THE ROLE OF THE AUTOPHAGY-LYSOSOME PATHWAY IN IN VITRO MODELS OF
NEURODEGENERATION
by
BURTON J. MADER
CANDACE L. FLOYD, COMMITTEE CHAIR
JOHN J. SHACKA
JOHN C. CHATHAM
ERIK ROBERSON
TALENE A. YACOUBIAN
A DISSERTATION
Submitted to the graduate facility of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Birmingham, ALABAMA
2014
Copyright by
Burton J. Mader
2014
THE ROLE OF THE AUTOPHAGY-LYSOSOME PATHWAY IN IN VITRO MODELS OF
NEURODEGENERATION
BURTON J. MADER
MOLECULAR AND CELLULAR PATHOLOGY
ABSTRACT
Recent studies have provided strong evidence that alterations in protein degradation
pathways, such as the autophagy-lysosome pathway (ALP), may contribute to neuronal
dysfunction and death which lead to clinical symptoms diagnosed with various
neurodegenerative diseases. Parkinson’s disease (PD) is characterized by neuromuscular
abnormalities resulting from the pathological loss of substantia nigra dopaminergic neurons and
widespread detection of Lewy bodies, intracellular protein inclusions composed primarily of αsynuclein. Additionally, altered gene function regulating the expression of α-synuclein has been
directly linked to the PD pathogenesis.
Autophagy is an intracellular degradation process that when altered can lead to the
accumulation neurotoxic proteins such as α-synuclein. Experimental inhibition of the ALP in
models of PD is effective at reproducing α-synuclein accumulation and cell death. Conversely,
stimuli that enhance ALP function are protective in PD and other models of neurodegenerative
disease. Macroautophagy is a subset of autophagy that regulates degradation by sequestering
cytosolic material within double-membraned vesicles, or autophagosomes, that are ultimately
degraded by the lysosome. The vacuolar type-ATPase (V-ATPase) regulates acidic vesicle pH,
and its inhibition by bafilomycin A1 (BafA1) is mediated through its interaction the V-ATPase
subunit ATP6V0C. BafA1-mediated inhibition of V-ATPase leads to elevated lysosome pH,
altered autophagy, accumulation of α-synuclein, and cell death.
iii
While autophagy is known to be important in maintenance of neuronal cell function, its
potential causative role in the progression of neurodegenerative diseases require further
investigation. We aim to (1) assess rotenone-induced alterations to the ALP that contribute to
neurodegenerative pathology; (2) characterize ALP function and neuronal toxicity after genetic
inhibition ATP6V0C under basal and stressed conditions; and (3) further understand low-dose
BafA1-mediated neuroprotection in models of neurodegeneration.
Using an in vitro model of PD, we treated differentiated neuronal cells with the
environmental pesticide rotenone, a known neurotoxin linked to PD. Although previous studies
report rotenone cause autophagosome accumulation, whether this accumulation is due to
increased autophagosome production or decreased degradation is not entirely clear. In our
studies we observed that rotenone induced accumulation of autophagosomes and α-synuclein that
resulted from decreased lysosome-mediated degradation. We provide evidence that rotenoneinduced lysosome dysfunction is caused by an elevation of pH concomitant with a reduction in
cellular energy levels prior to cell death. These studies conclude that rotenone has a pronounced
effect on macroautophagy completion that may contribute to its neurotoxic potential.
Lysosome dysfunction is linked to neuronal cell death in many neurodegenerative
diseases. As V-ATPase is a critical regulator of lysosome function, we investigated whether
knockdown of the V-ATPase subunit, ATP6V0C, could alter the ALP and cause PD-like cellular
pathologies. We found that knockdown of ATP6V0C alone caused an elevation in acidic vesicle
pH, increased autophagosome accumulation, and decreased autophagic turnover. These observed
alterations in the ALP occurred concomitantly with increased detection of α-synuclein.
Knockdown of ATP6V0C alone caused a decrease in neurite length which was further
exacerbated with BafA1 treatment. Although ATP6V0C knockdown alone did not alter cell
iv
viability, it caused a shift in the dose-response sensitizing cells to BafA1 toxicity. Together
these results indicate a role for ATP6V0C in maintaining constitutive and stress-induced ALP
function, in particular the metabolism of substrates that accumulate in age-related
neurodegenerative disease and may contribute to disease pathogenesis.
Our lab has previously shown low concentrations of BafA1 that do not inhibit V-ATPase
effectively attenuate ALP-related neuronal pathology by enhancing ALP function in in vitro
models of neurodegeneration. Additionally, low-dose BafA1 can protect dopaminergic cells from
α-synuclein toxicity. However, prior to these studies whether low-dose BafA1provided ALPdependent protection was not known. Using the lysosome inhibitor chloroquine to induce ALP
dysfunction and cell death, we investigated whether low-dose BafA1 protection could be
maintained in the absence of important ALP-related proteins. siRNA-mediated knockdown of
both ATP6V0C and the autophagy related protein Atg7 prevented the BafA1-mediated
attenuation of neuronal cell death induced by chloroquine, which induces cell death due to
lysosome dysfunction . We also provide evidence that low-dose BafA1 enhances autophagic flux
during periods of lysosome dysfunction, an effect that is inhibited by knockdown of ATP6V0C.
Our findings suggest that low-dose BafA1 confers neuroprotection directly through enhanced
ALP function and provides support for the therapeutic potential of methods which may enhance
autophagy to treat neurodegenerative pathologies.
Together, our findings highlight the importance of the ALP in maintenance of neuronal
function. As such the ALP may be a useful therapeutic target to attenuate or prevent neuron loss
in neurodegenerative disease. Current strategies that are intended to maintain neuronal cell
viability and in turn dopaminergic signaling by may prove to be extremely useful in slowing or
halting disease progression either alone or in combination with current therapeutic methods.
v
DEDICATION
I dedicate this work to my daughter Charlotte Sophia Mader whose very existence has inspired
and motivated me to persevere through my own perceived limitations. I would like to also
dedicate this work to my partner Christa Lyle whose patience, support, and sacrifice have
allowed me to pursue all of my interests and passions.
vi
ACKNOWLEDGEMENTS
The work in this dissertation was only accomplished with the help of a great many
people. First, I would like to thank my mentor Dr. John Shacka for taking me into his lab and
providing a nurturing environment for me to learn and grow as a basic research scientist. Under
his supervision and guidance I have learned how to formulate scientific hypotheses and design
appropriate experiments to explore those hypotheses. I will always be thankful for his patient
instruction and editorial skill as he helped me in my progression into a better scientific writer. It
was no small task to be sure.
I would also like to thank my committee, which includes Dr. Candace Floyd, Dr. John
Chatham, Dr. Erik Roberson, Dr. Talene Yacoubian, and Dr. Steven Carroll (former member).
Their questions and criticisms of my work over the past 4 years have been instrumental in
forcing me to grow and mature in the way I think about scientific problems.
Finally, thank you to the Shacka lab, including Leandra Mangieri, Chase Taylor, Dr.
Mike Nelson, Tonia Tse, Dr. Peter Liu, Hilary Flippo, Emily Milligan, Sara Stone, and Cailin
Thomas. It has been a pleasure working with all of you and your support and friendship is greatly
appreciated. I would also like to thank Angie Schmeckebier, Rhona Carr, and Barry Bailey for
their administrative assistance. Thank you to the UAB Department of Pathology and the
Graduate School of Biomedical Sciences. Finally, I would like to thank my family and friends
whose interest, love, and support have always inspired me to be a better person and strive to
achieve great things in my life.
vii
TABLE OF CONTENTS
Page
ABSTRACT ....................................................................................................................... iii
DEDICATION ................................................................................................................... vi
ACKNOWLEDGEMENTS .............................................................................................. vii
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES .............................................................................................................x
INTRODUCTION ...............................................................................................................1
Neurodegenerative disease.......................................................................................1
Parkinson’s disease ..................................................................................................2
The Autophagy-Lysosome Pathway ........................................................................4
Macroautophagy ......................................................................................................7
α-Synuclein Degradation .......................................................................................10
Lysosome Dysfunction ..........................................................................................12
The Lysosome and V-ATPase ...............................................................................12
Low-dose Bafilomycin-mediated Neuroprotection ...............................................14
Aims of this Dissertaion.........................................................................................15
ROTENONE INHIBITS AUTOPHAGIC FLUX PRIOR TO INDUCING
CELL DEATH ...................................................................................................................16
ATP6V0C REGULATES AUTOPHAGY-LYSOSOME PATHWAY FUNCTION
AND THE METABOLISM OF PROTEINS THAT ACCUMULATE IN
AGE RELATED NEURODEGENERATIVE DISEASE .................................................48
LOW-DOSE BAFILOMYCIN A1-MEDIATED NEUROPROTECTION IS
DEPENDANT ON AUTOPHAGY AND REQUIRES THE V-ATPASE SUBUNIT
ATP6V0C ........................................................................................................................101
DISCUSSION ..................................................................................................................132
GENERAL LIST OF REFERENCES .............................................................................142
viii
LIST OF TABLES
Table
Page
ATP6V0C KNOCKDOWN IN NEUROBLASTOMA CELLS ALTERS
AUTOPHAGY-LYSOSOME PATHWAY FUNCTION AND
METABOLISM OF PROTEINS THAT ACCUMULATE IN
NEURODEGENERATIVE DISEASE
1
Expression of ATP6V0C by quantitative real-time PCR ............................................60
ix
LIST OF FIGURES
Figure.
Page
INTRODUCTION
1
Illustrated Macroautophagy Pathway ............................................................................7
2
Structure of V-ATPase ................................................................................................12
ROTENONE INHIBITS AUTOPHAGIC FLUX PRIOR TO INDUCING
CELL DEATH
1
Rotenone induced SH-SY5Y cell death and caspase 3-like activity are concentration-
and time-dependent ......................................................................................................39
3
Rotenone causes accumulation of autophagic vacuoles by blocking their effective
degradation ...................................................................................................................41
4
Rotenone increases levels of α-synuclein ....................................................................43
5
Rotenone causes an increase in acidic vesicle pH .......................................................44
6
Rotenone does not induce lysosomal membrane permeabilization (LMP) .................45
7
Rotenone increases levels of LAMP-1 ........................................................................46
8
Rotenone exposure does not alter TFEB levels ...........................................................47
ATP6V0C KNOCKDOWN IN NEUROBLASTOMA CELLS ALTERS
AUTOPHAGY-LYSOSOME PATHWAY FUNCTION AND
METABOLISM OF PROTEINS THAT ACCUMULATE IN
NEURODEGENERATIVE DISEASE
1
Functional validation of ATP6V0C knockdown .........................................................92
2
ATP6V0C regulation of autophagy-lysosome pathway markers ................................93
3
ATP6V0C regulates autophagic flux ...........................................................................95
x
LIST OF FIGURES CONTINUED
4
ATP6V0C regulates basal and stress-induced metabolism of
alpha-synuclein ............................................................................................................97
5
ATP6V0C regulates basal and stress-induced metabolism of APP .............................99
6
ATP6V0C knockdown exhibit enhanced markers of cytotoxicity ............................101
LOW-DOSE BAFILOMYCIN A1-MEDIATED NEUROPROTECTION IS
DEPENDENT ON AUTOPHAGY AND REQUIRES THE V-ATPASE SUBUNIT
ATP6V0C
1
Validation of ATP6V0C knockdown.........................................................................127
2
ATP6V0C knockdown causes BafA1 sensitivity and inhibits low-dose BafA1-mediated
neuroprotection ............................................................................................................128
3
ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein ...................129
4
Low-dose BafA1 requires ATP6V0C to enhance autophagic flux ....................................130
xi
INTRODUCTION
Neurodegenerative Disease
Age-related neurodegenerative diseases such as Parkinson’s disease (PD) are
among the most devastating and widespread disorders facing our elderly population. The
financial toll associated with PD in the United States alone is estimated to cost in the tens
of billions of dollars annually in addition to the immeasurable emotional toll taken on the
patients and their families35 . Symptoms associated with neurodegenerative disease
diseases result from the dysfunction and/or death of neuronal cells that are critical to
maintaining normal sensory, movement, and/or cognitive function. These symptoms
range from memory loss to inhibition of neuromuscular control creating a great reduction
in a patient’s quality of life. It is therefore of the utmost importance to determine the
mechanisms that lead to neuronal dysfunction and/or death so that targeted therapies may
be developed to combat neurodegenerative disease.
A common characteristic of age-related neurodegenerative diseases is the brain
region-specific accumulation of cellular debris and toxic protein species incorporated into
large aggregates. Current research suggests that altered regulation of autophagy, a
cellular phenomenon where the cell degrades and recycles its intracellular material to
meet its energetic and nutritional needs, may play an important role in the pathogenesis
1
of several neurodegenerative diseases58 . Under normal conditions within neuronal cells
the presence of autophagic vesicles, termed autophagosomes, are difficult to detect
experimentally as they are efficiently degraded by the lysosome58. However, many
neurodegenerative diseases exhibit robust accumulation of autophagosomes, suggesting
alterations in autophagy may contribute to neuronal dysfunction and death58. While
dysfunctional autophagy is generally accepted as a central participant in pathologies
associated with various neurodegenerative diseases, the precise mechanisms which
ultimately lead to disease currently remain unclear. It is the goal of our research to
identify potential therapeutic methods that reduce the deleterious effects of
neurodegenerative disease through the identification of alterations in autophagy which
contribute to neurodegeneration.
Parkinson’s Disease
PD affects upwards of 1−2% of persons age 60 or older and is characterized by
neuromuscular abnormalities at a functional level and pathologically by the loss of
substantia nigra dopaminergic neurons and accumulation of termed Lewy bodies,
intracellular inclusions that contain filamentous aggregates of the protein α-synuclein as
their major component77. Symptoms from patients suffering from PD include resting
tremor (shaking), muscle rigidity, bradykinesia (slow movement), and postural
instability. PD represents a class of neurodegenerative diseases called
“synucleinopathies,” and are defined by the brain region-specific accumulation of αsynuclein-containing Lewy bodies. It is believed that the loss of substantia nigra
2
dopaminergic neurons leads to a profound deficit in levels of striatal dopamine required
for normal motor function25.
In 1997, PD was first linked to a mutation in SNCA, the gene that codes for αsynuclein, by the substitution of one amino acid64. Subsequently, there have been
numerous identified mutations and duplications of SNCA that are directly linked to the
accumulation of α-synuclein and PD pathology 65, 73. These genetic findings have paved
the way for the development of refined biological tools such as α-synuclein-specific
antibodies to accelerate the study of α-synuclein in PD. Lewy body formation is directly
linked to an overabundance of α-synuclein in PD brain. α-Synuclein accumulation is
dependent on its gene expression and protein biosynthesis in combination with efficient
removal by an intracellular degradation mechanism78. Histological analysis of autopsied
PD brains indicate that α-synuclein pathology is widespread and may even follow a
progressive migration pattern termed “Braak staging,” which describes detection of αsynuclein pathology that in the brain begins within the olfactory bulb and ends with late
stage α-synuclein detection within the cortex12. A recent in vivo study determined that
the viral over-expression of α-synuclein can cause a decrease in dopaminergic
neurotransmission coupled with neuritic swellings containing α-synuclein, both of which
are indicative of synaptic dysfunction51. While it is clear that α-synuclein plays a major
pathogenic role in the progression of PD, the specific mechanisms by which it causes
pathology remain largely unknown. Inhibition of cellular bioenergetics pathways that are
critically regulated by the mitochondria have been shown to induce neurodegeneration82.
A decreased function of complex I is reported in the substantia nigra of PD patients that
may lead to the generation of oxidative stress and a decrease in ATP production57.
3
Furthermore, a study has reported detecting small proportions of α-synuclein within the
mitochondria that may down-regulate the activity of the mitochondrial electron transport
chain complex I 22, suggesting a potential causal role for α-synuclein and mitochondrial
dysfunction in PD.
Widely used to model PD pathology, the drug rotenone is an environmental
pesticide that has been shown to induce oxidative stress, α-synuclein accumulation, and
dopaminergic neuron death through its selective pharmacological inhibition of
mitochondrial complex I 7, 32. Furthermore, exposure to environmental pesticides such as
rotenone is a known risk factor for PD7. Rotenone has also been shown in several studies
to cause accumulation of autophagosomes as a likely response to inhibition of
mitochondrial function and generation of oxidative stress14, 50, 52, 81, 90, 101. However at this
time, whether rotenone-induced accumulation of autophagosomes results from either
increased induction of macroautophagy or inhibition of lysosomal degradation is under
debate.
The Autophagy-Lysosome Pathway (ALP)
Autophagy, literally translated from Greek meaning “self-eating,” is an
intracellular lysosomal degradation pathway that breaks down cytoplasmic contents
including damaged proteins and organelles, and recycles their components for cellular
reuse. Christian de Duve first used the term “autophagy” in 1963 in his efforts to
establish nomenclature as a means to characterize a variety of cellular trafficking
pathways28, 40. Although much of the molecular research leading to the identification of
specific autophagy genes (ATG) was conducted in yeast, ATG homologs have been
4
found to be conserved in higher eukaryotes highlighting the importance of autophagy
from an evolutionary perspective33. These ATG genes code for numerous proteins (Atg)
involved in the regulation of the ALP and it is widely accepted that dysregulation of these
autophagic proteins can promote neuronal toxicity by causing deleterious effects on
autophagic processes.
Neurons are post-mitotic and as such they rely heavily upon the ALP to maintain
energy balance and organellar quality control without the putative protective effects of
cell division. The intracellular quality control that the ALP provides a neuron is essential
for its survival. Depletion of lysosome number is a prevalent finding in the aged brain
and may have a great impact on the brain’s ability to maintain a healthy intracellular
environment19. This depletion of lysosomes (and implied compromise in effective
autophagy) may account for elevated levels of putatively toxic proteins such as amyloid
beta and tau in AD, and α-synuclein in PD. In the past decade various signaling pathways
and modulators of the ALP have been proposed in numerous studies as promising targets
for the discovery of therapies to combat many various neurodegenerative diseases15.
Based on the cellular environment and nutrient levels within the cell at any given
time, autophagy can be classified as either “basal” (relatively low-level demand for
autophagy) or “stress-induced” (high level)16, 33. Basal, or constitutive autophagy, serves
as an essential intracellular quality control mechanism by removing aggregation-prone
proteins and damaged organelles and in turn maintains energy balance under normal
conditions. During periods of stress, autophagy can be upregulated to increase the
turnover of intracellular material to meet the cell’s metabolic needs. Autophagy provides
cells with the ability to adapt to environmental stresses and is thus considered to directly
5
contribute to maintenance of cellular viability15. Although there are recent descriptions of
very specific subsets of autophagy the ALP can be divided into three principal types
based on how the cargo is transported to the lysosome: microautophagy, chaperonemediated autophagy, and macroautphagy.
Microautophagy is the process of pinocytosis, the direct uptake of defined small
volumes of cytosol by the lysosome. This autophagic process happens without the
participation of vesicles or chaperone proteins as the lysosome appears to invaginate or
project arm-like protrusions to sequester the cytosolic constituents that are proximal to
the lysosome56. Microautophagy in mammalian cells has been described but the majority
of information learned about this pathway has resulted from studies of yeast 67, 85.
Chaperone-mediated autophagy is considered to be highly selective in its
degradation of cytosolic proteins. Chaperone-mediated autophagy degrades proteins
containing a KFERQ amino acid motif, which is recognized by the 70kDa cytosolic
chaperone heat shock protein, Hsc70. Subsequently the chaperone and its cargo are
transported to the lysosome where lysosome associated membrane protein type 2a
(LAMP-2a) mediates intraluminal substrate translocation for degradation. Diminished
levels of both LAMP-2a and Hsc70 have been previously documented in PD brain2.
Chaperone-mediated autophagy activity has been shown to decline with age in a variety
of tissues and is attributed to depleted LAMP-2a levels. Chaperone-mediated autophagy
has been reported in many studies to regulate the degradation of α-synuclein 53, 84, 91-93.
Furthermore, there is strong evidence that chaperone-mediated autophagy and
macroautophagy are closely interconnected in the process of α-synuclein degradation and
that blockage of one of these pathways causes an upregulation of the other54, 92.
6
The primary focus of our research is macroautophagy and its regulation is
explained in detail below, however the abbreviated description of the other types of
autophagy does not intend to diminish the impact chaperone-mediated autophagy,
microautophagy, and other specialized types of autophagy may have on cellular function
and survival.
Macroautophagy
Macroautophagy is a subset of autophagy that regulates intracellular degradation
by sequestering cytosolic material within double-membraned vesicles called
autophagosomes. Autophagosomes were first observed by electron microscopy in the
1950s, however most of what we have learned about macroautophagy has been published
within the past 15 years 97. These autophagosomes are transported along the microtubule
network from other parts of the cell by dynein ATP motors to perinculear lysosomes
where acidic hydrolases degrade the sequestered cellular material and subsequently
release their components for reuse 43.
The
initial phase of
macroautophagy is
termed “induction.”
