Am J Physiol Gastrointest Liver Physiol 286: G851–G862, 2004.
First published December 18, 2003; 10.1152/ajpgi.00175.2003.
Mitochondrial autophagy and injury in the liver
in ␣1-antitrypsin deficiency
Jeffrey H. Teckman,1 Jae-Koo An,1 Keith Blomenkamp,1 Bela Schmidt,2 and David Perlmutter2,3
1
Department 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
␣1-antitrypsin; autophagy; mitochondria; quality control; cyclosporin A
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 ␣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 ␣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 ␣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 ␣1-ATZ (32). However, in this
study, we report the presence of mitochondrial autophagy in
the liver in ␣1-AT deficiency and provide evidence for damage
to mitochondria that has characteristics that are unique and
specific to this genetic liver disease.
THE CLASSIC FORM of ␣1-antitrypsin (␣1-AT) deficiency, homozygous PIZZ ␣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 ␣1-anti-trypsin Z (␣1-ATZ) molecule (5). Polymerized
mutant ␣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 ␣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 ␣1-ATZ
within the ER is toxic to liver cells. The “accumulation”
mechanism is best demonstrated by studies of mice transgenic
for the human ␣1-ATZ gene (4, 9). In addition to periodic
Antibodies, labels, and cell lines. Antibodies included rabbit antihuman ␣1-AT (DAKO, Santa Barbara, CA), goat anti-human ␣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 ␣1-ATZ
and WT M have been previously described (24, 29, 30, 32, 35).
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.
http://www.ajpgi.org
METHODS
0193-1857/04 $5.00 Copyright © 2004 the American Physiological Society
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Teckman, Jeffrey H., Jae-Koo An, Keith Blomenkamp, Bela
Schmidt, and David Perlmutter. Mitochondrial autophagy and injury in the liver in ␣1-antitrypsin deficiency. Am J Physiol Gastrointest Liver Physiol 286: G851–G862, 2004. First published December
18, 2003; 10.1152/ajpgi.00175.2003.—Homozygous, PIZZ ␣1-antitrypsin (␣1-AT) deficiency is associated with chronic liver disease and
hepatocellular carcinoma resulting from the toxic effects of mutant
␣1-anti-trypsin Z (␣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 ␣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 ␣1-ATZ also involves the
mitochondria, with specific patterns of both mitochondrial autophagy
and mitochondrial injury seen in cell culture models of ␣1-AT deficiency, in PiZ transgenic mouse liver, and in liver from ␣1-ATdeficient 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 ␣1-AT
deficiency and suggest the possibility of mechanism-based therapeutic
interventions.
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MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
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䡠kg⫺1 䡠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.
RESULTS
Mitochondrial autophagy and injury in liver from ␣1-AT
deficient patients. We examined the livers from four ␣1-ATdeficient 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 ␣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,
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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 ␣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 ␮M 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
MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
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, electrondense body of the mitochondrion. These are the characteristics
of a late autophagic vacuole, or autolysosome. Finally, mitochondria that have almost completely transformed into elec-
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Fig. 1. Electron microscopy of mitochondrial autophagy and injury in human
liver. a: Transmission electron microscopy of human PIZZ, ␣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.
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tron-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).
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, ␣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 ␣1-AT deficiency, we used morphometry to compare the
livers from the same four PIZZ, ␣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
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MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
and this specific type of mitochondrial injury are uniquely
increased in liver from ␣1-AT-deficient patients (Fig. 2, A and
B). The two types of structural changes seen in PIZZ ␣1-ATdeficient 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 ␣1-ATdeficient patients (Fig. 2, c and d). These results suggest that
there are at least two morphologically distinct types of mitochondrial injury specific to ␣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.
Mitochondrial autophagy and injury in a transgenic mouse
model of ␣1-AT deficiency. Next, we used EM to examine liver
from the PiZ mouse. Previous studies have shown histological
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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.
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MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
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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.
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.
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
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.
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changes in the liver of this mouse that are characteristic of
␣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.
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
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MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
immunohistochemistry using antibody to activated caspase-3.
Figure 5B shows representative results in one of the normal
controls, one of the PIZZ ␣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 ␣1-AT deficiency.
Many previous examinations of liver from PIZZ ␣1-ATdeficient patients have shown that ␣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 ␣1-AT immunoreactivity. In Fig. 5C, we examined
the possibility that hepatocytes that stain intensely for activated
caspase-3 also stain positively for ␣1-AT in the ␣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 ␣1-AT in the PIZZ
liver. High-magnification merged images from these regions
show that both activated caspase-3 and ␣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
␣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 ␣1ATZ. HeLa cell lines were engineered for inducible expression
of WT ␣1-AT (HTO/M cell line) or mutant ␣1-ATZ (HTO/Z
cell line), which do not express ␣1-AT in the presence of
doxycycline (Dox). However, ␣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 ␣1-AT and the secretory defect
(intracellular retention) of mutant ␣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,
Fig. 5. Examination of caspase-3 activation by immunostain in liver expressing ␣1-antitrypsin Z (␣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 ␣1-AT (green) in ␣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 ␣1-AT; arrowheads, structures that immunostain positive for activated caspase-3. Bar ⫽ 10 ␮m.
