REGULATION OF NEURONAL DEATH BY THE AUTOPHAGY LYSOSOMAL
PATHWAY: IMPLICATIONS FOR PARKINSON DISEASE
by
VIOLETTA N. PIVTORAIKO
KEVIN A. ROTH, MENTOR
JOHN J. SHACKA, COMMITTEE CHAIR
STEVEN L. CARROLL
ELIZABETH SZTUL
W ANNE BURTON THEIBERT
TALENE ALENE YACOUBIAN
A DISSERTATION
Submitted to the graduate faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
BIRMINGHAM, ALABAMA
2011
Copyright by
Violetta N. Pivtoraiko
2011
REGULATION OF NEURONAL DEATH BY THE AUTOPHAGY LYSOSOMAL
PATHWAY: IMPLICATIONS FOR PARKINSON DISEASE
VIOLETTA N. PIVTORAIKO
NEUROSCIENCE GRADUATE PROGRAM
ABSTRACT
Parkinson
Disease
(PD)
is
the
second
most
common
age-related
neurodegenerative disorder and is characterized pathologically by the loss of
dopaminergic (DA) neurons in the stubstantia nigra pars compacta (SNpc). Mitochondrial
dysfunction, increased oxidative stress, and accumulation of aggregated α-synuclein (αsyn), an intracellular protein involved in synaptic function, are all pathological hallmarks
of PD have been implicated in PD pathogenesis. However, it is debated whether α-syn
aggregates themselves are responsible for neurodegeneration in PD, cellular pathways
involved in degradation of α-syn aggregates are believed to promote neuron survival. The
autophagy lysosomal pathway (ALP), a physiological mechanism for recycling of
intracellular components, has been shown to clear α-syn, its aggregates, and regulate
neuron survival in PD.
iii
The first part of this dissertation reviews the developments regarding the
contribution of the ALP to neuron survival and death regulation. It discusses the effects
of oxidative stress on ALP function and vice versa. The role of the lysosome, a cellular
organelle responsible for digestion of intracellular constituents delivered via ALP, in
regulation of neuron survival is highlighted.
Increased oxidative stress and α-syn aggregate accumulation has been reported in
PD models of mitochondrial dysfunction such as that induced by the insecticide rotenone.
Another part of this dissertation focused on rotenone’s effects on ALP function.
Rotenone-induced inhibition of lysosomal function has been observed. Our findings
suggested that lysosomal dysfunction may be responsible for α-syn accumulation and
neuron death observed in PD, implying that mechanisms improving lysosomal function
may be cytoprotective.
This hypothesis was tested in the second chapter of this dissertation using a
chloroquine (CQ) model of lysosome dysfunction. CQ, a known antimalarial agent,
induced α-syn accumulation and neuron death by inhibiting ALP function. Bafilomycin
A1, a plecomacrolide antibiotic, attenuated CQ-induced neuron death, an effect
accompanied by restoration of lysosomal function.
These studies expand our knowledge of the potential role of the ALP in PD
pathogenesis and suggest that further studies are needed to both decipher the
neuroprotective role of the ALP in preventing neuron death in response to PD-associated
stimuli, such as rotenone, and to identify novel ALP-related molecular targets for PD
therapy.
iv
DEDICATION
I would like to dedicate my dissertation to the memory of my father,
Nikolai M. Pivtoraiko
(1938 – 2011).
An extraordinary craftsman with a passion for discovery and research.
He will always be with me in memories…
v
ACKNOWLEDGEMENTS
I would like to extend my sincere gratitude to my committee members Drs. John
J. Shacka, Steven L. Carroll, Elizabeth Sztul, Anne Burton Theibert, and Talene Alene
Yacoubian for their suggestions and help, as well as their understanding and support
during these very difficult several months since my father’s passing.
My most sincere thanks are extended to my mentor, Dr. Kevin A. Roth for his
support, guidance, and patience, a lot of it! He has been training me to be independent
and learn from my mistakes; yet, he always made sure I was on the right track. I am
deeply thankful to Dr. Roth for helping me develop as a scientist. Dr. John J. Shacka
served as my co-mentor. It was convenient to have Dr. Shacka’s laboratory next door. I
am grateful to him for, among many other things, always being available to discuss
experimental results or brainstorm ideas.
I would also like to thank people who helped me accomplish the mission of
earning a PhD outside the lab. First and foremost, I would like to thank my brother,
Mihail N. Pivtoraiko, and mother, Zina Pivtoraiko. It was difficult to be so far away from
my family, but they supported me in so many ways through these five years. Thank you
so very much for all you have done for me! I look forward to coming back home! I would
like to thank my boyfriend, Dr. Pavel V. Kucheryaviy. He makes me laugh, and this
helps me keep positive attitude and stay focused on completing my work! Pavel has
supported and helped me in so many ways. I am so happy we found each other!
vi
I also extend many thanks to my friends in Birmingham, Alabama, who have
become like an extended family to me, and to my best friend Dr. Jennifer Vaughan, who
supported me in times of joy or frustration.
Lastly, I would like to extend a special thank you to the Neuroscience program
director, Dr. Lori McMahon. I enjoyed having her as an instructor, program director, and
just somebody to chat with.
All of these people contributed to my accomplishments as a graduate student at
UAB and helped me grow as a scientist and an individual. I am very grateful for having
you in my life!
vii
TABLE OF CONTENTS
Page
ABSTRACT.......................................................................................................... iii
DEDICATION .........................................................................................................v
ACKNOWLEDGEMENTS ................................................................................... vi
LIST OF FIGURES ............................................................................................... ix
INTRODUCTION ...................................................................................................1
Parkinson disease: clinical presentation and treatment ................................1
Genetic and environmental factors of PD ....................................................4
Animal models of PD...................................................................................6
Cell death mechanisms in PD ......................................................................8
Autophagy-lysosomal pathway regulation and implications in PD...........10
OXIDATIVE STRESS AND AUTOPHAGY IN THE REGULATION
OF LYSOSOME-DEPENDENT NEURON DEATH ...........................................19
LOW-DOSE BAFILOMYCIN ATTENUATES NEURONAL CELL
DEATH ASSOCIATED WITH AUTOPHAGY-LYSOSOME
PATHWAY DYSFUNCTION .............................................................................76
ROTENONE INDUCES AV ACCUMULATION BY ALTERING
LYSOSOMAL FUNCTION ................................................................................118
CONCLUSIONS AND DISCUSSION ...............................................................147
Regulation of autophagic flux by low dose BafA1 ..................................149
Oxidative Stress implications in the lysosomal function regulation ........153
LIST OF GENERAL REFERENCES .................................................................158
APPENDIX: INSTITUTIONAL ANIMAL CARE AND
USE COMMITTEE APPROVAL FORM ...........................................................169
viii
LIST OF FIGURES
Figure
Page
INTRODUCTION
1
Autophagy-lysosomal pathway and its regulation .......................................................18
OXIDATIVE STRESS AND AUTOPHAGY IN THE REGULATION
OF LYSOSOME-DEPENDENT NEURON DEATH
1
Convergence of the endosomal-lysosomal and autophagy-lysosomal degradation
pathways ......................................................................................................................71
2
Macroautophagy induction vs. inhibition in oxidative stress-induced lysosome
damage. ........................................................................................................................72
3
Chemical structure of chloroquine ...............................................................................73
4
Chloroquine-induced death of human SH-SY5Y cells follows alterations in the
processing of CD..........................................................................................................74
5
Oxidative stress, lysosomal membrane permeabilization and the induction of necrotic
vs. apoptotic death .......................................................................................................75
LOW-DOSE BAFILOMYCIN ATTENUATES NEURONAL CELL DEATH
ASSOCIATED WITH AUTOPHAGY-LYSOSOME PATHWAY DYSFUNCTION
1
Low-dose bafilomycin is not cytotoxic to SH-SY5Y cells ........................................105
2
Low-dose bafilomycin attenuates chloroquine-induced cell death and apoptosis .....106
3
Bafilomycin A1 attenuates chloroquine-induced inhibition of CD processing .........107
ix
4
Bafilomycin A1 attenuates chloroquine-induced increase in detergent-insoluble
endogenous α-syn oligomers .....................................................................................108
5
Bafilomycin A1 attenuates chloroquine-induced AV accumulation .........................109
6
Bafilomycin A1 attenuates chloroquine-induced inhibition of autophagic flux ........110
7
Bafilomycin attenuates the death of DA neurons in C. elegans following overexpression of wild-type human α-syn ........................................................................112
S1 Stimulus-induced death of SH-SY5Y cells................................................................113
S2 Low-dose bafilomycin attenuates stimulus-induced death of SH-SY5Y cells .........114
S3 Low-dose bafilomycin attenuates chloroquine-induced cell death of differentiated
SH-SY5Y cells ...........................................................................................................115
S4 Inhibition of autophagy induction does not attenuate chloroquine-induced
cell death ....................................................................................................................116
S5 Effects of bafilomycin and chloroquine on DA neuron death in C. elegans .............117
ROTENONE INDUCES AV ACCUMULATION BY ALTERING LYSOSOMAL
FUNCTION
1 Rotenone induced SH-SY5Y cell death and caspase 3-like activity is concentration
and time dependent ....................................................................................................139
2
Rotenone-induced SH-SY5Y cell death is accompanied by increased nuclear p53
accumulation but is not dependent on caspase activation ..........................................140
3
Rotenone induced concentration and time dependent primary telencephalic neuron
death that was attenuated by p53 deficiency..............................................................141
4
Rotenone induced a time-dependent increase in AV accumulation ..........................142
5
Rotenone inhibits autophagic flux .............................................................................143
6
Rotenone induces time-dependent p62 accumulation................................................144
x
7
Rotenone-induced SH-SY5Y cell death is accompanied by increases in volume of
acidic compartments ..................................................................................................145
8
Rotenone does not alter LAMP1 levels .....................................................................146
CONCLUSIONS AND DISCUSSION
1
Proposed model of the ALP regulation by CQ, rotenone, and low dose BafA1 .......157
xi
INTRODUCTION
Parkinson disease: clinical presentation and treatment
Parkinson
disease
(PD)
is
the
second
most
common
age-related
neurodegenerative disorder. It was originally described in 1817 by Dr. James Parkinson
in his monograph entitled “An Essay on the Shaking Palsy” [1]. The principal clinical
presentations of PD are resting tremor, rigidity, bradykinesia, and gait dysfunction. Other
non-motor symptoms including autonomic dysfunction (e.g., orthostatic hypotension),
pain and sensory disturbances (e.g., anosmia), mood disorders, sleep impairment, and
dementia are common but not always fully appreciated [2]. The mean age of PD onset is
55 years old; however, the incidence rate and severity of symptoms increases
dramatically with age [3]. PD affects about 2% of the population over 60 years old,
which constitutes 0.3% of the worldwide population [4]. Due to an increase in the aged
population, the number of PD patients is anticipated to go up dramatically in the coming
decades [5]. Most PD cases are considered sporadic, but approximately 10% of PD cases
are familial, being associated with a direct genetic defect [2].
PD is clinically diagnosed based on presentation of motor dysfunction and
responsiveness to standard PD treatment. However, definite diagnosis is given upon
neuropathological examination of post-mortem brain tissue. PD is characterized
pathologically by the loss of pigmented dopaminergic (DA) neurons in the stubstantia
nigra pars compacta (SNpc). SNpc is one of the basal ganglia nuclei located in the
midbrain. SNpc neurons modulate motor information transmitting pathways in the brain
1
by projecting most of their axons into the corpus striatum, a group of basal ganglia nuclei
consisting of putamen and caudate nuclei. It is hypothesized that loss of SNpc striatal
projections precedes the actual death of DA neurons. Likewise, onset of symptoms in
animal models of PD is associated with about 60% DA neuron loss in SNpc neurons and
approximately 80% DA depletion in striatum [3].
Another pathological hallmark of PD is the presence of intraneuronal inclusions,
termed “Lewy Bodies” (LB), in survived SNpc DA neurons and neurons in other brain
regions. LBs are spherical eosinophilic protein aggregates consisting predominantly of αsynuclein (α-syn); other proteins, such as ubiquitin, are also present in LB [2;6;7]. α-syn
is an intraneuronal protein that has been implicated in regulation of presynaptic vesicle
recycling and neurotransmitter release, especially for DA [8]. However, the exact role of
α-syn in normal neuronal physiology and in pathological conditions like PD is not
known. In PD patients, LBs are not localized strictly to SNpc. In fact, studies by Braak et
al. suggest that α-syn positive LBs appear first in the olfactory bulb and lower brainstem
areas before affecting basal ganglia and spreading into cortex in the later stages of the
disease [9]. This hypothesis is supported by the recent finding that appearance of
symptoms such as decreased olfaction, gastrointestinal dysfunction, and rapid eye
movement disorders are not just risk factors but indicators of early stage PD, when motor
symptoms are not yet present [2;10].
There is currently no cure for PD; all treatment options presently available are
largely symptomatic and do not halt the progression of the disease. The most effective
treatment regimen for PD patients is Levodopa (LD) therapy. LD is a DA precursor
which is converted into DA by amino acid decarboxylase (AADC) [11]. LD is usually
2
given in combination with peripheral AADCD inhibitors carbidopa and benserazide
which attenuate typical gastrointestinal (nausea, emesis) and cardiovascular (arrhythmia,
hypotension) side effects of levodopa treatment [2;11]. LD treatment substantially
enhances the quality of PD patients’ lives by improving their mobility. However, due to
the progressive nature of PD, the LD concentration required to obtain 50% of maximal
therapeutic effect progressively increases with duration of the disease [12]. Moreover,
long-term treatment with LD leads to development of adverse effects such as motor
fluctuations, dyskinesias, and neuropsychiatric complications [2].
The adverse motor effects of LD have been attributed to its short half-life (about
90 minutes when given in combination with carbidopa), which results in release of LDderived DA in short discrete boluses [12;13]. However, in healthy individuals, DA
receptors in striatum are generally tonically innervated. Pulsatile stimulation of striatum
by LD-derived DA has been suggested to alter the function of the basal ganglia causing
motor fluctuations and dyskinesias [14;15]. Therefore, use of therapies that ensure a more
continuous stimulation of striatum is the direction where PD care is progressing [2]. One
such therapy is deep brain stimulation (DBS) which delivers continuous low frequency
electrical stimulation of thalamic or basal ganglia nuclei [16]. Continuous infusion pump
therapy delivering LD into the duodenum is a promising future alternative to DBS
because it does not require neurosurgical intervention. Additionally, other drugs have
been used for treatment of motor PD symptoms in combination or instead of standard LD
regimen; they include DA agonists (e.g., pramipexole, ropinirole), centrally acting
antimuscarinic drugs (e.g., trihexyphenidyl, benztropine, ophenadrine), and monoamine
oxidase-B inhibitors (e.g., selegiline, rasagiline) [13].
3
Therapies such as LD or DBS can be quite successful in managing classical motor
PD symptoms. However, as the disease progresses the effectiveness of these therapies
diminishes. Moreover, nondopaminergic features of PD such as freezing, falling, and
dementia are not well controlled by either LD or DBS [2;16]. Therefore, treatment
approaches that could prolong survival of SNpc DA neurons (gene therapy) or replenish
these neurons (stem cell therapy) are becoming appealing alternative strategies for
management of PD. However, both gene and stem cell therapy remain experimental
approaches because of low efficacy, high cost and potential ethical issues [2;17;18].
Therefore, tremendous scientific effort is focused on understanding the key cellular and
molecular pathways regulating neuron death in PD. This knowledge will help make gene
and stem cell therapy approaches more efficacious and may lead to the discovery of novel
therapeutic interventions that could halt or slow down PD progression.
Genetic and environmental factors of PD
Genetic as well as environmental factors have been implicated in PD etiology.
Approximately 5% to 10% of PD cases are familial [19;20]. Mutations in α-synuclein
[21], uniquitin carboxyterminal hydrolase L1 (UCH-L1) [22], parkin [23], DJ-1 [24],
phosphatase and tensin homolog (PTEN)-induced novel kinase 1 (PINK1) [25], leucine
rich repeat kinase 2 (LRRK2) [26;27], high temperature requirement protein A2
(Omi/HtrA2) [28], and ATPase type 13A2 (ATP13A2 ) have been associated with familial
PD. These genes encode proteins regulating function of different cellular processes and
organelles such as proteasome (UCH-L1 and parkin), mitochondria (DJ-1, PINK1,
Omi/HtrA2, and LRRK2), lysosome (ATP13A2), and neurotransmitter vesicle recycling
4
and release (α-synuclein). Mutations in these genes are typically associated with an
earlier age of disease onset (<40 years old), neurodegeneration, and more extensive
aggregate accumulation than in sporadic PD patients [29]. LRRK2 mutations are
emerging as the most common cause of familial PD; they have also been reported in
patients with sporadic PD [30]. Nevertheless, epidemiologic studies suggest that genetic
factors do not play a major role in PD patients of 50 years old and older, who comprise
the vast majority of PD cases [31].