Nutrient deprivation
Fig. 1. Illustrated Macroautophagy Pathway
(mTOR-dependent),
oxidative stress, and infection are stimuli known signal the increased formation of
autophagosomes79. This induction phase leads into the maturation and expansion phase
7
with the formation of a cup-shaped membrane within the cytoplasm and is called a
phagophore (Fig. 1). While the origin of the phagophore remains unclear, recent studies
have suggested it may initiate from the endoplasmic reticulum, golgi, mitochondria, or
plasma membrane6. After phagophore formation multiple protein-protein and proteinlipid conjugation reactions continue to occur. As the membrane elongates and finally
completes its expansion the double-membraned autophagosomes sequesters cytoplasmic
cargo destined for degradation 28, 79.
The efficient regulation of autophagosome formation is maintained by two
interrelated ubiquitin-like conjugation systems that form the phagophore. The complexes
formed by these conjugation systems, Atg12-Atg5-Atg16 and LC3-PE (microtubuleassociated protein-light-chain-3-II -phosphatidylethanolamine), are essential for
phagophore maturation and autophagosome formation. Atg12 is activated by Atg7
(activating enzyme) and subsequently transported to Atg10 (a conjugating enzyme)
where Atg12 is covalently conjugated to Atg528. This Atg12-Atg5 duplex is conjugated
further with Atg16 and through a self-oligomerization reaction the Atg12-Atg5-Atg16
conjugate forms a tetramer and attaches itself to the expanding phagophore.
Simultaneously, LC3 is initially processed by the protease Atg4 and is activated by the
same Atg7 from the Atg12-Atg5-Atg16 pathway. Next, this modulated LC3 protein is
conjugated to the target lipid, PE, converting it from the cytosolic LC3-I into the
membrane bound LC3-II that is incorporated into the growing phagophore until the
autophagosome is finally formed28, 79. Several ALP-associated molecules have been
identified that are currently being investigated for their utility as therapeutic targets in
age-related neurodegenerative disease58. One study of particular interest demonstrated
8
PD-like symptoms in mice and was linked to deficiency of Atg7, including age-related
dopaminergic neuron loss concomitant with α-synuclein accumulation1, suggesting
autophagy is important for the degradation of putatively toxic proteins species.
Following complete formation of the autophagosome, the next step in the
macroautophagy pathway requires the hydrolysis of ATP to drive dynein motor- transport
of autophagosomes along microtubule networks to deliver their cargo to the lysosome for
degradation38, 45. Transport of these autophagosomes occurs from distal regions of the cell
towards the perinuclear region where lysosomes are located. Alterations in dynein that
affect transport are reported with the accumulation of autophagosomes and ALP
substrates associated with disease pathology seen in PD, Alzheimer’s disease and other
neurodegenerative diseases27. While direct links between dynein and neurodegeneration
are limited, there are a number of mutant genes that regulate dynein expression that are
found in PD and other neurodegenerative disease27.
Once delivered to lysosomes, autophagosomes tether, dock and then fuse with
lysosomal membranes in separately regulated events, independent of lysosomal
acidification55. Once fusion is complete, the autolysosome is formed and the autophagic
cargo will be degraded by lysosomal hydrolases. There is evidence that membraneassociated proteins such as the LAMP family and V-ATPase proteins may regulate these
steps leading to partial or complete fusion of the autophagosome to acidic vesicles 36, 42.
The mechanisms by which fusion is regulated by these lysosome membrane proteins
currently remain unclear.
Acidic hydrolases are essential for substrate degradation and reduction of their
functional activity has been shown to contribute to many disease processes. Pathology
9
can be caused by either genetic mutation in lysosomal storage diseases70 that lead to
robust substrate accumulation, or environmental stressors (inhibition of hydrolases or deacidification of the lysosome) that alter lysosome-mediated degradation. The products
of degradation are then sent back out into the cytosol for cellular reuse.
Although the ALP is believed to provide a pro-survival function at the cellular
level, dysfunction of the ALP may also potentially contribute to pathologies observed
concomitant with an overabundance of autophagosomes. Increased detection of
autophagosomes is found in several human neurodegenerative diseases including AD,
PD, and dementia with Lewy bodies 4, 20, 59.
While it remains controversial as to whether the accumulation of autophagosomes
indicates a causal role of the ALP in neuronal cell death, dysfunctional degradation
through inhibition of ALP function is widely considered a key player in the pathology of
age-related neurodegenerative disease. The ALP is described as the main degradation
pathway for α-syn oligomers and aggregates due to its large capacity when compared
with the proteosome, further implicating the potential for ALP dysfunction in PD
pathology47, 84, 93. Therefore, it is important to elucidate alterations in the ALP that
contribute to disease pathogenesis, giving special consideration to α-synuclein in PD and
metabolites of amyloid precursor protein in Alzheimer’s disease.
α-Synuclein Degradation
α-Synuclein is a 140 amino acid natively unfolded protein and in the nervous
system accounts for approximately 1% of cytosolic protein. α-Synuclein is detected in the
early stages of neuronal development when synapses are beginning to form. Recent
10
studies propose α-synuclein may regulate synaptic neurotransmission through its
association with the vesicles that release neurotransmitters30. The accumulation of αsynuclein, a presynaptic neuronal protein genetically linked to PD neuropathology seems
to occur through a combination of independent or interrelated factors including increased
expression, genetic mutation, post-translational modification, and decreased clearance by
degradation pathways78.
The ubiquitin-proteasome system and the ALP are considered the two main routes
by which α-synuclein is degraded 5, 78. It is reported that the UPS is primarily responsible
for degrading the α-synuclein monomer, while the ALP’s larger size capacity allows for
the degradation of the higher molecular weight species of α-synuclein suspected of
causing neuronal toxicity26. α-Synuclein degradation becomes even more complicated
under experimental conditions as it can inhibit both the ubiquitin-proteasome system and
the ALP, creating an environment that is primed for the emergence of a toxic feedback
loop in aberrant α-synuclein accumulation26, 75, 80. Although these degradation pathways
have been mostly accepted as the primary methods by which α-synuclein is degraded, the
specific mechanisms that regulate its degradation are still in question.
After experimentally inducing lysosome dysfunction, α-synuclein is reported to
be secreted in association with exosomes that may exert toxic potential upon neighboring
cells3. Induction of paracrine neurotoxicity toxicity is exacerbated by dysfunction of
autophagy18, 46. These studies highlight the importance of understanding the relationship
of the ALP to α-synuclein metabolism so that therapies can be developed to enhance
clearance of α-synuclein in a way that reduces neurotoxicity.
11
Lysosome Dysfunction
Lysosome dysfunction leads to a reduced capacity for cellular degradation
triggering the accumulation of cellular debris and large inclusions of aggregated proteins.
Acidiotropic drugs such as chloroquine are used experimentally to characterize lysosome
dysfunction. Chloroquine is a weak base that is attracted to acidic environments and can
move freely across the lysosome membrane. However, upon arrival in the lysosomal
lumen chloroquine molecules become trapped by the addition of a proton leading to its
intralysosomal accumulation and resultant elevation of lysosomal pH74. Inhibition of the
lysosomal enzymes that are dependent on acidic pH for optimal function can occur due to
increased lysosome pH. We have previously published data showing chloroquine causes
cell death and inhibition of autophagy with accumulation of proteins with neurotoxic
potential60, 72.
The Lysosome and V-ATPase
Drugs such as chloroquine and bafilomycin A1 (BafA1) are known induce pHdependent lysosome dysfunction through a variety of different mechanisms.
BafA1 is categorized in the plecomacrolide subclass of macrolide antibiotics and
is reported to selectively inhibit vacuolar typeATPase (V-ATPase, Fig. 2)11.Inhibition of VATPase is shown to be mediated by the binding of
BafA1 with high affinity to the c subunit of its V0
domain, or ATP6V0C 9, 10. Unlike chloroquine,
BafA1 causes lysosome dysfunction through its
Fig. 2. Structure of V-ATPase
12
inhibition of the V-ATPase pump, an essential component of lysosomal membrane
machinery that regulates proton movement to maintain lysosome pH.
At concentrations ≥10 nM, BafA1 inhibits V-ATPase and in turn increases
intravesicular pH99, thus mimicking the effects of chloroquine. Experimental inhibition of
V-ATPase by BafA1 has been shown to prevent the effective degradation of α-synuclein
and in turn promote the accumulation of α-synuclein associated with neurotoxic potential
3, 44, 60, 76, 87
.
As pharmacological inhibition of V-ATPase has been shown experimentally to
promote α-synuclein accumulation, further targeting this enzyme complex may provide
greater information regarding its role in regulating the basal vs. stress-induced
metabolism of α-synuclein. V-ATPase is a membrane-associated, multi-subunit protein
complex that functions as an ATP-driven proton-pump and is localized to many different
membranes of eukaryotic cells including lysosomes, endosomes, Golgi-derived vesicles,
secretory vesicles and in some cell types the plasma membrane29. V-ATPase maintains
the low pH of acidic vesicles through its regulation of proton pumping29.
The V-ATPase is a large complex that is comprised of two domains: the V1
domain, which can be found either associated with the full complex or disassociated
within the cytoplasm and is responsible for ATP hydrolysis; and the V0 domain, which
putatively rotates like a wheel to pump protons across the membrane into the lumen of
the lysosome98. Any cellular stress that impedes the ability of V-ATPase to maintain the
internal lysosomal pH may potentially harm the ALP’s degradative capacity thus
increasing the likelihood of cellular dysfunction and death.
13
Knockdown of ATP6V0C, a subunit of the V0 domain, has been shown recently
to inhibit vesicular acidification and sensitize cells to stress-induced cell death 13.
Alternatively, over-expression of ATP6V0C has been shown to contribute to the rescue
of dopaminergic signaling in a model of PD 39. Whether ATP6V0C itself is responsible
for regulating ALP function or the metabolism of substrates that accumulate in agerelated neurodegenerative diseases has not been previously investigated.
Low-dose Bafilomycin-mediated Neuroprotection
Our lab has demonstrated that the plecomacrolides BafA1, bafilomycin B1 and
concanamycin, in addition to their known inhibition of V-ATPase, all significantly
attenuate chloroquine-induced death of cerebellar granule neurons71, 72 at low
concentrations (≤ 1 nM) which do not inhibit V-ATPase11 or induce autophagosome
accumulation 72. These data suggest that low-dose BafA1-mediated neuroprotection is
independent of its inhibition of V-ATPase. Subsequent studies in different cell lines have
exhibited BafA1-mediated neuroprotection using concentrations of BafA1 that do not
alter neuronal viability or acidic vesicle pH 60. Low-dose BafA1 treatment also is shown
to attenuate α-synuclein oligomer accumulation that is triggered by chloroquine exposure
by some mechanism that attenuates the inhibition of autophagic flux60. Finally, BafA1mediated neuroprotection was confirmed through its inhibition of α-synuclein -associated
toxicity in C. elegans, a worm model used to study PD pathologies in dopaminergic
neurons60.
14
While these studies provide strong evidence of BafA1-mediated neuroprotection,
the mechanisms by which by which low-dose BafA1 affects the ALP or regulates αsynuclein clearance and neurotoxicity has not been investigated.
Aims of this Dissertation
The ALP and V-ATPase represent attractive targets for promoting the metabolism
and turnover of proteins that contribute to the pathogenesis of age-related
neurodegenerative disease such as α-synuclein in PD. The aims of this dissertation are (1)
assess rotenone-induced alterations to the ALP that contribute to neurodegenerative
pathology; (2) characterize ALP function and its contribution to neuronal toxicity after
genetic inhibition of the V-ATPase subunit ATP6V0C under basal and stressed
conditions; and (3) elucidate the mechanism that regulates low-dose BafA1-mediated
neuroprotection from pharmacologically-induced lysosome dysfunction.
Although it is currently accepted that alterations in the ALP contribute to
neurodegenerative disease there remains very little known about how this pathway
directly impacts cell survival within the human brain and causes disease. These studies
are intended to identify the dysfunctional mechanisms that may potentially occur in the
brains of PD patients or other patients diagnosed with neurodegenerative disease. The
ALP appears to be an important homeostatic mechanism in neuronal cells under normal
conditions, however under periods of stress the ALP’s role has been shown to provide
protection as well as to contribute to toxicity.
15
ROTENONE INHIBITS AUTOPHAGIC FLUX PRIOR TO INDUCING CELL DEATH
by
BURTON J. MADER, VIOLETTA N. PIVTORAIKO, HILARY M. FLIPPO, BARBARA J.
KLOCKE, KEVIN A. ROTH, LEANDRA R. MANGIERI AND JOHN J. SHACKA
ACS Chemical Neuroscience, 2012 Dec 19;3(12):1063-72
Copyright
2012
by
ACS Publications
Used by permission
Format adapted for dissertation
16
ABSTRACT
Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress,
α-synuclein accumulation and dopaminergic neuron death, principal pathological features of
Parkinson disease. The autophagy-lysosome pathway degrades damaged proteins and organelles
for the intracellular maintenance of nutrient and energy balance. While it is known that rotenone
causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not
been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with rotenone (10
µM) induced the accumulation of autophagic vacuoles at 6h and 24h as indicated by western blot
analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of
autophagic flux at these time points indicated that autophagic vacuole accumulation resulted
from a decrease in their effective lysosomal degradation, which was substantiated by increased
levels of autophagy substrates p62 and α-synuclein. Inhibition of lysosomal degradation may be
explained by the observed decrease in cellular ATP levels, which in turn may have caused the
observed concomitant increase in acidic vesicle pH. The early (6h) effects of rotenone on
cellular energetics and autophagy-lysosome pathway function preceded the induction of cell
death and apoptosis. These findings indicate that the classical mitochondrial toxin rotenone has
a pronounced effect on macroautophagy completion that may contribute to its neurotoxic
potential.
17
INTRODUCTION
Parkinson disease (PD) is an age-related neurodegenerative disorder that affects upwards
of 1-2% of persons age 60 or older and is characterized pathologically by the loss of substantia
nigra dopaminergic neurons and accumulation of intracellular protein inclusions termed Lewy
bodies1. Oxidative stress and a decreased capacity of neurons to degrade α-synuclein, a protein
reportedly involved in vesicular dopamine release and the major component of Lewy bodies, are
believed to be major contributors to Lewy body formation and neurodegeneration in PD2.
A decreased function of complex I of the mitochondrial electron transport chain is
reported in the substantia nigra of PD patients1 that may lead to the generation of oxidative stress
and a decrease in ATP production. One mechanism by which cells compensate for this
disruption in energy balance is through stimulation of the autophagy-lysosome pathway (ALP),
which shuttles outlived and/or damaged proteins and organelles to the lysosome for their pHdependent degradation and recycling3-5. In macroautophagy, a subset of the ALP, doublemembraned autophagic vacuoles shuttle cargo destined for degradation to lysosomes. Increased
numbers of autophagic vacuoles are detected in several human Lewy body diseases including PD
and dementia with Lewy bodies6-8 as well as in animal models of PD9. Moreover, the ALP is
believed to be the main route for degradation of α-synuclein oligomers and aggregates, further
implicating its dysfunction in PD pathology10.
Exposure to environmental pesticides such as rotenone, a selective inhibitor of complex I
of the mitochondrial electron transport chain, is associated with an elevated risk of developing
PD and is used experimentally to model PD pathophysiology11, 12. Rotenone induces
neurodegeneration and neuron death in association with caspase activation13, and causes α18
synuclein accumulation in experimental models of PD11, 14. Moreover, rotenone-induced
oxidative stress reportedly induces post-translational modifications of α-synuclein 15, which are
thought to promote its aggregation and toxicity16, 17.
Rotenone has also been shown in several studies to cause accumulation of autophagic
vacuoles8,18-21, as a likely response to inhibition of mitochondrial function and generation of
oxidative stress. However, it is not entirely clear if rotenone-induced accumulation of
autophagic vacuoles results from either an increased induction of macroautophagy or from the
inhibition of macroautophagy completion, which requires functional fusion of autophagic
vacuoles with lysosomes3. Such information may be important for designing rational
therapeutics that attenuate PD-associated neuronal dysfunction, in part through their maintenance
of ALP function. Thus the goal of this study was to delineate the mechanism by which
autophagic vacuoles accumulate in cultured neuronal cells following treatment with an acute,
death-inducing concentration of rotenone.
RESULTS AND DISCUSSION
It is well established that rotenone disrupts the ALP. However, the cause of
autophagic vacuole accumulation following rotenone treatment has not been carefully
investigated. The purpose of this study was to assess the manner by which rotenone, a potent
mitochondrial toxin that is used as an experimental model of PD, affects accumulation of
autophagic vacuoles using a neuroblastoma cell line (SH-SY5Y) that was differentiated to a
neuronal phenotype. We first assessed cell death, apoptosis and energetics following treatment
19
with rotenone. Next, using a death-inducing concentration of rotenone we assessed autophagic
flux to determine the manner by which autophagic vacuoles accumulate. We next assessed
vesicular acidification and the integrity of lysosomal membranes to determine the potential for
acidic vesicle dysfunction to contribute to changes observed in autophagic flux. Finally, we
correlated these observed effects of rotenone on macroautophagy by assessing relative levels of a
lysosomal membrane marker and a transcription factor that is responsible for its stress-induced
up-regulation.
Rotenone-induced neuronal cell death and apoptosis.
In retinoic acid-differentiated SH-SY5Y cells, rotenone induced concentration and timedependent cell death (Fig. 1A, C). A decrease in cell viability was observed beginning at 24h of
10µM rotenone treatment. About a 50% decrease in SH-SY5Y cell viability was observed after
48h of rotenone treatment, which progressed to 70% by 72h (Fig. 1C). Rotenone-induced SHSY5Y cell death was accompanied by a concentration-dependent increase in caspase 3-like
activity (Fig. 1B). A three to four-fold increase was also detected in caspase 3-like enzymatic
activity in 10µM rotenone treated SH-SY5Y cells vs. vehicle control at 48 and 72h, indicating
that rotenone-induced cell death was accompanied by caspase activation (Fig 1D). All
subsequent experiments utilized a concentration of 10 µM rotenone. To determine if caspase
activation was required for rotenone-induced neuron death, we measured cell viability in the
presence of a broad caspase inhibitor Boc-Asp(OMe)-FMK (Boc-FMK) (Fig 1E). Boc-FMK at a
concentration that completely inhibited rotenone-induced caspase-3-like activity (data not
shown) failed to inhibit rotenone-induced cell death. This data indicates that although rotenone
can induce robust apoptosis, caspase activation per se, is not required for cell death. To confirm
20
that a death-inducing concentration of rotenone affected cellular energetic, ATP levels were
measured at 6h and 12h following rotenone treatment (Fig 1F), time points that preceded the
induction of neuronal apoptosis and cell death (Figs 1C-1D). Rotenone caused a significant
reduction in ATP levels at both time points, due likely to the inhibition of complex I of the
mitochondrial electron transport chain and indicates the reliance of these cells on mitochondrial
ATP production as a source of energy.
Rotenone causes early and persistent accumulation of autophagic vacuoles by compromising
their lysosomal degradation.
Previous studies have identified accumulation of autophagic vacuoles in substantia nigra
neurons of PD brain and in in vitro models of rotenone-induced neuronal cell death6-8, 18, 19, 21,
suggesting that the ALP is involved in regulating dopaminergic neuron death. To further
delineate the involvement of macroautophagy in rotenone-induced neuronal cell death, we
assessed levels of autophagic vacuoles via western blot analysis for MAP-light chain 3-II (LC3II; Fig. 2), an accepted and selective marker of autophagic vacuoles3. Treatment with rotenone
induced a significant accumulation of autophagic vacuoles at both 6h (Fig. 2B) and 24h (Fig. 2C)
after treatment. Since the accumulation of autophagic vacuoles may result from either induction
of autophagy or from a decrease in their lysosomal degradation3, 22 we also assessed autophagic
flux (Fig. 2) via treatment with 100 nM bafilomycin A1, a selective inhibitor of vacuolar-type VATPase that completely blocks degradation via macroautophagy through its inhibition of
autophagic vacuole-lysosome fusion. Bafilomycin A1 was added to cells at 4h or 22h (2h prior
to collection at the 6h and 24h rotenone time points, respectively). At each time point tested, we
found no further increase in levels of LC3-II in bafilomycin A1-rotenone-treated cells compared
21
to cells treated only with 100nM bafilomycin A1 (Figs. 2B-2C). This result suggests that the
rotenone-induced increase in autophagic vacuoles resulted not from autophagy induction but
rather from a block in the lysosomal degradation of autophagic vacuoles. As a positive control
for autophagy induction, the addition of 100 nM BafA1 for the last 2h of a 24h rapamycin
treatment produced noticeably greater LC3-II immunoreactivity in comparison to 2h treatment
with BafA1 alone (data not shown).
Autophagic flux is also commonly assessed by detecting protein levels of
p62/A170/SQSMT1, a ubiquinating and LC3 binding protein that binds to ubiquitin aggregates
and promotes degradation via autophagy22. Thus decreases in p62 levels are associated with
enhanced autophagic flux as observed for example during serum starvation, whereas its
accumulation suggests autophagy degradation block22. We observed a significant increase in
p62 levels at both 6h and 24h following treatment with 10 µM rotenone (Figs. 2D-E). These
data support our assessment of autophagic flux (Figs. 2A-C) suggesting that rotenone disrupts
the degradation of autophagic vacuoles. Further evidence for rotenone-induced inhibition of
autophagic degradation in our model was supported by a significant increase in high-molecular
weight species of alpha synuclein at 48h following treatment (Fig. 3), as it is well established
that the ALP is important for α-syn degradation8, 23, 24. Levels of alpha synuclein following 6h
and 24h of rotenone treatment were not significantly different from vehicle control (data not
shown).
