mutation encoding substitution of lysine for glutamate-342 (31). This substitution confers polymerogenic properties on the mutant {alpha}1-anti-trypsin Z ({alpha}1-ATZ) molecule (5). Polymerized mutant {alpha}1-ATZ is retained in the endoplasmic reticulum (ER) rather than secreted in the body fluids where its function is to inhibit neutrophil proteases. Individuals with this deficiency have a markedly increased risk of developing emphysema by a loss-of-function mechanism, i.e., reduced levels of {alpha}1-AT in the lung to inhibit connective tissue breakdown by neutrophil proteases. A subgroup of PIZZ individuals develops liver injury and hepatocellular carcinoma by a gain-of-function mechanism, i.e., accumulation of polymerized mutant {alpha}1-ATZ within the ER is toxic to liver cells. The "accumulation" mechanism is best demonstrated by studies of mice transgenic for the human {alpha}1-ATZ gene (4, 9). In addition to periodic acid-Schiff-positive, diastase-resistant intrahepatic globules that represent ER dilated with the aggregated mutant protein, these mice develop liver injury and hepatocellular carcinoma. There are normal levels of antiproteases in these animals, as directed by endogenous genes; therefore, the liver injury cannot be attributed to a loss-of-function mechanism.

A variety of studies over the last 15 years have characterized the "quality control" system of the ER, a system responsible for management and degradation of mutant, misfolded, and unassembled proteins, and that in many experimental analyses has been shown to involve the proteasome proteolytic complex (23). Multiple proteolytic pathways appear to be involved in the quality control mechanism for {alpha}1-ATZ, including ubiquitin-dependent and -independent proteosomal pathways and one or more nonproteosomal pathways (24, 30). In fact, variation in the severity of the liver disease phenotype among PIZZ individuals appears to directly correlate with the efficiency of ER degradation of mutant {alpha}1-ATZ in genetically engineered fibroblast cell lines from carefully selected patients (35).

Nevertheless, there is still relatively limited information about the mechanism by which ER {alpha}1-ATZ retention leads to liver cell injury (11). We have found that there is an intense autophagic response in the liver of PIZZ individuals (32) and that autophagy is constitutively activated in the liver of the PiZ transgenic mouse model as well (28). Autophagy is an intracellular proteolytic pathway in which specialized vacuoles arise from ER membranes and engulf targets of degradation during times of stress, development, and nutrient deprivation. Autophagy may constitute a mechanism for protecting liver cells by degrading mutant {alpha}1-ATZ (32). However, in this study, we report the presence of mitochondrial autophagy in the liver in {alpha}1-AT deficiency and provide evidence for damage to mitochondria that has characteristics that are unique and specific to this genetic liver disease.


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Antibodies, labels, and cell lines. Antibodies included rabbit anti-human {alpha}1-AT (DAKO, Santa Barbara, CA), goat anti-human {alpha}1-AT (Cappel, Durham, NC), and antibody to full-length caspase-3, activated caspase-3, and caspase-3 blocking peptide (Cell Signaling, Boston, MA). Mitotracker green, mitotracker green FM, lysotracker red, Mitofluoro Red 594, and tetramethylrhodamine methyl ester (TMRM) were purchased from Molecular Probes (Eugene, OR) and employed as previously described (10, 19, 28, 32). HeLa cell lines, HTO/Z and HTO/M, engineered for inducible expression of {alpha}1-ATZ and WT M have been previously described (24, 29, 30, 32, 35). Pulse-chase experiments and analysis by immunoprecipitation and SDS-PAGE/fluorography were performed exactly as previously described (29, 35). For pulse-chase experiments, cells were incubated during the pulse and chase with cyclosporin A (CsA) or tacrolimus as shown. Each of these pulse-chase experiments was analyzed in triplicate with means ± SD of the half-time of disappearance of the 52-kDa {alpha}1-AT as determined by densitometry, exactly as previously described (24, 29, 30, 35). For other experiments, cells were harvested and subjected to immunoblot analysis for caspase-3 (using anticaspase-3— 8G10 from Cell Signaling Technology) according to the protocol described in the instructions from Cell Signaling Technology (24, 29, 30, 32, 35). For fluorescent automated cell sorting (FACS) analysis, cell suspensions were loaded with 15.6 nM TMRM (Molecular Probes) for 45 min at 37°C, and then 10,000 cells were analyzed at 575 nm emission wavelength in a FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA). Mitochondrial depolarization was accomplished in controls by a 20-min incubation with 20 µM CCCP (Sigma, St. Louis, MO; see Ref. 26).

Microscopy and quantitative analysis of microscopic features. Material for transmission electron microscopy (EM) was processed by standard techniques, as previously described (32). For each human liver specimen, the percentage of mitochondria undergoing autophagy and the percentage of mitochondria with internal injury were quantified by counting individual mitochondria in 10 photomicrographs, each showing a randomly selected hepatocyte with its nucleus. Rigorous, specific ultrastructural criteria for autophagy and injury were established before the analysis. Mitochondria were defined by the accepted description as structures with a smooth outer membrane, an inner membrane contiguous with cristae and containing a granular, moderately electron-dense internal matrix (3, 7). Mitochondria were deemed to be within an early autophagic vacuole by identification of the well-described, characteristic structure of a double, or multilamellar, smooth membrane completely surrounding the organelle in close proximity to, or in continuity with, rough ER (rER; see Ref. 32), that is, at least four distinct circumferential lipid bilayers were visible for any mitochondria considered to be within an early autophagic vacuole (2 for the EAP, 2 for the mitochondria). Mitochondria within vacuoles that had progressed to a late autophagic vacuole or autolysosome stage were identified by the appearance of the characteristic membrane-bound structure containing the compressed mitochondria, often with loss of visible cristae, as well as other electron-dense, often membrane-bound, material (32). Mitochondria were considered to have nonautophagic-related internal structural alterations if at least one accumulation of multilamellar membranes was visible within the outer limiting membrane of the mitochondria itself and/or if the internal cristae and matrix were heterogeneously distributed within the outer limiting membrane of the mitochondria, leaving open areas with membrane blebs in the organelle. Means and SDs were subjected to ANOVA using SigmaStat software (SPSS, Chicago, IL), which was also used in the other statistical analyses noted.