The development of PD symptoms in opiate addicts who received MPPP
contaminated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in
scientific attention being directed to environmental factors as potential contributors to PD
[32]. The importance of environment in PD pathogenesis was also supported by finding
of increased susceptibility of inhabitants of rural areas to develop PD, potentially through
chemical exposure [33-35]. Insecticide rotenone and pesticide paraquat have been most
studied in animal and cell culture models, where they cause some characteristics of PD
such as neuron death and α-syn aggregation. However, no single environmental factor
has been established to cause PD [2]. Therefore, it is currently believed that a
combination of genetic predisposition and environmental factors can initiate dysfunction
of multiple convergent or parallel cellular pathways, which when progressive, will
manifest PD pathology. This theory has been termed the multi-hit hypothesis [2].
5
Animal models of PD
Much has been learned about PD pathology from the study of human post-mortem
PD tissue. However, the use of animal and cell culture models of gene mutations
associated with familial PD or pharmacological agents that mimic PD pathology allow
the investigation of interactions between cellular and molecular pathways implicated in
PD. Numerous PD models have been generated utilizing non-human primates, small
rodents, and cell-culture systems. Each model mimics some aspects of human PD
pathology[36]. Among genetic models, α-syn animal models are the best characterized so
far. A substitution of an alanine for a threonine at position 53 (A53T) in the α-synuclein
gene was the first mutation identified in PD [37]. Two other α-synuclein mutations, A30P
and E46K, as well as duplications and triplications of the wild-type α-synuclein gene
have been identified in familial PD cases [38;39]. Based on these α-synuclein mutations
and gene multiplications, a number of animal models have been designed.
Overexpression of human mutant or wild-type α-synuclein in mice and rats causes
LB-like intracellular inclusion formation, depletion of DA, and appearance of subtle
locomotor deficits in old age but unlike human PD, these animals do not exhibit
neurodegeneration in SN [40]. However, viral-vector-mediated delivery of α-synuclein to
SN in rodent models more closely recapitulated the full spectrum of PD symptoms and
neuropathology. These rodents exhibited time-dependent α-syn aggregation, appearance
of swollen and dystrophic axons, and ultimately SN neuron loss [40]. Overexpression of
either mutant or wild-type α-synuclein induces motor dysfunction and degeneration of
DA neurons in fly (drosophila) and worm (C.elegans) models [41;42]. These invertebrate
6
organisms are commonly used for high throughput screening of potential neuroprotective
agents.
One of the best accepted and widely used in vivo PD models is MPTP-induced
neurotoxicity [43]. MPTP has been extensively used in monkeys and small rodents (rats
and mice) to induce PD symptoms. In all these animal models MPTP causes SN
neurodegeneration which is accompanied by motor dysfunction and accumulation of
protein aggregates resembling LB [2;44]. However, in rodents MPTP-induced permanent
DA neuron loss is associated with only transient PD-like behavioral symptoms such as
unsteady gait [45]. Nevertheless, MPTP has been extensively used to model PD in
rodents because it can easily pass the blood-brain barrier, permitting intra-peritoneal
administration and ease of experimental design.
The mechanism of MPTP-induced toxicity has been attributed to inhibition of
mitochondrial function. Once MPTP is taken up in the brain it is converted to MPP+, the
active compound, in astrocytes and enters catecholaminergic neurons via dopamine
transporter (DAT) [46]. MPP+ then accumulates in the mitochondria of DA neurons
where it inhibits complex I of the mitochondrial electron transport chain (METC)
[47;48]. A similar mode of neurotoxicity is observed with rotenone, a pesticide and
insecticide that targets mitochondria selectively by inhibiting complex I of METC [49].
Inhibition of METC complex I promotes the generation of reactive oxygen species (ROS)
which in turn can alter structure and function of many intracellular components [50] and
activate apoptotic machinery [51]. In contrast to MPP+, rotenone does not depend on
DAT for entry into DA neurons because it is highly lipophilic freely passing all
membranes. Therefore, rotenone exposure causes systemic inhibition of METC complex
7
I, which has been also observed in PD patients.
Selectivity of rotenone-induced
degeneration to DA neurons in SNpc is believed to be due to a unique sensitivity of these
neurons to complex I inhibition [52]. Growing evidence suggests that cell death induced
by METC inhibitors can be multifaceted in character involving not just apoptosis but also
induction of autophagy and possibly damage to lysosomal membranes [53-55] .
Cell death mechanisms in PD
Three major morphologic types of cell death have been described in PD:
apoptotic, necrotic, and autophagic [56]. Apoptosis is characterized by chromatin
condensation, nuclear fragmentation, and cytoplasmic blebbing [57]. Apoptosis is the
most extensively investigated form of cell death in the nervous system [56]. Necrotic cell
death is characterized by cell and organelle swelling or rupture of cell membranes
accompanied by spillage of intracellular contents [58]. Necrosis is usually considered to
be an accidental (i.e., non-programmed) form of cell death and is commonly observed
after trauma or infection [59]. However, necrosis has also been reported in PD and other
neurodegenerative diseases [60]. The molecular mechanisms that initiate necrotic cell
death in PD are not well understood, but may include excitotoxicity, intracellular Ca2+
increase, and ATP depletion [61]. Autophagic cell death is characterized by accumulation
of autophagic vacuoles (AVs) concomitant with markers of apoptosis or necrosis [62].
There is a growing awareness of a possible role for autophagic cell death in PD. It is also
becoming more evident that there is a complex interplay between these death pathways,
particularly between apoptosis and autophagy, the balance between which determines
either cell death or cell survival.
8
Apoptosis is a highly regulated process that can be activated by receptor-mediated
(extrinsic) or mitochondria-mediated (intrinsic) pathways that converge at cleavagedependent activation of aspartate-specific effector caspases (caspases-3, 6, and 7). Once
activated, effector caspases cleave many cellular components leading to degradation of
DNA and cytoskeletal proteins causing nuclear fragmentation, degradation of subcellular
components, and collapse of the cytoskeleton. Apoptosis allows a cell to die without
affecting the viability of neighboring cells and tissues [63]. Apoptosis is regulated in part
by the balance between pro- and anti-apoptotic members of the BCL-2 family of proteins.
This family consists of anti-apoptotic BCL-2 proteins such as BCL-2, BCL-xL, and
MCL-1 and pro-apoptotic proteins such as BAX, BAK, and BOK [64]. Various cell death
stimuli can cause conformational activation, oligomerization, and subsequent insertion of
pro-apoptotic BCL-2 family proteins into the outer mitochondrial membrane, which can
lead to cytochrome c release and induction of apoptotic cascade. This process is opposed
by anti-apoptotic BCL-2 family proteins which inhibit in outer mitochondrial membrane
permeabilization by interacting with pro-apoptotic BCL-2 family proteins [64].
p53 is a transcription factor and one of the most studied pro-apoptotic regulators. p53
has been reported to activate apoptosis via both transcription-dependent and –
independent pathways [65;66]. In response to a variety of toxic stimuli p53 undergoes
post-translational modification and accumulates in the nucleus where it induces
transcription of pro-apoptotic genes such as bax, and puma [67]. Transcriptionindependent cell death is mediated by cytoplasmic p53 via its interaction with Bcl-2
family members and induction of mitochondrial membrane destabilization and caspase
activation [68;69].
9
PD is associated with the loss of selective neuronal cell populations; therefore, the
possibility of apoptosis-associated molecules and processes being responsible for PD
pathogenesis has received significant attention. Elevated levels of Bax and caspase 3 have
been detected in PD human post-mortem studies and numerous PD animal and cell culture
studies [70;71]. Transcription-dependent and transcription-independent p53 activity has
also been implicated in DA neuron death regulation in a number of PD models [72;73].
Moreover, p53 deficiency or its pharmacological inhibition have been reported to protect
SN DA neurons in MPTP in vivo models of PD, suggesting that activation of p53dependent pathways may play a pathogenic role in the neurodegeneration observed in PD
[74;75]. However, implication of apoptosis as a general cell death mechanism in PD has
largely been supported by evidence from animal models and tissue culture studies, while
investigations on human postmortem brain have yielded conflicting results [76]. However,
identifying apoptotic neuron death in autopsied human brain can be difficult since
neurodegenerative processes represent chronic brain demise; while apoptotic cell death can
be executed within a few hours [77]. Overall, it is still not known if neurological
dysfunction observed in PD is a direct consequence of apoptotic neuron death or of
neuronal dysfunction occurring prior to frank neuron loss.
Autophagy-lysosomal pathway regulation and implications in PD
PD-associated neurodegeneration is accompanied by accumulation of protein
aggregates in LB [78]. Protein aggregates are thought to be formed as a result of toxic
gain of function mutations or modifications. It is debated whether soluble monomeric
aggregation-prone proteins, their oligomers or larger aggregates are most toxic [79].
10
However, in general, the protein’s capacity to aggregate correlates with its toxicity
(although not necessarily with the aggregates themselves). Two main systems are
responsible for clearance of proteins in cells; the ubiquitin-proteasome system (UPS) and
the autophagy-lysosomal pathway (ALP) [80].
The principal function of the ALP is to regulate intracellular energy balance by
recycling outlived and/or damaged cellular components such as protein complexes and
organelles. Three major types of autophagy have been defined: macroautophagy
(hereafter simply referred to as “autophagy”), microautophagy, and chaperone-mediated
autophagy.
Autophagy is initiated by generation of a double-membrane, phagophore, which
surrounds the cellular components targeted for degradation forming an AV [81].
Autophagy initiation is regulated in part by the activation of mammalian target of
rapamycin (mTOR) which inhibits autophagy input by phosphorilation-dependent
inactivation of autophagy-associated proteins (Atgs) regulating AV formation [82]. For
autophagy to be completed, the cargo of AVs has to be degraded and this is achieved by
fusion of AVs with lysosomes [82].
Growing evidence indicates that autophagy plays a critical role in protein
aggregate clearance and regulation of neuron death in PD [83]. Although α-syn clearance
is partially dependent on the UPS, wild-type α-syn can also be degraded via chaperonemediated autophagy and macroautophagy [84]. However, autophagy becomes the major
route of degradation for α-syn oligomers and aggregates that cannot be efficiently cleared
by the proteasome. The dependence of proteins on autophagy for their clearance
11
correlates with their propensity to aggregate [85;86]. For instance, inhibition of
autophagy has a much smaller effect on the clearance of wild-type α-syn than on the
clearance of the mutant aggregate-prone α-syn species [85;86]. Overexpression of wildtype α-syn has also been shown to alter autophagy induction by inhibiting AV formation
[87]. The pivotal role of autophagy in clearance of aggregate-prone proteins and their
aggregates is further supported by studies in mice lacking neuronal expression of Atg5 or
Atg7, genes responsible for AV formation and initiation of autophagy. These mice die as
young adults and exhibit accumulation of ubiquitin positive protein aggregates that
increase in size and number with age, and neuron loss in cerebrum and cerebellum
[88;89].
Inhibition of autophagy completion resulting from altered lysosomal function has
also been associated with neurodegeneration and α-syn accumulation [90;91]. For
instance, deficiency in cathepsin D, an aspartic lysosomal protease, leads to extensive
neuron death and is accompanied by accumulation of autophagosome/autolysosome-like
bodies containing ceroid lipofuscin as well as α-syn accumulation [90;92]. Mice with
combined deficiency of cathepsins B and L, lysosomal cysteine proteases, die during the
first four weeks of life; these animals manifest massive cell death of selected neurons in
the cerebral cortex and cerebellum. Neurodegeneration is accompanied by accumulation
of lysosomal bodies and by axonal enlargements, indicators of impaired degradation
capacity of the ALP in these mice [91]. A53T and A30P α-syn mutants have been
reported to inhibit lysosomal function by preventing intracellular substrates degradation
via lysosome [93]. Chronic inhibition of lysosome function can induce lysosomal
membrane permeabilization (LMP). This results in release of lysosomal proteases into the
12
cytosol and cytosolic acidification [94]. LMP can induce caspase-dependent apoptosis via
cathepsin B or D-mediated cleavage of Bid, pro-apoptotic Bcl-2 family protein. Caspaseindependent cell death has also been reported with LMP [95]. This is further reviewed in
Chapter 1.
Maintenance of the acidic environment in the lysosomal lumen is crucial for
proper functioning of lysosomal proteases [96]. Low pH in the lysosomal lumen is
maintained by vacuolar ATPases (V-ATPases) which pump H+ into lysosomal lumen
from cytosol. Several lysosomal membrane ion transporters have also been reported to
regulate lysosomal acidity and lysosomal enzyme function by providing neutralizing
current relieving positive charge accumulation in the lysosomal lumen generated by VATPase-mediated H+ translocation. One of the ion transporters implicated in lysosomal
pH regulation is ClC-7, a member of voltage-gated chloride channel (CLC) family. ClC-7
is localized to the lysosomal membrane where it acts as a Cl- /H+ exchanger, which
transports Cl- ions into and extrudes H+ out of lysosomal lumen. Mice lacking ClC-7
exhibit excessive bone mineralization (osteopertosis) and severe neurodegeneration in the
brain and retina that appears as early as 30 days of age [97-99]. Neurodegenration in
Clcn-7 knockout mice is preceded by accumulation of AVs and lysosomal storage
material in neuronal cell bodies [100]. However, cells lacking ClC-7 or expressing
mutant ClC-7 capable of conducting only Cl- ions maintain normal lysosomal pH
[97;99;101]. Therefore, although ClC-7 is undoubtedly important for proper ALP
function and neuronal cell survival, it has been suggested that efflux of other cations,
such as K+ and Na+, rather than Cl- influx, may be more important for maintenance of
13
proper lysosomal acidification [102]. Identification of these cation transporters and their
role in the regulation of ALP and neuron survival is the matter of future research efforts.
Discovery of a mutation in the ATP13A2 gene encoding a lysosome protein causing
familial early onset PD further highlights the importance of the ALP in PD pathology.
ATP13A2 encodes a lysosomal P-type ATPase and is involved in the maintenance of the
acidic environment of the lysosomal lumen. Interestingly, elevated levels of ATP13A2
expression have also been detected in the brains of sporadic PD patients, suggesting a
potential role for this protein and proper lysosomal functioning in idiopathic PD [96].
Mutations in another gene encoding a lysosomal protein, glucocerebrosidase (GBA), an
enzyme that catalyzes hydrolysis of membrane glycolipids, have also been associated
with PD [103]. Moreover, decreases in lysosomal markers such as LAMP1, a lysosomal
membrane-associated protein, suggests a decrease in lysosome numbers, as recently
reported in PD patients and MPTP mouse models of PD [104]. Lysosomal function has
been also shown to decline with age in the human brain, and thus, diminished autophagy
completion may contribute to age-related neurodegenerative disorders like PD [105]. In
Chapter 3 of this dissertation we report that ALP function is inhibited in rotenone model
of PD; our findings are in agreement with an earlier report using the MPTP model of PD
[104].
Although accumulation of AVs has been observed in affected neurons in PD and
numerous models of this disease, there is ongoing debate as to whether autophagy plays a
pro-survival or pro-death role in PD [83]. Indeed, autophagy is best known for its
homeostatic role mediating bulk degradation of cytoplasm, aggregate-prone proteins and
14
damaged organelles, such as mitochondria. These findings are often used to support the
argument that autophagy bears a pro-survival function [76]. Autophagy, as a cleansing
and recycling mechanism can only be effective if lysosomal degradation of AVs is
accomplished [91]. Therefore, a combination of factors that impair AV formation,
degradation, or overactivate AV formation relative to the degradative reserve of the cell
can lead to “cell death with autophagy”, which some investigators argue may be a more
precise term than autophagic cell death [106]. Likewise, factors that stimulate lysosomemediated AV degradation, increase autophagic flux, can provide neuroprotection. This
topic is described in Chapter 2 of this dissertation.
Based on our growing awareness of multiple pro-survival and pro-death
pathways, it seems likely that a single death pathway may not be solely responsible for
neuron loss in the context of PD. Instead, multiple pro-survival and cell death
mechanisms may interact to determine neuron fate [76]. Also, inhibition of one pathway
of cell death may not prevent neuron loss but instead, may recruit alternative death
mechanisms; e.g. inhibition of caspase activation may prevent apoptosis but stimulate
autophagic or necrotic cell death [107]. Therefore, increased research interest is aimed at
determining the interactions between apoptotic and autophagic death pathways.
There is a growing list of apoptosis regulators interacting with autophagic
machinery. For instance, Beclin1/Atg6, a protein involved in regulation of AV formation
and autophagy induction, has a Bcl-2 homology domain (BH-3-domain) and has been
shown to interact with pro-survival members of the Bcl-2 family of proteins. Bcl-2 and
Bcl-XL can bind to Beclin1 preventing it from interacting with the complexes involved in
15
AV formation, and in turn inhibit autophagy [108]. Therefore, the ratio of Bcl-2 to
Beclin1 is an important determinant of whether a cell will activate the pro-survival
autophagic pathway and/or a death-inducing apoptotic program.