Rotenone increases acidic vesicle pH but does not induce lysosomal membrane
permeabilization.
To determine if rotenone-induced inhibition of autophagic flux correlated with a
compromise in the function of acidic vesicles, we utilized a flow cytometric approach with the
22
acidotropic dye lysotracker red (LTR) to quantify acidic vesicle pH (Fig. 4). Rotenone induced a
significant, >30% loss in LTR mean fluorescence intensity vs. vehicle control-treated cells at
both 6h and 24h following treatment, time points that coincided with the observed decrease in
ATP production (6h; Fig. 1G) and inhibition of macroautophagy (Fig. 2). The effects of
rotenone on LTR mean fluorescence intensity were not as robust as those observed following
treatment with 100 nM BafA1, a potent inhibitor of acidic vesicle pH via its direct inhibition of
V-ATPase (refs). Epifluorescence microscopy images (Fig. 4C) corroborate our findings via
flow cytometry, where compared to vehicle control-treated cells there is a relative lack of LTR
fluorescence following treatment with rotenone or bafilomycin A1. These results indicate that
rotenone causes an early and persistent increase in acidic vesicle pH, which may be due possibly
to a net decrease in cellular ATP levels and in turn ATP-dependent acidification. Rotenone has
been shown previously to inhibit cathepsin D activity and decrease fluorescence emitted by the
acidotropic dye lysosensor green in cultured cell lines25, 26, evidence that corroborates our
findings.
We also determined if rotenone caused the onset of lysosomal membrane
permeabilization (LMP) by assessing the co-localization of cathepsin D and LAMP-1
immunoreactivity using confocal microscopy (Fig. 5). At 24h after treatment with rotenone, a
time point that corresponded to significant inhibition of autophagic flux (Fig. 2), a significant
decrease in LTR mean fluorescence intensity (Fig. 4) and the onset of cell death/apoptosis (Fig.
1), cathepsin D immunoreactivity co-localized with LAMP-1 similar to that of vehicle controltreated cells, suggesting a lack of LMP at this time point. In contrast, noticeable LMP (as
indicated by a diffuse staining pattern for cathepsin D that did not co-localize with LAMP-1) was
observed in cells treated for 24h with chloroquine (50 µM; Fig. 5), a lysosomotropic agent and
23
known inducer of LMP27. Together these results suggest that the inhibition of autophagic flux by
rotenone may be caused in part by its inhibition of vesicular acidification, perhaps via its potent
inhibition of intracellular ATP, but not because of the onset of LMP. It is possible that rotenone
may eventually induce LMP at later time points not tested in this study, as shown previously by
the dopaminergic neurotoxin MPP+9 and the oxidative stress-inducing agent hydrogen
peroxide28. However, the possibility still exists that rotenone-induced oxidative stress in our
model may compromise lysosomal membrane integrity that in turn may decrease the efficiency
of macroautophagy for clearing substrates5, but at a lower concentration and earlier time than
that is required for inducing LMP. Taken together, lysosome dysfunction corresponds with and
may contribute to a decreased turnover of autophagic vacuoles following rotenone treatment,
events that may be responsible for the accumulation of macroautophagy substrate and induction
of cell death.
Rotenone increased levels of LAMP-1 but not TFEB.
We also tested the effect of rotenone on levels of lysosomal membrane associated protein
1 (LAMP-1), a structural protein present on membranes of late endosomes and lysosomes.
LAMP-1 is best known for its regulation of lysosomal motility and endosomal-lysosomal fusion
with autophagic vacuoles29, and has been shown to fluctuate with changes in lysosomal volume
and/or number9, 30. Western blot analysis indicated an increase in LAMP-1 levels following
treatment with 10 µM rotenone (Fig. 6A), an effect that was significantly different vs. vehicle
control at 6h but not 24h (Fig. 6B). This increase in LAMP-1-suggests either an increase in the
number or size of acidic vesicles that may be a consequence of lysosome dysfunction (Fig. 4)
and inhibition of autophagic flux (Fig. 2). Conversely, LAMP-1 may also increase as a function
of de novo lysosome biogenesis, as shown recently under stressful conditions and as a response
24
to alterations in autophagy9, 30. To address this possibility the effects of rotenone on endogenous
levels of the transcription factor EB (TFEB) were assessed by western blot analysis, as TFEB has
been shown previously to regulate transcription of LAMP-1 in addition to other lysosomal
targets9, 30. Levels of TFEB at 6h and 24h following rotenone treatment were similar in rotenone
vs. vehicle control cells (Fig. 7), suggesting that the increase in LAMP-1 observed following
rotenone treatment does not result from lysosome biogenesis, but rather from the inhibition of
lysosomal degradation.
Our assessment of autophagic flux suggests that induction of macroautophagy is not the
major mechanism by which rotenone causes accumulation of autophagic vacuoles. In agreement
with this interpretation, previous reports have shown that cell death and markers of apoptosis
induced by 1-10 µM rotenone in SH-SY5Y are not influenced by siRNA knockdown of Atg5, a
gene that is critical for de novo synthesis of autophagic vacuoles19, 34. Interestingly, these studies
also showed that pre-treatment of SH-SY5Y cells with drugs that induce autophagy attenuate cell
death and apoptosis caused by rotenone post-treatment19, 34. These observations suggest that, in
addition to its ability to block the lysosomal degradation of autophagic vacuoles, rotenone may
also inhibit autophagy induction, an effect that is somehow circumvented upon pre-treatment
with drugs that stimulate formation of autophagic vacuoles. In support of this hypothesis, it has
been shown that rotenone causes de-phosphorylation of death-associated protein kinase (DAPK)
in association with mitochondrial dysfunction35. The “active” phosphorylated form of DAPK
has been shown to phosphorylate the BH3 domain of Beclin-1, thus disrupting the Bcl-XLBeclin-1 interaction and in turn promoting Beclin-1-dependent autophagy.
Alternatively, it is possible that rotenone inhibits autophagy induction through its effects
on microtubule assembly, as an intact microtubule network is also important for the formation of
25
de novo autophagic vacuoles33, 36. Rotenone has been shown previously to inhibit microtubule
assembly31, as well as increase levels of free tubulin in association with a decrease in
mitochondrial membrane potential32. An intact microtubule network is known to be important
not only for the formation of nascent autophagic vacuoles but also for the fusion of autophagic
vacuoles with lysosomes and endosomes33. Thus, if rotenone disrupts the function of
microtubules, it would not be surprising if autophagic vacuoles accumulate due to an effective
block in their trafficking to lysosomes for degradation. Reduced turnover of autophagic vacuoles
as a result of microtubule dysfunction may also contribute to persistent rotenone-induced
mitochondrial toxicity, as the ability of damaged mitochondria to be effectively recycled by
mitophagy (a type of macroautophagy selective for mitochondrial degradation) may be severely
compromised.
Other reports indicate that rotenone exhibits concentration-specific or even cell typespecific effects on autophagy induction. Treatment of mouse embryonic fibroblasts with 2 µM
rotenone caused accumulation of autophagic vacuoles concomitant with a reduction in levels of
p62, suggesting autophagy induction21. Concurrent treatment of SH-SY5Y cells with 100 nM
rotenone and either rapamycin, which induces autophagy, or 3-MA, which inhibits autophagy
induction, attenuated or exacerbated cell death respectively37, suggesting that autophagy
induction is not inhibited by low concentrations of rotenone. In addition, inhibition of autophagy
induction (3-MA vs Atg5 or Beclin-1 siRNA knockdown) in HEK 293 and U87 cells was shown
to attenuate cell death induced by 50 µM rotenone18, suggesting again that rotenone does not
inhibit autophagy induction and that autophagy induction may potentiate rotenone-induced cell
death at higher concentrations. We have observed previously using knockdown of Atg7 that
autophagy induction contributes to the onset of cell death induced by chloroquine, a
26
lysosomotropic agent that inhibits autophagic flux38, suggesting that autophagy induction can be
death-inducing in the face of lysosome dysfunction.
In conclusion, to our knowledge this is the first report indicating that rotenone inhibits the
lysosomal degradation of autophagic vacuoles. Inhibition of autophagic flux was observed at
both early and late time points, and may be caused by the rotenone-induced increase in acidic
vesicle pH and decrease in ATP levels. The induction of cell death and apoptosis was only
apparent at later time points, suggesting that the inhibition of autophagic turnover by rotenone
may contribute to cellular demise. In light of the relationship of rotenone as a neurotoxin model
of PD, preservation and/or enhancement of autophagic degradation should be considered a viable
therapeutic strategy to delay disease onset and/or progression.
METHODS
Cell Culture
SH-SY5Y human neuroblastoma cells were cultured in Minimum Essential Medium
Eagle (MEM) (Cellgro, Herndon, VA) and F12-K Nutrient Mixture (ATCC, Manassas, VA)
medium supplemented with 0.5% sodium pyruvate, 0.5% non essential amino acids (Cellgro,
Herndon, VA), 1% penicillin/streptomycin (Sigma, St. Louis, MO), and 10% Fetal Bovine
Serum (FBS) (HyClone, Logan, UT). SH-SY5Y cells were differentiated in 10% FBS feeding
media supplemented with 10µM retinoic acid (Sigma, St. Louis, MO) for 7-8 days. Medium
supplemented with retinoic acid was replaced every 2-3 days. For experiments, cells were seeded
at a density of 400/mm2 in media containing 2% B-27 supplement (Invitrogen, Grand Island,
27
NY) and 10µM retinoic acid. Rotenone (Sigma, St. Louis, MO) stock in DMSO was prepared
fresh for every experiment and added to differentiated SH-SY5Y cells for 3-72h.
Measurement of Cell Viability and Caspase-3-Like Activity
Cell viability was measured via Calcein AM fluorogenic conversion assay (Invitrogen,
Grand Island, NY) and caspase-3-like activity was detected via fluorogenic DEVD cleavage
assay and expressed relative to untreated controls as previously described in our laboratory39.
Measurement of ATP Levels
20,000 differentiated SH-SY5Y cells were plated per well of 96 well plates and after 24h
were treated with either DMSO vehicle or rotenone at a final concentration of 10 µM. At 6h and
12h following rotenone treatment levels of ATP were assessed using the ATPLite™
Luminescence ATP Detection Assay System (Perkin Elmer, Waltham, MA). Luminescence was
quantified using a BioTek Synergy 2 luminometer. ATP levels (µM/well) were calculated based
on a standard curve utilizing an ATP standard supplied by the assay kit. Mean ATP levels were
averaged from 6 wells per treatment per time point, and each time point was repeated for a total
of 3 independent experiments.
Western Blot Analysis
Whole cell lysates were prepared in buffer containing 1% SDS and 1% Triton X-100 as
previously described in our laboratory40. Protein concentrations were determined using the BCA
assay (Pierce, Rockford, IL). Equal amounts of protein were electrophoresed on SDSpolyacrylamide gels and subsequently transferred to PVDF membranes (BioRad, Hercules, CA).
Western blots were probed for LC3 (Abcam, Cambridge, MA), p62 (Abnova, Littleton, CO),
28
LAMP1 (1D4B; University of Iowa Hybridoma Bank) or alpha synuclein (Santa Cruz, Santa
Cruz, CA). GAPDH (Cell Signaling, Beverly, MA) or actin (Sigma, St. Louis, MO) were used as
loading controls. X-ray films of western blots were scanned for densitometric analysis using UNSCAN-IT gel 6.1 software (UN-SCAN-IT, Orem, UT).
Assessment of lysosomal membrane permeabilization (LMP)
To assess LMP, double-label immunocytochemistry was performed for cathepsin-D and
LAMP-1. Cells plated in 8-well chamber slides were treated for 24h with either DMSO vehicle,
rotenone (10 µM) or chloroquine (50 µM), then were fixed for 20 min with ice-cold 100%
methanol. Following PBS wash, fixed cells were incubated for 30 min with blocking buffer (5%
v/v horse serum in 1X PBS containing 1% Triton X-100) followed by overnight incubation in
blocking buffer without Triton X-100 and containing goat anti-cathepsin D antibody (Santa
Cruz, Santa Cruz, CA). After PBS wash, cells were incubated in blocking buffer containing
HRP-conjugated anti-goat IgG secondary antibody (ImmPRESS, Vector Laboratories,
Burlingame, CA) for 1h in the above blocking buffer, RT followed by PBS wash. Cells were
next subjected to a second fixative (4% paraformaldehyde, 15 min, 4°C) for 15 min to prevent
membrane rupture that occurs following exposure of methanol-fixed cells to the high salt
concentration of our detection reagent buffer. Following PBS wash, detection was performed
using Tyramide Signal Amplification (TSA; Perkin Elmer, Waltham, MA) by addition of Cy3conjugated tyramide in Plus Amp Buffer (30 min, RT) followed by PBS wash. At this point,
fixed cells were incubated in blocking buffer (1% BSA, 0.2% evaporated milk, 0.3% Triton X100 in PBS) for 30 min at RT then overnight in blocking buffer without Triton X-100 and
containing with the second primary antibody (mouse anti-human LAMP-1, U Iowa Hybridoma
29
Bank). Following PBS wash fixed cells were incubated with HRP-conjugated anti-mouse IgG
secondary antibody (ImmPRESS, Vector Laboratories, Burlingame, CA). Following PBS wash
detection was performed via TSA upon addition of FITC-conjugated tyramide in Plus Amp
Buffer (30 min, RT) followed by PBS wash. Nuclei were next counterstained using bisbenzimide (0.2 µg/ml, Sigma, St. Louis, MO) for 10 min, followed by PBS wash. After coverslipping, co-localization of cathepsin D and LAMP-1 was visualized using a Zeiss Observer.Z1
Laser Scanning Microscope (Thornwood, NY, USA) equipped with a Zeiss™ 40X 1.3 Oil DIC
M27 Plan-Apochromat objective and imaged using Zen™ 2008 LSM 710, V5.0 SP1.1 software.
Transmitted light images were also taken using DIC optics. Fluorescence filters were used to
observe bis benzimide (excitation 405 nm, emission 409–514 nm), FITC (excitation 488 nm,
emission 494–572 nm), and Cy3 (excitation 543 nm, emission 585–734 nm).
Measurement of Lysotracker Red Staining Intensity
Cells were plated at 1 million per well in a 6-well dish and were treated with DMSO
vehicle or rotenone (10 µM) for either 6 or 24h, or bafilomycin A1 (100 nM) for 4h prior to
analysis by flow cytometry. Cells were then incubated with LysoTracker® Red (100 nM final
concentration, Life Technologies, Eugene, OR) in Locke’s buffer (154mM NaCl, 5.6mM KCl,
3.6mM NaHCO3, 1.3mM CaCl2, 1mM MgCl2, 10mM HEPES, 1.009g/L glucose, pH 7.4) for 1h.
Cells were then harvested, pelleted and re-suspended in 1X PBS buffer before being passed
through a 70 μm nylon cell strainer (BD Falcon, Durham, NC) into collection tubes to provide a
single cell suspension. Cells were kept on ice and protected from light during immediate
transport to the flow cytometry facility (Joint UAB Flow Cytometry Facility; Enid Keyser,
Director) for analysis. 10,000 events were detected in each experimental condition using the BD
30
LSR II flow cytometer (Becton Dickinson, San Jose, CA). Further analysis was completed using
FlowJo software (Ashland, OR, licensed by UAB). LTR fluorescence was also visualized via
microscopy. Subets of cells were plated in 8 well glass chamber slides (Lab-Tek, Rochester,
NY) at a density of 60,000 per well for treatment with vehicle or rotenone (24h), or bafilomycin
A1 (4h).
Following 1h incubation with LTR was assessed via microscopy using a Zeiss
Axioskop® fluorescent microscope (Carl Zeiss Microimaging, LLC, Thornwood, NY) at 40X
magnification using bis-benzimide (0.2 µg/ml) for nuclear counter-stain. Transmitted light
images were also taken using phase contrast optics. Representative images were collected using
AxioVision 4.8 software (Carl Zeiss, Thornwood, NY).
Statistics
Significant effects of treatment were analyzed either by Student’s t-test (when two groups
were being compared); one-factor ANOVA (when effects of treatment or time were assessed
across multiple groups; or by two-factor ANOVA (when the effects of time vs. rotenone
treatment were assessed). Post hoc analysis was conducted using Bonferroni’s test. A level of p <
0.05 was considered significant.
31
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FIGURE 1
Fig. 1: Rotenone induced SH-SY5Y cell death and caspase 3-like activity are concentrationand time- dependent. Rotenone induced a concentration-dependent decrease in cell viability (A)
and increase in caspase 3-like activity (B), observed 24h after treatment. 10µM rotenone induced
a time-dependent decrease in SH-SY5Y cell viability (C) that was accompanied by an increase in
caspase 3-like activity (D) at 48 and 72h of treatment. Complete inhibition of rotenone-induced
39
caspase 3-like activity (not shown) with the broad caspase inhibitor Boc-FMK (Boc-Asp-FMK)
does not attenuate the decrease in cell viability after 48h treatment with 10 µM rotenone (E).
Rotenone (10µM) induced a significant decrease in cellular ATP levels (µM/well) at 6h and 12h
after treatment compared to CTL (F). Results represent mean ± standard deviation, and
experiments were repeated independently at least three times with similar results.* p<0.05 vs.
0µM rotenone CTL (A, B, E, F); * p<0.05 vs. 0h rotenone (C, D).
40
FIGURE 2
Fig. 2: Rotenone causes accumulation of autophagic vacuoles by blocking their effective
degradation. (A) Representative western blot of LC3-II (14 kDa) and actin (42 kDa) loading
control for SH-SY5Y lysates collected 6h and 24h following treatment with 10 µM rotenone
(ROT) in the presence or absence of 100 nM bafilomycin A1 (BafA1). Rotenone caused a
significant increase in LC3-II immunoreactivity at both 6h and 24h following treatment (A-C).
To measure autophagic flux, 100nM BafA1 was added for the last 2h of rotenone treatment prior
41
to preparing lysates. Quantification of LC3-II/actin ratios for each treatment is expressed
graphically as fold CTL for 6h rotenone (B) and 24h rotenone (C). Treatment with 10µM
rotenone induced a significant increase in AV accumulation, an effect that was not significantly
greater upon treatment with 100 nM BafA1. Representative western blot indicates that rotenone
increase levels of the autophagy substrate p62 at both 6h and 24h after treatment (D). Side bar in
(D) indicates higher p62-immunoreactive species following 6h and 24h treatment with rotenone
that suggests its enhanced ubiquitination. Blots were stripped and re-probed for actin.
Immunoreactivity for p62 (normalized to Actin) is quantified graphically in (E) and is expressed
as fold VEH CTL. Results = mean ± standard deviation from 3-5 independent experiments. * p
<0.05 vs. VEH CTL.
42
FIGURE 3
Fig. 3: Rotenone increases levels of α-synuclein. (A) Representative western blot analysis of
whole cell lysates indicates an increase in high molecular weight species of α-synuclein (>50
kDa) following 48h treatment with 10 µM rotenone. (B) Quantification of high-molecular
weight species of α-synuclein indicates a significant increase vs. vehicle control. Results = mean
± standard deviation obtained from 3 independent experiments. * p <0.05 vs. VEH CTL.
43
FIGURE 4
Fig. 4: Rotenone causes an increase in acidic vesicle pH. (A) Representative histogram of
alterations in acidic vesicle pH in SH-SY5Y cells after treatment with DMSO vehicle (VEH,
24h, orange line), rotenone (ROT, 10μM, 24h, blue line) or bafilomycin A1 (BafA, 100nM, 4h,
pink line) as determined by flow cytometry. Effects of rotenone treatment are expressed as a
leftward shift in fluorescence in the cell population when compared to vehicle control,
suggesting a loss of lysosomal/acidic vesicle pH. (B) Quantification of LTR mean fluorescence
intensity (MFI) indicates a >30% decrease following 6h and 24h treatment with rotenone. (C)
Phase contrast and fluorescence microscopy were used to image the rotenone-induced
44
attenuation of LTR fluorescence at 24h after treatment. Results = mean ± standard deviation
from 5 independent experiments * p <0.05 vs. VEH CTL.
FIGURE 5
Fig. 5: Rotenone does not induce lysosomal membrane permeabilization (LMP). Confocal
microscopy images obtained via double label immunocytochemistry for the soluble lysosomal
enzyme cathepsin D (red, Cy3) and the lysosomal membrane protein LAMP-1 (green, FITC)
following treatment for 24h with DMSO vehicle (top row), 10 µM rotenone (ROT, middle row)
or 50 µM chloroquine (CQ, bottom row). Punctate immunoreactivity for cathepsin D that colocalized to regions of the cell exhibiting intense LAMP-1 immunoreactivity was apparent in
45
vehicle and rotenone-treated cells. Diffuse staining for cathepsin D was observed in
chloroquine-treated cells that did not localize intracellularly to that of LAMP-1, suggesting the
onset of LMP. Images are representative of LMP assessment from 3 independent experiments.