In the mouse liver specimens, the definition of normal mitochondrial structure was identical to that for human liver described above. Injured mitochondria were defined by the presence of at least one accumulation of multilamellar membranes within the limiting membrane of the mitochondria and/or a change in the density of the internal structures of the mitochondria. However, if density change alone was the only change in a given individual mitochondrion, then there had to be open areas with bleb formation in the outer membrane for it to be considered injured. Although diffuse homogeneous density changes were seen in many mitochondria in the PiZ mice, this alteration was not by itself deemed to be sufficient for an abnormal designation. Calculation of means and SDs and statistical analysis were performed exactly as described for the human specimens above.

All of the quantitative analyses were performed by one examiner. Blinding of the examiner to the patients, disease controls, normal mice, and PiZ mice was not completely successful, because striking ultrastructural characteristics indicative of each disease were often evident. Blinding of analysis in mice ± CsA was performed. Repeat analysis of selected specimens showed <15% variability at different examination times. Tissue was prepared for immunohistochemistry, immunofluorescence, and fluorescence with vital dyes exactly as described previously (10, 19, 28, 29, 32). Immune EM for lamp1 was performed exactly as previously described (28, 32). Histological preparation and examination of specimens for light microscopy and quantitation of globules were performed exactly as previously described (28, 32). All human and animal protocols were approved by the Washington University Human Studies Committee and the Animal Studies Committee, respectively.

Fasting and CsA administration. For experiments in which mice were subjected to fasting, they were kept in their usual cages with inert gnawing material and water available ad libitum, but without nutrients. CsA (15 mg·kg-1·day-1) was administered enterally in water. Blood levels of CsA in selected animals were found to be similar to the human immunosuppressant therapeutic range (80–140 ng/ml for all animals examined). For experiments to quantitate autophagy, mice were treated with CsA for intervals of 6, 12, and 24 wk, as described. For fasting experiments, mice were pretreated with CsA for 1 wk and maintained on CsA during the fast.


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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial autophagy and injury in liver from {alpha}1-AT deficient patients. We examined the livers from four {alpha}1-AT-deficient patients with known liver disease but without cirrhosis on biopsy (2 adults and 2 children) by EM to determine if there were recognizable structures within the autophagic vacuoles. We hypothesized that the markedly increased autophagic activity present within hepatocytes in this disease might bring a variety of subcellular structures in the autophagosomes for degradation. Typical electron photomicrographs at low magnification used in these studies from a PIZZ {alpha}1-AT-deficient liver and a normal MM liver are shown in Fig. 1a. Rigorous ultrastructural definitions of mitochondria of autophagic vacuoles were employed as described in METHODS. Figure 1a shows a nest of multilamellar, autophagic vacuoles in the perinuclear region intertwined with rER cisternae and many nearby mitochondria in the PIZZ specimen, as we have previously described (28, 32). However, the rER cisternae are poorly organized and slightly dilated in the PIZZ specimen compared with the normal liver, and the mitochondria in the PIZZ specimen have a more heterogeneous electron density. When these mitochondria, and those in other cells from the PIZZ specimen, are examined at higher magnification, two distinct patterns of structural change are identified. First (Fig. 1, b-g), there is a pattern that is characteristic of mitochondrial autophagy. Mitochondria can be seen that are partially surrounded by an additional set of double membranes that arise from adjacent rER (Fig. 1b, black arrowheads show extension of ER membranes). In other areas of the same cell and in other cells, mitochondria are completely enveloped within circumferential ER membranes (Fig. 1, c-d), sometimes with preserved, normal morphology of intact cristae (white arrows) and normal electron density, whereas in other instances the mitochondrion has become compressed with increased electron density and is then surrounded by double, or multiple, smooth membranes (black arrows). These are the ultrastructural characteristic features of a mitochondrion being drawn in an early autophagic vacuole. Still other mitochondria (Fig. 1, f and g) appear to be within multilamellar membranes and exhibit highly compressed cristae, and the entire vacuoles are often seen in close proximity to, or actually fusing with, lysosomes. The box in Fig. 1f is reproduced at higher magnification in Fig. 1h, showing five distinct lipid bilayers surrounding the compressed, electron-dense body of the mitochondrion. These are the characteristics of a late autophagic vacuole, or autolysosome. Finally, mitochondria that have almost completely transformed into electron-dense debris within late autophagic vacuoles/autolysosomes are seen (Fig. 1g, degenerating mitochondria surrounded by multiple membranes shown by arrow and adjacent late autophagosome in top right).



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Fig. 1. Electron microscopy of mitochondrial autophagy and injury in human liver. a: Transmission electron microscopy of human PIZZ, {alpha}1-antitrypsin (AT)-deficient liver (ZZ), and normal MM human liver (NL) at low magnification. b-g show progressive mitochondrial autophagy, from early to late autophagic stages. Black arrowheads in b show early extensions of double endoplasmic reticulum (ER) membranes beginning to surround a mitochondrion. c and d show circumferential double ER membranes around the double membrane-bound mitochondrion (4 lipid bilayers, total). Black arrows in e-h show double, or multilamellar, smooth membranes. White arrows show mitochondrial cristae. Box in f is reproduced at higher magnification in h. i Shows immune electron microscopy of PIZZ specimen for lamp1 in which white arrow shows cristae and white arrowheads show gold beads conjugated to antibody indicating the presence of lamp1 protein. g-l Show progressive examples of internal mitochondrial injury from mild to severe in degree. AP, autophagic vacuole; rER, rough endoplasmic reticulum; M, mitochondria; Bar = 500 nm.