Pathways regulating induction of autophagy can also activate pathways that affect
apoptosis. For instance, PI3K/Akt-mediated phosphorylation of Bad, a BH3-only member
of the Bcl-2 family, leads to its dissociation from Bcl-2, thus allowing Bcl-2 to sequester
pro-apoptotic Bcl-2 family proteins such as Bax and preventing them from inducing
apoptosis. Akt also antagonizes the transcriptional activity of a number of pro-apoptotic
transcription factors, such as p53, which results in inhibition of pro-apoptotic gene
expression and promotion of cell survival [107]. Atg5, involved in AV formation and
conversion of LC3I to LC3II, can also influence apoptotic signaling pathways. Atg5 can
be cleaved following various apoptotic stimuli, forming an N-terminal product that
translocates to the mitochondrial membrane, interacts with Bcl-XL, and promotes
apoptosis. At the same time, Atg5 cleavage leads to autophagy inhibition, as a pool of
available Atg5 necessary for AV formation is decreased [107;109].
Recently, p53, a well-studied regulator of neuron apoptosis, was reported to also
modulate autophagy [110]. Interestingly, the effects of p53 on autophagy appear to be
dependent on its intracellular localization. Nuclear p53 can stimulate autophagy by
inducing transcription of damage-regulated-autophagy modulator (DRAM), a novel protein
believed to localize to the lysosomal membrane, or by inhibiting mTOR activity [110;111].
On the other hand, cytoplasmic p53 was shown to inhibit autophagy induction by activating
mTOR [110]. A number of studies have reported elevated protein and mRNA levels of p53
16
in postmortem PD brain tissue and in a number of PD animal and cell culture models,
suggesting that p53 may be involved in regulation of neuron loss in these pathologies
[112;113].
Several factors have been implicated in PD etiology; they include oxidative stress,
aberrant protein accumulation, and mitochondrial dysfunction [2]. Each of these factors
alone or in combination has been demonstrated to cause neuron death in PD models thus
mimicking PD pathology. Recent scientific interest in apoptotic and autophagic cell death
mechanisms and their involvement in PD has produced significant advances in our
understanding of the cellular and molecular processes controlling neuron life and death.
Despite these advances, numerous questions remain about the precise role of apoptosis
and autophagy in PD pathogenesis. Future investigations are necessary to devise
strategies for restoring function to injured neurons before they become committed to
death, regardless of the death pathway(s) being activated.
17
Figure 1. Autophagy-lysosomal pathway and its regulation. Autophagy-lysosomal
pathway (ALP) supplies neurons with energy and metabolic building blocks needed for
cellular maintenance by recycling outlived or damaged organelles and protein aggregates.
Therefore, it is thought to bear a pro-survival function. However, when the ALP integrity is
jeopardized, protein aggregate accumulation and cellular demise can occur. CB and CD are
cathepsin B and D, respectively, lysosomal proteases.
18
OXIDATIVE STRESS AND AUTOPHAGY IN THE REGULATION OF LYSOSOMEDEPENDENT NEURON DEATH
by
VIOLETTA N. PIVTORAIKO, SARA L. STONE, KEVIN A. ROTH AND JOHN J.
SHACKA
Journal of Antioxidants and Redox Signaling (2009) Vol. 11(3):481-96
Copyright 2009
by
Mary Ann Liebert, Inc.
Used by permission
Format adapted for dissertation
19
Abstract
Lysosomes critically regulate the pH-dependent catabolism of extracellular and
intracellular macromolecules delivered from the endocytic/heterophagy and autophagy
pathways, respectively. The importance of lysosomes to cell survival is underscored not
only by their unique ability to effectively degrade metalloproteins and oxidativelydamaged macromolecules, but also by the distinct potential for induction of both caspasedependent and –independent cell death with a compromise in the integrity of lysosome
function. Oxidative stress and free radical damage play a principal role in cell death
induced by lysosome dysfunction, and may be linked to several upstream and
downstream stimuli including alterations in the autophagy degradation pathway,
inhibition of lysosome enzyme function and lysosome membrane damage. Neurons are
sensitive to lysosome dysfunction and the contribution of oxidative stress and free radical
damage to lysosome dysfunction may contribute to the etiology of neurodegenerative
disease. This review provides a broad overview of lysosome function and explores the
contribution of oxidative stress and autophagy to lysosome dysfunction-induced neuron
death. Putative signaling pathways that either induce lysosome dysfunction and/or result
from lysosome dysfunction, and the role of oxidative stress, free radical damage and
lysosome dysfunction in pediatric lysosomal storage disorders (Neuronal Ceroid
Lipofuscinoses or NCL/Batten Disease) and in Alzheimer’s disease are emphasized.
20
Introduction
Lysosomes were discovered over 50 years ago by Christian de Duve in a series of
serendipitous experiments aimed originally at characterizing liver glucose 6phosphatase[1]. De Duve discovered the association of glucose 6-phosphatase with a
labile enzyme called acid phosphatase, which fractionated with populations of
mitochondria and microsomes. Upon further optimization of their fractionation protocols
a “light mitochondrial” fraction was discovered that was intermediate in sedimentation to
that of mitochondria and microsomes. Subsequent analysis of this purified fraction
delineated several more enzymes, one of which was cathepsin D (CD) that had acid pH
optima. Today the scientific community appreciates the lysosome as an organelle with
the critical function of regulating the pH-dependent degradation of intracellular
macromolecules. The ability of lysosomes to compartmentalize degradation within their
lumen protects the rest of the cell from the transient induction of oxidative stress and
cytoplasmic degradation. Under conditions of cell stress, however, lysosome function
and integrity may become compromised and can trigger regulated cell death.
Instrumental in this cell death induction are alterations in the vesicular recycling pathway
autophagy, which can induce lysosomal dysfunction and/or become compromised as a
result of lysosomal dysfunction. In addition, oxidative stress may cause direct,
intralysosomal damage or cause secondary lysosomal damage through the increased
production of damaged macromolecules or organelles. This review will provide an
overview of lysosome function and the role that oxidative stress and autophagy play to
lysosomal damage. Lysosomal death pathways will be explored in great detail, with
particular focus to their role in age-related neurodegenerative diseases including
21
Alzheimer’s disease and the pediatric neurodegenerative disease Neuronal Ceroid
Lipofuscinoses (NCL)/Batten Disease.
Lysosome Structure, Function and Assembly
Lysosomes serve an important intracellular role as the site for the terminal
proteolytic degradation of damaged proteins and organelles, which is accomplished in the
range of pH 4.5-5 via greater than 50 lysosomal hydrolases with acidic pH optima [2]
Morphologically, lysosomes are cytoplasmic dense bodies that are either spheroid, ovoid
or occasionally tubular in appearance[2]. Neuron lysosomes are typically less than 1
micron in size and are often situated in a perinuclear position [2]. Lysosomal hydrolases
are surrounded by a limiting membrane containing an abundance of glycosylated proteins
[3]. An intact lysosomal membrane provides the barrier necessary to maintain such a low
pH compared to the neutral pH of the surrounding cytosol. There are upwards of two
dozen cathepsins with specificities for different peptide bonds, including the cysteine
proteases cathepsins B (CB), H and L or the aspartic acid protease CD. Lysosomal
hydrolases catalyze the pH-dependent degradation of proteins into amino acid pools for
intracellular recycling. As will be discussed in subsequent sections, the increase in posttranslational oxidative modifications has been shown to decrease the effective
degradation of proteins by lysosomal hydrolases and may lead to an increase in protein
accumulation, which may contribute to the increase in autofluorescent lipopigment in
post-mitotic neurons[4].
22
Although lysosomal hydrolases reside at their terminal location in lysosomes,
their synthesis and transport to lysosomes requires a complex series of events that carries
them through many different organelles and vesicles (Fig. 1). As such, their localization
to lysosomes must be confirmed either by co-localization with lysosomal membrane
proteins such as LAMP-1 or LAMP-2[5] or by subcellular fractionation. Lysosome
synthesis begins initially in the endoplasmic reticulum (ER)[6], where newly synthesized
hydrolases contain an N-terminal, 20-25 amino acid signal peptide which allows their
translocation into the ER lumen. Upon cleavage of the signal peptide, oligosaccharides
are added onto the hydrolases, which allows the enzymes to be equipped with mannose6-phosphate (M6P) recognition markers in the trans Golgi network (TGN). This M6P
tag allows lysosomal hydrolases to recognize and bind to M6P receptors (M6PRs), and
the receptor-ligand complex subsequently exits from the TGN in clathrin-coated vesicles
as they deliver their contents directly to late endosomes or indirectly via delivery to early
endosomes, which are thought to mature into late endosomes. Endosomes exhibit an
acidic pH like lysosomes but can be distinguished from lysosomes in that lysosomes are
M6PR-negative.
The low pH of endosomes facilitates dissociation of lysosomal
hydrolases from M6PRs, which allows the vesicle-mediated recycling of M6PRs back to
the TGN. Concomitant with further maturation steps, including de-phosphorylation,
oligosaccharide trimming and proteolytic activation, lysosomal hydrolases arrive to the
lysosomes, events that are mediated most likely by a type of fusion event between the late
endosome and lysosome[7].
23
Autophagy
Intracellular macromolecules and organelles are delivered to lysosomes for
degradation and recycling by autophagy (Greek for “eat oneself”), and there are several
types of autophagy that dictate the manner in which macromolecules and organelles
arrive at the lysosome [8].
Arguably the best studied type of autophagy is
macroautophagy (Fig. 1), which involves the generation of a double-membraned
autophagosome that forms non-selectively around bulk cytoplasm, and the shuttling of
these contents through a series of vesicular fusion events to the lysosomes for pHdependent degradation by lysosomal hydrolases (for review see [9] ). Autophagosomes
may fuse either with late endosomes or lysosome[9] which both contain lysosomal
hydrolases in an acidic environment that facilitates degradation.
The fusion of
autophagosomes with endosomes forms single-membraned amphisomes[10],[11] which
fuse ultimately with lysosomes for terminal degradation. Macroautophagy is induced by
intracellular nutrient stress and/or energy depletion and is regulated at multiple levels by
upwards of 30 known autophagy-related gene (Atg) proteins, including signals that
stimulate autophagy induction, the initiation and completion of autophagic vacuole
formation, and the recycling of autophagic vacuoles (for review see [9]). Chaperonemediated autophagy (CMA) is a more selective form of autophagy in which specific
cytosolic proteins with “KFERQ” sequences are targeted by chaperone proteins such as
hsc70 to the lysosome, followed by internalization in lysosomes by the membrane-bound,
Lamp2a receptor[12]. Microautophagy is a less well-defined type of autophagy in which
lysosomes directly ingest cytosolic nutrients by membrane involution[13]. Although
microautophagy has been identified and studied in simple organisms such as yeast, its
24
occurrence and significance in mammalian cells is unclear.
Organelle-specific
macroautophagy (e.g. mitophagy, reticulophagy, etc.) has also been identified and may
selectively target
damaged
organelles
for
lysosomal
degradation[14],[15],[16].
Heterophagy by definition is distinct from autophagy because it involves the intracellular
degradation of extracellular material, which is mediated by endocytosis and the delivery
of material to lysosomes from endosomes[17].
Redox-Reactive Iron and Intralysosomal Damage
Lysosomes play a critical role in the breakdown of iron-containing
macromolecules upon their delivery to lysosomes by autophagy, and as such the
lysosome contains high levels of iron [18-21]. Metalloproteins such as ferritin have been
shown to rely on intact lysosome function for their effective degradation and removal of
iron, which is thought to provide an important source of free iron for essential
intracellular functions[22-25]. While the compartmentalization of high concentrations of
potentially redox-active iron within lysosomes is in theory a protective measure for the
rest of the cell, it may also increase the susceptibility for intralysosomal damage and the
induction of cell death[26]. The brain and neurons in particular contain relatively high
levels of iron and iron has been shown to accumulate in neurons with aging[27], which
further
implicates
the
potential
for
iron-mediated
damage
in
age-related
neurodegenerative disease. Ferric iron (containing at least one uncoordinated ligand)
may react with hydrogen peroxide in forming ferrous iron along with the deleterious
hydroxyl radical by the Fenton reaction[28]. The acidic pH of lysosomes in addition to
25
the presence of reducing equivalents such as cysteine provide a hospitable environment
for Fenton chemistry[29], and hydrogen peroxide may readily diffuse into the lysosomal
lumen from the cytoplasm, especially under conditions of oxidative stress. In addition,
lysosomes do not ordinarily contain reducing enzymes such as catalase or glutathione
peroxidase unless they are being degraded by autophagy, which exacerbates the potential
for reactive iron-induced damage in lysosomes[30]. Hydroxyl radical can oxidize a host
of macromolecules including lipids and proteins, which may not only inhibit their
degradation and contribute to the accumulation of intralysosomal lipofuscin as discussed
below, but they may also inhibit the function of lysosomal hydrolases, further decreasing
the degradative capacity of lysosomes[31;32]. In addition, the accumulation of oxidized
lipoproteins within lysosomes may negatively impact the integrity of lysosomal
membranes and provide a stimulus for the induction of lysosomal membrane
permeabilization (LMP), as discussed below.
Conversely, the autophagy of thiol-rich proteins including metallothioneins has
been proposed to counteract lysosomal damage by binding redox-active iron and other
transition metals such as zinc within lysosomes, thus decreasing the probability of Fenton
chemistry from occurring[33;34]. In addition, under some experimental conditions the
iron chelator desferrioxamine has been shown to attenuate cell damage and cell death
through its ability to localize within lysosomes and bind intralysosomal free iron[35-38].
Lipofuscin and Oxidative Stress
Lipofuscin is an intralysosomal waste material that accumulates in post-mitotic
cells such as neurons as a function of aging, or in dividing cells whose rate of
26
proliferation has been compromised (reviewed in [39] ). The makeup of lipofuscin is
chemically and morphologically amorphous, consisting of protein and lipid,
carbohydrates, transition metals and autofluorescent pigment[40]. The accumulation of
lipofuscin in post-mitotic cells is closely related to a compromise in its effective
degradation, combined with a lack of effective exocytosis[41]. Lipofuscin accumulation
is associated with age-related neurodegenerative diseases such as Alzheimer’s [42-44]
and in lysosomal storage disorders including NCL/Batten Disease[45], which may be
related in part to known alterations in the macroautophagy-lysosomal degradation
pathway that exist in these diseases. While it is clear that lipofuscin accumulation
correlates with lysosome dysfunction, it is not clear the extent to which its accumulation
directly contributes to the induction of neuron death, although adverse effects on cell
function have been reported[46], and an increased susceptibility of lipofuscin-loaded
fibroblasts to apoptosis[47].
Regardless, the finding that up to 75% of a neuron’s
perikarya may contain lipofuscin (reviewed in [48] ) suggests that altered lysosome
function may exacerbate the sensitivity to neurons to lysosomal death signals.
The inhibition of lipofuscin degradation may result from either the inhibition of
lysosomal hydrolases and/or an increase in oxidative stress. Lipofuscin accumulation has
been described experimentally by the chemical inhibition of lysosomal hydrolases, either
from treatment with protease inhibitors or from the lysosomotropic agent chloroquine[4951].
Age-related decreases in the activity of lysosomal hydrolases have also been
documented which may contribute to the age-related increase in lipofuscin with normal
brain aging[52;53]. Conversely, the overloading of cells with lipofuscin has been shown
to cause a decrease in the activity of lysosomal hydrolases[54], suggesting that lipofuscin
27
accumulation per se may also initiate a compromise in lysosome function. The ability of
oxidative stress to enhance lipofuscinogenesis has been documented in several cell
types[55-57].
Lipofuscinogenesis may be caused by proteins that are oxidatively
modified outside the lysosome and subsequently delivered to lysosomes for degradation,
or may be caused by the intralysosomal formation of reactive oxygen species (ROS), as
suggested by the potential for lysosomal lipoproteins to acquire oxidative cross-links[58].
The effect of either route would be a net increase in oxidatively modified lipofuscin with
an inherent compromise in its degradative capacity. The importance of oxidative stress in
lipofuscin accumulation is further emphasized by its decrease upon experimental
treatment with antioxidants or the iron chelator desferrioxamine[59]. In addition, the
inhibition of lysosomal hydrolases may exacerbate the oxidative stress-induced
accumulation of lipofuscin, since a compromise in intralysosomal enzymatic protein
degradation would provide greater opportunities for such proteins to acquire oxidative
modifications that contribute to lipofuscin accumulation. In support of this argument, the
accumulation of lipofuscin induced by combined oxidative stress and protease inhibition
was shown to be three times greater than that observed by either condition alone[60].
Lipofuscin is formed from a variety of intracellular sources that are delivered to
lysosomes by the autophagy degradation pathway (for review see [9] ). The induction of
macroautophagy may provide a potent stimulus for lipofuscin accumulation (Fig. 2).
