AUTOPHAGY: A CORE CELLULAR PROCESS WITH EMERGING LINKS TO
Running Title: Role of Autophagy in Lung Disease
Jeffrey A. Haspel1, 2, and Augustine M. K. Choi1,*
1 Division of Pulmonary and Critical Care Medicine, Department of Medicine; Brigham and
Women’s Hospital; 75 Francis Street; Boston, MA, 02115; USA.
2 Pulmonary and Critical Care Medicine Section, VA Boston Healthcare System, Boston, MA, USA.
Author Contributions: J.A.H and A.M.K.C co-wrote this review article.
Descriptor Number: 8.2 (Airway Pathology and Structure)
Funding Support: NIH T32 HL007633 and R03 HL097005.
Narrative Word Count: 4,981
Scientific Knowledge on the Subject: a growing body of literature indicates that autophagy is
linked to the pathogenesis of common pulmonary disorders.
What this Study Adds to the Field: this review provides an introduction to the field of autophagy
for clinical and research Pulmonologists previously unfamiliar with this topic. It also reviews
current evidence linking regulation of autophagy to common pulmonary diseases.
This article has an online data supplement, which is accessible from this issue's table of content
online at www.atsjournals.org
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AJRCCM Articles in Press. Published on August 11, 2011 as doi:10.1164/rccm.201106-0966CI
Copyright (C) 2011 by the American Thoracic Society.
Autophagy is a highly conserved homeostatic pathway by which cells transport damaged
proteins and organelles to lysosomes for degradation. Dysregulation of autophagy
contributes to the pathogenesis of clinically important disorders in a variety of organ
systems but, until recently, little was known about its relationship to diseases of the
lung. However, there is now growing evidence at the basic research level that
autophagy is linked to the pathogenesis of important pulmonary disorders such as
COPD, cystic fibrosis and tuberculosis. In this review, we provide an introduction to the
field of autophagy research geared to clinical and research Pulmonologists. We focus on
the best studied autophagic mechanism, macroautophagy, and summarize recent
studies that link the regulation of this pathway to pulmonary disease. Finally, we offer
our perspective on how a better understanding of macroautophagy might be utilized for
designing novel therapies for pulmonary disorders.
Abstract Word Count: 143
Keywords: autophagy, macroautophagy, lung, disease, COPD.
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At the cellular level, homeostasis is maintained by a series of deeply conserved
pathways of which “autophagy” is one central pillar. Autophagy refers to a collection of
catabolic pathways that transport components of the cytoplasm to lysosomes for
degradation (1). In addition to proteins, autophagy can target both carbohydrates (2, 3)
and lipids (4) for digestion as well as entire organelles, such as mitochondria and
peroxisomes (5-7). The products of digestion, such as free amino acids, are recycled
back to the cytoplasm for use in various biosynthetic pathways (8). Autophagy
contributes to cellular homeostasis via three basic mechanisms. First, autophagy
provides an alternative source of metabolic fuel (9). Second, autophagy removes
damaged cellular components, such as dysfunctional mitochondria and aggregated
proteins that would otherwise be toxic to the cell (7, 10, 11). Finally, autophagy is
entwined at the signal transduction level with the apoptotic pathway and impacts the
decision of a cell to undergo programmed cell death (12, 13). To study autophagy is to
study an aspect of the core operating system that enables eukaryotic cells to function.
Which begs the question: what does autophagy have to do with patients suffering from
pulmonary disease? Interestingly, the answer may be “quite a lot”. The purpose of this
review is twofold: to provide a basic introduction to the rapidly expanding field of
autophagy research with an emphasis on the most studied autophagic pathway
(macroautophagy), and also to review recent studies that link autophagic regulation to
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HOW DOES AUTOPHAGY WORK?
At present there are three known mechanisms by which autophagy can occur (Fig. 1).
The first mechanism is called Chaperone Mediated Autophagy (CMA) and was originally
described in lung fibroblasts (Fig. 1A) (14). This mechanism involves the direct
translocation of proteins across the lysosomal membrane via a complex that includes
Hsc70 and the lysosome transmembrane protein Lamp2A (15, 16). CMA is difficult to
monitor in vivo and at this point we don’t have any information about whether this
pathway is affected in pulmonary disease. However CMA plays an important role in the
pathogenesis of neurodegeneration and aging (17, 18), and is still an evolving field of
biomedical research. The second autophagic pathway is called microautophagy and
involves the direct invagination of cytosolic material into late endosomes or into
multivesicular bodies, which subsequently either degrade the material on site or deliver
the material to lysosomes for degradation (19). Until very recently nothing was known
about microautophagy at the molecular level and the existence of this process was only
suspected in mammalian tissues on the basis of electron micrographs. However it was
recently shown that microautophagy also employs Hsc-70 but, unlike CMA, it targets
proteins to late endosomal membranes through electrostatic interactions between this
chaperone and the lipid phospatidylserine, rather than by binding to LAMP2A (Fig. 1B)
(20). Microautophagy is similarly difficult to measure in vivo and so little is understood
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about its physiological significance at this point, although this may change as the
molecular mechanism is better defined.