 

To provide further evidence that these ultrastructural studies represented mitochondrial autophagy, we examined the PIZZ liver specimens by immune-label EM with antibody to the lysosomal membrane protein, lamp1. The lamp1 protein is also known to label late autophagosomes (3, 7, 28). The result (Fig. 1i) shows that, in regions of the cell similar to Fig. 1a containing many mitochondria and copious ER, there were easily identifiable vacuoles containing structures that appeared to be mitochondria. Cristae (white arrows) could be identified, and the vacuoles were positively labeled for the presence of lamp1 (white arrowheads). Other, more subtle differences in the ultrastructural appearance of Fig. 1, e-g, compared with Fig. 1i are likely the result of differences in the fixation and processing techniques required for conventional transmission EM vs. immune-label EM. The lamp1-positive structures surrounding mitochondria were not readily identified in the normal liver specimens. Taken together, these data suggest that mitochondrial autophagy is a frequent and ongoing process in the PIZZ, {alpha}1-AT deficient liver.

However, a second pattern of structural change was also observed in the PIZZ liver specimens in which mitochondria that are not surrounded by autophagic vacuoles still appear damaged or in various phases of degeneration. This damage is characterized by the formation of multilamellar structures within the limiting membrane (Fig. 1, j-l), condensation of the cristae and matrix, and, in some cases, dissolution of the internal structures, often leaving only electron-dense debris compressed in a thin rim at the periphery of the mitochondrion (Fig. 1l). Although this second type of damaged mitochondria may be clearly distinct from the mitochondria that are degenerating within autophagosomes, these mitochondria are sometimes seen in close proximity to, or even fusing with, autophagic vacuoles or autolysosomes (Fig. 1l).

To determine whether these structural changes are specific for {alpha}1-AT deficiency, we used morphometry to compare the livers from the same four PIZZ, {alpha}1-AT-deficient patients with livers from eight patients with other liver diseases (disease controls) and with four normal livers (normal controls). The disease controls included Wilson's disease, hepatic adenoma, hepatocellular carcinoma, cystic fibrosis, autoimmune hepatitis, sclerosing cholangitis, neonatal hepatitis, and nonalcoholic steatohepatitis. The results show that mitochondrial autophagy and this specific type of mitochondrial injury are uniquely increased in liver from {alpha}1-AT-deficient patients (Fig. 2, A and B). The two types of structural changes seen in PIZZ {alpha}1-AT-deficient patients were present in <1% of the mitochondria in the disease control and normal control livers and was found to be statistically significant. (In Fig. 2A, P < 0.04 for patients 1–4 compared with normal and disease controls and in Fig. 2B, P < 0.05 for patients 1, 2, and 4 compared with normal and disease controls.) Some of the liver disease control specimens showed ultrastructural changes that have been previously described for these diseases (3, 7, 27) but that are clearly different from the patterns of injury described here in the {alpha}1-AT-deficient patients (Fig. 2, c and d). These results suggest that there are at least two morphologically distinct types of mitochondrial injury specific to {alpha}1-AT-deficient human liver and that autophagy is involved in at least one of the injury processes. The fact that a relatively small proportion of the mitochondria shows evidence of injury (<20% of mitochondria undergoing autophagy and <2% with internal injury) may be indicative of the slow rate of progression of hepatocellular injury in this disease and/or may reflect the fact that liver specimens are obtained clinically at various stages in the disease and often late in the injury process.



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Fig. 2. Quantification of mitochondrial autophagy and injury in human PIZZ liver compared with normal and with liver disease controls. Results of the quantitative analysis are shown as means ± SD for mitochondrial autophagy in A and for mitochondrial internal injury in B. In each case, results for 4 different normal livers and 8 liver disease controls (including Wilson's disease, hepatic adenoma, hepatocellular carcinoma, cystic fibrosis, autoimmune hepatitis, sclerosing cholangitis, neonatal hepatitis, and nonalcoholic steatohepatitis) are shown. In A P < 0.04 for patients 1–4 compared with normal and disease control, and in B P < 0.05 for patients 1, 2, and 4 compared with normal and disease controls. c Shows an example of a normal mitochondrion from one of the normal liver controls, and d shows the classic appearance two abnormal mitochondria from the Wilsons's disease control.

 

Mitochondrial autophagy and injury in a transgenic mouse model of {alpha}1-AT deficiency. Next, we used EM to examine liver from the PiZ mouse. Previous studies have shown histological changes in the liver of this mouse that are characteristic of {alpha}1-AT deficiency (4, 9, 11), and recently we have shown marked, constitutively activated autophagy in the liver cells of this mouse (28, 32). Examination of livers from six different 9-mo-old PiZ mice showed increased mitochondrial injury. However, the predominant ultrastructural feature of the mitochondria was degeneration to different extents that did not appear to be occurring within autophagosomes (Fig. 3). These mitochondria have multilamellar structures within their limiting membrane and condensation of the matrix and cristae. In the most severely damaged mitochondria, there is almost complete dissolution of the internal architecture (Fig. 3, center) with electron-dense debris displaced toward the periphery (Fig. 3, right). Damaged mitochondria tended to be adjacent to rER membranes but were seldom engulfed in characteristic multilamellar autophagic vacuoles. The damage was seen in PiZ mice at the ages of 1, 2, 3, 4, 6, 9, 12, and 16 mo (2 mice at each age) without any apparent age-specific or developmental stage-specific features. Quantitative morphometry showed a mean of 16 ± 9% (mean ± SD) mitochondria with this sterotypical pattern of structural change in the hepatocytes of the 9-mo-old PiZ mice but only a mean of 1.5 ± 0.6% of the mitochondria in C57 black control mouse hepatocytes.