Nutrient deprivation and resultant oxidative stress are natural stimuli for macroautophagy
induction and as such may result in the increased delivery of undegradable, oxidatively
modified proteins to lysosomes that accumulate as part of lipofuscin. Along these lines,
ROS induced by starvation were found recently to critically regulate macroautophagy
28
induction through the cysteine-dependent activity of Atg4, an autophagy-specific protein
that regulates autophagosome formation[61]. The induction of mitophagy may also
increase the lysosomal delivery of oxidatively damaged mitochondrial membranes and
proteins in addition to superoxide anion, which is generated normally in mitochondria by
the electron transport chain[62].
In further support of mitophagy contributing to
lipofuscin accumulation, subunit c of mitochondrial ATP synthethase has been shown to
be a major component of lipofuscin, in particular in aged neurons[63]. An increase in
intralysosomal redox-active iron may also result from the autophagy-mediated
degradation of ferritin[64-67]. Under conditions of oxidative stress, the diffusion of
readily available hydrogen peroxide into the lysosomal lumen may drive Fenton
chemistry to form the highly reactive hydroxyl radical that would promote oxidative
cross-links that enhance lipofuscin accumulation, a hypothesis that has been previously
proposed and is further supported by the increase in lipofuscin accumulation upon
inhibition of lysosomal proteases[68]. The generation of intralysosomal free radicals
may cause peroxidation of membrane poly-unsaturated fatty acids to form relatively
stable and cytotoxic aldehydes, alkenals or hydroxyalkenals including malondialdehyde
or 4-hydroxy-nonenal (4-HNE);[69]. Treatment of purified protein with 4-HNE, for
instance, has been shown not only to form protein cross-links[70-72] and generate
protein-associated fluorescence similar to that found in the autofluorescent lipofuscin[7375] but also cause enzyme inactivation[69;76-80] that may further enhance lipofuscin
accumulation.
Inhibition of macroautophagy completion may also contribute to the accumulation
of lipofuscin (Fig. 2), as has been shown previously by treatment with the lysosomotropic
29
agent chloroquine or with protease inhibitors[51;81;82]. Treatment with lysosomotropic
agents and protease inhibitors has been shown to increase intralysosomal ferritin stability
and decrease the available pools of redox-active iron[83;84], which in contrast to
macroautophagy induction, may suggest a limited role for redox-active iron and Fenton
chemistry in the intralysosomal production of ROS following macroautophagy inhibition.
Rather, the inhibition of lysosomal hydrolases may initially play a more direct role in
lipofuscin accumulation following macroautophagy inhibition, since in this setting it
would be logical to predict a more direct compromise in lysosome function as the initial
stimulus for altered macroautophagy.
Since oxidized lipoproteins or lipofuscin
accumulation has been shown experimentally to decrease the activity of lysosomal
hydrolases[85;86], it is possible that lipofuscin accumulation per se may also initiate a
compromise in lysosome function that would lead to macroautophagy inhibition, perhaps
as a response to initial macroautophagy induction. This explanation is attractive for the
etiology of Alzheimer’s disease neuropathology, since it has been hypothesized
previously that macroautophagy is induced early in the course of AD onset which is
followed in later stages by macroautophagy inhibition[87].
Lysosomotropic Agents Generate Oxidative Stress
Christian De Duve coined the term “lysosomotropic” in 1974[88] to delineate a
group of uncharged compounds, typically amphiphilic weak bases, that are attracted to
acidic compartments within cells, or are in other words “acidotropic.” Such uncharged
molecules diffuse passively through the membranes of acidic organelles including
30
lysosomes, which have a typical pH range of 4.5-5[89]. Once inside lysosomes these
agents become protonated and their charge effectively precludes their transport across
lysosomal membranes, resulting ultimately in an effective increase in intralysosomal pH
and the impairment of lysosome-mediated degradation[90;91]. Accumulation of such
agents in lysosomes depends initially on the pH gradient between the intra- and extralysosomal compartments and can be prevented by the prior increase in intra-lysosomal
pH.
Chloroquine (7-chloro-4-(4-dimethylamino-1-methylbutylamino)quinoline; (see
structure, Fig. 3) is a well-known anti-malarial agent that has been used for many years to
investigate lysosome function.
Chloroquine exerts its anti-malarial effects by
concentrating in the acidic digestive vacuole of Plasmodium parasites, where it is
hypothesized to complex with ferric heme (ferriprotoporphyrin IX, FPIX) monomer[92]
that is produced upon parasitic degradation of host hemoglobin. By complexing with
FPIX, chloroquine promotes accumulation of the toxic, undimerized form of FPIX, which
increases susceptibility to iron-dependent peroxidation of lipid membranes[93], an effect
that has been observed upon treatment of liposomes with the chloroquine-FPIX
complex[94]. It is thus reasonable to predict that chloroquine also forms a similar type of
lipid peroxidation-generating complex with iron-containing proteins in the lysosomes of
mammalian cells. In support of this argument, chloroquine has been shown to effectively
inhibit the intralysosomal release of free iron from ferritin, which is known to require
intact lysosome function[95;96]. Regardless, chloroquine does induce lipid peroxidation
in mammalian cells[97-99] and future studies are needed to delineate if this occurrence is
specific for lysosomal membranes.
Because chloroquine effectively inhibits the
31
intralysosomal release of free iron from ferritin, Fenton chemistry may not play a
principal role in the induction of lysosomal damage mediated by chloroquine and
subsequent macroautophagy inhibition.
Alternatively, chloroquine-induced oxidative damage to lysosomal membranes
and the accumulation of oxidatively modified lipoproteins may result from
macroautophagy inhibition combined with its inhibition of lysosomal proteases[100-104]
mechanisms that may be responsible for its induction of lipofuscin as previously
described[105]. Chloroquine has also been shown recently to reduce intracellular levels
of glutathione[106], which could lead to an increased production of cytosolic hydrogen
peroxide and concomitant extralysosomal damage of macromolecules and organelle
membranes.
The intralysosomal accumulation of chloroquine has been shown to induce
profound alterations in lysosome function, including inhibition of both the proteolytic
maturation and enzyme activities of CB and CD [107-112], which may be secondary to
chloroquine-induced increase in intralysosomal pH and disruption of pH optima for these
enzymes. In our laboratory we have observed similar results in SH-SY5Y cells, such that
a death-inducing concentration of chloroquine markedly decreases maturation of CD as
measured by western blot (Fig. 4). However, recent reports also indicate that chloroquine
increased CD levels as measured by western blot, but it is unclear from these studies
which form of CD (pro vs. mature, “active” forms) was increased [113;114]. Earlier
studies reported an increase in lysosome size or swelling by chloroquine and other
lysosomotropic agents [115-117], which results from intralysosomal chloroquine
32
reaching isotonicity with levels in the cytosol and the subsequent increase in water flow
into the lysosome. Such “swollen” lysosomes may exhibit increased membrane fragility,
as indicated in isolated preparations by their increased latency to release lysosomal
enzymes[115;118] by an increase in lysosomal enzymes in purified cytosolic
preparations[119]. These findings clearly suggest the induction of LMP and may play a
significant role in the induction of cell death following chloroquine treatment, as
described below.
ROS, Autophagy and Lysosomal Membrane Permeabilization (LMP) as Death Stimuli
The susceptibility of lysosomes to oxidative stress and/or membrane
destabilization is thought to play a major role in the induction of LMP, which results in
the release of lysosomal enzymes into the cytosol and the potent induction of cell death.
Both macroautophagy induction[120] and inhibition[121] have been shown to potently
regulate cell death through the induction of LMP, which may involve the generation of
reactive oxygen species (Fig. 5). For many years it was believed that LMP-induced cell
death was un-regulated and necrotic in nature[122]. Today it is well-established that
LMP may induce both apoptosis and necrosis, which seems to depend in part on the
magnitude of LMP and the amount of proteolytic enzymes that are released into the
cytosol. Many studies have indicated that stimuli which produce LMP tend to induce
apoptosis at lower concentrations and necrosis at higher concentrations[123]. Since
multiple types of cell death can be induced following LMP, it is not surprising that the
inhibition of apoptosis following LMP has been shown to shunt the type of death to a
33
more necrotic nature[124]. To this end, we have also shown that the inhibition of Baxdependent neuron death following lysosome dysfunction does not attenuate the degree of
neuron loss or neurodegeneration[125].
The cysteine protease CB and the aspartic acid protease CD are two of the most
ubiquitous lysosomal enzymes[126] and as such they have been shown to play a major
role in the stimulus-specific induction of cell death following LMP. Since lysosomal
hydrolases possess optimal activation at acidic pH, it is fair to question their ability to
function once released into the cytosol. However, in vitro studies have shown that
lysosomal proteases can function for several minutes to over an hour at neutral pH[127] ,
confirming their potential for activation outside of lysosomes. In addition, recent studies
have indicated that the cytoplasmic pH is lowered in the course of cell death[128],[129],
which increases the potential for lysosomal proteases to directly influence cell death
following LMP.
Many studies have utilized hydrogen peroxide to generate oxidative stressinduced LMP and apoptosis, in both neural [130] and non-neural cell types [131;132]. In
addition, studies have indicated the induction of LMP by other stimuli that indirectly
induce hydrogen peroxide, including TNF-α [133] and lipopolysaccharide[134]. The
induction of LMP by hydrogen peroxide is believed to occur through its ability to freely
diffuse from the cytosol into iron-rich lysosomes, where it utilizes Fenton chemistry to
induce production of the highly reactive hydroxyl radical[135].
In addition, both
hydrogen peroxide and stimuli known to indirectly produce hydrogen peroxide (such as
TNF-α) have been shown to induce activation of phospholipase A2 (PLA2), which in
34
theory stimulate the degradation of membrane lipids that could potentially increase
lysosome destabilization and LMP[136].
LMP-induced apoptosis has also been
evidenced following treatment with other oxidative stress-inducing compounds, including
napththazarin [137]which generates ROS through redox cycling, and hypochlorous acid,
shown recently to induce lysosome destabilization in cultured cortical neurons[138].
ROS-induced LMP is a potent stimulus that has been shown in many studies to
precede the induction of mitochondrial-dependent apoptosis[139], which has also been
indicated by treatment with lysosomotropic agents or other agents that mediate indirect
production of ROS[140]. In addition, several studies have shown that CB[141] and CD
[142] mediate mitochondrial apoptosis, findings which strongly implicate LMP in the
“lysosomal-mitochondrial axis” theory of cell death as previously described[143].
Further proof of this paradigm came from an elegant study whereby the cytosolic
microinjection of CD induced caspase-dependent death, an effect that was inhibited by
combined microinjection of CD with its inhibitor pepstatin A[144].
Conversely,
lysosomal enzymes have been shown to increase production of mitochondrial ROS,
which may result in further lysosomal destabilization as part of a deleterious feedback
loop[145].
Recent studies have shown that one mechanism by which cytosolic cathepsins
induce mitochondrial apoptosis is through direct effects on Bcl-2 family members (Fig.
5). This concept was first suggested by Stoka et al in 2001, which reported cleavage of
the pro-apoptotic Bcl-2 family member Bid by lysosomal extracts, and the ability of this
cleavage product to induce cytochrome c release from mitochondria[146]. Bid cleavage
35
along with induction of mitochondrial apoptosis was first shown to be mediated via the
cysteine protease caspase-8[147] . A follow-up study confirmed that CB is directly
responsible, at least in part, for Bid cleavage and induction of mitochondrial
apoptosis[148]. Another study has suggested that CD plays a role in apoptosis mediated
by Bid cleavage following treatment with ceramide[149]. In addition, recent evidence
has shown that following the induction of LMP, cytosolic CD interacts directly with proapoptotic Bax in the promotion of mitochondrial apoptosis by a variety of stimuli
including treatment with hydrogen peroxide[150]. This CD-Bax-mitochondrial death
pathway has also been shown to stimulate downstream mitochondrial release of
apoptosis-inducing factor (AIF)[151], a mitochondrial flavoprotein that upon release
from mitochondria is implicated in caspase-independent apoptosis and necrosis[152].
Thus the interaction of cytosolic cathepsins with Bcl-2 family members has the potential
to induce multiple types of cell death, and future studies are warranted to determine if this
pathway also plays an important role in the induction of neuron death in acute injury or
neurodegenerative disease.
For many years it was widely believed that the regulation of cell death by Bcl-2
family members was due solely to their manipulation of mitochondrial membrane
integrity. However there have been several intriguing studies over the last few years
suggesting that other organelles, including the ER and lysosomes, may also be regulated
by Bcl-2 family members in the induction of cell death[153]. The first reports of Bcl-2
family-mediated regulation of lysosome function were from the laboratory of Ulf Brunk,
which suggested that lysosome-localized Bcl-2 attenuated hydrogen peroxide-induced
apoptosis, at least in part by promoting lysosome stabilization[154]. Subsequent studies
36
have shown that pro-apoptotic Bax not only localizes to lysosomal membranes following
stressful stimuli but also regulates the induction of LMP[155]. The BH3 domain-only
molecules Bim and Bad were also shown to localize to lysosomes following a death
stimulus and regulate the induction of LMP, although their induction of LMP required the
presence of Bax[156]. Together these findings support the potential for the additional
“upstream” influence of Bcl-2 family members in the regulation of lysosome-dependent
neuron death (Fig. 5), and as a result, the potential for their regulation of multiple types
of neuron death.
Chloroquine-Induced Neuron Death
One of the most striking observations following treatment of cells or tissues with
chloroquine is the massive accumulation of autophagic vacuoles that results from the
inhibition in completion of the macroautophagy-lysosomal degradation pathway. We and
others have shown that sustained incubation with chloroquine potently induces cell death
that is characterized by morphological and biochemical markers of apoptosis and is
preceded by autophagic vacuole accumulation[157-159]. In our laboratory chloroquineinduced cell death has been evidenced in a variety of cell types, including immature and
fully-differentiated primary neurons, neural precursor cells and a variety of neural cell
lines (Fig. 4).
At present, whether the accumulation of AVs directly mediates
chloroquine-induced neuron death has not been thoroughly investigated, although the
ability of macroautophagy inhibition to induce cell death has been clearly indicated
previously in the literature[160;161]. Death induced by macroautophagy inhibition may
37
result from a compromise in homeostatic organelle turnover, thus increasing the
accumulation of damaged organelles with compromised function which could trigger the
initiation or completion of death pathway signaling.
Certainly the accumulation of
undegradable oxidized lipoproteins may cause associated damage to lysosomal
membranes.
Mitochondrial dysfunction appears to play a major role in chloroquine-induced
cell death, as indicated previously by a decrease in mitochondrial membrane potential
and an attenuation of cell death by the targeted disruption of pro-apoptotic bax or bcl-2
over-expression, and the exacerbation of cell death following the targeted disruption of
anti-apoptotic bcl-x [162-164].
Although chloroquine induces robust activation of
caspase-3 the targeted genetic disruption of caspase-3 or treatment with general caspase
inhibitors does not attenuate chloroquine-induced neuron death[165;166]. Together these
findings suggest either that the commitment point for chloroquine-induced neuron death
lies upstream of caspase activation, or indicates the potential for both caspase-dependent
and-independent death pathways triggered by disruption of the macroautophagylysosomal degradation pathway. We have also shown that chloroquine-induced death of
immature neurons is attenuated by the protooncogene p53, an effect that was not
observed in cultures of post-mitotic neurons (68, 70) which suggests that p53-dependent
autophagic cell death may be cell-type and/or differentiation dependent.
As such,
chloroquine-induced, p53-dependent autophagic death is being actively investigated as a
potential therapeutic target in several types of cancers, including glioblastomas[167].
38
We have shown recently that the plecomacrolide antibiotic bafilomycin A1
(BafA1) and other structurally similar compounds, significantly attenuates chloroquineinduced neuron death[168], at concentrations (≤ 1 nM) shown previously not to inhibit
vacuolar-type ATPase[169]. While a previous study suggested that a high dose of 100
nM BafA1 attenuated cell death induced by hydroxychloroquine by attenuating the pHdependent fusion of chloroquine into the lysosome[170], our results suggest that
“neuroprotective” concentrations of BafA1
≤ 1( nM) do not alter the ability of
chloroquine
to
inhibit
macroautophagy,
since
AVs
still
accumulate
in
chloroquine+BafA1-treated cells, concomitant with an absence of apoptotic morphology,
and that the chloroquine-induced inhibition of long-lived protein degradation was not
affected by 1 nM BafA1[171]. Ongoing studies in our laboratory are delineating the
potential mechanisms by which plecomacrolide antibiotics attenuate neuron death
induced by lysosomotropic agents and whether cell death induced by other disruptions in
lysosome function can also be attenuated by plecomacrolides.