FIGURE 6
Fig. 6: Rotenone increases levels of LAMP-1. Representative western blot (A) for LAMP-1
(~110 kDa) along with actin (42 kDa) loading control for lysates obtained from SH-SY5Y cells
treated with vehicle control (VEH CTL) or 10 µM rotenone (ROT) for 6h or 24h. Quantification
of LAMP-1 signal averaged from 4 independent experiments indicated that levels of LAMP-1
were significantly greater at 6h following rotenone. * p<0.05 vs. vehicle CTL.
46
FIGURE 7
Fig. 7 Rotenone exposure does not alter TFEB levels. (A) Representative western blot analysis
for TFEB (53kDa) and GAPDH (37kDa) loading control following 6h and 24h treatment with
vehicle control (CTL) or 10 µM rotenone (ROT). (B) Quantification of TFEB/GAPDH ratios
following 6h and 24h treatment of rotenone are expressed graphically as fold CTL. Results =
mean ± standard deviation obtained from 6 independent experiments.
47
ATP6V0C KNOCKDOWN IN NEUROBLASTOMA CELLS ALTERS AUTOPHAGYLYSOSOME PATHWAY FUNCTION AND METABOLISM OF PROTEINS THAT
ACCUMULATE IN NEURODEGENERATIVE DISEASE
by
LEANDRA R .MANGIERI*, BURTON J. MADER*, CAILIN E. THOMAS, CHARLES A.
TAYLOR, AUSTIN M. LUKER, TONIA E. TSE, CARRIE HUISINGH AND JOHN J.
SHACKA
*
these authors contributed equally to this work.
Under review at PLOS ONE
Format adapted for dissertation
48
ABSTRACT
ATP6V0C is the bafilomycin A1-binding subunit of vacuolar ATPase, an enzyme
complex that critically regulates vesicular acidification. We and others have shown previously
that bafilomycin A1 regulates cell viability, autophagic flux and metabolism of proteins that
accumulate in neurodegenerative disease. To determine the importance of ATP6V0C for
autophagy-lysosome pathway function, SH-SY5Y human neuroblastoma cells differentiated to a
neuronal phenotype were nucleofected with non-target or ATP6V0C siRNA and following
recovery were treated with either vehicle or bafilomycin A1 (0.3-100 nM) for 48h. ATP6V0C
knockdown was validated by quantitative RT-PCR and by a significant decrease in
Lysostracker® Red staining. ATP6V0C knockdown significantly increased basal levels of
microtubule-associated protein light chain 3-II (LC3-II), α-synuclein high molecular weight
species and APP C-terminal fragments, and inhibited autophagic flux. Enhanced LC3 and
LAMP-1 co-localization following knockdown suggests that autophagic flux was inhibited in
part due to lysosomal degradation and not by a block in vesicular fusion. Knockdown of
ATP6V0C also sensitized cells to the accumulation of autophagy substrates and a reduction in
neurite length following treatment with 1 nM bafilomycin A1, a concentration that did not
produce such alterations in non-target control cells. Reduced neurite length and the percentage
of propidium iodide-positive dead cells were also significantly greater following treatment with 3
nM bafilomycin A1. Together these results indicate a role for ATP6V0C in maintaining
constitutive and stress-induced ALP function, in particular the metabolism of substrates that
accumulate in age-related neurodegenerative disease and may contribute to disease pathogenesis.
49
INTRODUCTION
Vacuolar-ATPase (V-ATPase) is a membrane-associated, multi-subunit protein complex
that functions as an ATP-driven proton-pump 43. V-ATPase is organized into two coordinately
operating multi-subunit domains: the peripheral V1 domain that performs ATP hydrolysis and the
integral V0 domain that allows for proton translocation across the membrane. The V1 and V0
domains are connected to each other by a central “stalk” of shared subunits. The rotary action of
the stalk subunits has been proposed to drive proton translocation across the membrane upon V1
hydrolysis of ATP.
V-ATPase is localized to many different membranes of eukaryotic cells including
lysosomes, endosomes, Golgi-derived vesicles, secretory vesicles and in some cell types the
plasma membrane 43. V-ATPase has well documented functions, including maintenance of both
acidic vesicle and cytosolic pH and vesicle fusion with vacuoles 49, 50. V-ATPase-dependent
maintenance of acidic pH in lysosomes and endosomes is important for optimal function of their
proteolytic enzymes, whereas V-ATPase-dependent vesicle fusion serves a variety of functions
including neurotransmitter release from synaptic vesicles, transport of Golgi-derived lysosomal
enzymes and membrane proteins, and effective fusion of autophagosomes with lysosomes and
endosomes 8, 11, 43, 49, 50.
Pharmacologic inhibition of V-ATPase was first reported in 1988 by the use of antibiotic
drugs coined “bafilomycins” derived from Streptomyces soil bacteria. Bafilomycin A1 and
structurally related compounds have in common a 16-18 membered macrolactone ring linked to
a unique side chain and together represent the plecomacrolide subclass of macrolide antibiotics 4.
Bafilomycin A1 has been shown to inhibit V-ATPase with high affinity, at concentrations ≥
10nM 4. Bafilomycin A1 and similarly structured compounds are widely used as pharmacologic
50
tools to inhibit lysosome acidification and inhibit autophagy-lysosome pathway (ALP) function
by preventing autophagosome-lysosome fusion, thus promoting the robust accumulation of
autophagosomes 5, 18, 38, 49, 50. It is believed that V-ATPase-dependent vesicle fusion also requires
the maintenance of acidic pH, though recent studies have indicated that fusion may occur in a
pH-independent manner 1, 2.
It is widely believed that inhibition of ALP function contributes to the aberrant
accumulation of protein species in age-related neurodegenerative disease that not only define
disease-specific neuropathology but also may contribute to disease pathogenesis, including αsynuclein in Parkinson’s disease and metabolites of amyloid precursor protein (APP) in
Alzheimer’s disease 10, 28, 32, 39. Several ALP-associated molecules have been identified that are
currently being investigated for their utility as therapeutic targets in age-related
neurodegenerative disease 10, 15, 17, 25, 27, 29, 34, 35, 39, 44. Experimental inhibition of V-ATPase by
bafilomycin A1 prevents the effective degradation of α-synuclein that in turn promotes
accumulation of α-synuclein soluble oligomeric and insoluble aggregate species with neurotoxic
potential 9, 19, 21, 41, 47, 48. Bafilomycin A1-mediated inhibition of V-ATPase also effectively
inhibits the rapid degradation of full-length APP and its metabolites, C-terminal fragments
(CTFs) that are formed initially upon cleavage of full-length APP by β-secretase 13, 40, 45.
Subsequent cleavage by γ-secretase can promote the generation of toxic Aβ species, whereas
subsequent cleavage by α-secretase or γ-secretase can generate the putatively toxic APP
intracellular domain (AICD) 40, 45. As such, the ALP and putatively V-ATPase represent
attractive targets for promoting the metabolism of proteins that contribute to the pathogenesis of
age-related neurodegenerative disease.
51
Through analysis of bafilomycin A1-resistant strains of the fungus Neurospora crassa it
was discovered that bafilomycin A1 inhibition of V-ATPase activity is mediated by binding with
high affinity to the c subunit in the V0 domain, or ATP6V0C 3. Knockdown of ATP6V0C has
been shown recently to inhibit vesicular acidification and sensitize cells to stress-induced cell
death 6, 7, 51, while ATP6V0C-deficient mice are embryonic lethal 42. However, whether
ATP6V0C itself is responsible for regulating ALP function, as well as the metabolism of
substrates that accumulate in age-related neurodegenerative diseases has not been previously
investigated. In the present study we found that knockdown of ATP6V0C in neuronal cells
adversely affected ALP function concomitant with accumulation of ALP-associated substrates
including α-synuclein and APP-CTFs, and exacerbated stress-induced cell death. Our findings
suggest an important role for ATP6V0C in maintenance of ALP function that may portend
relevance to age-related neurodegenerative disease.
MATERIALS AND METHODS
Cell Culture
Naïve SH-SY5Y human neuroblastoma cells (ATCC, CRL-2266) were maintained in T75 flasks (Corning, 430641) at 37˚C and 5% CO2 in Minimum Essential Media (Cellgro, 10-010CV) and F12-K media (ATCC, 30-2004) supplemented with 0.5% sodium pyruvate (Cellgro, 25000-CI), 0.5% non-essential amino acids (Cellgro, 25-025-CI), 1% penicillin/streptomycin
(Invitrogen, 15140-122), and 10% heat-inactivated fetal bovine serum (FBS; Thermo Scientific,
SH30109.03). Naïve cells received full media change every two days. Prior to their use in
experiments, naïve SH-SY5Y cells were differentiated for seven days to a post-mitotic state in
52
10% FBS media supplemented with 10 μM retinoic acid (RA; Sigma, R2625). Complete RAsupplemented media was replenished every two days.
Nucleofection with siRNA
Small-interfering RNA (siRNA) specific for human ATP6V0C was obtained from
Thermo Scientific. The Amaxa Nucleofector™ system (Lonza, VVCA-1003), a modified
electroporation system, was utilized to transiently knock down ATP6V0C. First, RAdifferentiated SH-SY5Y cells were re-suspended in the Lonza™ Nucleofector reagent. Next,
400nM of re-suspended ATP6V0C siRNA (Thermo Scientific, M-017620-02-0010), or nontarget siRNA (Thermo Scientific, D-001206-13-05), (in siRNA buffer with 20 mM KCl, 6 mM
HEPES-pH 7.5, and 0.2 mM MgCl2) was added to the cell suspension. After electroporation,
500 μL of FBS-containing media was immediately added to the cell suspension. Following a 10
min recovery period, nucleofected cells were transferred to T-75 flasks and incubated overnight
in 10% FBS differentiation media for 24h. The next day nucleofected cells were plated for
experiments (24h after nucleofection and 24h prior to drug treatment) in either eight well glass
chamber slides (LabTek, 154941), six well plates (Corning 3516) or 60 mm dishes (Corning,
430166) at a density of 500/mm2 in 0% FBS differentiation media containing 2% B-27
supplement (Invitrogen, 17504044). All experimental endpoints ended at 96h after
nucleofection.
Treatment with Bafilomycin A1
Following nucleofection, recovery and plating for experiments (i.e. 48h after
nucleofection), media was exchanged for fresh 0% FBS differentiation media containing 2% B-
53
27 supplement with either DMSO vehicle (0 nM control) or bafilomycin A1 (0.3-100 nM; AG
Scientific, B-1183). Bafilomycin A1 was prepared as a 10 mM stock solution in DMSO (Sigma,
D8418-1L) and stored at -20˚C. Unless otherwise noted, all bafilomycin A1 treatments were for
48h, with experiments ending 96h after initial nucleofection.
Quantitative Real Time PCR
Cells nucleofected with ATP6V0C siRNA or non-target siRNA were processed for total
RNA extraction with the use of TRIzol reagent (Invitrogen, 15596-026). cDNA was generated
with oligo (dT) from four µg of RNA using the SuperScript III First Strand Synthesis System
(Invitrogen #18080-051). Primers used for detection of ATP6V0C mRNA were ATP6VOC 5’ATGTCCGAGTCCAAGAGCGGC-3’ and ATP6VOC 5’-CTACTTTGTGGAGAGGATGAG3’. Primers for actin were 5’-GCTCGTCGTCGACAACGGCTC-3’ and 5’CAAACATGATCTGGGTCATCTTCTC-3’. Template for cDNA (2 µL) was added along with
1X Fast SYBR Green Master Mix (ABI #4385610) and water to a final volume of 20μL/well.
Assays were performed in triplicate on a Bio-Rad CFX96 instrument using the following
conditions: (95°C for three minutes followed by 95°C for 15 seconds and 60°C for one minute
for 45 cycles. The comparative cycle threshold (Ct) method (ddCT; 14) was used to determine
expression of ATP6V0C in each sample relative to endogenous actin control, which was then
used to determine knockdown in ATP6V0C siRNA samples as a percentage of non-target siRNA
control.
54
Measurement of Acidic Vesicle pH
After treatment for 48h with DMSO vehicle, cells plated in 60 mm dishes were incubated
with LysoTracker Red® (LTR; Life Technologies™, L7528; 100 nM final concentration) for 30
min in Locke’s buffer (15mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 1.3mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, 1.009 g/L glucose, pH 7.4). Cells were then harvested, pelleted, and resuspended in 1 X PBS then passed through a 70 μm nylon cell strainer (BD Falcon, 08-771-2)
and into collection tubes to provide a single cell suspension. Cells were kept on ice and protected
from light during immediate transport to the Joint UAB Flow Cytometry Facility (Enid Keyser,
Director) for analysis. A total of 1 × 104 events were detected in each experimental condition
using the BD LSR II flow cytometer (Becton Dickinson). Further analysis was performed using
FlowJo software (licensed by UAB) to assess mean fluorescence intensity. LTR fluorescence
was also visualized via microscopy in cells plated in eight well glass chamber slides and treated
for either 48h with DMSO vehicle or with 100 nM bafilomycin A1 for the last 3h of the 48h time
course. Following treatment with LTR, fluorescent images were captured using a Zeiss Axioskop
fluorescent microscope (Carl Zeiss Micro-imaging, LLC) at 40X magnification. Transmitted
light images were also taken using phase contrast optics.
Western Blot Analysis
After treatment for 48h, nucleofected cells in 60 mm dishes were incubated for seven
minutes at 37˚C with Accutase (Innovative Cell Tech., AT104) to detach cells from their
substrate. Whole cell lysates were collected and protein concentrations were determined using
BCA protein assay (Fisher Scientific, PI-23227) as previously described 38. Equal amounts of
each protein sample were then electrophoresed using 12% Tris/Glycine SDS-polyacrylamide
55
gels, or for APP CTFs using 16.5% Tris-Tricine pre-cast gels (Bio-Rad Laboratories, 456-3064)
and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, 162-0177).
Following a 30 minute incubation at room temperature with 5% blocking milk (Bio-Rad, 1706404) in 1X tris-buffered saline with tween (TBST), membranes were incubated overnight with
primary antibodies for immunodetection of the following proteins: LAMP1 (University of Iowa
Hybridoma Bank, H4A3 ); LC3 (rabbit anti-LC3, Abcam, ab51520); α-syn (Santa Cruz
Biotechnology, Inc., SC7011); and APP CTF (Covance Inc, SIG-39152). Membranes were
washed with 1X TBST containing 0.1% Tween 20 and then incubated with secondary IgG-HRP
conjugated antibody for 1h at room temperature. After washing blots, SuperSignal West Femto
Chemiluminescent Substrate (Thermo Scientific, PI-34096) was used to detect α-syn and
Enhanced chemilumescence (ECL; Thermo Scientific, PI-32106) was used to detect LAMP-1,
LC3 and APP. Blots were then stripped with Restore™ Western Blot stripping buffer (Thermo
Scientific, 21059) and probed for actin (Sigma, A1978) to normalize for gel loading. Films with
detected bands of interest were scanned using Adobe® Photoshop® and band intensities were
calculated using UN-SCAN-IT gel digitizing software (Silk Scientific, Inc.).
Assessment of Autophagic Flux
Following nucleofection, plating for experiments and 44-46h following media change,
subsets of cells in six well plates were treated with 100 µM chloroquine (Sigma, C6628) plus
200 µM leupeptin (Sigma, L9783) to inhibit lysosome function. At 48h following initial media
change (equaling 2-4h following treatment with lysosome inhibitors) lysates were processed as
above for western blot analysis of LC3-II and actin loading control.
56
Assessment of Autophagosome-Lysosome Fusion
Following nucleofection with ATP6V0C siRNA or non-target control siRNA, cells plated
in eight well glass chamber slides were treated for 48h with DMSO and subsequently fixed for
15 min at 4˚C using Bouin’s fixative (75% saturated picric acid, 23.8% of a 37% w/v
formaldehyde solution, 4.7% glacial acetic acid) in the presence of 0.5% saponin (Sigma,
47036). Following incubation for 30 min with 1X PBS blocking buffer containing 1% bovine
serum albumin, 0.2% non-fat dry milk and 0.3%Triton X-100, cells were incubated overnight at
4˚C in blocking buffer without Triton X-100 and containing rabbit anti-LC3 antibody (Sigma,
L7543) to detect autophagosome punctae. Following PBS wash, fixed cells were incubated for
1h with SuperPicTureTM (Invitrogen, 879263) anti-rabbit IgG secondary antibody. Following
PBS wash, fixed cells were fixed cells were subjected to tyramide signal amplification
(incubation for 30 min with Cy3 plus tyramide, Perkin EImer, FP1170) to detect LC3. Next,
fixed cells were incubated for 10 min with 3% hydrogen peroxide to neutralize residual
peroxidase from the first secondary antibody. After PBS wash and 30 min incubation with
blocking buffer, fixed cells were incubated overnight at 4˚C in blocking buffer containing the
second primary antibody, mouse anti-human LAMP-1 (University of Iowa Hybridoma Bank,
H4A3) to detect lysosomes. Following subsequent PBS wash, fixed cells were incubated for 1h
with Vector Impress (Vector Labs, MP-7402) anti-mouse IgG. Following PBS wash, tyramide
signal amplification was performed (incubation for 30 min with FITC plus tyramide, Perkin
Elmer, FP1168) to label LAMP-1. Following PBS wash, fixed cells were last incubated with
bis-benzimide (0.2 µg/ml in PBS, Sigma) to label nuclei. Images were captured using a Zeiss
Observer.Z1 laser scanning microscope equipped with a Zeiss 40X Plan-Apochromat objective
and imaged using Zen 2008 LSM 710, V5.0 SP1.1 software.
57
Assessment of Cell Death Morphology and Neurite Length
After treatment for 48h with 0-10 nM bafilomycin A1, nucleofected cells plated on eight
well glass chamber slides were fixed with Bouin’s fixative for 15 min, 4˚C. Cell death
morphology was visualized via transmitted light and DIC optics on a Zeiss Observer.Z1 laser
scanning microscope equipped with a Zeiss 40X 1.3 DIC M27 Plan-Apochromat objective and
imaged using Zen 2008 LSM 710, V5.0 SP1.1 software. Neurite length was assessed by first
capturing images using Olympus BX51 microscope (Olympus of the Americas) and a 100X
UPlan FLN objective. Neurites were measured using the tracing tool within Neurolucida
software (MBF Bioscience, version 5.65). Cells identified for measurements had neuritic
processes that did not form a synapse with neighboring cells. Data files collected using
Neurolucida were quantified using the Nueurolucida Explorer program to determine average
neurite length per cell.
Quantification of Percent Cell Death
Cell death was quantified using propidium iodide (PI; Life Technologies, V13242)
staining and flow cytometry. Following treatment for 48h cells with 0-100 nM bafilomycin A1,
nucleofected cells were incubated with PI for 15 min at a final concentration of 1μg/mL. Cells
were then prepared for flow cytometry as above for assessment of acidic vesicle pH and
quantified as the percentage of cells that were PI-positive relative to vehicle control using
FlowJo software.
58
Statistical Analysis and Figure Preparation
Quantitative real time PCR data were expressed as mean ± SEM percent knockdown for
ATP6V0C. LTR staining was presented as mean ± SEM fluorescence intensity. Western blot
band intensities for LAMP-1, LC3-II, α-syn and APP-CTFS were normalized to actin loading
control, and were expressed (mean ± SEM) either as such to assess bafilomycin A1
concentration responsiveness, or further normalized for each ATP6V0C siRNA condition as
mean ± SEM fold change relative to its companion non-target control to determine effects of
genetic manipulation. Neurite length was presented as mean ± SEM average length (µm) per
cell. Cell death was quantified as mean ± SEM percentage of PI-positive cells. To determine
effects of genetic manipulation, between groups comparisons of Non-target vs. ATP6V0C
siRNA were evaluated for significance using either one-sample t-test (LTR data; quantitative
real-time PCR data; western blot data normalized as fold change relative to the companion nontarget control) or two-sample t-test (assessment of neurite length and percent cell death). In
addition, to assess concentration responsiveness, within group comparisons of absolute (raw)
data were evaluated for significance using one-way ANOVA. Significant ANOVAs were
followed by post hoc analysis using Bonferroni’s Multiple Comparison Test. For all tests,
statistical significance was set a priori at p < 0.05. Graph Pad Prism® was used to perform
statistical analysis and generate graphs, and Adobe® Photoshop® was used to assemble figures. A
minimum of three independent replicates were performed for each experimental endpoint.
59
RESULTS
Validation of ATP6V0C knockdown
We first set out to confirm siRNA-mediated knockdown of ATP6V0C. Quantitative real
time PCR analysis was performed on cDNA that was generated from RNA in samples isolated
from differentiated SH-SY5Y human neuroblastoma cells harvested 96h following nucleofection
with non-target or ATP6V0C siRNA. Using the comparative cycle threshold (Ct) method
(ddCT; 14) with actin mRNA expression serving as an internal standard, we determined that
ATP6V0C mRNA was significantly reduced by 95.01 ± 2.33% (p<0.05 vs. Non-target siRNA
using one sample t-test) in ATP6V0C siRNA samples compared to non-target control (Table 1).