The final pathway, macroautophagy (21), receives the most attention in the literature
and its contribution to human disease is the best explored of the three pathways. In
fact, macroautophagy so much better studied that it is often referred to in many papers
as simply “autophagy”, even though it is only one of the pathways involved in lysosome-
dependent degradation. This is in large part because macroautophagy can be visualized
at both the light microscopic level (using fluorescent fusion proteins), and at the EM
level making it relatively easy to detect (22). In macroautophagy, a vesicular membrane
is constructed around a volume of cytoplasm that is intended for degradation (Fig. 1C).
This novel structure, called an autophagosome, is distinct from other vesicles on
electron micrographs because it contains a double-unit limiting membrane (21).
Autophagosomes then deliver their cargo for degradation by fusing with late
endosomes and lysosomes, gradually losing their distinctive membrane structure (23).
The products of digestion, such as free amino acids, are recycled back to the cytoplasm
via lysosomal permeases (8). Of the three known autophagic pathways,
macroautophagy is notable for its ability to process large intracellular structures such as
organelles (5, 6), invasive bacteria in the cytoplasm (24, 25), and large protein
aggregates (10). Macroautophagy is sensitive to nutrient availability (19, 26), and is
altered in a variety of non-pulmonary diseases, such as neurodegeneration (27),
myopathy (28, 29), and cancer (30). All of the current literature about the role of
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autophagy in pulmonary disease focuses on macroautophagy, so we will focus on this
mechanism for the remainder of this review.
The last decade has seen a dramatic increase in our understanding of the molecular
mechanisms underlying macroautophagy (Fig. 2). The current paradigm is that
macroautophagy is mediated by a highly intricate mechanism that requires the
contribution of at least 20-30 core proteins that are physically grouped into several
distinct multi-protein complexes, each with a distinct functional specialization (31). The
most upstream complex described so far is composed of the proteins ATG1, FIP200,
ATG101 and ATG-13 and possesses serine/threonine kinase activity (32, 33). This
complex is regulated by mTOR and AMPK, which sense nutrient and intracellular ATP
availability and, through phosphorylation of ATG1, adjust the rate of macroautophagy to
the metabolic needs of the cell (34). The primary function of the ATG1-containing
complex is to control the activity and localization of another structure called the vps34
multi-protein complex, although the mechanism by which this is accomplished is
unknown. The vps34 complex has phosphatidylinositol-3 kinase (PI3K) activity and the
production of PI3 moieties is required for autophagosome formation (35, 36). One
important constituent of the vps34 complex is the protein ATG6/Beclin-1, which is an
adaptor protein that is crucial for the participation of the vps34 complex in
autophagosome formation (37, 38). In yeast, the vps34 complex localizes to a distinct
peri-nuclear structure called the phagophore assembly site (PAS; (39)) whereas in
mammalian cells this complex localizes to many sites distributed throughout the cell
(40). The precise location of these sites is controversial but there is microscopy data to
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suggest the vps34 complex can nucleate autophagosomes on the surface of both the ER
(40), and mitochondria (41), depending on the cell type and experimental conditions.
The PI3-phosphate moieties laid down by vps34 recruit binding proteins that are critical
for autophagosome maturation and may also intrinsically produce local changes in the
membrane physical properties that make it easier to mold (42-44). The result is a cup-
like structure that has been termed the “omegasome” (40, 45, 46). The final group of
proteins is an ubiquitin-like ligase system composed of the proteins ATG3, ATG7, and
ATG10, and a trimeric complex composed of ATG5, ATG12 and ATG-16L that localizes to
the omegasome (47, 48). The function of this system is to conjugate a protein called
ATG8/LC3b to the lipid phosphatidylethanolamine (PE), which is abundant in
autophagosome membranes (49). The PE-conjugated form of LC3b (called LC3b-II) is
inserted into both sides of the autophagosome membrane (50), and is required for
autophagosome membrane elongation (51, 52). LC3b is actually just one of at least 5
homologous proteins in mammalian cells (53, 54), all of which are targeted to the
autophagosome by PE conjugation and cooperate in autophagosome maturation and
sealing (52). Luminal-facing LC3b-II is degraded by lysosomal hydrolases (55), while
protein on the cytosolic-facing side of the autophagosome is recovered via de-lipidation
(Fig. 3) (56). The cytoplasmic, non-lipidated form of LC3b, called LC3b-I, can then be
recruited for the construction of new autophagosomes (50, 56). LC3b also plays an
important diagnostic role in macroautophagy research: because it is targeted uniquely
to autophagosome membranes it serves as a convenient marker for this structure (50).