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Fig. 3. Electron microscopy of mitochondria in liver of C57BL and PiZ transgenic mice. Transmission electron microscopy of hepatocyte mitochondria from C57BL mice (left) and PiZ mice (middle and right). Arrows show condensates of multilamellar membranes within mitochondria. Bar = 500 nm.

 

Effect of CsA on mitochondrial ultrastructure and capacity to tolerate fasting in the transgenic mouse system. CsA has been shown to reduce mitochondrial injury in vivo and also to inhibit starvation-induced mitochondrial autophagy via blockade of mitochondrial permeability transition (10, 13, 16, 18, 19). Therefore, we examined the effect of administering CsA to PiZ mice. PiZ mice were given pharmacological doses of CsA by gavage for intervals of 6 wk (2 mice), 12 wk (2 mice), and 24 wk (2 mice). Controls included untreated PiZ mice (3 mice), untreated C57 black mice (3 mice), and C57 black mice treated with CsA (3 mice). Quantification of the pooled mean injured mitochondria in the hepatocytes for each group (Fig. 4) showed that CsA treatment mediated a marked decrease in the mitochondrial structural alterations described. There were no detectable differences between the 6-, 12-, and 24-wk CSA treatment intervals (data not shown). As an additional control, tacrolimus, which has immunosuppressive properties similar to CsA but without an effect on the mitochondrial permeability transition (13, 18, 19), was administered for 12 wk to PiZ mice (3 mice). Analysis showed that tacrolimus had no effect on mitochondrial structure [14 vs. 16 ± 10% (mean ± SD) injured mitochondria in tacrolimus-treated compared with baseline PiZ mice, respectively; P > 0.2]. Morphometric quantification of steady-state general autophagy not involving mitochondria using previously described techniques revealed no difference between the PiZ mice at baseline and those treated with CsA [2.0 vs. 1.7 ± 0.4% (mean ± SD) cytoplasm occupied by autophagic vacuoles in PiZ mice at baseline vs. CsA treated, P > 0.1; see Ref. 28]. These data are consistent with the hypothesis that CsA does not inhibit autophagy in general, but may act specifically on the permeability transition of mitochondria, to inhibit the unique mitochondrial injury in the PiZ mice.



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Fig. 4. Effect of cyclosporin A (CsA) on mitochondrial injury in C57BL and PiZ mice. Quantitative analysis (pooled mean for each group ± SD) of percent injured mitochondria as determined by electron microscopy in C57BL mice and PiZ mice both at baseline and after treatment with CsA. P < 0.05 for baseline PiZ mice compared with the 3 other groups.

 

Next, we examined the effect of CsA on the capacity of the PiZ mice to tolerate fasting. We have previously reported that PiZ mice suffer increased mortality associated with liver injury during fasting compared with C57 black mice, and fasting is known to induce increased hepatic autophagy, including mitochondrial autophagy (8, 17, 25, 28). We subjected groups of five PiZ mice, C57 black mice, and PiZ mice treated for 1 wk with CsA to a 72-h fast. None (0%) of the PiZ mice was able to survive, whereas all (100%) of the C57 black and all (100%) of the CsA-treated PiZ mice survived. Our previous report showed that fasting of short duration (18 h) induced increased steatosis in PiZ mouse liver compared with the wild type (WT), but in the longer, 72-h fasting experiments reported here, we noted that fat droplets had disappeared from hepatocytes in mice that could not survive the fast. CsA treatment appeared to delay the disappearance of steatosis from the 72-h-fasted PiZ mice.

Because CsA has many other effects on cellular function, it is not entirely possible to conclude that these protective effects are the result of prevention of mitochondrial injury and/or autophagy. We did, however, examine one potential nonmitochondrial mechanism for a protective effect of CsA, an effect on the accumulation of {alpha}1-ATZ in the ER. First, we examined the liver for periodic acid-Schiff-positive, diastase-resistant globules, the characteristic histological correlate of aggregated {alpha}1-ATZ retained in the ER (11, 31). There was no change associated with CsA treatment [28 vs. 31 ± 7% (mean ± SD) of hepatocytes containing globules, P > 0.1 and 4.4 vs. 4.1 ± 2.1 µm (mean ± SD) globule diameter, P > 0.2 in PiZ mice at baseline vs. CsA treated, respectively]. Second, we examined the ER using EM. There was no difference in the dilated ER observed in CsA-treated compared with untreated PiZ mice (data not shown). These data militate against the possibility that there is less mitochondrial injury and less mortality associated with CsA treatment as a result of CsA-mediated decreased ER accumulation of {alpha}1-ATZ protein.

Evidence for caspase activation in {alpha}1-AT-deficient liver. Previous studies have shown that mitochondrial permeability transition precedes mitochondrial autophagy and is inhibited by CsA (3, 10, 13, 18). The permeability transition is also involved in caspase activation and other signal transduction pathways (12, 13, 18). To determine whether the caspase cascade is activated in {alpha}1-AT-deficient liver, we used immunofluorescence with antibody to activated caspase-3 to analyze liver from the PiZ mouse (Fig. 5A). The results show no hepatocyte immunostaining in the WT C57 black mice but diffuse cytoplasmic staining with focal areas of higher signal throughout hepatocytes in the PiZ mouse liver. Administration of CsA to the PiZ mice was associated with a reduction in staining for caspase-3 to the level observed in the WT C57 black mouse liver, and the specific fluorescent signal could be blocked in the untreated PiZ liver by preincubation of the activated caspase-3 antibody with caspase-3 blocking peptide. Next, we examined three specimens of liver from normal humans, six specimens of liver from {alpha}1-AT-deficient patients, and the liver disease control specimens described above by immunohistochemistry using antibody to activated caspase-3. Figure 5B shows representative results in one of the normal controls, one of the PIZZ {alpha}1-AT-deficient livers, and one of the disease controls (Wilson's disease). There is no detectable activated caspase-3 signal within the normal human hepatocytes or in the specimen with Wilson's disease but a diffuse cytoplasmic pattern of staining, with focal areas of more intense staining, in the PIZZ specimen. Widespread hepatocellular death was not apparent by conventional light microscopic examination in any of the specimens, nor were any of the patients in clinical liver failure at the time the biopsies were obtained (data not shown). In the liver disease controls, there were liver cells with intense immunostaining for activated caspase-3 corresponding with features of apoptosis under conventional microscopy, but none of these specimens had the widespread diffuse cytoplasmic-activated caspase-3 signal that was characteristic of {alpha}1-AT deficiency.