Table 1
Expression of ATP6V0C by quantitative real-time PCR
%
Sample
Amplicon
Ct1
dCt2
ddCt3
% ATP6V0C expression4
knockdown5
ATP6V0C siRNA
ATP6V0C
27.58
4.99
4.32
4.99%
95.01% +/2.33%*
ATP6V0C siRNA
Actin
22.59
Non-target siRNA
ATP6V0C
24.29
Non-target siRNA
Actin
23.62
0.67
1
Ct is number of cycles to reach threshold, or threshold cycle
dCt = average Ct(ATP6V0C) – average Ct(Actin)
3
ddCt = dCt(ATP6V0C siRNA) – dCt(Non-target siRNA)
4
% ATP6V0C expression = 2 –ddCt
5
% Knockdown = 100% - 4.99%
*p<0.05 via one-sample t-test compared to Non-target siRNA control; n=3 replicates
2
To determine if ATP6V0C knockdown regulates vesicular acidification as previously
reported 51 we performed flow cytometric analysis of LTR staining at 96h following
60
nucleofection (Fig. 1). Representative images indicated a noticeable reduction in LTR
fluorescence following knockdown with ATP6V0C siRNA following vehicle treatment (Fig. 1A,
B) compared to non-target control (Fig. 1D, E). As a comparative control, treatment for 3h with
100 nM bafilomycin A1 appeared to completely ameliorate LTR fluorescence for both non-target
control cells and following knockdown with ATP6V0C (Fig. 1C, 1F). Quantification of LTR
mean fluorescence intensity in vehicle-treated cells (Fig. 1G) indicated a significant reduction
following ATP6V0C knockdown (0.54 ± 0.04) expressed as fold-change relative to non-target
control (Fig. 1B), confirming in our knockdown model the importance of ATP6V0C for the
maintenance of acidic vesicle pH.
ATP6V0C knockdown increases levels of lysosome marker LAMP-1 levels by a low
concentration of bafilomycin A1
Levels of LAMP-1 (lysosome-associated membrane protein-1) have been shown
previously to increase following lysosomal stress 20, 24, 37. To further determine the effects of
ATP6V0C knockdown on acidic vesicles we performed western blot analysis to assess LAMP-1
levels at 48h following treatment (Fig. 2A, C, E). Levels of LAMP-1 were assessed following
treatment with vehicle control or following treatment with bafilomycin A1 at concentrations
shown previously to have either little effect on inhibiting V-ATPase or vesicular pH (1 nM) or
cause profound inhibition of V-ATPase and vesicular acidification at ≥10 nM 4, 31, 38. In nontarget siRNA control cells, significant increases in LAMP-1/actin ratios were observed following
treatment with 10 nM (2.89 ± 0.41) and 100 nM (2.92 ± 0.39) bafilomycin A1 compared to
treatment with 0 nM (0.90 ± 0.23) or 1 nM (1.22 ± 0.31) concentrations (Fig. 2C, open columns).
In ATP6V0C siRNA cells (Fig. 2C, filled columns), the concentration responsiveness of
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bafilomycin A1 shifted such that significantly higher LAMP-1/actin ratios were observed at all
three concentrations tested (1 nM: 2.03 ± 0.28; 10 nM: 2.52 ± 0.33; 100 nM: 2.18 ± 0.32)
compared to that observed in 0 nM vehicle control (0.76 ± 0.18). When effects of ATP6V0C
siRNA knockdown were compared directly to that of non-target siRNA control (Fig. 2E), a
significant 2.06 ± 0.30 fold increase in relative LAMP-1/actin was observed at the 1 nM
concentration, whereas fold changes by 0 nM vehicle control (0.89 ± 0.16), 10 nM (1.03 ± 0.24)
or 100 nM (0.79 ± 0.11) concentrations of bafilomycin A1 were not significantly different from
non-target control. Together these findings indicate that ATP6V0C knockdown shifted the
concentration responsiveness of cells to bafilomycin A1 by increasing LAMP-1 levels at a lower
concentration not ordinarily associated with lysosome inhibition.
ATP6V0C knockdown increases basal and stress-induced accumulation of autophagosomes.
To determine if ATP6V0C deficiency affected the basal vs. bafilomycin A1-induced
accumulation of autophagosomes we performed western blot analysis for LC3-II (Fig. 2B, D, F).
Assessing bafilomycin A1 concentration responsiveness in non-target siRNA control cells (Fig.
2D, open columns), treatment with 10 and 100 nM bafilomycin A1 produced significantly
greater LC3-II/actin ratios (10 nM: 1.99 ± 0.45; 100 nM: 2.16 ± 0.36) compared to 0 nM vehicle
control (0.40 ± 0.10), while LC3-II/actin ratios following treatment with 1 nM bafilomycin A1
(0.79 ± 0.10) were significantly different only in comparison to the 100 nM concentration.
While a significant one-way ANOVA was obtained in assessing the bafilomycin A1
concentration responsiveness resulting from ATP6V0C knockdown (Fig. 2D, filled columns),
post hoc analysis revealed no significant differences in LC3-II/actin ratios in knockdown cells
with respect to any concentration of bafilomycin A1. When effects of ATP6V0C siRNA
62
knockdown were compared directly to that of non-target siRNA control (Fig. 2E), treatment with
both 0 nM vehicle control (1.55 ± 0.10) and 1 nM bafilomycin A1 (2.36 ± 0.39) exhibited
significantly higher fold changes in relative LC3-II/actin in ATP6V0C knockdown cells
compared to non-target siRNA control. Fold change differences in relative LC3-II/actin
following ATP6V0C knockdown and treatment with 10 nM bafilomycin A1 (1.08 ± 0.09) or 100
nM bafilomycin A1 (1.01 ± 0.09) were not significantly different relative to non-target siRNA
control. These findings indicate that ATP6V0C knockdown not only increased the basal
accumulation of autophagosomes but also enhanced their accumulation at a “low” concentration
of bafilomycin A1. The lack of significant concentration responsiveness following ATP6V0C
knockdown may reflect the observed increase in autophagosomes following 0 nM vehicle and 1
nM bafilomycin A1 treatment.
ATP6V0C knockdown inhibits autophagic flux
To determine the manner by which knockdown of ATP6V0C caused the
accumulation of autophagosomes we assessed autophagic flux by western blot analysis of LC3II, following inhibition of lysosome function for the last 2-4h of a 48h treatment period by
treatment with a high concentration of the lysosomotropic agent and weak base chloroquine, and
the protease inhibitor leupeptin (Fig. 3A-B) as previously reported 18. Treatment of non-target
siRNA control cells with lysosome inhibitors caused a robust and significant increase in LC3II/actin (1.39 ± 0.26) compared to vehicle treatment alone (0.69 ± 0.06). When the effects of
ATP6V0C siRNA knockdown were compared relative to non-target siRNA control, a significant
fold-change increase was observed following vehicle treatment (1.51 ± 0.19), while the foldchange difference in relative LC3-II/actin following treatment with lysosome inhibitors for
63
ATP6V0C siRNA knockdown (1.14 ± 0.13) was not significantly different from non-target
control.
To further investigate the manner by which autophagic flux was inhibited we
performed double-label immunocytochemistry for LC3 and LAMP-1 18 in vehicle-treated nontarget siRNA control cells (Fig. 3C-E) and ATP6V0C siRNA knockdown cells (Fig. 3F-H).
Representative images from vehicle-treated non-target siRNA control cells indicate a relative
lack of co-localization with LC3 and LAMP-1 punctae (Fig. 3E). In comparison, there were
several instances of LC3 and LAMP-1 punctae co-localizing in vehicle treated ATP6V0C
knockdown cells (Fig. 3H). Together these results suggest that ATP6C0C knockdown inhibits
autophagic flux in a manner caused in part by the accumulation of dysfunctional LC3 and
LAMP-1-positive autolysosomes.
ATP6V0C knockdown induces basal and stressed accumulation of high molecular weight αsynuclein species
It is well-established that lysosome function is important for the effective metabolism of
substrates including α-synuclein that accumulate in age-related neurodegenerative diseases 10, 31,
44
. We have shown previously that treatment of differentiated SH-SY5Y cells with agents that
disrupt ALP function causes the accumulation of high molecular weight (MW) α-synuclein
species that are associated with PD pathology 24, 31. Specifically, inhibition of V-ATPase with
bafilomycin A1 has been shown using western blot analysis to increase levels of α-synuclein
high MW species 21. Thus western blot analysis was used to determine if knockdown of
ATP6V0C altered levels of endogenous high MW α-synuclein species (defined as greater than
50 kDa) under basal (vehicle) conditions or following treatment with 1-100 nM bafilomycin A1
(Fig. 4). Representative western blot analysis suggests that treatment with bafilomycin A1
64
induced the accumulation of α-synuclein high MW species (Fig. 4A). However, one-way
ANOVA of pooled data (Fig. 4B) was not significant, thus suggesting a lack of concentration
dependence between groups of non-target siRNA control cells treated with 0 nM (2.74 ± 0.81), 1
nM (3.24 ± 0.81), 10 nM (4.98 ± 1.53) or 100 nM (5.46 ± 1.55) bafilomycin A1, or between
groups of ATP6V0C siRNA knockdown cells treated with 0 nM (3.68 ± 0.87), 1 nM (4.38 ±
1.17), 10 nM (4.94 ± 1.40) or 100 nM (4.65 ± 1.02) bafilomycin A1. This lack of significant
concentration-dependence, at least for non-target siRNA control cells may be explained by the
wide rage in absolute values for α-synuclein/actin ratios observed between different experiments.
Regardless, when effects of ATP6V0C siRNA knockdown were compared directly to that of
non-target siRNA control (Fig. 4D), treatment with both 0 nM vehicle control (1.90 ± 0.38) and
1 nM bafilomycin A1 (1.75 ± 0.32) exhibited significantly higher fold changes in relative αsynuclein high MW species/actin compared to non-target siRNA control. Fold changes between
observed following treatment with 10 nM (1.31 ± 0.24) or 100 nM (1.12 ± 0.16) bafilomycin A1
were not significantly different. Levels of α-synuclein monomer (17 kDa) were also assessed in
non-target siRNA control and ATP6V0C siRNA knockdown cells (Fig. 4A). Similar to αsynuclein high MW species (Fig. 4B), one-way ANOVA was not significant, suggesting a lack
of bafilomycin A1 concentration responsiveness for ratios of α-synuclein monomer/actin (Fig.
4C) between groups of non-target siRNA control cells treated with 0 nM (0.79 ± 0.19), 1 nM
(0.95 ± 0.21), 10 nM (0.91 ± 0.21) or 100 nM (0.89 ± 0.21) bafilomycin A1, or between groups
of ATP6V0C siRNA knockdown cells treated with 0 nM (1.03 ± 0.23), 1 nM (1.12 ± 0.25), 10
nM (0.78 ± 0.20) or 100 nM (0.65 ± 0.18) bafilomycin A1. Interestingly, when the effects of
ATP6V0C siRNA knockdown were compared directly to that of non-target siRNA control (Fig.
4E), a significant fold-change decrease in α-synuclein monomer was observed in ATP6V0C
65
siRNA knockdown cells treated with 100 nM bafilomycin A1 (0.75 ± 0.10), even though
concomitant increases in α-synuclein high MW species were not observed at this concentration.
Significant differences in α-synuclein monomer were not observed in ATP6V0C knockdown
cells treated with vehicle control (0 nM: 1.38 ± 0.19) or other concentrations of bafilomycin A1
(1 nM: 1.21 ± 0.10; 10 nM: 0.95 ± 0.13). In summary, while treatment with bafilomycin A1 did
not cause concentration-dependent increases in α-synuclein, our findings suggest that ATP6V0C
deficiency alters both the basal and stress-induced metabolism of α-synuclein.
ATP6V0C knockdown induces basal and stress-induced accumulation of APP CTFs
The importance of intact ALP function for the effective degradation of APP and its
enzymatic cleavage products, termed C-terminal fragments (CTFs) is also well documented 29, 40,
45
. Treatment with bafilomycin A1 and other inhibitors of lysosome function have been shown
previously to promote the accumulation of APP CTFs 40, 45. To determine if ATP6V0C regulated
the basal vs. stressed accumulation of endogenous APP CTFs we performed western blot
analysis using an antibody that recognizes both full-length APP and its CTFs (Fig. 5). CTFs
were defined in our study as ≤ 15 kDa based on previously published reports 12 and were
quantified as such. APP CTFs observed in our study correspond in size to the C99 fragment (14
kDa), formed by sequential β-and γ-secretase cleavage of APP in the “amyloidogenic” pathway;
the C83 fragment (12.5 kDa), formed by β-and α-secretase cleavage of APP in the “nonamyloidogenic” pathway; and the APP intracellular domain (AICD), formed by both
amyloidogenic and non-amyloidogenic pathways 12, 13, 40, 45. Bafilomycin A1 concentration
responsiveness was first assessed for ratios of APP-CTFs/actin (Fig. 5B) in non-target siRNA
control cells (open columns) and ATP6V0C siRNA knockdown cells (filled columns). Results
66
of one-way ANOVA and post-hoc analysis revealed significant increases in ratios of APP
CTFs/actin following treatment with bafilomycin A1 at 10 nM (5.15 ± 0.62) and 100 nM (5.71 ±
1.16) concentrations, compared to that of treatment with either 0 nM (0.47 ± 0.16) or 1 nM (0.76
± 0.32) concentrations. For ATP6V0C siRNA knockdown cells, significant differences in foldchange APP CTFs/actin were observed at 10 nM (5.33 ± 1.23) and 100 nM (5.31 ± 1.45)
concentrations of bafilomycin A1 when compared to those in the 0 nM vehicle control group
(0.74 ± 0.22) but not following treatment with 1 nM bafilomycin A1 (2.13± 0.60). When the
effects of ATP6V0C siRNA knockdown were compared directly to non-target siRNA control
(Fig. 5C), significant increases in ratios of APP CTFs/actin relative to siRNA control were
revealed for both 0 nM (1.64 ± 0.12) and 1 nM (3.33 ± 0.71) concentrations of bafilomycin A1,
while comparisons between 10 nM (0.99 ± 0.13) and 100 nM (0.92 ± 0.16) concentrations were
not significantly different. These results suggest that ATP6V0C plays a functional role in the
effective degradation of APP CTFs, some of which exhibit documented neurotoxic potential.
ATP6V0C deficiency induced markers of neurotoxicity.
As ATP6V0C deficiency was shown previously to exacerbate stress-induced cell death 6,
7, 51
we next determined if knockdown of ATP6V0C was similarly toxic to differentiated SH-
SY5Y neuroblastoma cells by investigating different markers of cytotoxicity (Fig. 6).
Representative cell morphology is pictured for non-target siRNA cells (Fig. 6A-D) or for
ATP6V0C siRNA knockdown cells (Fig. 6E-H). DMSO vehicle-treated non-target control cells
(Fig. 6A) and ATP6V0C knockdown cells (Fig. 6E) both exhibited normal appearing cell soma
and neurites. Neuritic processes imaged in our study appear similar to those observed previously
in SH-SY5Y cells 26, 33. Non-target control cells treated with 1 nM bafilomycin A1 (Fig. 6B)
67
also appeared morphologically similar to vehicle-treated non-target cells (Fig. 6A), but looked
healthier than ATP6V0C knockdown cells treated with 1 nM bafilomycin A1 (Fig. 6F), which
exhibited comparatively shorter neurites. Treatment with 3 nM bafilomycin A1 caused a
noticeable reduction in neurite length as well as increased pyknosis in both non-target control
cells (Fig. 6C) and ATP6V0C knockdown cells (Fig. 6G), although neurite length in ATP6V0C
knockdown cells treated with 3 nM bafilomycin A1 appeared shorter than non-target control
cells treated at the same concentration. Treatment with 10 nM bafilomycin A1 caused
pronounced pyknosis and neurite loss in both non-target control cells (Fig. 6D) and ATP6V0C
knockdown cells (Fig. 6H). To quantify our morphological observations we measured neurite
length (Fig. 6I). One-way ANOVA was performed followed by post hoc analysis to determine
bafilomycin A1 concentration responsiveness for non-target siRNA control-treated cells (Fig. 6I,
open columns) or ATP6V0C siRNA knockdown cells (Fig. 6I, filled columns). For non-target
siRNA control cells, vehicle control neurite length (87.77 ± 7.80) was significantly greater than
that observed at 3 nM (55.22 ± 4.38) or 10 nM (23.58 ± 2.35). Neurite length in non-target
control cells following treatment with 10 nM bafilomycin A1 was also significantly less than in
cells treated with 1 nM (70.42 ± 6.18) or 3 nM bafilomycin A1. In ATPV0C knockdown cells,
neurite length following treatment with DMSO vehicle (67.15 ± 6.54) was significantly greater
than that measured following treatment with all other concentrations of bafilomycin A1 (1 nM:
48.38 ± 5.38; 10 nM: 21.61 ± 1.61; 10 nM: 20.29 ± 1.67). In addition, neurite length in 1 nM
bafilomycin A1-treated cells was significantly greater than that of 3 and 10 nM concentrations.
When neurite lengths in ATP6V0C knockdown cells were compared to those in non-target
control cells using two-sample t-test, significant reductions in neurite lengths were observed in
68
ATP6V0C knockdown cells at 0 nM, 1 nM and 3 nM concentrations of bafilomycin A1but not at
the 10 nM concentration.
Flow cytometric analysis was also used to quantify the effects of ATP6V0C deficiency
on cell death by assessing the percentage of PI-positive cells that accumulated 48h after
treatment with DMSO vehicle or 0.3, 1, 3, 10 or 100 nM bafilomycin A1 (Fig. 6J). Percent cell
death (mean ± SEM) was measured as follows for non-target control cells (open columns):
DMSO vehicle: 16.75 ± 2.24; 0.3 nM: 18.94 ± 2.09; 1 nM: 20.35 ± 2.36; 3 nM: 37.30 ± 4.10; 10
nM: 81.17 ± 0.36; 100 nM: 81.3 ± 1.48. Following a significant one-way ANOVA and
subsequent post hoc analysis, significant increases in percent cell death were observed following
treatment with concentrations ≥ 3 nM bafilomycin A1 compared to treatment with 0 nM vehicle
control or 0.3 and 1 nM concentrations. Percent cell death following treatment with bafilomycin
A1 was also significantly greater at 10 and 100 nM compared to treatment with 3 nM. Percent
cell death for ATP6V0C knockdown cells (filled columns) were as follows: DMSO vehicle:
17.31 ± 2.07; 0.3 nM: 23.30 ± 1.08; 1 nM: 27.97 ± 4.89; 3 nM: 66.98 ± 5.63; 10 nM: 79.07 ±
2.48; 100 nM: 82.57 ± 2.42. Similar to non-target control, results of one-way ANOVA were
found to be significant, with post hoc analysis indicating significant increases in percent cell
death following treatment with concentrations ≥ 3 nM bafilomycin A1, compared to treatment
with 0 nM vehicle control or 0.3 nM and 1 nM concentrations. However, unlike non-target
siRNA-treated cells, percent cell death in 3 nM bafilomycin A1-treated ATP6V0C knockdown
cells treated was not different than that observed at 10-100 nM concentrations. Asterisks in Fig.
6J indicate significant differences with respect to concentration of bafilomycin A1. When
percent cell death following ATP6V0C knockdown was compared directly to non-target control
using two-sample t-test, a significant increase in percent cell death was observed specifically in
69
ATP6V0C knockdown cells treated with 3 nM bafilomycin A1, as indicated by the hash tag (#)
in Fig. 6J. Together these findings corroborate our other findings by indicating an increased
sensitivity of cells to bafilomycin A1-induced cytotoxicity following knockdown of ATP6V0C.
DISCUSSION
V-ATPase regulates the proper function of acidic vesicles including endosomes and
lysosomes, in part through its maintenance of vesicular pH. This is important not only for the
effective transport and delivery of lysosome-associated structural and functional molecules to
their pre-destined location, but also for protein degradation pathways that rely on the effective
fusion between endosomes, autophagosomes and/or lysosomes. While several studies have
indicated the ability of V-ATPase inhibitors including bafilomycin A1 to regulate autophagylysosome pathway function and substrate degradation, it is unknown whether the molecular
target that bafilomycin A1 binds, the ATP6V0C subunit of V-ATPase, directly mediates these
events. Results of the present study show how knockdown of ATP6V0C increases vesicular pH
and inhibits basal autophagic flux as well as basal and stress-induced metabolism of autophagy
markers/substrates, suggesting that ATP6V0C contributes to the function of the autophagylysosome pathway under constitutive conditions and under conditions of stress. In addition,
results of our cytotoxicity assays indicate the contribution of ATP6V0C in the maintenance of
cell survival under basal and stressed conditions.