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While the above molecular mechanism proposed for macroautophagy is now
impressively detailed, much of this information comes from genetically deleting
individual pathway members and then observing the results. As such we know a great
deal about the hierarchal relationships between macroautophagy proteins, but we have
little insight into how the functional groups cooperate on a biochemical level. It is not
even certain that macroautophagy represents a single unified process, which is the
current paradigm, or a collection of processes that happen to produce similar looking
vesicles under EM, but only partially overlap on the molecular level. Indeed,
autophagosomes have been observed in mammalian cells even when critical proteins in
the “canonical pathway” described above have been genetically knocked out (57). That
said, it is clear from genetic deletion of macroautophagy-related (ATG) proteins in mice
that protein turnover via this process has physiologic consequences that are relevant to
human disease, including pulmonary disease.
HOW IS MACROAUTOPHAGY MEASURED?
In the current literature a diverse and somewhat confusing variety of approaches are
used to examine the macroautophagy pathway (22). They can be grouped into 3 basic
approaches: microscopy (Table S1), static biochemical measurements (such as western
blot analysis; Table S2), and dynamic biochemical measurements (Table S3).
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Microscopy was the first technique used to study macroautophagy and the discovery of
autophagosomes on EM images is what gave rise to the field in the first place (58). At
the ultrastructural level, early-stage autophagosomes (AVis) can be distinguished by
their double unit-membrane structure and by the consistency of its luminal material,
which resembles that of the adjacent cytoplasm (59). However, EM image analysis is
less sensitive for picking up late stage autophagosomes (AVds), as they tend to have lost
their distinguishing physical characteristics. Fluorescence-based methods get around
this problem by tagging specific proteins that are found on both early and late-stage
autophagosomes. The most commonly used reporter is the GFP-LC3 fusion protein
(22). Upon induction of macroautophagy by nutrient starvation, GFP-LC3 changes its
cellular localization from a diffuse cytosolic pattern to a punctate pattern as it is
recruited to newly formed autophagosomes (22). This phenomenon can be observed
both in mammalian tissue culture cells and in vivo using GFP-LC3 over-expressing
transgenic mice (60). Fluorescence based reporter systems can be used in fixed cells or
observed dynamically using video microscopy (61).
The most widely used assay to examine macroautophagy is western blot analysis of
LC3b protein (62). During the autophagosome maturation process LC3b is conjugated to
the lipid phosphatidylethanolamine (PE) and then inserted into the inner and outer
leaflets of the autophagosome membrane. Despite its higher molecular weight the PE-
conjugated form of LC3b (called LC3b-II), migrates more rapidly in SDS-PAGE gels than
the un-conjugated version (LC3b-I), and can be easily distinguished on western blot (Fig.
3). Levels of LC3b-II on western blot were shown to correlate with autophagosome
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abundance and so this method provides a convenient alternative to microscopy and can
also be applied to tissue biopsies (50, 63). Other macroautophagy markers that are
amenable to western blot analysis include p62 (64), which is an adaptor protein that
targets ubiquinated proteins to the autophagosome by interacting with LC3b-II (65).
Under starvation conditions, the steady state level of p62 tends to fall as a result of
increased consumption of this protein within autophagosomes (64), and so levels of p62
are generally assumed to inversely correlate with macroautophagic activity (or flux).
A common feature of the assays mentioned so far is that they are primarily static
estimates of the levels of autophagosomes or macroautophagy proteins at a given point
in time. Recently however, some authors have begun to question the assumption that
such static measurements reliably equate with increased protein turnover (66,
67), citing examples where increased amounts of autophagosomes and LC3b-II proved
instead to represent a decrease in the fusion of autophagosomes with lysosomes and
hence a lower rate of flux (68, 69). As such, there has been an increased emphasis on
dynamic “activity assays” that directly measure macroautophagic flux.