Chloroquine-induced cell death was shown previously to involve LMP, as
indicated immunocytochemically by the diffuse cytosolic immunoreactivity of the
lysosomal protease CB in chloroquine-treated cells[172]. LMP was suggested as an
upstream mediator of mitochondrial cell death, since selective inhibition of CB
significantly attenuated chloroquine-induced mitochondrial dysfunction concomitant with
an increase in viability[173]. Interestingly, chloroquine also enhances the extracellular
secretion of many lysosomal enzymes including β-hexaminodase, CB and CD[174],
which effectively blocks the delivery of newly synthesized lysosomal hydrolases to
lysosomes. It will be important in future studies of chloroquine-induced LMP to confirm
39
results of immunocytochemistry with rigorous biochemical analyses indicating an
increased appearance of lysosomal enzymes in purified cytosolic fractions via western
blot.
Chloroquine has also been shown to inhibit the activities of sphingolipid
metabolizing enzymes including sphingomyelinase and acid ceramidase[175;176] which
are localized to lysosomes and most likely reflects the deleterious alterations in lysosome
function that are induced upon chloroquine treatment.
Inhibition of these lipid
metabolizing enzymes causes the accumulation of ceramide and sphingosine, two highly
reactive lipid mediators that have been shown to mediate oxidative stress-induced
apoptosis[177;178]. Sphingosine has been shown to exhibit detergent-like properties
towards lysosome membranes which may contribute to chloroquine-induced LMP and
subsequent apoptosis[179]. Together these results suggest that the aberrant production of
reactive lipid metabolites may not only mediate cell death induced as a result of lysosome
dysfunction mediated during macroautophagy inhibition but may also further exacerbate
lysosome dysfunction and stimulate LMP-induced cell death.
It should be noted,
however, that inhibition of sphingolipid metabolizing enzymes also increases levels of
the anti-apoptotic sphingolipid sphingosine-1-phosphate concomitant with pro-apoptotic
sphingolipids[180], which suggests a potential balance of pro- vs. anti-apoptotic lipid
mediators that must be addressed appropriately to understand the net contribution of lipid
mediators in neuron death regulation.
40
Oxidative Stress, Autophagy and Lysosome Dysfunction in CNS Aging and Alzheimer’s
Disease
There are several properties of the aging brain that make it uniquely susceptible to
age-related oxidative damage. First, neurons are post-mitotic, thus over their lifespan
age-related macromolecular damage accumulates and compromises their function. This
is evidenced by the age-related increase in lipofuscin, which may provide both cause and
effect for age-related declines in lysosome function and autophagy signaling [51;181183]. Second, neurons have high energy demands compared to other cell types and as
such they may be more vulnerable to the deleterious effects of mitochondrial dysfunction,
combined with the fact that the electron transport chain of oxidative phosphorylation
generates ROS [184].
Third, the brain is composed of high amounts of lipids and
transition metals including iron[185-187], which increases the probability of age-related
lipid peroxidation. Lastly, the aging brain contains fewer reducing equivalents that in
theory would contribute to an increase in oxidative stress[188]. Age-related oxidative
stress in the cytoplasm may cause macroautophagy induction, in particular as cytoplasmic
macromolecules or organelles become damaged and are delivered to lysosomes for
degradation.
Conversely, age-related oxidative stress in the lysosome may lead to
macroautophagy inhibition if the result of sustained oxidative stress is a net compromise
in lysosome function.
Many studies have indicated pronounced alterations in the endosomal-lysosomal
pathway in human AD brain, which are some of the earliest reported abnormalities in AD
brain neurons and precede the onset of both Aβ-containing plaque and tangle
41
neuropathology[189]. Enlarged, Aβ-immunoreactive endosomes have been reported in
brains of AD patients[190] prior to Aβ deposition, which suggests a potential for
endosomal-mediated Aβ secretion and deposition. Increased levels of CD have also been
localized to endosomes of AD patients[191;192]. Endosomal CD may be linked to Aβ
formation in that CD possesses inherent β- and γ-secretase activity, enzymes that are
responsible for the cleavage of amyloid precursor protein into Aβ [193] although APP
processing was shown previously to be unaffected by CD deficiency in mice[194].
Increased levels and activity of CB and CD in AD brain have been shown to occur
concomitant with lysosome proliferation[195-199] and may reflect a compensatory
response to altered macroautophagy, but it is not clear if such alterations serve a
beneficial role to promote protein degradation or death signaling. In addition, both CB
and CD have been localized extracellularly to amyloid plaque, which may indicate their
potential to regulate plaque deposition[200;201]. In support of this argument, CB has
been shown to effectively decrease levels of the more amyloidogenic Aβ1-42, and CB
deficiency in mice was shown to cause an increase in extracellular Aβ deposition[202].
Alterations in the autophagy-lysosomal degradation pathway have been indicated
in AD by pathological increases in autophagic vacuoles (AVs) observed in cortical
biopsies obtained from AD brain[203;204]. Accumulating AVs in AD brain have been
found to localize in large part to dystrophic neurites, which may be related to alterations
in intracellular trafficking that either cause AV accumulation or result from AV
accumulation.
Both immature, double-membraned autophagic vacuoles and mature,
single-membraned autophagolysosomes have been shown to accumulate in AD
dystrophic neurites, which implicates both macroautophagy induction and inhibition in
42
AD and may reflect both an early and late autophagic response of individual neurons to
AD-associated stress. Recent evidence suggests the localization of Aβ in AVs both in
human AD brain and in experimental models of AD, and that the processing of APP into
Aβ may even occur within AVs[205]. The intracellular accumulation of Aβ has also
been reported following the chemical inhibition of macroautophagy completion mediated
via in vivo and in vitro treatment with chloroquine[206;207], providing further evidence
that macroautophagy plays a vital role in Aβ processing and degradation.
Taken together, evidence suggests that alterations in both the endosomallysosomal and autophagy-lysosomal degradation pathways play an intimate role in the
generation of AD neuropathology, and oxidative stress may play a major role in inducing
alterations in intracellular recycling pathways. Oxidative stress has been proposed to
play a major role in the onset and progression of AD.
Many reports of increased
oxidative damage have been reported in AD brain (reviewed in[208;209], which may
have important ramifications on the macroautophagy-lysosome degradation pathway. An
increase in mitophagy has been reported in AD brain[210], which is likely a response of
autophagy to clear oxidatively damaged mitochondria in post-mitotic neurons. Treatment
of neuronal cells under conditions of oxidative stress was shown recently to induce
macroautophagy of Aβ and promote its localization in lysosomes[211], which may reflect
a stimulus early in the progression of AD to clear intracellular levels of Aβ. The effects
of oxidative stress on lysosomal function in experimental models of AD have not been
directly tested, although as described below, treatment with Aβ produces profound effects
on lysosomal function that may be related in part to the generation of oxidative stress.
43
Aβ-Induced Neuron Death
While it is obvious that there is definite progression of neuron loss in AD, studies
of AD brain have in large part shown inconsistent findings regarding a role for apoptosis
as an important mechanism of neuron death (reviewed in [212] ). This variability of
results may be explained by the inherent heterogeneity of the human AD population at
the time of tissue biopsy and by differences in the processing of postmortem tissue. In
addition, there is an inherent challenge in proving with great confidence the relevance of
neuron death mechanisms in age-related neurodegenerative disease, since only a small
number of neurons succumb to cell death at any one point in time. Nevertheless, there
are countless studies focused on delineating the mechanisms of Aβ-mediated neuron
death as a contributing factor to neuron loss in human AD brain.
Results of in vitro studies indicate a clear link between Aβ, oxidative stress and
cell death.
Many studies have shown that Aβ-induced cell death and apoptosis is
mediated by oxidative stress[213], effects that in many cases were inhibited upon
treatment with antioxidants[214]. Treatment with Aβ has been shown to increase free
radical production and markers of oxidative stress[215], and the induction of oxidative
stress has been shown to induce the intracellular accumulation of Aβ [216]. As discussed
above, the autophagy-lysosomal degradation pathway is a sensitive target for oxidative
stress-induced damage, and treatment with soluble forms of Aβ at death-inducing
concentrations has been shown to result in its intralysosomal localization[217] and
induction of intralysosomal oxidative stress[218] and the induction of LMP[219]. Other
alterations in lysosome function, including alterations in levels of CD have also been
44
reported following Aβ treatment[114]. While many studies of Aβ-induced cell death
suggest a role for apoptosis[220], our laboratory has previously shown that Aβ-induced
neuron death is Bax-dependent but caspase-independent[221]. Even though we observed
activation of caspase 3 following treatment with Aβ, neither inhibition of caspase-3 nor
the targeted genetic disruption of caspase-3 attenuated Aβ-induced neuron death[222].
Together these findings suggest that Aβ may play a significant role in oxidative stressinduced neuron death in AD brain, although the role of caspase-dependent apoptosis is
still controversial. The likely disruption of the macroautophagy-lysosomal degradation
pathway in AD brain, however, suggests the potential contribution of multiple types of
neuron death, both caspase-dependent and –independent, to AD neuropathology.
Regulation of neuron death in CD-deficient mice as a model of NCL/Batten Disease
NCL is a heterogeneous group of pediatric lysosomal storage disorders known
collectively as Batten Disease. Clinical features of NCL/Batten disease include seizures
and progressive blindness with eventual loss of motor control and ultimate death[223].
NCLs were classified originally by their age of onset, and include congenital (at birth),
infantile (INCL, within 1 year of birth); late infantile (LINCL, 2 to 4 years); juvenile
(JNCL, 4 to 7 years); or the very rare adult form (ANCL). Presently there are seven
known gene mutations in humans that cause NCL (CLN1, CLN2, CLN3, CLN5, CLN6,
CLN8 and CD) which produce distinct biochemical alterations in lysosome function and
are also defined by the type of storage protein that accumulates as a result of lysosome
dysfunction[224].
45
A major focus of our laboratory is the study of CD deficiency-induced neuron
death as a model of lysosome dysfunction in congenital NCL/Batten disease. It was not
until 2006 that human CD mutations were first reported in two separate studies[225]. In
one study a complete loss of CD function was reported in four patients with congenital
NCL [226],and these patients exhibited perinatal seizures before dying by two weeks of
age. In the other study, a partial loss of CD enzymatic activity was observed in an
adolescent patient that was diagnosed with NCL-like symptoms at early school-age [227].
Prior to the finding of CD mutations in humans, however, sporadic mutations in CD
resulting in characteristic NCL-like phenotype were reported in sheep [228] and more
recently in American bulldogs [229]. In 1995 the effects of experimental CD deficiency
in mice were initially reported in an attempt to further characterize the role of CD in
lysosome function[230]. CD deficiency was found to inhibit bulk proteolysis and CDdeficient mice succumbed to death by postnatal day 26 from a plethora of morbidities
including intestinal necrosis, thromboembolia and seizures[231].
An increase in seizures and blindness in CD-deficient mice led to the subsequent
analyses of brain function in these mice, when it was determined that CD deficiency
resulted in robust neurodegeneration characterized by the massive accumulation of
lipofuscin-laden AVs[232] and significant neuron loss[233]. AVs in CD-deficient mice
were found to accumulate as early as postnatal day 8[234] and their accumulation has
been shown to precede the induction of apoptosis[235], which occurs as early as postnatal
day 16 [236]. While a role for oxidative stress has not been directly verified in the
induction of AV accumulation, the accumulation of lipofuscin and likely inhibition of
autophagy are strong indicators that the over-production of oxidative stress plays a
46
prominent role in CD deficiency-induced neuropathology, and further studies are needed
to confirm this. Previous studies have suggested that the induction of nitrosative stress in
CD-deficient brain accelerates CD deficiency-induced neuropathology, arising potentially
from an increase in nitric oxide and peroxynitrite from microglial activation[237].
Treatment of CD-deficient mice with inhibitors of nitric oxide synthase attenuated the
appearance of apoptotic neurons but not neurons exhibiting AV accumulation and lacking
apoptotic morphology[238]. In particular, such apoptotic neurons were found in many
cases to be localized adjacent to neurons undergoing such “autophagic stress”, which led
to the hypothesis that cells undergoing autophagic neurodegeneration induced microglial
activation which in turn resulted in nitric oxide-dependent apoptotic death of neighboring
neurons[239]. This hypothesis, if correct, would explain subsequent findings in our
laboratory indicating inactivation of pro-survival Akt and activation of pro-apoptotic
GSK-3β in CD-deficient neurons with a time course related to that of apoptosis
induction[240], along with findings in our laboratory indicating inactivation of Akt and
apoptosis induction in cultured cells upon treatment with peroxynitrite[241].
To further investigate the role of apoptosis in CD deficiency-induced neuron
death, we generated mice deficient for both CD and the pro-apoptotic molecule Bax
[242]. While Bax deficiency clearly reduced the induction of apoptosis following CD
deficiency, there was no decrease in neuron loss, neurodegeneration or autofluorescent
storage material[243]. Together these results suggest that while CD deficiency induces
apoptosis, the resultant lysosome dysfunction contributes to the induction of multiple
types of neuron death and that apoptosis plays a limited role in the neurodegenerative
phenotype induced by CD deficiency. While the relative contribution of apoptosis to
47
neuron death and neurodegeneration is obviously disease-specific, it is clear that in the
present day study of neuron death, a whole host of cell death mechanisms should be
considered, including apoptotic vs. non-apoptotic, or with different types of apoptosis
that are either caspase-dependent vs. –independent.
Perspectives
Lysosome dysfunction is quickly emerging as a prominent area of research in
which to study potential mechanisms of neuron death. Multiple types of neuron death
have been delineated in the literature that appear to involve at some level the disruption
of lysosome function, and both the induction of oxidative stress and altered autophagy
signaling have the capacity to regulate neuron death through the lysosome. Potential
therapies such as the phosphodiesterase inhibitor zaprinast, the lysosomal “modulator” ZPhe-Ala-diazomethylketone (PADK), plecomacrolide antibiotics such as BafA1, calpain
inhibitors, cathepsin inhibitors and metal chelators[244] may act through their direct
attenuation of oxidative stress or indirect attenuation of oxidative stress-induced damage,
which in theory would promote the stabilization of lysosome membranes and decrease
the onset of LMP in neurons. As such, the use of these agents will undoubtedly receive
greater prominence in the near future as the lysosome in turn receives greater attention as
a therapeutic target in the onset and progression of neurodegenerative disease.
48
Acknowledgements
We wish to thank Angela Schmeckebier and Barry Bailey for expert technical assistance
in preparation of this manuscript. We also thank the UAB Neuroscience Core Facilities
(NS47466 and NS57098) for technical assistance. This work is supported by grants from
the National Institutes of Health (NS35107 and NS41962), a pilot grant from the UAB
Alzheimer’s Disease Research Center, and a VISN7 Career Development Award from
the Birmingham VA Medical Center.
Author Disclosure Statement
No competing financial interests exist for all authors.
Abbreviations
Aβ
beta amyloid
AD
Alzheimer’s disease
AIF
apoptosis-inducing factor
APP
amyloid precursor protein
Atg
autophagy-related gene
AV
autophagic vacuole
BafA1
bafilomycin A1
CB
cathepsin B
49
CD
cathepsin D
CMA
chaperone-mediated autophagy
ER
endoplasmic reticulum
FPIX
ferriprotoporphyrin IX
4-HNE
4-hydroxy-nonenal
LMP
lysosomal membrane permeabilization
M6P
mannose-6-phosphate
NCL
Neuronal Ceroid Lipofuscinoses
PLA2
phospholipase A2
ROS
reactive oxygen species
TGN
trans-Golgi network
50
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Fig. 1. Convergence of the endosomal-lysosomal and autophagy-lysosomal
degradation pathways. Lysosomal hydrolases are produced in the endoplasmic
reticulum (ER) and upon delivery to the trans-Golgi network (TGN) are transported in
vesicles by recognition of mannose-6-phosphate receptors (M6PRs) to the late endosome
(or to the early endosome which then matures to form the late endosome). The late
endosome is then thought to deliver lysosomal hydrolases via a type of fusion event to
their terminal location, the lysosome, which is M6PR negative. Damaged organelles and
macromolecules are surrounded by a limiting membrane from the ER to form a preautophagosomal structure (PAS), which matures to form the double-membraned
autophagosome. The pH of autophagosomes is not sufficient to degrade their
intralumenal contents and fusion with lysosomes (forming the autophagolysosome) or
with endosomes (forming an amphisome), which both contain pH-dependent acid
hydrolases, must take place for autophagosomal contents to be effectively degraded.
Please refer to text for further details.
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Fig. 2. Macroautophagy induction vs. inhibition in oxidative stress-induced
lysosome damage. The induction of lysosomal membrane damage, LMP and cell death
may be directly influenced by both the aberrant induction vs. inhibition of
macroautophagy, which can lead to the induction of intralysosomal oxidative stress. It
has also been proposed that an initial over-induction of macroautophagy induction may
lead to an eventual inhibition of macroautophagy, which also may be related in part to
the induction of oxidative stress. Please see text for further details.
72
Fig. 3. Chemical structure of chloroquine.
Chloroquine (7-chloro-4-(4dimethylamino-1-methylbutylamino)quinoline) represents the class of fluoroquinolones.