Basal vesicular acidification was significantly attenuated following knockdown of
ATP6V0C in SH-SY5Y cells differentiated to a neuronal phenotype, which is similar to previous
findings in MCF-7 breast cancer tumor cells 51. This disruption in vesicular acidification is
likely responsible for the basal inhibition of autophagic flux observed in our study, which we
70
further characterized by the accumulation of LC3 and LAMP-1 positive punctate co-localizing
together that likely represent a population of autolysosomes with a decreased functional capacity
for autophagic degradation. Although bafilomycin A1-induced inhibition of V-ATPase has been
shown previously to inhibit autophagosome-lysosome fusion 49, and mutants of V0 subunits of VATPase inhibit vacuole fusion in a pH-independent manner 1, our results suggest that basal
knockdown of ATP6V0C in the absence of pharmacological inhibition by V-ATPase inhibition
has the capacity to inhibit autophagic flux through its inhibition of lysosomal pH in a manner
that does not require fusion block.
While vesicular acidification and autophagic flux were significantly inhibited, and LC3-II
levels significantly increased following knockdown of ATP6V0C under basal conditions, we did
not observe a corresponding increase in basal LAMP-1 levels. Although we observed an
increase in LAMP-1 and LC3 co-localization that we attributed to inhibition of autophagic flux,
it is possible that residual ATP6V0C molecules persisting after knockdown allowed for an
incomplete inhibition of basal autophagy that would preclude LAMP-1-positive lysosomes from
accumulating in size and/or number. Alternatively, it is possible that basal ATP6V0C deficiency
disrupts the participation of V-ATPase in a recently identified, complex signaling network
involving mTORC1 and TFEB (transcription factor EB) 30, 52. TFEB is considered a master
regulator for the transcription of lysosome-and autophagy associated genes 36. Thus a disruption
in TFEB signaling could also explain why basal LAMP-1 levels were not increased following
knockdown of ATP6V0C, a possibility that is worthy of future investigation.
The increase in basal levels of α-synuclein and APP observed following knockdown
suggests a potential contribution of ATP6V0C and V-ATPase to the onset and progression of
age-related neurodegenerative disease. To date, alterations in ATP6V0C have yet to be reported
71
in human neurodegenerative disease. A previous study has indicated increased expression of the
E3 ubiquitin ligase protein RNF182 in Alzheimer’s disease brain 23. RNF182 was shown using
in vitro studies to bind ATP6V0C with high affinity and target it for degradation 23, thus
suggesting the potential for disruption of ATP6V0C function in Alzheimer’s disease. As such,
future investigation of ATP6V0C expression patterns in brains of neurodegenerative disease
patients is warranted. Even if ATP6V0C expression patterns are found to be unaltered in human
neurodegenerative disease, recent studies of ATP6V0C over-expression suggest the potential for
its therapeutic benefit. Over-expression of ATP6V0C in mouse substantia nigra was shown
recently to attenuate behavioral deficits following treatment with a dopaminergic neurotoxin 16,
suggesting its potential utility as a therapeutic target for preventing neurodegeneration.
Furthermore, over-expression of ATP6V0C has been shown in vitro to induce HIF-1α gene
expression in a pH-independent manner 22 providing further proof of principle that overexpression of one subunit of V-ATPase has functional consequence.
In several of our experimental endpoints, knockdown of ATP6V0C consistently and
significantly enhanced the sensitivity of cells to low concentrations of bafilomycin A1. Levels
of LAMP-1, LC3-II, α-synuclein and APP CTFs were all significantly higher, and neurite
lengths shorter in knockdown cells following treatment with 1 nM bafilomycin A1, a
concentration we have shown previously to have little if any influence on autophagy-associated
markers or vesicular pH in non-transfected cells 38, and induce protection against chloroquineinduced death and accumulation of autophagic substrates 31. ATP6V0C knockdown also caused
a significant further reduction in neurite length and induction of cell death following treatment
with 3 nM bafilomycin A1. The most plausible explanation for these findings is the incomplete
nature of ATP6V0C knockdown, with a resultant decrease in residual binding sites causing a
72
shift in bafilomycin A1 concentration responsiveness that would allow lower concentrations of
bafilomycin A1 to effectively inhibit V-ATPase and in turn inhibit autophagic degradation and
induce cell death. It would be interesting for future studies to determine if knockdown of
ATP6V0C prevents the ability of low concentrations of bafilomycin A1 to attenuate ALPassociated dysfunction, as we have shown previously in our laboratory 31.
Although we observed an approximate 95% reduction in ATP6V0C mRNA, it is possible
that heightened sensitivity of knockdown cells to bafilomycin A1 could be explained by residual
pools of ATP6V0C protein that were translated prior to knockdown. Our laboratory has tried to
detect ATP6V0C protein using several commercially available antibodies and to date we have
been unable to reliably detect its protein levels, thus limiting our current assessment of
ATP6V0C knockdown to its mRNA levels. Alternatively, these results may be explained in part
by the existence of different bafilomycin A1 binding sites within V-ATPase, such as ATP6V0A.
Isoforms of ATP6V0A have been shown previously to regulate V-ATPase-dependent H+
transport 43 and are sensitive in yeast to bafilomycin A1-dependent inhibition 46. Future study of
ATP6V0A could provide meaningful insight with respect to the V-ATPase subunit specificity for
bafilomycin A1 and its derivatives for regulating ALP-associated degradation.
While 1 nM bafilomycin A1 did not enhance cell death in ATP6V0C knockdown cells,
its ability to shorten neurite length suggest its cytotoxic potential that could have resulted in cell
death had measurements been taken at later time points. As bafilomycin A1 inhibits V-ATPase
completely at 10 nM 4, 38, it is understandable why treatment of knockdown cells with a ~threefold lower concentration of bafilomycin A1 (i.e. 3 nM) shown previously to partially inhibit VATPase 4 would be more effective than a ten-fold lower concentration (i.e. 1 nM) in causing
neurite shortening and inducing cell death. Timing may also explain why an enhanced
73
knockdown effect was not observed with higher concentrations of bafilomycin A1 (10-100 nM).
Had measurements of autophagy substrates and cytotoxicity following treatment with “high”
concentrations of bafilomycin A1 been made at time points preceding 48h, the possibility exists
for an unmasking of knockdown-specific effects at these concentrations.
The only knockdown-specific alteration for ATP6V0C observed in the present study
following treatment with ”high, toxic” concentrations of bafilomycin A1 was a significant
decrease in α-synuclein monomeric species. This result is difficult to interpret, especially
considering that α-synuclein high MW species did not concomitantly increase in knockdown
cells following treatment with 100 nM bafilomycin A1. This result may be explained by an
enhancement of ubiquitin proteasome system (UPS) function as a consequence to inhibition of
the ALP that would in turn enhance degradation of α-synuclein monomer 10 . In support of this
argument, cross-talk and compensation of the ALP or the UPS has been demonstrated recently
for the degradation of α-synuclein following separate inhibition of either pathway 9.
Interestingly, treatment with a cytotoxic concentration of bafilomycin A1 has been shown
recently to inhibit the formation of α-synuclein aggregates 19, which could explain why the
bafilomycin A1 concentration dependence for formation of α-synuclein high MW species in our
study was not more robust. However, caution must be used in comparing results between these
two studies, as our study assessed levels of endogenous α-synuclein levels and theirs assessed αsynuclein aggregation following over-expression 19, as it has been shown recently how relative
levels of α-synuclein can influence the functional capacity of its degradation by the ALP and the
UPS 9.
In summary, results of the present knockdown study in neuronal cells indicate a
contribution of ATP6V0C towards the basal vs. stress-induced function of the ALP and ALP-
74
associated substrate degradation. Future studies are warranted to validate the utility of
ATP6V0C as a therapeutic target to enhance substrate degradation in age-related
neurodegenerative disease.
ACKNOWLEDGEMENTS
We wish to thank Barry R. Bailey for expert administrative assistance in preparation of
this manuscript. We also wish to thank UAB Neuroscience Core facilities (NS047466 and
NS057098) for technical assistance in the optimization of experiments performed for this
manuscript.
75
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FIGURE 1
Figure 1. Functional validation of ATP6V0C knockdown. Vesicular acidification following
siRNA-mediated knockdown of ATP6V0C was assessed using Lysotracker Red® (LTR). LTRpositive punctae were imaged in differentiated SH-SY5Y cells at 96h following nucleofection of
either Non-target siRNA control (A-C) or ATP6V0C siRNA (D-F). DMSO vehicle or 100 nM
bafilomycin A1 (BafA1) was added to cells at 3h prior to the addition of LTR. Scale bar = 20
µm. Inset boxes in panels A and D are magnified in panels B and E respectively, with arrows
92
indicating LTR-positive punctae. (G) Flow cytometric quantification of LTR relative mean
fluorescence intensity (MFI) in vehicle-treated cells at 96h following nucleofection with Nontarget vs. ATP6V0C siRNA. Data are expressed as mean ± SEM and are represented by six
independent experiments. #p<0.05 vs. Non-target siRNA using one sample t-test.
FIGURE 2
Figure 2. ATP6V0C regulation of autophagy-lysosome pathway markers. Representative
western blots for lysosome marker LAMP-1 (A) or autophagosome marker LC3-II (B) from
lysates collected following nucleofection with Non-target or ATP6V0C siRNA and subsequent
93
treatment for 48h with 0-100 nM bafilomycin A1 (BafA1). Blots were stripped and re-probed
for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments
are presented graphically in panels C-F. Within group comparisons of BafA1 concentration
responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right)
were determined for LC3-II (C) or LAMP-1 (D) by expressing mean ± SEM band intensities
relative to actin loading control. All lines above columns indicate significant within-group
differences with respect to concentration (*p<0.05 using one-way ANOVA and Bonferroni’s
post-hoc test). Comparisons between groups (Non-target vs. ATP6V0C siRNA) for LC3-II (E)
and LAMP-1 (F) were determined for each concentration of BafA1 by expressing mean ± SEM
fold changes for each ATP6V0C siRNA condition relative to its companion Non-target control.
#
p<0.05 vs. corresponding Non-target siRNA control using one-sample t-test.
94
FIGURE 3
Figure 3. ATP6V0C regulates autophagic flux. Representative western blot for
autophagosome marker LC3-II (A) to assess autophagic flux by treating in the presence or
absence of the lysosome inhibitors ( “LI” in panel A and “LYSO INH” in panel B) chloroquine
(100 µM) plus leupeptin (200 µM) for the last 2-4h of a 48h time course. Blots were stripped
and re-probed for actin (42 kDa) to normalize for gel loading. Data from seven independent
95
experiments are presented graphically in panel B. Comparisons between groups (Non-target vs.
ATP6V0C siRNA) were determined by normalizing bands to actin loading control and
expressing as mean ± SEM fold change for each ATP6V0C siRNA condition (filled columns)
relative to its companion Non-target control (open columns). #p<0.05 vs. corresponding Nontarget siRNA control using one-sample t-test. (C-H) Representative confocal microscopy images
of fixed cells following nucleofection with either Non-target siRNA control (C-E) or ATP6V0C
siRNA (F-H) and subsequent treatment with DMSO vehicle for 48h. LC3 immunoreactivity
(red, C, F) and LAMP-1 (green, D, G) are shown separately and as merged (E, H). Nuclei are
stained in panels E and H with bis-benzimide (blue). Co-localization of LC3 and LAMP-1
immunoreactivity (arrows, panels E, H) suggests inhibition of autophagic flux resulting from
accumulation of dysfunctional autolysosomes. Scale bar = 50 µm.
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FIGURE 4
Figure 4. ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein.
Representative western blot for α-syn (A) from lysates following nucleofection and subsequent
treatment for 48h with 0-100 nM bafilomycin A1 (BafA1), indicating α-syn high molecular
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weight (HMW) species (>50 kDa, suggesting multimeric species) and α-syn monomer (~17
kDa). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data
from at least six independent experiments are presented graphically in panels B-E. Within
groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns,
left; ATP6V0C siRNA, filled columns, right) were determined for α-syn HMW species (C) or
monomer (D) by expressing mean ± SEM band intensities relative to actin loading control
(results of one-way ANOVA were not significant, p>0.05). Comparisons between groups (Nontarget vs. ATP6V0C siRNA) for α-syn HMW species (D) or monomer (E) were determined for
each concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C
siRNA condition relative to its companion Non-target control. #p<0.05 vs. corresponding Nontarget siRNA control using one-sample t-test.
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FIGURE 5
Figure 5. ATP6V0C regulates basal and stress-induced metabolism of APP. Representative
western blot (A) for amyloid precursor protein C-terminal fragments (APP CTFs) from
nucleofected cells following 48h treatment with 0-100 nM bafilomycin A1 (BafA1). An
antibody was used that recognized both full-length APP (~110 kDa) and APP CTFs (≤ 15 kDa).
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Sizes indicated for CTFs are predicted relative to migration of molecular weight marker and
correlate to sizes as previously published (please see results section for further information). In
addition to a short-exposure (5 min) blot, a long-exposure (2h) blot is shown to indicate APP
CTFs in vehicle-treated cells. Blots were stripped and re-probed for actin (42 kDa) to normalize
for gel loading. APP CTFs from long-exposure blots quantified from four independent
experiments are expressed graphically (B-C). Within groups comparisons of BafA1
concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled
columns, right) were determined (B) by expressing mean ± SEM band intensities relative to actin
loading control. All lines above columns indicate significant within-group differences with
respect to concentration (*p<0.05 using one-way ANOVA and Bonferroni’s post-hoc test).
Comparisons between groups (Non-target vs. ATP6V0C siRNA) were determined for each
concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C siRNA
condition relative to its companion Non-target control. #p<0.05 vs. corresponding Non-target
siRNA control using one-sample t-test.
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FIGURE 6
Figure 6. ATP6V0C knockdown exhibit enhanced markers of cytotoxicity. Representative
confocal microscopy images obtained from differentiated SH-SY5Y cells following
nucleofection with Non-target (A-D) or ATP6V0C (E-H) siRNA and subsequent treatment for
48h with 0 (A, E), 1 (B, F), 3 (C, G) or 10 (D, H) nM bafilomycin A1 (BafA1). Arrows indicate
neuritic processes. Scale bar = 50 µm. Neurite length (µm) is expressed graphically (I) and
represents results (mean ± SEM) from three independent experiments (10 cells per condition in
each experiment). Percent cell death (percentage of propidium iodide (PI)-positive cells
quantified using flow cytometry) is expressed graphically (J) as mean ± SEM with data obtained
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from a total of nine independent experiments. All lines above columns indicate significant
within-group differences with respect to concentration (*p<0.05 using one-way ANOVA and
Bonferroni’s post-hoc test). Comparisons between groups (Non-target vs. ATP6V0C siRNA)
were determined for each concentration of BafA1 using two-sample t-test (#p<0.05).
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LOW-DOSE BAFILOMYCIN A1-MEDIATED NEUROPROTECTION IS DEPENDENT ON
AUTOPHAGY AND REQUIRES THE V-ATPASE SUBUNIT ATP6V0C
by
BURTON J. MADER, CHARLES A. TAYLOR, TONIA E. TSE, LEANDRA R. MANGIERI
AND JOHN J. SHACKA
In preparation for submission
Format adapted for dissertation
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ABSTRACT
The autophagy-lysosome pathway (ALP) is an essential intracellular homeostatic
pathway that transports intracellular materials to the lysosome for degradation and cellular reuse.
Preservation of ALP function is critical to neuronal cell viability especially in periods of stress.
Inhibition of autophagic flux resulting from either inhibited autophagosome formation or
inhibited lysosome-dependent degradation, can induce neuronal dysfunction and death associated
with neurodegenerative disease. Agents, such as Bafilomycin A1 (BafA1), and other structurally
similar plecomacrolide antibiotics are widely reported to inhibit vacuolar ATPase (V-ATPase)
and have high affinity for binding to the V-ATPase subunit, ATP6V0C. BafA1, when used at
≥10 nM, has been shown to cause lysosome dysfunction with autophagosome accumulation and
cell death. Similarly, chloroquine (CQ) is a lysosomotropic agent that accumulates in lysosomes
causing an elevation in lysosome pH leading to lysosome dysfunction and cell death.
Our lab has previously demonstrated that low concentrations of BafA1 ( ≤1nM), which
do not inhibit V-ATPase, significantly attenuate CQ-induced death of cerebellar granule neurons
and differentiated SH-SY5Y neuroblastoma cells. BafA1-mediated neuroprotection was likely
regulated through a mechanism that improves autophagic flux. Furthermore, a bafilomycin
derivative significantly and dose-dependently attenuated dopaminergic neuron death in C.
elegans caused by the overexpression a neurotoxic protein dependent on ALP-mediated
degradation. Our current study tested the hypothesis that low-dose BafA1 neuroprotection is
dependent on autophagy and requires ATP6V0C. We report that ATP6V0C knockdown, in
addition to elevation of lysosome pH, inhibited the protective effects of low-dose BafA1 in our
in vitro neuronal model of CQ-induced lysosome dysfunction. We also found that low-dose
BafA1-mediated neuroprotection was dependent on autophagy, and was ameliorated in cells
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nucleofected with Atg7 siRNA, which inhibits autophagy at the point of autophagosome
formation. Finally, we observed low-dose BafA1 rescued cells from CQ-induced degradation
block, an effect that was not seen after ATP6V0C knockdown. Together, our findings suggest
that low-dose BafA1 confers neuroprotection through a mechanism that affects both upstream
and downstream ALP modulators. Low-dose BafA1 and other therapies that enhance autophagic
flux in periods of stress have great therapeutic potential in neurodegenerative pathologies that
involve ALP dysfunction and cell death.
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INTRODUCTION
The autophagy-lysosome pathway (ALP) is an essential intracellular homeostatic
pathway that mediates the delivery of intracellular macromolecules to the lysosome for
degradation. The ALP consists of many sub-types of autophagy, including microautophagy,
macroautophagy and chaperone-mediated autophagy (CMA). Macroautophagy is characterized
by double-membraned autophagosomes that form around bulk cytoplasm and organelles that are
ultimately degraded by the lysosomes 11, 29. There are conjugation pathways in macroautophagy
that form the Atg5-Atg12-Atg16 complex and convert LC3-I to LC3-II, which are activated
upstream by Atg7. These conjugation reactions form the autophagosome double membrane29.
Inhibition of Atg7 prevents these pathways from forming new autophagosome s and as a result
macroautophagy is impaired at an early stage. The accumulation of autophagosome s is a sign of
intracellular stress, which may result from either enhanced ALP induction or the inhibition of
efficient autophagosome clearance, often secondary to lysosome dysfunction18. Drugs that
disrupt lysosome function, such as chloroquine (CQ) and Bafilomycin A1 (BafA1) are widely
used as agents to study ALP dysfunction. Treatment with these agents has been shown by us and
others to promote the accumulation of autophagosome s as well as other ALP substrates12, 19.
CQ is a widely-prescribed antimalarial agent and has also been used to treat autoimmune
diseases. CQ is attracted to acidic environments as a weak base and can move freely across the
lysosome membrane28. However, upon arrival in the lysosomal lumen, CQ molecules are trapped
by the addition of a proton, which results in CQ accumulation and elevated lysosomal pH. CQinduced death has been described in many cell types including immature neurons26 and neuronal
cell lines in combination with robust accumulation of autophagosomes and ALP substrates19.
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BafA1 is a macrolide antibiotic that was characterized initially for its selective inhibition
of vacuolar-type ATPase (V-ATPase), a two domain complex that is involvled in sensing and
altering lysosome pH6. The peripheral V1 domain hydrolyses ATP to drive the rotary action of
the pore-like V0 domain, allowing for proton translocation across the membrane. BafA1 and
similarly structured “bafilomycins” are widely used to inhibit the V-ATPase through their
interaction with ATP6V0C causing lysosome dysfunction and has been shown to cause neuronal
cell death1. Nanomolar concentrations of BafA1 are known to disrupt the vesicular proton
gradient and increase the pH of acidic vesicles30. This disruption of vesicular acidification by
both BafA1 and CQ has been proposed to prevent the fusion of autophagosomes. High
concentrations of BafA1 are known to inhibit V-ATPase completely2, induce vacuolar
deacidification3, 4, 30, and promote apoptosis10, 17.
Our lab has previously demonstrated that low concentrations of BafA1 ( ≤1nM), which
do not inhibit V-ATPase, significantly attenuate CQ-induced death of cerebellar granule neurons
and differentiated SH-SY5Y neuroblastoma cells25, 27. BafA1-mediated protection is optimal a
low concentrations (≤1 nM) which do not inhibit V-ATPase2 or induce AV accumulation27.
These data suggest that low-dose BafA1-mediated neuroprotection is independent of its
inhibition of V-ATPase. This protection attenuates the accumulation of putatively toxic proteins
and associated cell death linked with neurodegenerative disease20. However, the mechanisms by
which LDB protects the cell from CQ-induced lysosome dysfunction is still unclear.
The role of ATP6V0C in low-dose BafA1-mediated protection has not yet been fully
characterized. In one of our recent studies, ATP6V0C knockdown sensitized neuronal cells to
BafA1 treatment thereby lowering the concentration of BafA1 necessary to promote toxicity.
ATP6V0C knockdown also caused an increase in toxic protein species associated with
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neurodegenerative disease. Taken together it is apparent that ATP6V0C provides a prosurvival
function in the maintenance of cellular pH and its inhibition is associated with decreased survival
in the presence of additional stress.