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Fig. 5. Examination of caspase-3 activation by immunostain in liver expressing {alpha}1-antitrypsin Z ({alpha}1-ATZ). A: immunofluorescent staining for activated caspase-3 in liver of C57 black mice (C57BL), PiZ mice (PiZ), PiZ mice treated with CsA (PiZ + CsA), and PiZ mice with activated caspase-3 antibody preincubated with activated caspase-3 blocking peptide (PiZ + block). B: immunohistochemical staining for activated caspase-3 in normal human liver, PIZZ ZZ human liver, and in Wilson's disease liver (Wilson's). C: immunofluorescent staining for activated caspase-3 (red) of normal [wild-type (WT)] human liver (top left) and then double-label immunofluorescent staining for activated caspase-3 (red) and {alpha}1-AT (green) in {alpha}1-AT PIZZ liver at low magnification in the same hepatic lobule (top right and bottom left) and merged at high magnification (bottom right). Arrows, globules of dilated ER that immunostain positive for {alpha}1-AT; arrowheads, structures that immunostain positive for activated caspase-3. Bar = 10 µm.

 

Many previous examinations of liver from PIZZ {alpha}1-AT-deficient patients have shown that {alpha}1-AT immunostain-positive globules are only present in some of the hepatocytes and that there are regions with significantly less, or even complete absence, of {alpha}1-AT immunoreactivity. In Fig. 5C, we examined the possibility that hepatocytes that stain intensely for activated caspase-3 also stain positively for {alpha}1-AT in the {alpha}1-AT-deficient liver. The result again shows no signal in control, normal WT liver but that there are regions with focal intense staining for activated caspase-3 in the hepatic lobule that colocalize with areas having intense focal staining for {alpha}1-AT in the PIZZ liver. High-magnification merged images from these regions show that both activated caspase-3 and {alpha}1-AT-positive areas are present within the same hepatocytes, but have distinct, separate subcellular localizations.

Mitochondrial autophagy and injury in a cell line model of {alpha}1-AT deficiency. To provide further evidence for mitochondrial functional abnormalities, and perhaps to better understand the sequence of events involved in the development of mitochondrial dysfunction, autophagy, and caspase activation, we examined a model cell line with inducible expression of {alpha}1-ATZ. HeLa cell lines were engineered for inducible expression of WT {alpha}1-AT (HTO/M cell line) or mutant {alpha}1-ATZ (HTO/Z cell line), which do not express {alpha}1-AT in the presence of doxycycline (Dox). However, {alpha}1-AT expression is induced beginning 3 days after withdrawal of Dox from the culture media and continues in a time-dependent manner (Fig. 6A). Pulse-chase studies show that these cell lines recapitulate the physiological secretion of WT {alpha}1-AT and the secretory defect (intracellular retention) of mutant {alpha}1-ATZ (Fig. 6B), exactly as has been described previously (24, 30, 31, 35). In the HTO/M cells studied 7 days after withdrawal of Dox, partially glycosylated 52- and 55-kDa polypeptides disappear from the intracellular compartment over 60–120 min of the chase period, coincident with the progressive appearance of the 55-kDa, mature glycoprotein in the extracellular fluid. In HTO/Z cells, the disappearance of the 52-kDa polypeptide in the intracellular compartment is significantly slower, with ~50% remaining at 120 min, and only trace amounts are secreted in the extracellular fluid.



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Fig. 6. Analysis of inducible expression and secretion of {alpha}1-AT in HTO/M and HTO/Z cells. A: 35S biosynthetic radiolabeling in aliquots of HTO/M (WT) and HTO/Z (Z) cells 0, 3, and 7 days after induction of gene expression by removal of doxycycline (Dox) from the culture medium. The intracellular fractions were isolated, immunoprecipitated with antibody to {alpha}1-AT, and analyzed by SDS-PAGE fluorography with molecular weight markers as shown. Mr, relative molecular weight. B: pulse-chase studies of HTO/M and HTO/Z cells out of Dox for 7 days subjected to 35S radiolabeling and then chased for the time points shown. The intracellular fraction (IC) and extracellular media (EC) were harvested and analyzed by immunoprecipitation with {alpha}1-AT antibody and SDS-PAGE fluorography with molecular weight markers as shown.

 

First, we used EM to determine whether there were mitochondrial ultrastructural changes in the HTO/Z cell line (Fig. 7A). The results show that HTO/Z cells maintained in the presence of Dox (baseline, no {alpha}1-AT expression) had normal-appearing, intact mitochondria, but HTO/Z cells maintained in the absence of Dox showed not only mitochondrial autophagy but also a pattern of internal injury similar to that observed in the PiZ mice. Clusters of mitochondria that were in close proximity to rER and autophagosomes were found to be swollen with blebs in the outer membrane (Fig. 7A, arrows) and to have areas of matrix and cristae in various phases of dissolution.