73
Fig. 4. Chloroquine-induced death of human SH-SY5Y cells follows alterations in
the processing of CD. A) Treatment of human SH-SY5Y cells with chloroquine (50
µM) significantly attenuates cell viability at 48h vs. vehicle control but not at 24h.
*p<0.05 vs. vehicle control (student’s unpaired t-test). B) By 24h, chloroquine induces a
modest decrease in the mature “active” form of CD, migrating at approximately 30 kDa,
along with a marked increase in the inactive, “pre-pro” fragment migrating at
approximately 50 kDa, in comparison to vehicle control. After 48h of chloroquine
treatment, levels of the mature active form of CD appear to be further reduced in
comparison to 24h. Levels of β-tubulin (migrating at approximately 50 kDa) serve as the
loading control.
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Fig. 5. Oxidative stress, lysosomal membrane permeabilization and the induction of
necrotic vs. apoptotic death. Agents that promote the direct or indirect production of
oxidative stress may lead to lysosome membrane permeabilization (LMP) and cell death.
It is thought that the induction of total LMP favors the onset of necrosis, whereas partial
LMP favors the onset of apoptosis. LMP is associated with the release of lysosomal
cathepsins into the cytosol and the interaction with pro-apoptotic Bcl-2 family members,
which leads to the induction of mitochondrial apoptosis. Pro-apoptotic Bcl-2 family
members may also act directly at the lysosomal membrane as a stimulus for LMP. For
further details, please see the text.
75
LOW-DOSE BAFILOMYCIN ATTENUATES NEURONAL CELL DEATH
ASSOCIATED WITH AUTOPHAGY-LYSOSOME PATHWAY DYSFUNCTION
by
VIOLETTA N. PIVTORAIKO, ADAM J. HARRINGTON, BURTON J. MADER,
AUSTIN M. LUKER, GUY A. CALDWELL, KIM A. CALDWELL, KEVIN A. ROTH
AND JOHN J. SHACKA
Journal of Neurochemistry (2010) Vol. 114(4):1193-204
Copyright 2009
by
Wiley-Blackwell Publishing
Used by permission
Format adapted for dissertation
76
Abstract
We have shown previously that the plecomacrolide antibiotics bafilomycin A1 and B1
significantly attenuate cerebellar granule neuron death resulting from agents that disrupt
lysosome function. To further characterize bafilomycin-mediated cytoprotection, we
examined its ability to attenuate the death of naïve and differentiated neuronal SH-SY5Y
human neuroblastoma cells from agents that induce lysosome dysfunction in vitro, and
from in vivo dopaminergic neuron death in C. elegans.
Low-dose bafilomycin
significantly attenuated SH-SY5Y cell death resulting from treatment with chloroquine,
hydroxychloroquine amodiaquine and staurosporine. Bafilomycin also attenuated the
chloroquine-induced reduction in processing of cathepsin D, the principal lysosomal
aspartic acid protease, to its mature “active” form. Chloroquine induced autophagic
vacuole accumulation and inhibited autophagic flux, effects that were attenuated upon
treatment with bafilomycin and were associated with a significant decrease in
chloroquine-induced accumulation of detergent-insoluble α -synuclein oligomers.
In
addition, bafilomycin significantly and dose-dependently attenuated dopaminergic neuron
death in C. elegans resulting from in vivo over-expression of human wild-type α synuclein. Together, our findings suggest that low-dose bafilomycin is cytoprotective in
part through its maintenance of the autophagy-lysosome pathway, and underscores its
therapeutic potential for treating Parkinson Disease and other neurodegenerative diseases
that exhibit disruption of protein degradation pathways and accumulation of toxic protein
species.
77
Introduction
The autophagy-lysosome pathway (ALP) is responsible for the highly-regulated
recycling of intracellular contents, whereby macronutrients and damaged organelles are
enclosed within double-membraned autophagic vacuoles (AVs) and delivered to
lysosomes for pH-dependent degradation by lysosomal hydrolases. It is well known that
intact lysosome function is critical for effective completion of the ALP (reviewed in
(Pivtoraiko et al. 2009) and (Shacka et al. 2008). Lysosome dysfunction can potently
inhibit autophagy completion as demonstrated by robust AV accumulation followed by
the induction of cell death (Zaidi et al. 2001); (Boya et al. 2005); (Shacka et al. 2006b).
While the ALP is ordinarily cytoprotective, it is unclear whether AV accumulation
resulting from lysosome dysfunction contributes to cell death.
Chloroquine is an anti-malarial drug and potent lysosomotropic agent. As a weak
base chloroquine accumulates in acidic vesicles and raises their pH (reviewed in
Pivtoraiko et al. 2009). We and others have shown that chloroquine disrupts lysosome
function and inhibits autophagy completion as demonstrated by AV accumulation, and
induces both apoptotic and non-apoptotic cell death (Zaidi et al. 2001); (Boya et al.
2003); (Boya et al. 2005); (Shacka et al. 2006b); (Pivtoraiko et al. 2009).
Growing evidence suggests that the ALP is also altered in age-related
neurodegenerative diseases including Parkinson disease (PD), the most common
neurodegenerative movement disorder. Alterations in autophagy were reported initially
by the aberrant accumulation of AVs in substantia nigra neurons of PD patients (Anglade
et al. 1997). Mutations in several PD-specific genes, including α -synuclein, LRRK2,
78
Parkin and ATP13A2, are known to adversely affect autophagy and/or lysosome function
(reviewed in Shacka et al.
2008 and Pan et al.
2008).
α -Synuclein is a major
component of Lewy bodies in PD brain and α-synuclein accumulation is thought to play
an important causal role in the onset and progression of PD. Lysosomes are important for
α -synuclein clearance, and cathepsin D (CD), the principal lysosomal aspartic acid
protease, is the main lysosomal enzyme involved in the degradation of α -synuclein
(Sevlever et al. 2008). Consistent with this hypothesis, CD deficiency has been reported
to enhance α -syn toxicity (Qiao et al. 2008); (Cullen et al. 2009), and several studies
indicate the therapeutic potential for autophagy induction in promoting α -synuclein
clearance in PD (Webb et al. 2003); (Spencer et al. 2009); (Yang et al. 2009a); (Yu et
al. 2009). Together, these findings suggest that ALP-targeted therapies may be effective
in maintaining α -synuclein clearance and in general inhibiting neurodegenerative
disease-associated neuropathology.
Bafilomycin A1 represents the plecomacrolide subclass of macrolide antibiotics
and was characterized initially by its selective inhibition of vacuolar type (V)-ATPase
(Bowman et al. 1988). V-ATPase maintains the low pH of acidic vesicles through its
regulation of proton pumping (Forgac 2007). At concentrations ≥ 10 nM, bafilomycin
A1 inhibits V-ATPase and in turn increases intravesicular pH (Yoshimori et al. 1991),
thus mimicking the effects of chloroquine. However, we have shown previously that the
plecomacrolides bafilomycin A1, bafilomycin B1 and concanamycin all significantly
attenuate chloroquine-induced death of cerebellar granule neurons (Shacka et al. 2006b);
(Shacka et al. 2006a), at low concentrations (≤ 1 nM) which do not inhibit V-ATPase
(Bowman et al. 1988) or induce AV accumulation (Shacka et al. 2006b). These data
79
suggest that bafilomycin-mediated neuroprotection is independent of its inhibition of VATPase.
However, whether bafilomycin affects the ALP or regulates α -synuclein
clearance and neurotoxicity has not been investigated.
In the current study, we extended our analysis of chloroquine -induced death and
bafilomycin A1 neuroprotection to SH-SY5Y, a human neuroblastoma cell line
commonly used to model dopaminergic neurons. We also assessed the cytoprotective
effects of bafilomycin in the nematode C. elegans that over-express human wild-type α synuclein, which we have previously shown to induce both age- and dose-dependent
neurodegeneration in vivo (Cao et al. 2005); (Hamamichi et al. 2008). Our results
indicate that bafilomycin attenuates neuronal cell death in all of these models consistent
with its ability to maintain ALP function and reduce α -synuclein neurotoxicity.
Materials and Methods
Cell Culture. Naïve SH-SY5Y human neuroblastoma cells were cultured in Minimum
Essential Medium Eagle (MEM) (Cellgro, Herndon, VA) and F12-K (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). Cells were plated at 200 cells/mm 2 and
grown for 24h in media containing 1% FBS prior to treatment. Cells were treated from 048h in media containing 1% FBS. SH-SY5Y cells were differentiated for 7-8 days in
complete media supplemented with 10µM retinoic acid (Sigma).
Retinoic-acid-
supplemented media was replaced every 2-3 days. Unless otherwise noted, differentiated
80
cells were plated at 400/mm2 in differentiation media containing 2% B-27 (Invitrogen,
Carlsbad, CA) and were treated in this media for up to 48h.
Cells were treated with chloroquine, hydroxychloroquine, amodiaquine,
staurosporine or 3-methyladenine (Sigma), and either
bafilomycin A1 (Sigma) or
bafilomycin B1 (A.G. Scientific, San Diego, CA).
Measurement of SH-SY5Y Cell Viability and Caspase-3-Like Activity. Cell viability was
measured using calcein AM fluorogenic conversion assay (Invitrogen). Caspase-3-like
activity was detected via fluorogenic DEVD cleavage assay and expressed relative to
untreated controls. These assays were performed using previously published protocols
(Nowoslawski et al. 2005).
Tandem fluorescent-tagged LC3 (tfLC3) Assay of Autophagic Flux. Differentiated SHSY5Y cells were transiently transfected with ptfLC3 (mammalian expression vector in
plasmid DH5a; Entrez ID of insert U05784) (Addgene, Cambridge, MA) using the
Amaxa Nucleofector™ II device protocol (Lonza, Cologne, Germany). Two million cells
were pelleted and resuspended in 100 µl of Lonza™ Nucleofector reagent followed by
addition of ptfLC3 DNA (2 µg/reaction). Transfer of the reaction mixture was completed
by electroporation in the Amaxa Nucleofector™ II device. 500 µl of neutralization
media was immediately added followed by transfer of the full volume to a fresh tube,
where cells recovered for 10 min, RT. Transfected cells were plated in poly-L-lysine
coated glass chamber slides (LabTek) at 120,000 cells/600 µl media per well and kept at
81
370C prior to treatment. Media was exchanged for fresh 4h after plating to remove
transfection reagents in addition to cells that did not survive the transfection reaction.
Cells were treated with 50 µM chloroquine in the presence or absence of 1 nM BafA1 for
8h. After treatment slides were fixed with 3% paraformaldehyde for 45 min at room
temperature, washed with 1X PBS and cover-slipped. Cells were visualized using a
Zeiss™ Observer.Z1 Laser Scanning Microscope (Thornwood, NY) 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. Fluorescence filters were used to observe bis benzemide
(excitation 405 nm, emission 409-514 nm), EGFP (excitation 488, emission 494-572
nm), and mRFP (excitation 543, emission 585-734).
Generation and analysis of C. elegans. Isogenic C. elegans strain UA44 (baIn11; Pdat1::α-syn,
Pdat-1::GFP) that co-express α-synuclein and GFP in dopamine neurons and
exhibit age-dependent α-synuclein-induced neurodegeneration were age synchronized by
bleaching as previously described (Lewis and Fleming 1995) and placed in 1 ml of water
containing 5% methanol (with or without bafilomycin B1) for 24h at 20ºC with gentle
agitation. After incubation, worms were washed with M9 buffer three times, transferred
to NGM plates, and grown at 20ºC. For each trial, 30 worms were immobilized with 3
mM levamisole, transferred onto a 2.5% agarose pad, and analyzed for neuroprotection at
both day 7 and day 10 post-hatchings. Worms were considered wild-type (WT) when
there were four intact CEP type DA neurons and two ADE type DA neurons without any
signs of degeneration. Each bafilomycin B1 treatment was analyzed in triplicate (90
worms per concentration).
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To determine if increased concentrations of bafilomycin B1 induced DA neuron
cell death, the C. elegans strain BY200 (Pdat-1::GFP) (Nass and Blakely 2003), which
express GFP in the DA neurons without degeneration, was synchronized, treated with
high concentrations of bafilomycin B1, and analyzed as described above. To study the
effect of chloroquine on DA neurons, C. elegans strain UA44 was crossed into knockout
strain NL131 [pgp-3(pk18)], shown previously to be sensitive to chloroquine (Broeks et
al. 1995) to generate the isogenic strain UA146 [baIn11; pgp-3(pk18)]. This strain was
synchronized, treated with chloroquine using methods similar to bafilomycin B1, and
analyzed as described above.
Statistics. Significant effects of treatment were analyzed either by one-factor ANOVA
(three or more groups) or by unpaired, two-tailed t test (two groups). Post hoc analysis
was conducted using Bonferroni’s test. A level of p < 0.05 was considered significant.
Results
Bafilomycin B1 is a structural analog of bafilomycin A1 and both compounds
were shown previously at high doses and with equal potency to inhibit V-ATPase
(Bowman et al. 1988), whereas we have shown that low doses of each are equally
effective in attenuating chloroquine-induced death of cerebellar granule neurons (Shacka
et al. 2006b). Treatment of naïve SH-SY5Y cells for 48h with bafilomycin A1 (Fig. 1A)
or bafilomycin B1 (Fig. 1B) did not alter cell viability when added alone at ≤ 1 nM ,
whereas both significantly reduced cell viability at concentrations ≥ 6nM, effects similar
83
to those reported in cerebellar granule neurons (Shacka et al. 2006a; Shacka et al.
2006b). To determine if bafilomycin-induced death of SH-SY5Y cells was apoptotic,
caspase-3-like activity was measured following treatment for 48h with 0.1-10 nM
bafilomycin A1 (Fig. 1C). Significant increases in caspase-3 like activity were observed
only at bafilomycin A1 concentrations ≥ 6 nM, suggesting that cell death induced by
high concentrations of bafilomycin A1 (Fig. 1A) was apoptotic. These results confirm
that bafilomycin A1 and bafilomycin B1 neither induced SH-SY5Y cell death or
apoptosis at concentrations ≤1 nM.
Naïve SH-SY5Y cells were treated with cell death stimuli 24h after plating, and
cell viability was measured 48h after treatment (Supplemental Fig. 1). Chloroquine
significantly decreased cell viability at concentrations ≥ 20 µM (Supplemental Fig. 1A),
and cell death was maximal at 60-80 µM. Upon treatment of cells with 50 µM
chloroquine, significant decreases in cell viability were observed from 24-48h following
treatment (Supplemental Fig. 1B), and was maximal at 48h. The chloroquine analogs
amodiaquine (Supplemental Fig. 1C) and hydroxychloroquine (Supplemental Fig. 1D)
significantly reduced cell viability in a concentration-dependent manner similar to that of
chloroquine, although amodiaquine was 2-3 times more potent in reducing cell viability
by 50% (~15 µM) in comparison to chloroquine (~40 µM) and hydroxychloroquine (~50
µM). In addition, treatment for 48h with staurosporine, a classical apoptotic stimulus
shown previously to disrupt lysosome function (Bidere et al. 2003); (Kagedal et al.
2005), significantly decreased SH-SY5Y cell viability at all concentrations tested (0.0050.1 µM), and was maximally effective at 0.1 µM (Supplemental Fig. 1E).
84
When bafilomycin A1 or bafilomycin B1 were added at 0.1-1nM concurrently for
48h with 50 µM chloroquine, we observed significant attenuation of chloroquine-induced
cell death (Fig. 2A-B), an effect that was maximal at 0.6-1 nM for bafilomycin A1 (Fig.
2A) and 0.3-1nM for bafilomycin B1 (Fig. 2B). Bafilomycin A1 (1nM) also significantly
attenuated the loss in cell viability following 48h treatment with amodiaquine (15 µM) or
hydroxychloroquine (50 µM; Supplemental Fig. 2A) or staurosporine (0.1 µM,
Supplemental Fig. 2B). High-dose bafilomycin has been shown previously to directly
inhibit chloroquine localization within lysosomes (Boya et al. 2003). To rule out this
potential interaction of low-dose bafilomycin against chloroquine-induced cell death,
cells were pre-treated with bafilomycin A1 for either 12h (Fig. 2C) or 24h (Fig. 2D) and
following its wash-out post-treated with 50 µM chloroquine for 48h. Both 12h and 24h
pre-treatment with bafilomycin A1 significantly attenuated cell death induced by
chloroquine post-treatment, supporting our argument that the cytoprotective effects of
low-dose bafilomycin are not from its inhibition of chloroquine localization to lysosomes.
We have shown previously that chloroquine induces robust neuron apoptosis
(Zaidi et al. 2001); (Shacka et al. 2006b) and that bax deficiency significantly attenuates
chloroquine-induced death of cultured neurons, suggesting the importance of the intrinsic
apoptotic pathway in regulating chloroquine-induced death (Zaidi et al. 2001); (Shacka
et al. 2006b). We have also shown that low-dose bafilomycin attenuates chloroquineinduced apoptosis of cultured neurons (Shacka et al.