Currently the most popular method for measuring macroautophagic flux is the “LC3b
turnover assay”, in which cells are cultured in the presence or absence of an inhibitor of
lysosome proteases for a fixed length of time (67). Because LC3b-II is inserted on the
luminal surface of autophagosomes as well as the exterior, a portion of LC3b-II is
degraded when autophagosomes fuse with lysosomes (Fig. 3). By comparing the
difference in the amount of LC3b-II on western blot in the presence versus the absence
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of lysosome inhibitors, macroautophagic flux can be measured as the amount of LC3b-II
degraded per unit time (67). Other groups have produced turnover assays that are
based on specific substrates of macroautophagy besides LC3b-II (64, 70-72).
In summary, there are a wide variety of ways to examine individual components of the
macroautophagy pathway as well as the proteolytic flux that is a product of this
pathway. What is missing from the field is any kind of consensus about what
represents the minimal acceptable set of measurements upon which conclusions should
be based. For basic researchers interested in pulmonary disease the problem is even
more complex because the lung is a very heterogenous organ composed of up to 40
different cell types (73). As a result, changes in the macroautophagy system that occur
in a subset of cell types can be obscured when analyzing whole lung homogenates.
Even when a stimulus globally alters the macroautophagy machinery in the lung the
physiologic significance of that regulation might vary by cell type (see the section on
COPD below). Our recommendation is to carefully define the cell type being studied
(e.g. respiratory epithelia, fibroblasts), and to include at least one macroautophagic flux
measurement in addition to static measurements of autophagosomes or LC3b-II. Given
recent improvements in methodology and the pioneering studies described below, the
stage is now set to rapidly explore the significance of macroautophagy in pulmonary
WHY STUDY MACROAUTOPHAGY IN THE LUNG?
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As mentioned above macroautophagy is just one catabolic pathway among several that
can degrade proteins in lysosomes. For the Pulmonologist it is fair to ask if there’s any
evidence that would justify focusing on macroautophagy as opposed to any other
catabolic pathway. Recently, Inoue et al. (74) described the first lung-specific
macroautophagy knockout mouse, in which atg7 is specifically deleted from Clara Cells.
They found that atg7 deletion produced severe abnormalities in airway epithelial cells
that included cellular swelling, a loss of rough ER, loss of cilia, and abnormal looking
mitochondria (74). The cellular swelling was physiologically significant because it
produced increased airway resistance after challenging the mice with methacholine
(74). This effect was related to exaggerated changes in airway caliber in the atg7
deleted lungs rather than a true asthma phenotype, as there was no evidence of
inflammation or airway remodeling characteristic of that disease. Nevertheless, these
results suggest that macroautophagy is critical for maintaining the internal organization
of small airway epithelia, which has important implications for human airway diseases
such as asthma. Whether macroautophagy is also important for the basal functioning
of other parenchymal cells, such as alveolar epithelium, will hopefully be addressed with
new tissue specific knockout mice currently in development. In summary, there is now
evidence to support a crucial role specifically for macroautophagy in pulmonary cellular
physiology. Moreover, macroautophagy has also been linked to the pathogenesis of
several important adult pulmonary disorders such as COPD, Cystic Fibrosis and
tuberculosis, as described below.
CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)
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COPD is among the most prevalent adult disorders worldwide (75), and at present, is
also the pulmonary disorder with the most intensively studied link to macroautophagy.
It's important to first note that, at the histological level, COPD is an umbrella term
meant to capture a range of pathologies that result in irreversible obstructive
ventilatory deficits in patients. At one histological extreme is a chronic bronchitis
picture characterized by small airway remodeling, chronic inflammatory cell infiltration,
goblet cell hyperplasia and frequent bacterial colonization (76). At the other extreme is
the emphysematous phenotype characterized by alveolar membrane destruction and air
space enlargement without inflammatory infiltrates (76). It is presumed that most
patients fall on a continuum between these stereotypical presentations. Much of the
macroautophagy literature has primarily been connected to emphysema as this
phenotype can be easily reproduced in mice (77).
The first indication that macroautophagy was regulated in COPD patients came from
western blot analysis of lung biopsy specimens, which showed elevated levels of LC3b-II
protein in patients with COPD compared to non-COPD control patients (78). The
amount of LC3b-II induction correlated positively with clinical severity as measured by
GOLD score (78). To further investigate this, our laboratory turned to smoke exposure
models in tissue culture cells and in mice, given that cigarette smoke represents the
major risk factor for COPD development. Our group as well as others found that
exposure of lung epithelial cell lines and fibroblasts to cigarette smoke extract (CSE)
induced the accumulation of autophagosomes on electron micrographs and enhanced
levels of LC3b-II protein, similar to what was seen in lung biopsies from COPD patients
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(79, 80). The appearance of autophagosomes occurred quickly, on the order of a few
hours, and preceded cell death, which is seen with prolonged exposure to CSE (79).