While these studies provide strong evidence for BafA1-mediated cytooprotection, the
mechanism by which low-dose BafA1 modulates ALP function and maintains cell viability has
not been investigated. To determine if low-dose BafA1 -mediated protection is dependent on
ATP6V0C and/or ALP function we genetically manipulated a differentiated neuronal cell line
and measured effects on viability and the ALP. We report that low-dose BafA1 confers
protection that is dependent on functional autophagy and preservation of lysosome function.
MATERIALS AND METHODS
Cell Culture
Naïve SH-SY5Y human neuroblastoma cells (ATCC, CRL-2266) were maintained in T75 flasks (Corning, 430641) at 37˚C and 5% CO2 in Minimum Essential Media (Cellgro, 10-010CV) and F12-K media (ATCC, 30-2004) supplemented with 0.5% sodium pyruvate (Cellgro, 25000-CI), 0.5% non-essential amino acids (Cellgro, 25-025-CI), 1% penicillin/streptomycin
(Invitrogen, 15140-122), and 10% heat-inactivated fetal bovine serum (FBS; Thermo Scientific,
SH30109.03). Naïve cells received full media change every two days. Prior to their use in
experiments, naïve SH-SY5Y cells were differentiated for seven days to a post-mitotic state in
10% FBS media supplemented with 10 μM retinoic acid (RA; Sigma, R2625). Complete RAsupplemented media was replenished every two days.
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Nucleofection with siRNA
Small-interfering RNA (siRNA) specific for human ATP6V0C was obtained from
Thermo Scientific. The Amaxa Nucleofector™ system (Lonza, VVCA-1003), a modified
electroporation system, was utilized to transiently knock down ATP6V0C. First, RAdifferentiated SH-SY5Y cells were re-suspended in the Lonza™ Nucleofector reagent. Next,
400nM of re-suspended ATP6V0C siRNA (Thermo Scientific, M-017620-02-0010), or nontarget siRNA (Thermo Scientific, D-001206-13-05), (in siRNA buffer with 20 mM KCl, 6 mM
HEPES-pH 7.5, and 0.2 mM MgCl2) was added to the cell suspension. After electroporation,
500 μL of FBS-containing media was immediately added to the cell suspension. Following a 10
min recovery period, nucleofected cells were transferred to T-75 flasks and incubated overnight
in 10% FBS differentiation media for 24h. The next day nucleofected cells were plated for
experiments (24h after nucleofection and 24h prior to drug treatment) in either eight well glass
chamber slides (LabTek, 154941), six well plates (Corning 3516), 48 well plates (Corning ) or
60 mm dishes (Corning, 430166) at a density of 500/mm2 in 0% FBS differentiation media
containing 2% B-27 supplement (Invitrogen, 17504044). All experimental endpoints ended at
either 72h or 96h after nucleofection.
Quantitative Real Time PCR
Cells nucleofected with ATP6V0C siRNA or non-target siRNA were processed for total
RNA extraction with the use of TRIzol reagent (Invitrogen, 15596-026). cDNA was generated
with oligo (dT) from four µg of RNA using the SuperScript III First Strand Synthesis System
(Invitrogen #18080-051). Primers used for detection of ATP6V0C mRNA were ATP6VOC 5’ATGTCCGAGTCCAAGAGCGGC-3’ and ATP6VOC 5’-CTACTTTGTGGAGAGGATGAG-
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3’. Primers for actin were 5’-GCTCGTCGTCGACAACGGCTC-3’ and 5’CAAACATGATCTGGGTCATCTTCTC-3’. Template for cDNA (2 µL) was added along with
1X Fast SYBR Green Master Mix (ABI #4385610) and water to a final volume of 20μL/well.
Assays were performed in triplicate on a Bio-Rad CFX96 instrument using the following
conditions: (95°C for three minutes followed by 95°C for 15 seconds and 60°C for one minute
for 45 cycles. The comparative cycle threshold (Ct) method (ddCT; 5) was used to determine
expression of ATP6V0C in each sample relative to endogenous actin control, which was then
used to determine knockdown in ATP6V0C siRNA samples as a percentage of non-target siRNA
control.
Measurement of Acidic Vesicle pH
After treatment for 48h with DMSO vehicle, cells plated in 60 mm dishes were incubated
with LysoTracker Red® (LTR; Life Technologies™, L7528; 100 nM final concentration) for 30
min in Locke’s buffer (15mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 1.3mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, 1.009 g/L glucose, pH 7.4). Cells were then harvested, pelleted, and resuspended in 1 X PBS then passed through a 70 μm nylon cell strainer (BD Falcon, 08-771-2)
and into collection tubes to provide a single cell suspension. Cells were kept on ice and protected
from light during immediate transport to the Joint UAB Flow Cytometry Facility (Enid Keyser,
Director) for analysis. A total of 1 × 104 events were detected in each experimental condition
using the BD LSR II flow cytometer (Becton Dickinson). Further analysis was performed using
FlowJo software (licensed by UAB) to assess mean fluorescence intensity. LTR fluorescence
was also visualized via microscopy in cells plated in eight well glass chamber slides and treated
for either 48h with DMSO vehicle or with 100 nM bafilomycin A1 for the last 3h of the 48h time
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course. Following treatment with LTR, fluorescent images were captured using a Zeiss Axioskop
fluorescent microscope (Carl Zeiss Micro-imaging, LLC) at 40X magnification. Transmitted
light images were also taken using phase contrast optics.
Treatment with Bafilomycin A1 and Chloroquine
Following nucleofection, recovery and plating for experiments (i.e. 48h after
nucleofection), media was exchanged for fresh 0% FBS differentiation media containing 2% B27 supplement in and treated as follows: 0 nM (control), 1, 10, 100 nM BafA1 (AG Scientific, B1183), 50 μM CQ (Sigma) in the presence or absence of 1 nM BafA1. CQ was prepared at 5
mM stock solutions in 0% FBS media and stored at -20°C. BafA1 was prepared at 10 mM stock
solutions in DMSO and stored at -20°C. All cells were maintained at 37°C and 5% CO2
throughout this protocol. Bafilomycin A1 was prepared as a 10 mM stock solution in DMSO
(Sigma, D8418-1L) and stored at -20˚C. Unless otherwise noted, all bafilomycin A1 treatments
were for 48h, with experiments ending 96h after initial nucleofection.
Western Blot Analysis
After treatment for 48h, nucleofected cells in 60 mm dishes were incubated for seven
minutes at 37˚C with Accutase (Innovative Cell Tech., AT104) to detach cells from their
substrate. Whole cell lysates were collected and protein concentrations were determined using
BCA protein assay (Fisher Scientific, PI-23227) as previously described 26. Equal amounts of
each protein sample were then electrophoresed using 12% Tris/Glycine SDS-polyacrylamide
gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, 162-0177).
Following a 30 minute incubation at room temperature with 5% blocking milk (Bio-Rad, 170-
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6404) in 1X tris-buffered saline with tween (TBST), membranes were incubated overnight with
primary antibodies for immunodetection of the Atg7 (rabbit anti-Atg7, Cell Signaling).
Membranes were washed with 1X TBST containing 0.1% Tween 20 and then incubated with
secondary IgG-HRP conjugated antibody for 1h at room temperature. After washing blots,
Enhanced chemilumescence (ECL; Thermo Scientific, PI-32106) was used to detect Atg7. Actin
(Sigma, A1978) was used to normalize for gel loading. Films with detected bands of interest
were scanned using Adobe® Photoshop® and band intensities were calculated using UN-SCANIT gel digitizing software (Silk Scientific, Inc.).
Measurement of Cell Viability
Viability of SH-SY5Y cells was determined after 48h treatment using MTT (3-(4, 5Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric reduction assay. MTT
stock solution was prepared using sterile PBS (2mg/mL). 50 μL of MTT stock solution was then
added to each well containing 200 uL cells plus media (final volume = 250 μL or .4 mg/mL) and
incubated for 2h at 37°C and 5% CO2. Note: MTT stock solution was added to a blank well
containing 200 μL media for subtracting background values. Following the initial incubation,
MTT plus media solution was carefully aspirated from each well, including the blank well noted
above, and replaced with 200 μL of DMSO and incubated for 30 minutes at 37°C. Individual
treatment groups will be quantified at a wavelength of 570 nm using a spectrophotometer.
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Assessment of Autophagosome-Lysosome Fusion
Following nucleofection with ATP6V0C siRNA or non-target control siRNA, cells plated
in eight well glass chamber slides were treated for 48h with DMSO and subsequently fixed for
15 min at 4˚C using Bouin’s fixative (75% saturated picric acid, 23.8% of a 37% w/v
formaldehyde solution, 4.7% glacial acetic acid) in the presence of 0.5% saponin (Sigma,
47036). Following incubation for 30 min with 1X PBS blocking buffer containing 1% bovine
serum albumin, 0.2% non-fat dry milk and 0.3%Triton X-100, cells were incubated overnight at
4˚C in blocking buffer without Triton X-100 and containing rabbit anti-LC3 antibody (Sigma,
L7543) to detect autophagosome punctae. Following PBS wash, fixed cells were incubated for
1h with SuperPicTureTM (Invitrogen, 879263) anti-rabbit IgG secondary antibody. Following
PBS wash, fixed cells were fixed cells were subjected to tyramide signal amplification
(incubation for 30 min with Cy3 plus tyramide, Perkin EImer, FP1170) to detect LC3. Next,
fixed cells were incubated for 10 min with 3% hydrogen peroxide to neutralize residual
peroxidase from the first secondary antibody. After PBS wash and 30 min incubation with
blocking buffer, fixed cells were incubated overnight at 4˚C in blocking buffer containing the
second primary antibody, mouse anti-human LAMP-1 (University of Iowa Hybridoma Bank,
H4A3) to detect lysosomes. Following subsequent PBS wash, fixed cells were incubated for 1h
with Vector Impress (Vector Labs, MP-7402) anti-mouse IgG. Following PBS wash, tyramide
signal amplification was performed (incubation for 30 min with FITC plus tyramide, Perkin
Elmer, FP1168) to label LAMP-1. Following PBS wash, fixed cells were last incubated with
bis-benzimide (0.2 µg/ml in PBS, Sigma) to label nuclei. Images were captured using a Zeiss
Observer.Z1 laser scanning microscope equipped with a Zeiss 40X Plan-Apochromat objective
and imaged using Zen 2008 LSM 710, V5.0 SP1.1 software.
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Statistical Analysis and Figure Preparation
Quantitative real time PCR data were expressed as mean ± SEM percent knockdown for
ATP6V0C. LTR staining was presented as mean ± SEM fluorescence intensity. For assays of
viability (MTT reduction), values within a single experiment will be generated by averaging
replicates from at least three different wells and will be expressed as percent nontransfected/non-target/vector control for any given treatment.
Western blot band intensities Atg7 was normalized to actin loading control, and was
expressed (mean ± SEM) either as such to assess the effects of Atg7 siRNA. Neurite length was
presented as mean ± SEM average length (µm) per cell. To determine effects of genetic
manipulation, between groups comparisons of Non-target vs. ATP6V0C siRNA were evaluated
for significance using either one-sample t-test (LTR data; western blot data normalized as fold
change relative to the companion non-target control) or two-sample t-test (assessment of neurite
length and percent cell death. For all tests, statistical significance was set a priori at p < 0.05.
Graph Pad Prism® was used to perform statistical analysis and generate graphs, and Adobe®
Photoshop® was used to assemble figures. A minimum of three independent replicates were
performed for each experimental endpoint.
RESULTS
Validation of ATP6V0C knockdown
We measured siRNA mediated knockdown of ATP6V0C using quantitative real time
PCR analysis on cDNA generated from RNA in samples isolated from differentiated SH-SY5Y
human neuroblastoma cells harvested 96h following nucleofection with non-target or ATP6V0C
siRNA. Using the comparative cycle threshold method (Ct)5 with actin mRNA acting as an
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internal standard. We found that ATP6V0C message was reduced significantly with only 5.6%
remaining when compared to non-target controls (Fig. 1A). Next we tested lysosome acidity by
LTR as a measure of functional alterations after ATP6V0C knockdown. LTR is attracted to
acidic cellular compartments and will accumulate in a pH dependent manner that is observed as
an intense red fluorescence in acidic environments that will decrease as pH increase. We found
that ATP6V0C knockdown markedly attenuated LTR fluorescence, suggesting ATP6V0C
regulates lysosome pH (Fig. 1B).
Low-Dose BafA1-mediated neuroprotection requires ATP6V0C
Cell viability was measured by MTT assay 96h post-nucleofection and 48h after
treatment with BafA1 (1-100nM), CQ (50 µM), or both. ATP6V0C knockdown causes
sensitivity to BafA1 (1-10nM) that results in a significant reduction in cell viability (Fig 2,
p*≤0.05). There are no ATP6V0C-depedent effects on cell viability at either the highest
concentration of BafA1 (100nM) or with CQ treatment (50µM). However, when non-target
control cells were treated with both CQ and low-dose BafA1 (1nM) we observed the
preservation of cell viability when compared to CQ treatment alone (Fig 2, p**≤0.05). This
protective effect was attenuated and cell viability was not significantly different from CQ
treatment in cells nucleofected with ATP6V0C siRNA (Fig 2, n.s.).
Low-Dose Bafa1-Mediated Neuroprotection Is Dependent On Autophagy
First we confirmed siRNA-mediated knockdown of Atg7 at 96h post-nucleofection. Atg7
protein levels were reduced compared to their non-target scrambled controls in both nutrient rich
and depleted conditions (Fig. 3A). We quantified this knockdown caused by Atg7 siRNA to be
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37.4% of the amount found in non-target control cells in complete media (Fig. 3B). Knockdown
of Atg7 almost completely ameliorated the detection of LC3-II conversion, an indicator of
autophagosome levels, in both complete media and starvation media (Fig. 3A). Finally we tested
the effects of Atg7 knockdown on low-dose BafA1-mediated neuroprotection against CQinduced lysosome dysfunction and found that protection was lost cells with knockdown when
compared to their non-target control (Fig. 3C). This suggests that the ALP must function
uninhibited to maintain low-dose BafA1-mediated neuroprotection.
Autophagic Flux is Enhanced by Low-Dose Bafa1 in a ATP6V0C-Dependent Manner
To determine the effects of low-dose BafA1 treatment on autophagic flux we used
immunocytochemistry techniques to label the autophagosome marker LC3 (cy3 red) and the
lysosome membrane marker LAMP-1 (FITC green) and observed their interaction in cells
nucleofected with either non-target siRNA or ATP6V0C siRNA. We found that there no
noticeable co-localization (seen as yellow) in untreated cells (Fig. 4A, 4B). However, in
ATP6V0C knockdown cells we see more co-localization of LC3 and LAMP-1 signal (Fig. 4D)
when compared to their non-target control (Fig. 4C), suggesting decreased lysosome
degradation. CQ induced robust co-localization of LC3 and LAMP-1 indicating strong inhibition
of autophagic flux, but no ATP6V0C-dependent differences were detected (Fig. 4E, 4F). In nontarget control cells we observed an attenuation of CQ-induced co-localization of LC3 and
LAMP-1 (Fig. 4G) indicating improved autophagic flux, an effect that is not maintained after
knockdown of ATP6V0C (Fig. 4H).
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DISCUSSION
The goal of this study was to better elucidate the mechanism by which low-dose BafA1
confers neuroprotection against CQ-induced lysosome dysfunction. We have provided the first
evidence that low-dose BafA1 requires ATP6V0C for its reported ability to attuenuate CQinduced neuronal cell death (Fig. 2). Furthermore, we found that inhibition of the ALP by
siRNA-mediated knock down of Atg7 attenuates neuroprotection against lysosome dysfunction
affored by low-dose BafA1 (Fig. 3). We found that inhibition of ATP6V0C increased ALPrelated sensitivity to BafA1 even at low concentrations and was demonstrated by increased
LAMP-1 and LC3 co-localization (Fig. 4), which suggests an inhibition of autophagic flux that
likely negated low-dose BafA1’s pro-survival stimuili in the presence of CQ.
Our siRNA-mediated knockdown significantly reduced ATP6V0C mRNA levels (Fig
1a). Thus, to date our lab has not been able validate the utility of an antibody that can identify
ATP6V0C by western blot. As such it is not clear how much residual ATP6V0C is present
during the course of our experiments. However, we have previously demonstrated consistent
microscopic changes associated with ATP6V0C knockdown including a decrease in neurite
length and a decrease in lysosome acidification (Fig 1b), both of which likely contribute to the
deleterious consequences of knocking down ATP6V0C. Lysosome acidification is also inhibited,
although not to the degree that is seen with 100nM BafA1 treatment for 3-4h (data not shown),
thus suggesting that partial acidification remains.
Low-dose BafA1-mediated protection against lysosome dysfunction was attenuated by
nucleofecting cells with siRNA targeting ATP6V0C. ATP6V0C knockdown causes sensitivity to
BafA1 after knockdown of ATP6V0C that closely resembles the increased toxic effects of anticancer drugs on cancer cells previously unresponsive to treatment (REF). In agreement with our
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previous data (pg. 89) we report that knockdown of ATP6V0C reduces cell viability following
treatment with 1nM BafA1 when compared to non-target control cells (Fig. 2). The V0
proteolipid ring is made of different subunits that closely resemble each other in structure and
function, and are known to exhibit conformational changes that can affect lysosome acidification
and V-ATPase assembly21. There is evidence in the literature that BafA1 may potentially bind
other sites32 thus it is possible that knockdown of ATP6V0C causes conformational changes in
the V0 protelipid ring that could expose other potential binding sites.
When we tested the effects of ATP6V0C knockdown on low-dose BafA1-mediated
protection against CQ-induced toxicity we found, as expected, loss of protection. The potential
mechanism by which ATP6V0C mediates low-dose BafA1 protection against CQ-induced
toxicity remains unclear. However, in combination with our previously data, which indicates that
low-dose BafA1 attenuates markers of ALP dysfunction19 (AV accumulation; inhibition of
autophagic flux; increase in α-synuclein) it would be logical to predict that low-dose
BafA1confers protection through its enhancement of ALP function. While loss of protection may
be a caused, in part, by lysosome dysfunction, the possibility that BafA1 could be exerting its
pro-survival effects by some alternate mechanism cannot be ruled out. There is evidence that
BafA1 and ATP6V0C mediate transcripition through interaction with hypoxia inducible factor
1(HIF-1α) 16, a transcription factor that is known to participate in cell survival functions. Other
studies supporting a cytoprotective role for ATP6V0C include the report that its expression is
enhanced in cancer cells that are resistant to chemotherapeutic-induced death, and that its RNAimediated knockdown in cancer cells exacerbates stimulus-induced cell death9, 31. Furthermore, in
the context of neurodegenerative disease ATP6V0C over-expression in vivo was able to regulate
neurotransmission and improve results on behavioral tests8. The direct contribution of ATP6V0C
118
to cellular viability is unclear however these studies demonstrate that it does provide potentially
numerous avenues for further exploration. And so, we are also interested in the potential for
over-expression of ATP6V0C as a method of inducing low-dose BafA1-like effects on ALP
function that also increase cell viability, without the potential for toxicity that is associated with
BafA1.
To determine if low-dose BafA1-mediated protection requires the ALP we conducted
siRNA-mediated knockdown of Atg7 to prevent autophagosome formation. We specifically
focused on Atg7, a known mediator of both conjugation pathways necessary for autophagosome
formation, to determine if basal ALP activity is critical to low-dose BafA1protection against
lysosome dysfunction. The downstream effects of Atg7 inhibition were seen functionally in
reduced levels of LC3-II conversion (Fig. 3), which indicates decreased autophagosome
formation. Our previously published data indicate that ALP enhancement after low-dose BafA1
treatment effectively blocks the deleterious effects of CQ, however in this present study we
found that low-dose BafA1 requires the uninhibited formation of autophagosome s to confer its
protective effects (Fig. 3). After independent 48h treatments with BafA1 and CQ there was no
evidence of Atg7-dependent alterations in neuronal toxicity, however after 48h dual treatment
any protection afforded by low-dose BafA1 was lost in cells that had undergone Atg7
knockdown. This result suggests the ALP is necessary, specifically in the formation of
autophagosomes, for the protection against lysosome dysfunction that is conferred by low-dose
BafA1. This result provides proof of principle that low-dose BafA1requires the ALP to reduce
cellular toxicity in periods of stress, specifically.
Additionally we used immunocytochemical detection methods and confocal microscopy
to determine if low-dose BafA1exhibited ATP6V0C-dependent alterations in autophagic flux
119
(Fig. 4). Co-localization of LC3 and LAMP-1 was decreased in non-target cells following
treatment with low-dose BafA1 and CQ when compared to the ATP6V0C knockdown cells,
suggesting inhibition of autophagic flux. We demonstrate no ATP6V0C-dependent interaction of
LC3 and LAMP1 under basal conditions. However, after ATP6V0C knockdown and treatment
with1nM BafA1 there was qualitatively more colocalization of LC3 and LAMP-1, an effect that
is interpreted as a block in autophagic flux. Combined treatment with low-dose BafA1and CQ
reduced LAMP-1 detection in the non-target controls compared to ATP6V0C knockdown cells
suggesting ATP6V0C maintains autophagic flux in a way that confers improved viability.