2006b).
We next sought to
determine the effects of chloroquine ± low-dose bafilomycin A1 on the induction of SHSY5Y apoptosis.
Treatment for 24h with 50 µM chloroquine significantly induced
apoptosis as measured by enzymatic caspase-3-like activity (Fig. 2E), an effect that was
85
significantly attenuated by treatment with bafilomycin A1 and was maximally protective
at 0.3-3 nM.
To determine if caspase activation was a commitment point for
chloroquine-induced death of SH-SY5Y cells, we measured the effects of chloroquine ±
low-dose bafilomycin A1 on cell viability in the presence of the general caspase inhibitor
BOC-Asp (OMe)-FMK (Fig. 2F). This caspase inhibitor at 30 µM completely blocked
chloroquine-induced caspase-3 activity but neither attenuated chloroquine-induced cell
death nor enhanced bafilomycin-mediated cytoprotection. Together these data obtained
in SH-SY5Y cells corroborate our previous findings in cerebellar granule neurons
(Shacka et al. 2006b); (Shacka et al. 2006a) suggesting that 1) bafilomycins potently
attenuate cell death and apoptosis induced by chloroquine and structurally similar analogs
when used at concentrations ≤1 nM; and 2) bafilomycin regulation of chloroquineinduced cell death occurs upstream of caspase activation.
We have shown recently that chloroquine-induced death of SH-SY5Y cells
correlated with a compromise in the maturation of CD (Pivtoraiko et al.
2009).
Treatment for 24 hour with 50 µM chloroquine significantly induced levels of the
unprocessed, 50 kDa pre-pro form of CD (Fig. 3A, B) and significantly decreased levels
of the mature active 32 kDa form of CD (Fig. 3A, D). These effects were observed as
early as 12h following chloroquine treatment (data not shown). The 47 kDa pro-form of
CD was not significantly affected by treatment (Fig. 3C). When added by itself, 1 nM
bafilomycin A1 did not alter CD processing.
However, 1 nM bafilomycin A1
significantly attenuated the chloroquine-induced decrease in the mature form of CD (Fig.
3D), suggesting the ability of bafilomycin A1 to partially recover lysosomal enzyme
function at concentrations that significantly attenuates chloroquine-induced cell death.
86
To determine if neuronal differentiated SH-SY5Y cells exhibited protective
effects of bafilomycin against chloroquine-induced cell death similar to that of naïve
undifferentiated cells, naïve SH-SY5Y cells were differentiated for 7-8 days with retinoic
acid.
Chloroquine induced a concentration-dependent decrease the viability of
differentiated cells (Supplemental Fig. 3A) in differentiated cells similar to that of naïve
cells (Supplemental Fig. 1A), an effect that was significantly attenuated by the addition
of 1 nM bafilomycin A1 (Supplemental Fig. 3B). We next sought to determine if
chloroquine affects the clearance of endogenous α -synuclein oligomers in neuronal SHSY5Y cells, since it is well known that intact lysosome function plays an important role
in
-synuclein degradation (Lee et al. 2004); (Qiao et al. 2008); (Sevlever et al. 2008);
(Cullen et al. 2009). Triton X-soluble vs. -insoluble fractions were prepared from
lysates of differentiated SH-SY5Y cells following 48h treatment with chloroquine (50
µM) in the presence or absence of low-dose bafilomycin A1 (1 nM). Treatment with
chloroquine significantly increased levels of endogenous detergent-insoluble α -synuclein
oligomers (Fig. 4A-B). By itself, 1 nM bafilomycin A1 did not significantly alter levels
of insoluble α -synuclein oligomers (Fig. 4A-B). In contrast, 1 nM bafilomycin A1
significantly attenuated the chloroquine-induced increase in detergent-insoluble α synuclein oligomers present in the detergent-insoluble fraction (Fig. 4A-B). Chloroquine
and/or bafilomycin had no effect on endogenous levels of detergent-soluble α -synuclein
(data not shown). Together, these data indicate that low-dose bafilomycin regulates the
clearance of detergent-insoluble forms of endogenous α -synuclein oligomers.
We have shown previously that chloroquine-induced death of cerebellar granule
neurons was accompanied by the robust accumulation of AVs (Shacka et al. 2006b). To
87
determine the effects of chloroquine ± low-dose bafilomycin on AV accumulation in
neuronally differentiated SH-SY5Y cells, western blot analysis was performed on
detergent-soluble vs. –insoluble protein fractions (Fig. 5). Levels of LC3-II, which were
used as a selective marker of AVs, were not increased following treatment with
bafilomycin A1 (1 nM) or in vehicle-treated control cells. Treatment with chloroquine
(50 µM) for 48h significantly induced AV accumulation as measured by LC3-II/actin
ratios vs. control-treated cultures (Fig. 5A-C). In contrast, co-treatment with chloroquine
and bafilomycin A1 significantly decreased AV accumulation compared to chloroquine
treatment alone (Fig. 5A-C).
To further investigate whether attenuation of chloroquine-induced death by lowdose bafilomycin was associated with alterations in ALP function we utilized the tandem
fluorescent-tagged LC3 (tfLC3) assay (Fig. 6), an in vitro fluorescence measure of
autophagic flux (Kimura et al. 2007); (Kimura et al. 2009). Over-expression of the
tfLC3 plasmid results in tandem expression of both mRFP-LC3 and GFP-LC3. Under
basal conditions, GFP-LC3 loses fluorescence due to the acidic nature of lysosomes.
However, a compromise in lysosome stabilizes GFP-LC3 fluorescence and in turn
increases GFP-LC3 and mRFP-LC3 co-localization. Cultures of tfLC3-transfected cells
were treated for 8h with chloroquine (50 µM) in the presence or absence of 1nM
bafilomycin A1. Both vehicle control and low-dose bafilomycin-treated cells exhibited
basal RFP and GFP fluorescence and little if any co-localization (Fig. 6A, B). Treatment
with chloroquine induced the appearance of both GFP-LC3 and RFP-LC3 fluorescent
punctae and their apparent co-localization, (Fig. 6C), effects that appeared to be
dramatically reduced in chloroquine plus bafilomycin A1-treated cultures (Fig. 6D).
88
Together, these data suggest that low dose bafilomycin may preserve lysosome function
and thus, maintenance of autophagic flux following chloroquine exposure.
We also attempted to determine if inhibition of autophagy induction by treatment
of cultures with 3-methyladenine, an inhibitor of class-III PI3-K, attenuated chloroquineinduced death. Treatment of differentiated SH-SY5Y cells with either 50 µM or 5 mM 3methyladenine did not attenuate chloroquine-induced death. However, 3-methyladenine
was toxic in its own right to SH-SY5Y cells compared to vehicle control (Supplemental
Fig. 4), suggesting the importance of basal autophagy induction in promoting cell
survival. These data suggest that autophagy induction is not important for regulating
chloroquine-induced cell death.
To determine if plecomacrolides protected against α -synuclein- induced DA
neurodegeneration in vivo, isogenic worms over-expressing human wild-type α synuclein in dopamine neurons were acutely exposed to bafilomycin B1 for 24h during
larval development, and subsequently scored for dopaminergic neuron loss at either 7 or
10 days post-hatching (4 and 7 day adults, respectively). Acute exposure of animals to
50-150 µg/ml bafilomycin B1 showed significant protection against α-synuclein- induced
dopaminergic degeneration (Fig. 7). Exposure of these worms to lower concentrations of
bafilomycin B1 did not significantly protect against dopaminergic neuron degeneration at
day 7 (Fig. 7A), whereas higher concentrations induced DA neuron death in a majority of
the animals. At day 10, animals exposed to either 50 or 100 µg/ml bafilomycin B1 still
exhibited significant protection against neurodegeneration (Fig. 7B). Worms lacking αsyn over-expression were also treated with bafilomycin B1and the percentage of worms
exhibiting DA neurons at either 7 or 10 days following treatment was not significantly
89
different from vehicle control at concentrations ranging from 100-300 µg/ml
(Supplemental Fig. 5A). However, treatment of worms with 400-500 µg/ml bafilomycin
B1 was lethal to the embryos, thus precluding neuron counts from these worms and
suggesting the importance of intact V-ATPase function for worm survival. Together
these results provide in vivo evidence that acute exposure of bafilomycin protects
dopaminergic neurons against α -synuclein-induced neurodegeneration.
Since it is possible that low-dose bafilomycin exerted a “pre-conditioning effect”
on DA neuron survival, we sought out to determine if treatment with low doses of
chloroquine also protected against DA neuron death in worms. However, since worms
are naturally resistant to some natural toxins, including chloroquine (Broeks et al. 1995),
we crossed α-synuclein over-expressing worms into those mutant for pgp-3. PGP-3
encodes a P-glycoprotein transmembrane protein that has been predicted to export toxins
such as chloroquine from cells and thus increase resistance to chloroquine toxicity
(Broeks et al. 1995). Worms were treated with eleven concentrations of chloroquine
ranging from 0.0005-10 mg/ml (as with bafilomycin-treated worms) and assayed for
protection against α-synuclein- induced DA neurodegeneration (Supplemental Fig. 5B).
Significant differences between treated or non-treated DA neurons were not observed at
day 7 or 10, although there was a general trend of DA neurodegeneration with the highest
concentrations of chloroquine. These results suggest that bafilomycin is specific in
exerting a possible pre-conditioned effect
cytotoxicity.
90
in
attenuating α-synuclein-induced
Discussion
The function of bafilomycins was defined originally by its selective inhibition of VATPase (Bowman et al. 1988), which effectively increases the pH of acidic vesicles.
Bafilomycin-mediated inhibition of V-ATPase results in the inhibition of lysosomal
enzyme function and/or processing (Ishidoh and Kominami 2002); (Singh et al. 2006),
induction of lysosome membrane permeabilization (Nakashima et al. 2003), inhibition of
AV-lysosome fusion (Yoshimori et al.
1991) as well as potent inhibition of
macroautophagy completion followed by induction of cell death (Shacka et al. 2006b).
In contrast, results of the present study suggest that bafilomycins significantly attenuate
neuronal cell death caused by agents that disrupt lysosome function and by overexpression of wild-type human α -synuclein, when used at concentrations that do not
inhibit V-ATPase (Bowman et al. 1988) or affect vesicular pH (Shacka et al. 2006b).
Importantly, the cytoprotective concentrations of bafilomycins used in our study (≤ 1
nM) do not disrupt the ALP when used alone, and actually attenuate markers of ALP
dysfunction (decrease in CD maturation and AV accumulation; inhibition of autophagic
flux; increase in detergent-insoluble α-synuclein) caused by the lysosomotropic agent
chloroquine. Together, these findings delineate a potentially novel mechanism of action
for bafilomycins as ALP preservation agents, and may serve to identify future
therapeutics capable of delaying the onset and/or progression of neurodegenerative
diseases including PD.
Bafilomycin A1 significantly and dose-dependently attenuated SH-SY5Y cell
death induced by the lysosomotropic agents chloroquine, hydroxychloroquine or
amodiaquine (Fig. 2; Supplemental Fig. 2). Amodiaquine exhibited a three-fold higher
91
potency than chloroquine and hydroxychloroquine (Supplemental Fig. 1), a disparity
documented previously in other cell lines (Basque et al. 2008) and may be due to
structural differences in amodiaquine (phenolic substitution in its side chain) and/or its
known uptake by an active transporter (Hayeshi et al.
2008).
In addition, both
bafilomycin A1 and bafilomycin B1 provided similar concentration-dependent protection
against chloroquine-induced cell death (Fig. 2).
Cytoprotective concentrations of
bafilomycin A1 (≤ 1 nM) neither induced AV accumulation (Fig. 5) nor affected CD
processing (Fig. 3). The effects of low-dose bafilomycin are similar to those found
previously in cerebellar granule neurons, which indicated a lack of effect on pHdependent lysotracker red fluorescence and AV accumulation (Shacka et al. 2006b).
Low-dose bafilomycin also significantly attenuated staurosporine-induced cell
death (Supplemental Fig. 2), although its relative protection was modest in comparison to
that of chloroquine. Staurosporine has been shown to induce lysosome dysfunction in
different cell types (Bidere et al. 2003); (Kagedal et al. 2005), as indicated by lysosomal
membrane permeabilization, an increase in cytosolic cathepsins and induction of
apoptosis.
Staurosporine’s effects on lysosome function are most likely indirect
compared to chloroquine, and staurosporine has been shown previously to disrupt several
different protein kinase signaling pathways (Ruegg and Burgess 1989). These reasons
may explain why bafilomycin-mediated cytoprotection is less robust against
staurosporine vs. chloroquine and other lysosomotropic agents.
Our previous
investigations indicated that low-dose bafilomycin did not attenuate staurosporineinduced death of cerebellar granule neurons (Shacka et al. 2006b). This discrepancy
92
may be due to subtle cell-type-specific differences in the timing of and/or sensitivity to
staurosporine-induced cell death.
The general caspase inhibitor BOC-Asp (OMe)-FMK neither attenuated
chloroquine-induced cell death nor enhanced the attenuation of chloroquine-induced cell
death by bafilomycin A1 (Fig. 2F), at a concentration that completely inhibited
chloroquine-induced caspase-3-like activity.
Thus bafilomycin A1 may attenuate
chloroquine-induced cell death at a point either upstream and/or independent of caspase-3
activation. Our previous studies in cerebellar granule neurons also demonstrated that
chloroquine-induced cell death did not require caspase-3 activation or expression, but
was attenuated by the targeted deletion of bax, which regulates apoptosis upstream of
caspase activation (Shacka et al.
2006b). However, Bax deficiency did not further
attenuate chloroquine-induced cell death upon treatment with bafilomycin A1, suggesting
that bafilomycin A1 may regulate both Bax-dependent and –independent cell death
pathways resulting from lysosome dysfunction.
The nematode C. elegans is a useful model organism to study neurodegeneration
in vivo caused by either chemical (Nass et al. 2002); (Cao et al. 2005) or genetic factors
(Cooper et al. 2006); (Hamamichi et al. 2008). In addition, C. elegans contains only
eight readily identified dopaminergic neurons, six in the anterior [two pairs of cephalic
(CEP) and one pair of anterior deirid (ADE)] and two in the posterior body segments
[one pair of posterior deirid (PDE)], making them a powerful model to study
dopaminergic neuron degeneration in particular, allowing an unprecedented level of
accuracy in quantifying effects of modifiers. Bafilomycin B1 significantly attenuated
dopaminergic neuron death in C. elegans following over-expression of wild-type human
93
-synuclein (Fig. 7) where an inverted “U” shaped dose response curve was observed ten
days after initial treatment. The maximal protective concentration of bafilomycin B1 in
C. elegans was approximated at 100 µg/ml or 161 µM, a 160-fold higher concentration
for optimal cytoprotection (1 nM) in cultured cells (Fig. 3); (Shacka et al. 2006b);
(Shacka et al. 2006a). However, C. elegans has a protective cuticle layer that most
likely compromised bafilomycin diffusion and penetration, an effect that is well
characterized for other compounds (Holden-Dye and Walker 2007; Rand and Johnson
1995). In addition, the amount of active bafilomycin capable of affecting dopaminergic
neurons in C. elegans may be further lowered upon metabolism within the worm, as has
been demonstrated previously with other compounds (Rand and Johnson 1995), thus
necessitating a higher effective concentration range than optimal for cultured cells. Overexpression of wild-type ATP13A2, a gene expressing a lysosomal ATPase and mutations
in which are associated with a juvenile-onset hereditary parkinsonism (Klein and
Lohmann-Hedrich 2007), attenuates neuron death induced by α -synuclein overexpression in C. elegans (Gitler et al. 2009), further implicating the importance of intact
lysosome function in regulating α -synuclein-induced neurotoxicity.
Thus, it is
conceivable that bafilomycin B1 protected against over-expression of wild-type αsynuclein in C. elegans in part through its preservation of lysosomal function and
promotion of α -synuclein clearance. Importantly, results in C. elegans suggest that
bafilomycin attenuates dopaminergic neuron death following a stimulus (α -synuclein
over-expression) that is, on one hand distinct from treatment with lysosomotropic agents
in vitro yet may produce the same end result (disruption of the ALP).
94
Chloroquine treatment increased levels of high molecular weight, oligomeric
forms of endogenous detergent-insoluble α -synuclein (Fig. 4), an effect that was
significantly attenuated by low-dose bafilomycin A1. Aggregated α -synuclein is the
most abundant protein composing Lewy bodies in PD, dementia with Lewy bodies, and a
Lewy body variant of Alzheimer disease (Trojanowski and Lee 1998). Whether α synuclein oligomerization and aggregation are cytotoxic or cytoprotective is
controversial, and evidence for both has been suggested (Hasegawa et al. 2004); (Rochet
et al. 2004); (Ruan et al. 2009); (Yang et al. 2009b); (Yu et al. 2009). However, it is
generally accepted that enhanced α -synuclein clearance is cytoprotective (Yu et al.