Importantly genetic depletion of two macroautophagy pathway members, Beclin-1 and
LC3b, reduced the rate of cell death in CSE exposed cells (78, 79), and mice deficient in
LC3b were resistant to emphysematous changes caused by chronic cigarette smoke
exposure (81). The overall interpretation was that the macroautophagy pathway
facilitated lung epithelial and fibroblast cell death in response to cigarette smoke, which
then contributed to the development of emphysema. Given that cigarette smoke
exposure stimulated the formation of the death inducing signaling complex (DISC), a
complex containing the protein Fas Receptor, we concluded that macroautophagy must
mediate emphysematous change through programmed cell death (79).
While our group focused on the role of macroautophagy in lung epithelial cells during
COPD, others have focused on alveolar macrophages. Macrophages derived from COPD
patients often have impaired antimicrobial activities (82, 83), but at the same time they
are prone to hyper-secrete pro-inflammatory cytokines when presented with a
stimulant such as bacterial endotoxin (84). The combination of these two functional
alterations in macrophages may help to make COPD patients vulnerable both to airway
bacterial colonization and chronic bronchitis (85). In a recent article, Monick et al. (86)
examined the role of macroautophagy in alveolar macrophages from actively smoking
patients (with greater than a ten pack-year smoking history), or macrophages of non-
smokers that were exposed to CSE. Importantly, their study employed direct
measurements of macroautophagic flux and so could directly examine the effect of
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smoking on protein turnover via this pathway. They found that macroautophagic flux
was strongly inhibited in the alveolar macrophages of COPD patients, and this effect
could be reproduced in the macrophages of non-smokers by exposing them to CSE (86).
The level of inhibition was at a downstream step in the pathway: autophagosomes
could form but then weren't degraded as fast as normal, leading to an overall
accumulation of autophagosomes. The inhibition of macroautophagy by smoke
exposure coincided with alterations in the energy metabolism of alveolar macrophages:
including increased numbers of depolarized, non-functional mitochondria, decreased
ATP levels and a greater dependence on anaerobic respiration for ATP production (86).
An identical metabolic phenotype can be produced, in the absence of smoke exposure,
by inhibiting macroautophagy through genetic or chemical means (87-89). Taken
together, the results of Monick et al. (86) suggest that a loss of macroautophagic flux is
at least partly responsible for the metabolic disturbances seen in the alveolar
macrophages of smokers and COPD patients. Moreover, since mitochondrial damage
has been implicated in immune dysfunction in septic patients (90), their results may
help to explain the increased susceptibility of COPD patients to infection.
Macroautophagy inhibition also may help to explain why macrophages from COPD
patients secrete higher amounts of IL-1β compared to healthy individuals. Multiple
groups have found that inhibiting macroautophagy leads to excessive secretion of
multiple cytokines when cells are challenged with microbe-derived compounds such as
endotoxin (91-93). We and others recently investigated the mechanism by which
macroautophagy inhibition leads to increased cytokine production by focusing on
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caspase-1 activation, which is the rate limiting step in the secretion of IL-1β and IL-18 in
macrophages (88, 89). We found that, in macroautophagy-deficient macrophages, the
mitochondrial population was dysfunctional because of a failure in organelle quality
control. Mitochondria from macroautophagy-deficient cells produce excessive
amounts of reactive oxygen species and are more prone to undergo membrane
permeabilization when stressed by endotoxin exposure (89). As a result internal
components of mitochondria, such as mitochondrial DNA, leak into the cytoplasm and
lead to caspase-1 activation, followed by excessive IL-1β and IL-18 secretion (89). Our
overall conclusion was that macroautophagy exerts a moderating effect on at least
some aspects of the innate inflammatory response by maintaining mitochondrial health.
Given that smoking inhibits macroautophagy and impairs mitochondrial homeostasis,
it’s tempting to speculate that the inhibition of macroautophagy contributes to the
excessive secretion of pro-inflammatory cytokines observed in some COPD patients.