Increased LAMP-1 and LC3 colocalization is seen in neuroblastoma cells and has also been
correlated with lysosome dysfunction prior to cell death 7. Agents such as rapamycin (mTORdependent) and trehalose (mTOR-independent) are known to induce autophagy have been used
effectively to restore ALP function leading to improved cell viability in models of
neurodegeneration24.
Additionally, it would be of particular utility to further determine the effects of low-dose
BafA1 on upstream autophagy signaling regulators such as mTOR and its downstream targets,
which may participate in neuronal survival. mTOR and V-ATPase have been recently linked to
interact as modulators of autophagic signalling and rely on transcription factor EB (TFEB) to
control a network of genes which regulate autophagy and lysosome biogenesis. TFEB is
inhibited by mTOR, but under conditions of lysosome dysfunction TFEB is translocated to the
nucleus22 through inhibition of mTOR, an effect that can be induced by rapamycin. If the lowdose BafA1 effect is shown to be transcriptionally regulated it would open the door to therapies
that have similar effects on the upstream signaling pathways that promote autophagic flux but
without the potential for toxicity through use of BafA1.
120
Neurite length, as an indicator of neuronal structural stability, after ATP6V0C
knockdown has been shown to be reduced under basal conditions and BafA1 treatment (1nM,
3nM) when compared to appropriate non-target controls (pg. 89). Neurites are particularly
dependent on efficient ALP-mediated degradation as they rely on retrograde transport of
autophagic cargo to maintain their synaptic integrity13-15, 23. Accumulation of autophagosomes
within neurites is reported to contribute to synaptic dysfunction and degradation. Because we
observed shortened neurites after ATP6V0C knockdown, we predict that low-dose BafA1 would
not likely preserve neuritic structure without basal levels of ATP6V0C. We are presently
conducting experiments that focus on ALP dysfunction and its promotion of neurite retraction or
shortening, and whether or not low-dose BafA1may preserve neurite length in an ATP6V0Cdependent way as a further indication of its mechanism of protection.
Our data is consistent with the belief that low-dose BafA1 -mediated protection is
directly linked to maintenance and/or enhancement of ALP function in periods of lysosome
dysfunction. Through the elucidation and characterization of the actual mechanism of low-dose
BafA1-mediated protection, our lab and others will be able to develop strategies to replicate this
protection through methods not normally associated with cellular toxicity.
121
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FIGURE 1
Figure 1. Validation of ATP6V0C knockdown. Quantitative real-time PCR was used to assess
ATP6V0C mRNA levels (A). Vesicular acidification following siRNA-mediated knockdown of
ATP6V0C was assessed using Lysotracker Red® (LTR). LTR-positive punctae were imaged in
differentiated SH-SY5Y cells at 96h following nucleofection of either Non-target siRNA control
or ATP6V0C (B). Scale bar = 20 µm. #p<0.05 vs. Non-target siRNA using one sample t-test.
127
FIGURE 2
Figure 2. ATP6V0C knockdown causes BafA1 sensitivity and inhibits low-dose BafA1mediated neuroprotection. Percent cell viability (MTT assay) is expressed graphically (Fig. 2)
as mean ± SEM with data obtained from at least 9 independent experiments in cells 96h postnucleofection and 48h after treatment. The line above columns indicate significant within-group
differences after low-dose BafA1-mediated neuroprotection (**p<0.05 using one-way ANOVA
and Bonferroni’s post-hoc test). Comparisons between groups (Non-target vs. ATP6V0C
siRNA) were determined for each concentration of BafA1 using two-sample t-test (#p<0.05).
128
FIGURE 3
Figure 3. ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein.
Representative western blot for Atg7 (A) from lysates following nucleofection and subsequent
incubation in complete media throughout 48h prior to collection compared with cells incubated
in starvation media (PBS) for the final 2h (Fig. 3A). Both nutrient rich and starvation conditions
after knockdown displayed reduced levels of LC3-II when compared to non-target controls (Fig.
3A) Blots were stripped and re-probed for actin to normalize for gel loading. Data from 4
129
independent experiments in complete media after Atg7 knockdown demonstrate significant
reduction of Atg7 protein levels (Fig. 3B). Comparison of low-dose BafA1 and CQ treatments
alone after knockdown indicated no Atg7 dependent on neuronal viability. However, after
knockdown of Atg7, cells treated with CQ and low-dose BafA1 in combination were
significantly less viable when compared to their non-target controls (Fig. 3C, *p<0.05 vs.
corresponding Non-target siRNA control using 2-sample t-test).
FIGURE 4
Figure 4. Low-dose BafA1 requires ATP6V0C to enhance autophagic flux. Representative
confocal microscopy images of fixed cells following nucleofection with either Non-target siRNA
control (A, C, E, G) or ATP6V0C siRNA (B, D, F, H) and subsequent treatment with DMSO
vehicle for 48h. LC3 immunoreactivity (red) and LAMP-1 (green) are as merged in all panels as
an indicator of autophagic flux. Nuclei are stained with bis-benzimide (blue). There does not
appear to be a block in autophagic flux in either set of untreated cells (A, B). Colocalized LC3
130
and LAMP-1 immunoreactivity (panels D, E, F, H) suggests inhibition of autophagic flux
resulting from accumulation of dysfunctional autolysosomes. Decreased co-localization of LC3
and LAMP-1 indicate low-dose BafA1 restores autophagic flux non-target cells (G). Scale bar =
20 µm
131
DISCUSSION
The aims of our studies were to test the global hypothesis that alterations in the
ALP contribute to neuronal dysfunction and death in models of neurodegenerative
disease. First we tested the effects of rotenone, a drug known to cause PD-like
pathologies, on ALP function to elucidate the mechanism that leads to α-synuclein
accumulation. Next, we genetically inhibited ATP6V0C, a V-ATPase membrane
associated subunit, by siRNA-mediated knockdown to better its role in the maintenance
of ALP function under basal and stressed conditions. Finally we examined the role of
ATP6V0C in the protection seen previously with low-dose BafA1 on chloroquineinduced lysosome dysfunction. Our in vitro models utilized pharmacological stressors
and genetic manipulation to elucidate the involvement of the ALP in PD-associated
pathologies. In developing a better understanding of the ALP’s role in neuronal
dysfunction and degeneration we are more able to identify potential therapeutic targets to
combat neurodegenerative disease.
Maintenance of acidic lysosomal pH is a necessary homeostatic cellular function
that provides an optimal environment for the activity of acidic hydrolases responsible for
ALP-mediated degradation. Previous studies provide conflicting data on whether
regulation of vesicular pH is dependent on ATP6V0C. Knockdown of ATP6V0C has
been previously reported in one study to increase acidic vesicle pH 100, while in another it
did alter vesicular pH 13. In our study we observed an elevation of acidic vesicle pH after
knockdown of ATP6V0C that did not alter neuronal viability but did sensitize the cells to
further pharmacological treatment with BafA1. siRNA-mediated inhibition of ATP6V0C
132
did not have any associated toxicity and it is probable that basal ATP6V0C remained due
to the transient nature of our genetic inhibition. However, while we have been unable to
detect ATP6V0C at the protein level, our results indicating a functional consequence of
ATP6V0C suggest knockdown at the protein level. It will be important in future studies
to quantify protein levels following knockdown to more accurately assess degree of
knockdown. Although cell death was not increased after knockdown alone, toxicity was
dramatically increased when combined with BafA1 treatment (1nM-3nM). This result
was due likely to genetic inhibition of ATP6V0C causing increased sensitivity of VATPase to inhibition by BafA1. The ATP6V0C binding pocket for BafA1 was found
through site-directed mutagenesis, which prevented inhibition of the V-ATPase complex
8
. However, it appears that knockdown of ATP6V0C, leading to its absence from the V-
ATPase complex, may be promoting its sensitivity to BafA1. The V-ATPase is an
essential regulator of lysosome acidification and numerous reports of its inhibition link
altered V-ATPase function with cellular dysfunction and decreased viability 3, 44, 60.
Alternatively, these results may be explained in part by the existence of different BafA1
binding sites, such as ATP6V0A. Isoforms of ATP6V0A have been shown previously to
regulate V-ATPase-dependent H+ transport and are sensitive in yeast to bafilomycindependent inhibition 86. In addition, other ion transporters at the lysosomal membrane
could be modulated to compensate for the inhibition of V-ATPase limiting its deleterious
effects, an area of research worthy of further exploration.
It is reported that rotenone inhibits complex I of the mitochondrial electron
transport chain inducing toxic levels of oxidative stress and increased ALP induction50, 52.
Rotenone is also implicated in causing lysosome dysfunction, but the mechanism by
133
which rotenone causes lysosome dysfunction is not entirely clear. Our results suggest that
rotenone induces lysosome dysfunction by causing an elevation in pH that occurs through
cellular depletion of ATP. Thus, we propose that in periods of low ATP availability the
V-ATPase machinery cannot properly acidify the lysosome leading to the accumulation
of ALP substrates. To our knowledge this is the first report of rotenone’s effects on
lysosome pH in association with depleted ATP levels, suggesting a potential mechanism
for inhibiting the proton-motive function of V-ATPase at the lysosomal membrane.
The V-ATPase provides a common intersection where multiple cellular pathways
cross, and can be targeted in numerous ways for therapeutic benefit. A recent study
concluded that the V-ATPase membrane-integrated V0 domain, the pore-like structure
containing ATP6V0C that transports protons across various membranes in regulation of
vesicular and cellular pH, also acts as a sensor of vesicular pH that controls exocytosis
and synaptic neurotransmission via the reversible dissociation of the V1 domain 63. We
would be particularly interested in the future to determine if BafA1 interaction with the
V0 domain could potentially be used as a method to rid the cell of toxic undegraded
materials through exocytosis. Furthermore, the V0 domain has also been linked to
SNARE-dependent exocytosis 23 where a conformational change in V0 proteolipids are
reported to stimulate fusion by creating a hydrophobic crevice that promotes lipid
reorientation and formation. The idea of BafA1 regulating V-ATPase assembly and
disassembly as a method of inducing vesicle exocytosis was recently bolstered by
evidence that BafA1 limited recruitment of the V1 domain, in turn keeping it
disassociated from the V0 domain95. This could be further explored as a method for the
cell to remove vesicular waste that cannot be efficiently degraded intracellularly.
134
The accumulation of autophagosomes in neuronal cells that is associated with a
wide range of neurodegenerative diseases is caused by either elevated autophagic
induction or inhibition of lysosome-dependent degradation. Biochemical detection of
LC3-II by western blot analysis is considered the gold standard for assessment of
autophagosome levels41. Our first evidence of alterations in the ALP was observed as
elevated autophagosome accumulation in lysates from differentiated neuronal cells
stimulated by either rotenone exposure or siRNA-mediated knockdown of ATP6V0C.
Since there were relatively moderate increases autophagosome detection after both
stressors, it was logical to predict that there was either a slight elevation in autophagic
induction or inhibition of degradation. Our autophagic flux assay did not demonstrate
evidence of increased induction in knockdown cells nor those treated with rotenone.
However it did suggest that their existed a partial degradation block after both stressors.
Rotenone treatment increased biochemical detection of the ALP substrate p62, an
autophagic cargo linker protein that is commonly used to measure ALP-dependent
degradation, that when detected at elevated levels indicates decreased lysosome
degradation. In a previous study rotenone was shown to induce the accumulation of
p6231, an autophagic cargo linker protein that is commonly used to measure of ALPdependent degradation, while in another it caused a reduction of p62 concomitant with
increased detection of autophagosomes 94. Although p62 levels were increased in our
rotenone model of PD, indicating inhibition of autophagy, it would be interesting to
further characterize p62 localization within the cell and its association with other ALP
substrates. Colocalization of α-synuclein and p62 has been previously reported in a
similar model, suggesting their lysosome-mediated degradation; however their
135
persistence would likely be due to defective ALP87. If these proteins were found to be
colocalized in our hands it would be of particular utility to determine what proportion of
aggregates were also colocalized with LC3, which would indicate autophagosomemediated sequestration but inefficient degradation41. Using our rotenone model we would
predict that α-synuclein would likely colocalize with both p62 and LC3 after rotenone
treatment due to inefficient degradation resulting from elevated lysosome pH.
Pharmacological methods to enhance autophagy have been used experimentally to
prevent rotenone-induced death and may be able to provide protection in periods of
lysosome dysfunction21, 31, 66, 90.
Neuronal function is dependent upon neurites that project out from their cell body
and form synapses with other cells. Neurite length is dependent on multiple stimuli and
reduced neurite length is reported due in part to dysfunctional autophagy62. Furthermore,
neurites that do not form synapses or retract from synapses are evidence of neuronal
dysfunction and have been shown to accumulate autophagosomes in models of
neurodegeneration 17. As previously published studies have not investigated the role of
V-ATPase, or its subunit ATP6V0C, in the regulation of neurite length we decided to
investigate neurite length after knockdown of ATP6V0C. Interestingly we detected a
reduction in neurite length that occurs after knockdown of ATP6V0C alone, and
sensitizes cells to further BafA1-mediated reduction. The loss of neurite length in our
studies is believed to be caused by inhibition of lysosome degradation, accompanied by
the accumulation of autophagosomes. Prevention of autophagosome accumulation is
reported to preserve neuritic structure 62. The decrease in neurite length observed in our
hands is in agreement with a recent study of lysosome dysfunction that reported altered
136
neurite morphology concomitant with inhibition of autophagy and the accumulation of
proteins with neurotoxic potential48. Therapies developed to enhance lysosome function
and improved the clearance of neurotoxic proteins could be used to effectively preserve
the neuritic architecture that is necessary for neuronal homeostasis.
Autophagic machinery and lysosomal biogenesis are coordinated through a gene
network that is sensitive to lysosome inhibition 19. This gene network is regulated by the
transcription factor EB (TFEB), which is phosphorylated (inactivated) by and colocalizes with mTOR on the lysosomal membrane during periods of suppressed
autophagy, but is activated and subsequently translocated to the nucleus stimulating
transcriptional activation after lysosome disruption or induction of autophagy69.
Following rotenone treatment we detected increased lysosomal volume or number by
elevation of LAMP-1, a lysosomal membrane protein that is best known for its regulation
of lysosomal motility and endosomal-lysosomal fusion with autophagic vacuoles36. This
increase in LAMP-1 suggests an increase in either the number or size of acidic vesicles
that may be a consequence of lysosome dysfunction 96. Conversely, LAMP-1 may also be
altered as a function of de novo lysosome biogenesis, as shown recently under stressful
conditions and as a response to alterations in autophagy 19. To address this possibility the
effects of rotenone on endogenous levels of TFEB were assessed as it regulates
transcription of LAMP-1 in addition to other lysosomal targets. Levels of TFEB
following rotenone treatment were unchanged, suggesting that the increase in LAMP-1
observed following rotenone treatment does not result from lysosome biogenesis, but
rather from the inhibition of lysosomal degradation. Agents that promote lysosome
dysfunction may also play a role in influencing gene expression of a coordinated network
137
of lysosomal and autophagy genes 68. It is important to note that while levels of TFEB
may be unchanged the localization of TFEB may be of greater importance as its
translocation to the nucleus promotes transcription of lysosome-associated genes. Thus,
TFEB could be exogenously expressed and/or activated as a protective response to stressinduced lysosome dysfunction. Genetic manipulation of TFEB in our model may provide
a useful complimentary approach in future studies to further characterize the putative role
of ATP6V0C in regulating the clearance of ALP substrates.
The aggregation of proteins with neurotoxic potential, such as α-synuclein in PD,
is considered a central theme in numerous neurodegenerative diseases and is believed to
contribute to the disease pathogenesis. Here we report that α-synuclein high molecularweight (HMW) species (indicative of α-synuclein aggregation) putatively dependent on
the ALP for clearance were increased biochemically after rotenone treatment, and
ATP6V0C knockdown under basal or stressed conditions. It is reasonable to predict that
the size of these aggregates may preclude proteasome-mediated degradation probably
because these aggregates are too large to enter the proteasome barrel 83. In light of this
data, lysosomal degradation becomes even more important to cell viability because
aggregated proteins would be preferentially routed to the ALP for degradation 47, 84, 93.
Failure to degrade these aggregates could lead to cellular toxicity that may lead to further
ALP dysfunction as α-synuclein has been linked with inhibition of the ALP, at stages of
formation and completion 75, 80, 88.
While our data indicate lysosome dysfunction as a major contributor to αsynuclein accumulation, the causes of decreased lysosome-dependent degradation of
autophagosomes and other ALP substrates may be potentially due to defects in their
138
microtubule-dependent vesicular transport structure or machinery. Deficits in this
transport mechanism have been associated with increased autophagosome accumulation,
increased p62 detection, and lysosomal fusion block 49. Therefore restoration or
enhancement of dynein-dependent transport could provide an effective strategy to reduce
autophagosome accumulation through improved delivery to the lysosome for
degradation37.
The protective effects of low-dose BafA1 on chloroquine -induced lysosome
dysfunction were first described by Shacka et al. 2006 by its ability to alleviate dosedependent inhibition of autophagy, apoptotic signaling, and cell death72. Low-dose
BafA1 also attenuated the chloroquine-induced reduction in processing of the mature
'active' form of cathepsin D61, the principal lysosomal aspartic acid protease responsible
for the degradation of α-synuclein, suggesting BafA1 inhibition of neuronal cell death is
a potentially novel mechanism of action apart from its ability to inhibit V-ATPase.
Chloroquine was shown to induce autophagosome accumulation and inhibited autophagic
flux, effects that were attenuated upon treatment with low-dose BafA1 and were
associated with a significant decrease in chloroquine-induced accumulation α-synuclein
aggregates. In addition, a bafilomycin derivative significantly and dose-dependently
attenuated dopaminergic neuron death in C. elegans resulting from in vivo overexpression of human wild-type α-synuclein 60. However, it is currently unknown whether
BafA1-mediated protection is dependent on the ALP for the execution of its therapeutic
effects. Our current study reports that low-dose BafA1-mediated protection against cell
death is dependent on the autophagy protein Atg7 and the V-ATPase subunit ATP6V0C.
After independent experiments siRNA-mediated knockdown of Atg7 and ATP6V0C we
139
observed the attenuation of protection afforded by low-dose BafA1 against chloroquine induced death. ATP6V0C knockdown could be expected to inhibit protection by lowdose BafA1 due to its high affinity and interaction with BafA1. We observed a shift in
the dose response of BafA1 that sensitized cells to increased autophagosome
accumulation and cell death associated with lysosome dysfunction so we did not expect
low-dose BafA1 to provide protection since the ALP was likely already compromised.
Atg7 knockdown-mediated attenuation of low-dose BafA1 protection is more difficult to
interpret. Our present data suggests low-dose BafA1 protection is dependent on Atg7, the
ALP activator protein necessary for the dual conjugation pathways’ regulation of
autophagosome formation. Atg7 knockdown consistently inhibited the detection of LC3II by western blot in both basal and stressed conditions, confirming that autophagic
induction at an early stage of autophagosome formation is inhibited. Knockdown of Atg7
ameliorated low-dose BafA1-mediated protection, suggesting protection is dependent on
autophagosome formation. Autophagic flux appears to be restored as indicated by the
reduction in colocalization of LC3 and LAMP-1 following treatment with low-dose
BafA1. This result is in agreement with our previous data suggesting low-dose BafA1
confers protection by enhancing autophagic flux60. Together, our findings suggest that
low-dose bafilomycin is cytoprotective in part through its maintenance of the ALP, and
underscores its therapeutic potential for treating PD and other neurodegenerative diseases
exhibiting disruption of protein degradation pathways and accumulation of toxic protein
species.
Our studies aimed to test the hypothesis that alterations in the ALP cause a block
in flux and induce lysosome dysfunction, both of which contribute to neuronal
140
dysfunction and death in neurodegenerative disease. We have demonstrated that stimuli
that increase lysosome pH trigger autophagosome accumulation. There are a multitude of
studies that indicate lysosome dysfunction is integral to the pathogenesis in many
neurodegenerative diseases. Our studies have shown that low-dose BafA1-mediated
improvements in neuronal viability require functional autophagy and basal levels of
ATP6V0C. Our manipulation of the ALP by either pharmacological or genetic means has
highlighted the importance of autophagic degradation by the lysosome as a homeostatic
and protective mechanism that, when compromised, may lead to the promotion of toxic
protein aggregation and cellular dysfunction and death.
Current treatment methods for PD include dopamine replacement, dopamine
agonists, neuronal precursor transplantation, and by surgical means with deep brain
stimulation24, 34, 89. All these methods represent reactive treatments for patients already
experiencing symptoms due to robust loss of nigral dopaminergic neurons and
subsequent decrease in dopamine neurotransmission. Our studies provide strategies
intended to support dopaminergic neuron viability which may provide early stage
protection against PD progression. Models using pharmacological intervention as well as
gene therapies which elevate ALP function have been shown to promote neuronal
survival concomitant with decreased cellular pathology, and provide proof of principle
for future potential use in clinical settings. Strategies to improve the efficiency and
capacity of the ALP are necessary to preserve neuronal function and viability in the
context of PD and other autophagy-related neurodegenerative diseases.
141
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