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Fig. 7. Mitochondrial injury and autophagy in HTO/Z cells. A: transmission electron microscopy of mitochondria in HTO/Z cells maintained in Dox without {alpha}1-AT expressed (baseline) and maintained out of Dox ({alpha}1-ATZ expression induced). Arrow shows mitochondria with bleb in outer membrane near autophagic vacuole and dissolution of internal mitochondrial structures. B: FACS analysis of HTO/M cells (WT) and HTO/Z cells (Z) at baseline (dark gray curve), with {alpha}1-AT induced (black curve) and control with mitochondria depolarized by CCCP (light gray curve). C: confocal, double-label fluorescent microscopy in 0.5-µm slices of HTO/Z cells at baseline (baseline), HTO/M cells with {alpha}1-AT WT induced (WT), and HTO/Z cells with mutant Z induced (Z) labeled with 500 nM MitoTracker Green and 500 nM Mitofluoro Red 594 for 30 min. Arrows show green fluorescent structures. D: confocal, double-label fluorescent microscopy in 0.5-µm slices of HTO/Z cells at baseline (baseline), HTO/M cells with {alpha}1-AT WT induced (WT), HTO/Z cells with mutant Z induced (Z), and HTO/Z cells with mutant Z induced in the presence of CSA (Z + CSA). Cells were labeled with 500 nM MitoTracker Green and 1 µM Lysotracker Red for 30 min; arrows show yellow areas of dye colocalization.

 

Next, we examined the possibility that there were also functional changes in the mitochondria associated with induction of mutant {alpha}1-ATZ expression in this model cell line. HTO/M and HTO/Z cells were maintained in Dox or out of Dox for 7 days, a time associated with robust induction of WT {alpha}1-AT or mutant {alpha}1-ATZ protein synthesis. We employed the reagent TMRM, which will specifically label mitochondria in living cells and fluoresce at 575 nm (10, 19) but lose fluorescence if the mitochondria depolarize. We labeled HTO/M cells at baseline with TMRM and quantified fluorescence in 10,000 cells by FACS (Fig. 7B, left, dark gray curve). Identical analysis in another aliquot of HTO/M cells after expression of {alpha}1-AT has been induced showed no change in TMRM fluorescence (Fig. 7B, left, black curve). Treatment of HTO/M cells with the mitochondrial poison, CCCP (Fig. 7B, left, light gray curve), induced depolarization and a significant drop in fluorescence, as would be predicted. Analysis of HTO/Z cells at baseline was identical to HTO/M cells (Fig. 7B, right, dark gray curve). However, when mutant {alpha}1-ATZ expression is induced in HTO/Z cells, there is a reduction in fluorescence indicative of mitochondrial depolarization (Fig. 7B, right, black curve shifted left of dark gray curve). The effect of inducing {alpha}1-ATZ expression was not as great as the effect of CCCP (Fig. 7B, right, light gray curve).

We further examined the mitochondrial depolarization in these cells using confocal microscopy and fluorescent vital dyes (Fig. 7B). Again, HTO/M and HTO/Z cells were used either in Dox or out of Dox for 7 days. Living cells were then stained with MitoTracker Green, which labels all mitochondria in cells green under fluorescence regardless of membrane potential, and Mitofluoro Red 594, which labels mitochondria with red fluorescence but which will loose fluorescence if the mitochondria depolarize. This technique will label normal mitochondria yellow (green + red). In the HTO/M and HTO/Z cells at baseline, numerous yellow fluorescent structures are seen throughout the cells, which is similar to the appearance of HTO/M cells with WT {alpha}1-AT induced. However, in HTO/Z cells with {alpha}1-ATZ induced, many green structures are visible, consistent with depolarized mitochondria. Taken together, these FACS and confocal microscopy data indicate that, when {alpha}1-ATZ gene expression is induced and the mutant protein accumulates in the ER, there is a specific effect on mitochondrial function with depolarization and opening of the mitochondrial permeability transition pore. Mitochondrial depolarization has been shown to initiate mitochondrial autophagy in experimental hepatocellular systems (10).

Therefore, we next examined the possibility that ER retention of {alpha}1-ATZ induced in the HTO/Z cell line is associated with the movement of mitochondria in acidic vacuoles that is suggestive of mitochondrial autophagy. We again stained live cells with MitoTracker Green to label the mitochondria with green fluorescence and LysoTracker Red, which labels all acidic compartments with red fluorescence. Autophagic vacuoles acidify early in their biogenesis (8, 17, 25) and have been previously shown to fluoresce red with this technique (28, 32). HTO/M and HTO/Z cells in the presence of Dox (Fig. 7D, left, baseline) and out of Dox for 7 days (Fig. 7D, center) or HTO/Z cells treated with CsA, a known inhibitor of mitochondrial autophagy in other systems (Fig. 7D, right), were studied. The results show in the HTO/Z cells at baseline that green, filamentous structures, consistent with mitochondria, and numerous punctuate red structures, consistent with acidic vacuoles including lysosomes, endosomes, and autophagosomes are easily identified and distinct from each other. After induction of {alpha}1-AT expression, HTO/M cells have a similar appearance. However, when {alpha}1-ATZ expression is induced, the HTO/Z cells show significant numbers of yellow punctuate structures (Fig. 7D, arrows), suggesting colocalization of the dyes as mitochondria move in acidic autophagic vacuoles. The yellow colocalization signal is markedly decreased by treatment with CsA. These data indicate that, when {alpha}1-ATZ accumulates in the ER of these cells, there is a specific effect on mitochondria with movement of mitochondria in acidic vacuoles suggestive of mitochondrial autophagy.