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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 ␣1-ATZ in the ER. First, we examined
the liver for periodic acid-Schiff-positive, diastase-resistant
globules, the characteristic histological correlate of aggregated
␣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 ␮m (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 ␣1-ATZ protein.
Evidence for caspase activation in ␣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 ␣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 ␣1-AT-deficient patients,
and the liver disease control specimens described above by
MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
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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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 ␣1-AT expression) had normalappearing, intact mitochondria, but HTO/Z cells maintained in
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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.
Next, we examined the possibility that there were also
functional changes in the mitochondria associated with induction of mutant ␣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
␣1-AT or mutant ␣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
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Fig. 6. Analysis of inducible expression and secretion of ␣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 ␣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
␣1-AT antibody and SDS-PAGE fluorography with molecular weight markers
as shown.
analysis in another aliquot of HTO/M cells after expression of
␣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 ␣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 ␣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 ␣1-AT induced. However, in HTO/Z
cells with ␣1-ATZ induced, many green structures are visible,
consistent with depolarized mitochondria. Taken together,
these FACS and confocal microscopy data indicate that, when
␣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 ␣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 ␣1-AT expression, HTO/M cells have a similar appearance.
However, when ␣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 ␣1-ATZ accumulates in
the ER of these cells, there is a specific effect on mitochondria
MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
G859
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 ␣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
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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 ␣1-AT expressed (baseline)
and maintained out of Dox (␣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 ␣1-AT induced
(black curve) and control with mitochondria depolarized by
CCCP (light gray curve). C: confocal, double-label fluorescent
microscopy in 0.5-␮m slices of HTO/Z cells at baseline (baseline), HTO/M cells with ␣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, doublelabel fluorescent microscopy in 0.5-␮m slices of HTO/Z cells at
baseline (baseline), HTO/M cells with ␣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 ␮M
Lysotracker Red for 30 min; arrows show yellow areas of dye
colocalization.
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MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
in the accumulation of ␣1-ATZ in the ER. In fact, these results
indicate that CsA mediates a significant decrease in the rate of
degradation of ␣1-ATZ in cell culture model systems.
DISCUSSION
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 ␣1-ATZ, these results
suggest that changes in mitochondrial permeability precede
activation of the caspase cascade and are therefore consistent
with previously described data.
Finally, we examined the effect of CsA on degradation of
␣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 ␣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 ␣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 ␣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 ␣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 ␣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 ␣1-ATZ secreted in the
extracellular fluid. A dose-response for CsA was observed for
this effect of reduced ␣1-ATZ intracellular disappearance, with
⬍5 ␮M CsA showing no effect (1.8 ⫾ 0.4 h, P ⬎ 0.2 for 5
␮M) and increased inhibition with increased dose up to 50 ␮M
(2.4 ⫾ 0.3 h for 10 ␮M, 3.5 ⫾ 0.2 h for 25 ␮M, and 4.25 ⫾
0.2 h for 50 ␮M). Similar results were obtained when this same
experiment was repeated in Hepa1,6 cells and in human skin
fibroblasts engineered for expression of ␣1-ATZ (4.1 ⫾ 0.2 h,
P ⬍ 0.05, and 4.0 ⫾ 0.3 h, P ⬍ 0.05, respectively, with 50 ␮M
CsA). Thus there is no evidence that CsA mediates a decrease
Fig. 9. Effect of CsA and tacrolimus on intracellular degradation of ␣1-ATZ.
Pulse-chase biosynthetic labeling experiments for the time points shown to
determine the effect of CsA and tacrolimus on the disappearance of ␣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 ␣1-AT antibody and SDS-PAGE fluorography.
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Fig. 8. Caspase-3 activation in HTO/Z and HTO/M cells 7 days after induction
of ␣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).
Most of what is known about ␣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
␣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 ␣1-AT-deficient patients, in
a genetically engineered mouse model of ␣1-AT deficiency,
and in a cell line that models ␣1-AT deficiency. In the liver of
␣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 ␣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 ␣1-ATZ and only occur after
induction of ␣1-ATZ gene expression.
There are at least two possible explanations for this mitochondrial damage. In the first, accumulation of ␣1-ATZ in the
ER is by itself responsible for mitochondrial dysfunction.
MITOCHONDRIAL AUTOPHAGY AND LIVER INJURY IN ␣1-AT DEFICIENCY
activated in the liver in ␣1-AT deficiency or by secondary
effects of end-stage liver disease. Liver disease in ␣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 ␣1-ATZ is
directly responsible for mitochondrial damage and dysfunction
in the ␣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
␣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 ␣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.
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