Taken together, there are now multiple studies to suggest that macroautophagy plays a
significant and complex role in COPD pathogenesis. In lung epithelial cells,
macroautophagy may contribute to apoptosis and thus promote the emphysematous
phenotype, while in alveolar macrophages macroautophagy inhibition by cigarette
smoke may contribute to airway inflammation and bacterial infection. From a clinical
perspective, the contributions of macroautophagy to COPD development have potential
therapeutic and diagnostic implications. From a therapeutic standpoint, the possibility
that macroautophagy may play different physiologic roles depending on the cell type
may mean that simply providing a chemical stimulator of macroautophagy to COPD
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patients could have unpredictable consequences, improving some symptoms of this
disease while making others worse. From a diagnostic standpoint, the fact that
macroautophagy marker proteins like LC3b are increased prior to the onset of apoptosis
suggests that they might prove useful as early biomarkers of COPD progression.
Future research will focus how we can rationally apply what we've learned about
macroautophagy in COPD pathogenesis to ameliorate the clinical consequences of this
CYSTIC FIBROSIS (CF)
It now well enshrined in textbooks that the lung pathology caused by cystic fibrosis is
primarily a consequence of impaired mucocilary clearance caused by mutations in
the cftr chloride channel, leading to chronic bacterial colonization, bronchiectasis and
obstructive physiology (94). Based on this paradigm, the arena where the crucial steps
in CF pathogenesis occur is in the extracellular space- within the lumen of small
airways. As such, the idea that CF is also a disease of defective intracellular
housekeeping within airway epithelial cells, may come as a surprise. However, this is
the conclusion of a recent series of cell biology articles that propose that impaired
macroautophagy is a factor in epithelial cell dysfunction in CF.
The basis of these articles derives from research showing that inactivating cftr mutations
lead to exaggerated pro-inflammatory cytokine secretion in airway epithelial cells when
challenged with Pseudomonas aeruginosa (95). To investigate why CFTR-deficient cells
are intrisinicaly prone to airway inflammation, Mauiri et al. (96) focused on the most
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common cftr mutant, ∆508, which folds improperly and is therefore retained in the
ER. Expression of ∆508 mutant protein led to ER stress and elevated ROS production
followed by activation of the enzyme tissue transglutamindase (TG2), an enzyme that
can act as a “natural fixative” by forming covalent bridges between lysine and glutamine
residues (96). TG2 activation in this context led to the production of protein aggregates
that sequestered the anti-inflammatory regulatory proteins PPAR-γ and iκbα (96, 97). It
was proposed that the loss of these proteins explained the pro-inflammatory phenotype
seen in epithelial cells expressing mutant CFTR.
Intracellular protein aggregates are typically cleared by macroautophagy, and the failure
to do so is critical for the pathogenesis of important CNS diseases such as Huntington’s
disease (98). Therefore in a recent article, Luciani et al. (99) examined why protein
aggregates induced by the ∆508CFTR mutant were not disposed of by
macroautophagy. They found that that expressing ∆508CFTR in epithelial cells inhibited
macroautophagic flux (99). The level of inhibition was at the level of autophagosome
formation and was caused by depletion of the protein Beclin-1. Beclin-1 is a
multifunctional adaptor protein that is a critical component of the vps34 complex that is
responsible for autophagosome membrane initiation (100). Luciani et al. found that
Beclin-1 was a substrate for TG2 and, in the context of ∆508CFTR expression, was cross-
linked by TG2 and sequestered in protein aggregates similar to PPAR-γ (99). This led to
the sequestration of the entire vps34 complex and inhibition of macroautophagy
(99). The loss of macroautophagy was significant because this pathway appears to be
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responsible for degrading CFTR protein that becomes aggregated within the ER
(101). As such macroautophagy inhibition leads to a vicious cycle where ∆508CFTR
protein accumulates unchecked and leads to continual TG2 activity, protein aggregation
in the cytoplasm, and excessive inflammation. Importantly, restoring macroautophagy
by over-expressing Beclin-1 led to protein aggregate clearance in the cytoplasm and
marked reduction in airway inflammation in a mouse model of CF (99). Presumably,
restoring macroautophagy somehow led to improved folding of the ∆508 mutant in the
ER, since Beclin-1 over-expression allowed some of this mutant protein, which is
otherwise functional, to reach the cell surface.
Taken together, there is rapidly emerging evidence at the basic research level that
impaired macroautophagy may be important to airway disease in CF
patients. Translating this knowledge into novel CF therapies is complicated because of
the nature of macroautophagy blockade in this disease: simply providing a chemical
stimulant of the process like rapamycin is unlikely to be effective. However, a gene-
therapy approach in which macroautophagy genes are over-expressed in airway
epithelia might be feasible and perhaps might enhance the effectiveness of prior
attempts at gene-targeted therapy for CF.