Next, we examined whether caspase activation occurs before or after mitochondrial depolarization in the model cell line. For this, we examined aliquots of intracellular lysate from HTO/M and HTO/Z cells for caspase-3 cleavage 7 days after induction of {alpha}1-AT gene expression (Fig. 8). The results show that caspase-3 is not cleaved in either the HTO/M or HTO/Z cells in the presence or absence of Dox. Cleavage of caspase-3 is seen in the positive control cells treated with staurosporine. With the use of actin as a control, there was no evidence of unequal loading as an explanation for the absence of the caspase-3 cleavage product in HTO/M or HTO/Z cells. Because our previous studies have shown that mitochondrial depolarization and movement in acidic vacuoles can be detected by 7 days after the induction of {alpha}1-ATZ, these results suggest that changes in mitochondrial permeability precede activation of the caspase cascade and are therefore consistent with previously described data.



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Fig. 8. Caspase-3 activation in HTO/Z and HTO/M cells 7 days after induction of {alpha}1-AT expression. Immunoblot for caspase-3 (full length and cleaved on top) and actin as a loading control (bottom) of the same intracellular fractions of HTO/M and HTO/Z cells, with molecular weight markers as shown, in Dox or out of Dox for 7 days and with or without exposure to staurosporine (Staur) as noted. Black arrowhead shows full-length caspase-3; white arrowhead shows activated (cleaved) caspase-3. Identical results were obtained when the experiment was performed with the cells out of Dox for 4 days (data not shown).

 

Finally, we examined the effect of CsA on degradation of {alpha}1-ATZ in the HTO/Z cell line to exclude the possibility that the positive effect of CsA on mitochondria is the result of an acceleration of {alpha}1-ATZ degradation, which would decrease the upstream pathological state. HTO/Z cells maintained in the absence of Dox were treated with either CsA or tacrolimus (Fig. 9). The result shows that, in the control, a 52-kDa {alpha}1-ATZ polypeptide is present intracellularly at time 0 and disappears progressively over 4 h with a half-life of 1.75 ± 0.3 h. This polypeptide corresponds to {alpha}1-ATZ with immature glycosylation retained within the ER lumen and has a half-life similar to previously published data (24, 35). However, in the presence of CsA, the 52-kDa {alpha}1-ATZ polypeptide disappears much more slowly (half-life 4.25 ± 0.2 h, P < 0.04), with a significant amount still present after 8 h of the chase period. There was no difference in the disappearance of {alpha}1-ATZ in cells treated with tacrolimus (1.6 ± 0.3 h, P > 0.2) compared with control (Fig. 9, bottom), and there was no difference among all three conditions in the trace amounts of {alpha}1-ATZ secreted in the extracellular fluid. A dose-response for CsA was observed for this effect of reduced {alpha}1-ATZ intracellular disappearance, with <5 µM CsA showing no effect (1.8 ± 0.4 h, P > 0.2 for 5 µM) and increased inhibition with increased dose up to 50 µM (2.4 ± 0.3 h for 10 µM, 3.5 ± 0.2 h for 25 µM, and 4.25 ± 0.2 h for 50 µM). Similar results were obtained when this same experiment was repeated in Hepa1,6 cells and in human skin fibroblasts engineered for expression of {alpha}1-ATZ (4.1 ± 0.2 h, P < 0.05, and 4.0 ± 0.3 h, P < 0.05, respectively, with 50 µM CsA). Thus there is no evidence that CsA mediates a decrease in the accumulation of {alpha}1-ATZ in the ER. In fact, these results indicate that CsA mediates a significant decrease in the rate of degradation of {alpha}1-ATZ in cell culture model systems.



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Fig. 9. Effect of CsA and tacrolimus on intracellular degradation of {alpha}1-ATZ. Pulse-chase biosynthetic labeling experiments for the time points shown to determine the effect of CsA and tacrolimus on the disappearance of {alpha}1-ATZ from the intracellular fraction of HTO/Z cells maintained out of Dox. The intracellular fraction and extracellular media were harvested and analyzed by immunoprecipitation with {alpha}1-AT antibody and SDS-PAGE fluorography.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Most of what is known about {alpha}1-AT deficiency suggests that a gain-of-function mechanism is responsible for liver injury and for the predilection for development of hepatocellular carcinoma. However, there has been very little consideration of the mechanism by which retention of a mutant glycoprotein in the ER results in liver cell injury. By carefully examining the ultrastructural changes in the liver in this disease, we recently found that autophagy is a very prominent component of the hepatic lesion (32). We have also shown that a mouse model of {alpha}1-AT deficiency is characterized by a unique and specific state in which the autophagic response is constitutively activated (28). Here we describe the surprising discovery that there is a striking degree of mitochondrial autophagy and significant mitochondrial injury in the liver of {alpha}1-AT-deficient patients, in a genetically engineered mouse model of {alpha}1-AT deficiency, and in a cell line that models {alpha}1-AT deficiency. In the liver of {alpha}1-AT-deficient patients and of PiZ mice, there is morphological evidence for mitochondrial autophagy, mitochondrial injury, and caspase-3 activation that is prevented by CsA but not by tacrolimus. Here, autophagy was defined exclusively by ultrastructural criteria. Although LC3 has recently been identified as a specific marker for autophagy (15), an antibody that permits consistent detection of the endogenous LC3 protein in mammalian tissues by immunofluorescence has not yet been developed. In the model cell line, induction of mutant {alpha}1-ATZ expression and accumulation in the ER is associated with mitochondrial autophagy and injury, as shown by morphological and functional effects, including mitochondrial depolarization and movement into acidic vacuoles that is also inhibited by CsA. Moreover, mitochondrial functional defects in the model cell line are specific for mutant {alpha}1-ATZ and only occur after induction of {alpha}1-ATZ gene expression.

There are at least two possible explanations for this mitochondrial damage. In the first, accumulation of {alpha}1-ATZ in the ER is by itself responsible for mitochondrial dysfunction. Autophagy might therefore only be activated secondarily to remove already damaged organelles in a way consistent with the known housekeeping activity of autophagy. There is now ample evidence in the literature that mitochondria are closely apposed to the cisternae of the ER physically (22) and that there are functional interactions between these two organelles (1). Recent studies show that specific signals are transmitted between these two intracellular compartments (2, 33). Potentially even more relevant studies have shown that there is mitochondrial dysfunction, including release of cytochrome c and caspase-3 activation, when there is ER dilatation and/or "ER stress" induced by brefeldin A, tunicamycin, and thapsigargin (14, 34). It is not yet known, however, whether the mitochondrial dysfunction that was described in these last studies was the result of ER dilatation and/or ER stress or the result of an independent drug effect.

A second possible explanation envisages mitochondrial dysfunction as a direct result of the autophagic response to ER retention of {alpha}1-ATZ. In this scenario, mitochondria are recognized nonspecifically by the autophagic response, which is constitutively activated to remove and degrade areas of the ER that are distended by aggregated mutant protein. Several lines of evidence from the studies reported here militate against this possible explanation. First, when {alpha}1-ATZ gene expression is induced under tightly controlled conditions in a genetically engineered cell line, mitochondria are directly depolarized. Second, CsA inhibits movement of mitochondria in acidic vacuoles and inhibits mitochondrial injury in the PiZ mouse in vivo without affecting autophagy in general. Nevertheless, it is not possible at this juncture to completely exclude the possibility that CsA has other mechanisms of action in vivo (6, 10, 13, 18, 19). Definitive resolution of this issue will require investigation of the effect of {alpha}1-ATZ accumulation in the ER on mitochondrial function in an autophagy-deficient context.

The results of experiments with CsA are also noteworthy for their therapeutic implications. CsA mediated a marked and specific decrease in degradation of mutant {alpha}1-ATZ. Although this could be the result of the effect of CsA on autophagy, recent studies have shown that CsA also inhibits the proteosomal degradation pathway (20, 21) and enhances calcium uptake in the ER (26). In either case, the implication of the studies in the transgenic mouse in vivo, taken together with the studies in cell culture, is that CsA can prevent mitochondrial damage even under circumstances in which {alpha}1-ATZ is continuing to accumulate in the ER. Thus these results provide a proof in principle for mechanism-based therapeutic approaches to liver disease in {alpha}1-AT deficiency, i.e., pharmacological intervention directed at distal steps in the pathobiological pathway that leads to liver injury, such as the "mitochondrial" step, without correction of the primary defect and/or the more proximal steps in the pathobiology of this liver disease.

Several results of this study will need further investigation. First, although there is a widespread and diffuse pattern of caspase-3 activation in the hepatic lobule of {alpha}1-AT-deficient liver and also evidence for caspase-9 activation in the PiZ mouse model (Rudnick D., Perlmutter D., Teckman J., unpublished observation), which is not present in normal human liver or in liver disease controls, it is not yet clear why other signs of hepatocellular death are not prominent in these liver specimens. One possibility is that regulatory effects that inhibit the caspase cascade distal to caspase-3 (13, 17, 18) are also activated in the liver in {alpha}1-AT deficiency or by secondary effects of end-stage liver disease. Liver disease in {alpha}1-AT deficiency is relatively slowly progressive, so it is also possible that small numbers of hepatocytes go onto cell death over relatively long intervals of time and therefore the signs of cell death fall below the limits of detection at any single point in time. In this respect, it is noteworthy that hepatocyte proliferation is increased in the PiZ mouse at baseline compared with the C57 black mouse by bromodeoxyuridine (BrDU) incorporation (D. Rudnick, D. Perlmutter, J. Teckman, unpublished observation). These data imply that there is also significant ongoing cell death in the liver of the PiZ mouse and, moreover, that it occurs at a relatively low rate because the increase in BrDU incorporation is on the order of 2.0 ± 0.3% compared with 0.4 ± 0.1% in normal hepatocytes (P < 0.001).

A second area for further investigation will be to determine why there is less mitochondrial autophagy in the PiZ mouse liver than in the PIZZ human liver. There is a significant increase in mitochondrial injury in the PiZ mouse liver compared with C57/BL liver shown here, and our previous studies have shown a marked increase in autophagy in general in the PiZ mouse liver (28, 32). This could reflect the overall lesser degree of liver injury in the PiZ mouse compared with the human PIZZ liver, which may be the result of human clinical liver specimens being examined only at late stages in the disease progression and predominantly obtained in the subgroup of PIZZ individuals susceptible to liver disease. Alternatively, it might reflect a difference in the rate of autophagosome formation and/or turnover. In either case, the fact that there is mitochondrial injury with a relatively low mitochondrial autophagy in the PiZ mouse liver provides additional support for the idea that ER accumulation of {alpha}1-ATZ is directly responsible for mitochondrial damage and dysfunction in the {alpha}1-AT-deficient liver.

In conclusion, these experimental results raise the possibility for the first time that mitochondrial damage and caspase activation are involved in the mechanism for liver cell injury in {alpha}1-AT deficiency. Further studies will be needed to examine the effects of CsA, particularly the effects of long-term CsA administration in vivo, and to examine the possibility that other agents that prevent mitochondrial dysfunction or autophagy have therapeutic effects in {alpha}1-AT deficiency.


    ACKNOWLEDGMENTS
 
This work was made possible with support from the National Institutes of Health Grants DK-52526, HL-37784, DK-67960, and DK-52574, the March of Dimes, the Alpha-1 Foundation, the American Liver Foundation, and the generosity of the "Alpha1" Community.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Teckman, Dept. of Pediatrics, Washington Univ. School of Medicine, 660 South Euclid Ave., Box 8208, St. Louis, MO 63110.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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