Mycobacterium Tuberculosis (MTb)
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Mycobacterium tuberculosis (MTb) remains one of the most prevalent infectious agents
worldwide and represents a highly significant disease for Pulmonologists. Despite the
high disease burden imposed by MTb it’s important to note that the majority of
immunocompetent patients successfully resolve their infection. Recent studies suggest
that macroautophagy contributes to the eradication of this pathogen.
The initial stages of MTb infection occur within alveolar macrophages that encounter
inhaled droplets containing the organism. These previously unstimulated macrophages
succeed in internalizing MTb bacteria by phagocytosis. However, the phagocytic
vesicles containing MTb fail to fuse with lysosomes (102), due to MTb virulence factors
that alter the phospholipid structure of the phagosome membrane (103-105). As such
the vesicles become protected niches that support unchecked MTb replication, which
leads to the eventual lysis of the infected macrophages often within mediastinal lymph
nodes. In immunocompetent hosts this ultimately leads to an inflammatory response
directed by Th1 polarized CD4+ T-cells, which contains or eliminates the MTb infection
(106). Crucial to the success of the cellular immune response to MTb is the elaboration
of IFN-γ, which activates macrophages and enables them to kill MTb bacteria or else
sequester them in granulomas (107). Macrophages stimulated by IFN-γ acquire several
antimicrobial activities not observed in naïve cells, but most notably they are able to
overcome the block in fusion between MTb containing phagocytic vesicles and
lysosomes (108). As a result IFN-γ activated macrophages are able to kill the MTb
bacteria they ingest, which contributes to clinical resolution.
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Interestingly, the ability of IFN-γ to stimulate the clearance of MTb organisms within
macrophages requires an intact macroautophagy pathway. IFN-γ was shown to
stimulate macroautophagy and inhibition of this process by chemical or genetic means
leads to elevated bacterial MTb titers within macrophages (108, 109). Conversely,
activation of macroautophagy by means other than IFN-γ exposure (such as nutrient
starvation or rapamycin), similarly leads to bacterial clearance (108, 110). Interestingly
the bulk macroautophagy pathway alone is insufficient to promote MTb clearance.
Rather autophagosomes export specific cytosolic proteins to the MTb containing vesicle,
which is mediated by the adaptor protein p62 (109). Among the proteins that are
delivered are ubiquitin and a ribosomal protein (rps30) that, when cleaved by lysosomal
hydrolases, are degraded to cationic peptides that are potent MTb antibiotics (109,
111). This novel mechanism of reprocessing cytosolic proteins to generate endogenous
antimicrobial peptides may apply to other infections besides MTb.
Furthermore there is genetic data to suggest that macroautophagy contributes to MTb
immunity in humans. IFN-γ appears to activate macroautophagy by upregulating a
47kDa GTPase called IRGM-1 (108, 112-114). IRGM-1 is required for macroautophagy
stimulated by both IFN-γ and starvation and appears to work by manipulating the life
cycle and redox state of mitochondria (114). Interestingly, polymorphisms in the IRGM-
1 gene were recently linked to increased susceptibility to MTb infection, at least in some
cohorts (115, 116). Other polymorphisms in the same gene were also linked to Crohn’s
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disease, another disorder in which macroautophagy is thought to influence
How can this knowledge be exploited to improve MTb therapeutics? One possibility is
to employ drugs that stimulate macroautophagy in persistently infected individuals with
the goal of reducing latent infection or the development of drug resistance. While some
of these drugs have undesirable immunosuppressive effects (such as rapamycin), there
may be other candidates with better side effect profiles (such as metformin (118) or
carabmazepine (119)). Alternatively, since the role of macroautophagy is to bring anti-
microbial peptides into contact with MTb, administering these protein fragments
exogenously may have therapeutic value as it would bypass the block in phagosome
maturation. It will be interesting to see in the coming years if our growing knowledge
of macroautophagy influences new approaches to MTb therapies.
In summary, autophagy plays an important role in many diseases relevant to
Pulmonologists. While progress has been made in elucidating the role of
macroautophagy in some pulmonary diseases, research into other autophagic pathways
(CMA and microautophagy) is still very much in its infancy. As with any other core
cellular process, turning basic science knowledge about autophagy into therapies is
difficult because of the interdependent nature of biochemical pathways. For the
Page 22 of 51
practicing Pulmonologist, autophagy based approaches to therapy are not yet on the
horizon, but there is now sufficient evidence to speculate that exploiting this pathway
will lead to novel therapies for important pulmonary disorders. For the pulmonary
scientific community, research into the role of autophagy in pulmonary diseases is just
beginning and will yield new insights and perhaps even challenge paradigms that shape
our approach to pulmonary disease.
We thank Joshua Englert, Hilaire Lam, Stephan Ryter, and Robyn Haspel for their critical
review of this manuscript.
Page 23 of 51
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Figure 1: Schematic depiction of the three known autophagy pathways. A. Chaperone
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C. Macroautophagy based on Mizushima (121).
Figure 2: Proposed molecular mechanism for autophagosome formation, based on
Itakura et al. (122), Hyashi-Nisino et al. (45, 46), and Weidberg et al. (52).
Figure 3: Schematic of the macroautophagy pathway. Red circles depict LC3b; orange
ovals depict mitochondria; yellow hexagons and black dots depict ribosomes and
cytosolic proteins respectively. A representative western blot of LC3b-I and LC3b-II is
displayed on the lower left.
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Figure 1 - Schematic depiction of the three known autophagy pathways. A. Chaperone mediated
autophagy based on Dice (120). B. Microautophagy based on Sahu et al. (20). C. Macroautophagy
based on Mizushima (121).
215x279mm (300 x 300 DPI)
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Figure 2 - Proposed molecular mechanism for autophagosome formation, based on Itakura et al.
(122), Hyashi-Nisino et al. (45, 46), and Weidberg et al. (52).
215x279mm (300 x 300 DPI)
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Figure 3 - Schematic of the macroautophagy pathway. Red circles depict LC3b; orange ovals depict
mitochondria; yellow hexagons and black dots depict ribosomes and cytosolic proteins respectively.
A representative western blot of LC3b-I and LC3b-II is displayed on the lower left.
215x279mm (300 x 300 DPI)
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AUTOPHAGY: A CORE CELLULAR PROCESS WITH EMERGING LINKS TO
Jeffrey A. Haspel, and Augustine M. K. Choi
Online Data Supplement
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SUPPLEMENTARY TABLE 1
EM quantification of
per cell correlates
are being formed.
Labor intensive and
number does not
flux in all models.
When used as a
can precipitate as
aggregates in the
cytosol, so not all
half-life is similar in
how this readout
GFP-LC3 from a
diffuse to punctate
pattern of staining
Applicable to both
systems and mice.
Can indentify cell-
tissues or samples.
Similar to GFP-LC3 Can differentiate
(green+, red+) and
late (green-, red+)
As a result the rate
lysosome fusion can
GFP-LC3 cluster life
span (time lapse
are identified as
they appear and
then are followed
using time-lapse live
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flux can then be
expressed in terms
of the average life-
span of GFP-LC3
rate at which GFP-
disappear per cell.
Applicable to tissue
culture models only.
Not all GFP-LC3
Table S1: Microscopy-based methods for monitoring macroautophagy.
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SUPPLEMENTARY TABLE 2
LC3-II abundance on
western blot (87)
LC3b-II levels on
p62 is incorporated
binding to LC3b-II
constant, a decline
in static p62 levels
Easy to perform
Can be applied both
to cells and tissue
p62 ferries protein
therefore is a
marker of selective
or quality control
Does not reliably
western blot (89)
analysis of mRNA
levels to exclude
regulation as a
cause of changes in
Table S2: Static biochemical measurements for monitoring macroautophagy
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SUPLEMENTARY TABLE 3:
turnover assay (26,
37, 41, 168)
Cells or tissues are
loaded with radio-
amino acids. The
rate of protein
turnover is then
measured in terms
of the amount of
liberated from TCA
stores over time.
Gold standard for
Wide linear range of
concomitant use of
3-MA to determine
the component of
Does not distinguish
but the quantitative
turnover and bulk
protein turnover is
selective and non-
selective forms of
assays (95-98, 169,
LC3b-II inserted into
LC3b-II can be
flux can then be
expressed as the
difference in LC3b-II
signal on western
blot in the presence
versus the absence
Similar to LC3b-II
Can be applied in
vitro and in vivo.
p62 turnover assays
(89, 90, 95)
Can measure rate of
Does not measure
Does not measure
the turnover of
information on the
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that are cleared by
repeats do not.
expressed in cells
and luminescence is
lower ratio of
clearance of the
protein by macro
higher ratio of
Cells or tissues are
leupeptin to inhibit
are purified via
is expressed in
terms of the
amount of cytosolic
clear an exogenous
load of protein
Includes an internal
protein to provide
normalization of the
Can be utilized in
vitro and in vivo.
Validated for in vivo
use only in skeletal
muscle so far.
assays. (100, 171)
perform on multiple
Best suited for
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