2009). The ALP and the ubiquitin-proteasomal system (UPS) are both involved in α synuclein clearance (Webb et al. 2003); (Lee et al. 2004); (Vogiatzi et al. 2008) and
alterations in both are associated with α -synuclein aggregation in PD brain (Chu et al.
2009). While the relative importance of the ALP vs. the UPS on α -synuclein clearance
under normal physiological conditions and during a pathological process such as PD is
not completely understood, recent data suggest that ALP inhibition has a more profound
role in accumulation of wild-type α -synuclein (Vogiatzi et al. 2008) and that these
distinct protein degradation mechanisms can act in a compensatory manner (Pandey et al.
2007). It has also been shown that CD is the main lysosomal protease responsible for α synuclein degradation (Sevlever et al. 2008) and that α -synuclein aggregation and
toxicity are significantly impacted by relative expression levels of CD (Qiao et al. 2008).
In SH-SY5Y cells, attenuation of chloroquine-induced cell death by bafilomycin A1 was
associated with a partial restoration of mature CD and a decrease in endogenous,
oligomeric detergent-insoluble α -synuclein.
95
Together, our data suggest that the
bafilomycin cytoprotection is mediated, at least in part, by preventing accumulation of
potentially toxic oligomeric detergent-insoluble α -synuclein forms through restoration of
the ALP.
Additional evidence supporting the ability of low-dose bafilomycin to “preserve”
ALP function following chloroquine treatment is indicated by its ability to attenuate the
chloroquine-induced decrease in the mature, “active” form of CD (Fig. 3), decrease the
chloroquine-induced accumulation of AVs (Fig. 5) and attenuate the chloroquine-induced
inhibition of autophagic flux (Fig. 6). CD is synthesized on ough ER and undergoes
initial processing in the Golgi, in particular from the pre-pro to pro forms. However, the
mature form of CD is generated from proteolytic cleavage in the low pH environment of
lysosomes (Marquardt et al. 1987), thus suggesting the deleterious effects of chloroquine
(and hence the protective effects of bafilomycins) on CD maturation are directly related
to their respective effects on lysosome function. Chloroquine-induced inhibition of CD
processing from pre-pro to pro forms (Fig. 5) may be due to a negative feedback
mechanism aimed to prevent excessive CD synthesis upon inhibition of its maturation in
lysosomes, a hypothesis that requires further investigation.
Our previous assessment of chloroquine-induced AV accumulation in cerebellar
granule neurons (Shacka et al. 2006b) indicated little effect of bafilomycin, leading us to
speculate that the cytoprotective effects of bafilomycin A1 were independent of AV
accumulation. Our newest evidence however indicates that bafilomycin A1 attenuation
of chloroquine-induced death correlates with a significant decrease in AV accumulation
and preservation of autophagic flux in differentiated SH-SY5Y cells, differences that may
be explained by cell type-specific effects of chloroquine and bafilomycin.
96
The molecular target for low-dose bafilomycin-mediated neuroprotection remains
unresolved, since our cumulative findings predict that it is independent of V-ATPase
inhibition (Shacka et al. 2006b). One potential target is hypoxia inducible factor (HIF)1α, shown previously to compete with Von Hippel-Lindau tumor suppressor protein for
binding to the c subunit of the V0 sector of V-ATPase (ATP6V0C) in a pH-independent
manner (Lim et al. 2007). HIF-1α is well known to regulate trans-activation of several
genes including heme oxygenase-1, an enzyme complex shown recently to regulate both
the ALP (Zukor et al. 2009) and degradation of wild-type α -synuclein (Song et al.
2009). Thus bafilomycins may regulate neuronal survival through maintenance of the
ALP that is in part independent of V-ATPase inhibition.
In summary, we have shown that low-dose bafilomycin protects neuronal cells
against both chloroquine and wild-type α -synuclein-induced cell death in a manner
consistent with a potential preservation of the ALP. The ability of bafilomycin to regulate
the oligomerization of detergent-insoluble endogenous α -synuclein may be related in
part to its ability to preserve CD maturation and decrease the accumulation of detergent
insoluble AVs, which may ultimately correspond to an attenuation of neuronal cell death.
Further study of plecomacrolide antibiotics such as bafilomycins A1 and B1 is warranted
to better understand the role of the ALP in regulating neuron death in neurodegenerative
diseases such as PD. Rigorous pharmacokinetics studies are also warranted to determine
the relative ability of bafilomycins to cross the blood brain barrier following peripheral
administration and reach therapeutic levels in the brain, and if structural analogs can be
developed that effectively penetrate the CNS and exhibit a marked decrease in V-ATPase
inhibitory activity.
97
Acknowledgements
We thank Sara L. Stone for expert technical assistance, Shawn Williams and the UAB
High Resolution Imaging Facility for assistance with confocal imaging, and the UAB
Neuroscience Core Facilities (NS47466 and NS57098) for technical assistance.
We also
thank the Caenorhabditis Genetics Center (CGC) for kindly providing the C. elegans
mutant strains, as well as the C. elegans Gene Knockout Project at OMRF for generating
mutant strains. Finally, we thank Rhonda Carr and Barry R. Bailey for assistance in
manuscript preparation. This work is supported by grants from the National Institutes of
Health (NS35107, NS41962, and CA134773) (Roth), the Howard Hughes Medical
Institute and Michael J. Fox Foundation (Caldwell) and a pilot grant from the UAB
Alzheimer’s Disease Research Center (Shacka). The co-authors wish to acknowledge no
conflicts of interest with any aspect of this manuscript.
98
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104
Fig. 1. Low-dose bafilomycin is not cytotoxic to SH-SY5Y cells. 48h treatment with
bafilomycin A1 (BafA1, A) or bafilomycin B1 (BafB1, B) decreases cell viability at
concentrations ≥ 6 nM for BafA1 and ≥ 3 nM for BafB1. (C) BafA1 significantly
increases caspase-3-like activity at concentrations ≥ 6 nM. Results represent mean ± SD
from at least three independent experiments. *p<0.05 vs. 0 µM vehicle CTL; #p<0.05
vs.0-3 nM BafA1 (A) or 0-1 nM BafB1 (B).
105
Fig. 2. Low-dose bafilomycin attenuates chloroquine-induced cell death and
apoptosis. 48h treatment with bafilomycin A1 (BafA1, A) or bafilomycin B1 (BafB1, B)
significantly attenuates chloroquine (CQ, 50 µM)-induced cell death from 0.1-6 nM for
BafA1 (A) and from 0.1-1 nM for BafB1 (B). (C-D) Pretreatment with 1 nM BafA1 for
either 12h (C) or 24h (D) significantly attenuates the reduction in viability following 48h
post-treatment with 50 µM CQ. (E) 24h treatment with BafA1 (1 nM) significantly
attenuates CQ-induced increase in caspase-3-like activity. (F) Co-treatment with the
general caspase inhibitor BOC-Asp (OMe)-FMK (Boc-FMK, 30 µM) neither attenuates
CQ-induced cell death nor enhances the cytoprotective effects of BafA1 against CQinduced cell death. *p<0.05 vs. 0 µM vehicle CTL; ** p<0.05 vs. vehicle pretreatment/CQ post-treatment (C-D) or CQ (F); #p<0.05 vs.0-3 nM & 3-6 nM BafA1 (A),
0-0.1 nM & 3-10 nM BafB1 (B), 0-0.1 nM & 6-10 nM BafA1 (C); ##p<0.05 vs. CQ+BocFMK.
106
Fig. 3. Bafilomycin A1 attenuates chloroquine-induced inhibition of CD processing.
(A) Whole cell lysates of SH-SY5Y cells were collected 24h following treatment with
chloroquine (CQ, 50 µM) and/or bafilomycin A1 (BafA1, 1 nM), and subjected to
western blot analysis for CD (pre-pro form, 50 kDa; pro form, 47 kDa; mature “active”
form, 32 kDa). Blots were stripped and re-probed for GAPDH (37 kDa) loading control.
Levels of (B) pre-pro CD, (C) pro CD and (D) mature CD were normalized to levels of
β-tubulin. Results represent mean ± SD from at least three independent experiments.
*p<0.05 vs. 0 µM vehicle CTL; **p<0.05 vs. CQ.
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Fig. 4. Bafilomycin A1 attenuates chloroquine-induced increase in detergentinsoluble endogenous α-syn oligomers. (A) Representative western blot analysis of
endogenous α-syn high molecular weight oligomers (34 & 51 kDa) in detergent–insoluble
fractions, prepared from lysates of differentiated SH-SY5Y cells collected 48h after
treatment with chloroquine (CQ, 50 µM) and/or bafilomycin A1 (BafA1, 1 nM). Blots
were stripped and re-probed for actin (42 kDa) loading control. (B) Levels of insoluble
α-syn high molecular weight oligomers were quantified and normalized to levels of actin.
Results represent mean ± SD from five independent experiments. *p<0.05 vs. 0 µM
vehicle CTL; **p<0.05 vs. CQ.
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Fig. 5. Bafilomycin A1 attenuates chloroquine-induced AV accumulation. (A)
Representative western blot analysis for LC3-I (cytosolic, 16 kDa) vs. LC3-II (AV
membrane-specific, 14 kDa) in detergent-soluble (LEFT) vs. –insoluble (RIGHT)
fractions prepared from differentiated SH-SY5Y cells 48h after treatment with
chloroquine (CQ, 50 µM) and/or bafilomycin A1 (BafA1, 1 nM). Blots were stripped
and re-probed for actin (42 kDa) loading control. Levels of LC3-II were quantified and
normalized to actin for detergent-soluble (B) and detergent-insoluble (C) fractions. Note
only detergent soluble fractions exhibited LC3-I immunoreactivity. Results represent
mean ± SD from at least four independent experiments. *p<0.05 vs. 0 µM vehicle CTL;
**p<0.05 vs. CQ.
109
110
Fig. 6. Bafilomycin A1 attenuates chloroquine-induced inhibition of autophagic
flux. Differentiated SH-SY5Y were transiently transfected to over-express tfLC3 and
following 24h recovery were treated for 8h with chloroquine (CQ, 50 µM) and/or
bafilomycin A1 (BafA1, 1 µM) to observe effects of low-dose BafA1 on chloroquineinduced co-localization (merged image, right panel) of mRFP-LC3 (left panels) and
eGFP-LC3 (center panels) fluorescent punctae, suggesting inhibition of autophagic flux.
Cells were fixed and imaged via confocal microscopy as described in the Methods
section. Panels a-c from CTL (A), BafA1 (B), CQ (C) and CQ+BafA1 are low
magnification images; the box in each of these panels indicates higher magnification
inset panels (d-f) for each. Images are representative of three independent experiments.
Scale bar in Ac and Af = 10 µM.
111
Fig. 7. Bafilomycin attenuates the death of DA neurons in C. elegans following
over-expression of wild-type human α-syn. (A, B) Worms over-expressing α-syn in
DA neurons were acutely exposed to bafilomycin B1 (BafB1, 0-200 µg/ml) for 24h
during larval development, and then subsequently scored for DA neuron loss at either
seven days (A) or ten days (B) post-hatching (4 and 7 day adults, respectively). Results
represent mean ± SD from at least three independent experiments, where 30 worms were
analyzed for each experiment (n=90). *p<0.05 vs. 0 µg/ml vehicle CTL.
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Supplemental Fig. 1. Stimulus-induced death of SH-SY5Y cells. Naïve SH-SY5Y
cells were treated with chloroquine (CQ, A, B), amodiaquine (ACQ, C),
hydroxychloroquine (HCQ, D) or staurosporine (STS, E) for 48h. Cell viability was
assessed using fluorogenic calcein conversion assay. All death stimuli induced
significant and concentration-dependent decreases in cell viability, and the effects of CQ
were temporally specific (B). Results represent mean ± SD obtained from at least three
independent experiments. *p<0.05 vs. 0 µM vehicle CTL; #p<0.05 vs. 0-50 µM CQ (A),
0-36h (B), 0-15 µM ACQ (C), 0-50 µM HCQ (D) and 0-0.01 µM STS (E).
113
Supplemental Fig. 2. Low-dose bafilomycin attenuates stimulus-induced death of
SH-SY5Y cells. 48h treatment with bafilomycin A1 (BafA1, 1 nM) significantly
attenuates the reduction in cell viability following treatment with A) HCQ (50 µM) vs.
ACQ (15 µM) or B) staurosporine (STS, 0.1 µM). *p<0.05 vs. 0 µM vehicle CTL; **
p<0.05 vs. HCQ; #p<0.05 vs. 0.1 µM STS; ##p<0.05 vs. ACQ.
114
Supplemental Fig. 3. Low-dose bafilomycin attenuates chloroquine-induced cell
death of differentiated SH-SY5Y cells. (A) Chloroquine (CQ, 50 µM) induces
concentration-dependent decrease in viability of retinoic acid-differentiated SH-SY5Y
cells, as measured by fluorogenic calcein conversion assay 48h after treatment. (B)
Bafilomycin A1 (BafA1, 1 nM) significantly attenuates CQ-induced decrease in cell
viability. Results represent mean ± SD obtained from at least three independent
experiments. *p<0.05 vs. 0 µM vehicle CTL; **p<0.05 vs. CQ.
115
Supplemental Fig. 4. Inhibition of autophagy induction does not attenuate
chloroquine-induced cell death. Differentiated SH-SY5Y cells were treated with either
50 µM or 5 mM 3-methyladenine (3-MA), a classical class-III PI3-K inhibitor, in the
presence or absence of 50 µM chloroquine. 3-MA was added during the last 24h of the
48h incubation with 50 µM chloroquine to avoid additional 3-MA-specific toxicity.
Viability was measured at 48h after chloroquine treatment. *p<0.05 vs. 0 µM vehicle
CTL.
116
Supplemental Fig. 5. Effects of bafilomycin and chloroquine on DA neuron death
in C. elegans. (A) Worms over-expressing GFP (but not α-syn) in DA neurons were
acutely exposed to 0-500 µg/ml BafB1 for 24h and then scored for DA neuron loss as in
Fig. 7 (A, B). Note that the 400-500 µg/ml concentrations of BafB1 were lethal to C.
elegans embryos thus precluding neuron counts from these worms. (B) Worms overexpressing α-syn in DA neurons were crossed into worms mutant for pgp-3, a strain of
worms that enhances CQ toxicity due to a predicted decrease in toxin export from cells.
Worms were treated with 0.0005-10 mg/ml chloroquine and assessed for DA neuron loss
as for BafB1 (Fig. 7 A, B). Treatment with chloroquine at any concentration did not
alter the percentage of worms exhibiting WT DA neurons. Results represent mean ± SD
obtained from at least three independent experiments, where 30 worms were analyzed for
each experiment (n=90). *p<0.05 vs. 0 µg/ml vehicle CTL.
117
ROTENONE INDUCES AV ACCUMULATION BY ALTERING LYSOSOMAL
FUNCTION
by
VIOLETTA N. PIVTORAIKO, BARBARA KLOCKE, KEVIN A. ROTH, JOHN J.
SHACKA
In preparation
Format adapted for dissertation
118
Abstract
Parkinson disease (PD) is a debilitating neurological disorder associated with progressive
neurodegeneration of dopaminergic neurons in substantia nigra. Rotenone, a selective
inhibitor of complex I of the mitochondrial electron transport chain, is commonly used to
model PD in vitro and in vivo as it induces oxidative stress, α-synuclein accumulation,
and neuron death, main pathological features of PD. Autophagy, a physiological process
characterized by recycling of outlived and/or damaged proteins and organelles in doublemembrane autophagic vacuoles (AVs), has recently been shown to regulate rotenoneinduced neuron death. However, the cause of rotenone-induced AV accumulation has not
been investigated. Here we report that rotenone alters lysosomal function and decreases
autophagic flux. Our data indicate that the autophagy lysosomal pathway (ALP) is highly
sensitive to rotenone toxicity, which is demonstrated by a rapid and sustained increase in
LC3 II levels, a marker of AVs, and volume of acidic intracellular compartments, as
demonstrated by increase in red AO fluorescence intensity, as well as increased levels of
p62, a protein that is degraded through the ALP. We hypothesize that these rotenoneinduced changes in ALP function, if prolonged, can contribute to rotenone-induced
neuron death.
119
Introduction
Parkinson
Disease
(PD)
is
the
second
most
common
progressive
neurodegenerative disorder affecting approximately 2% of the general population at 50
years old; the incidence rate increases with age[1;2]. Although the etiology of the vast
majority of PD cases is still not known, pathologically PD is primarily characterized by
the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and accumulation of
intracellular protein inclusions termed Lewy bodies (LB)[1;3]. Oxidative stress and
hindered capacity of the cell to dispose of misfolded and/or aggregated α-synuclein (αsyn), a protein thought to be involved in DA-containing vesicle release and the major
component of LB, are believed to be among the major factors responsible for LB
formation and neurodegeneration in PD[4].
The decrease in function of complex I of mitochondrial electron transport chain
(METC) has been reported in SN of PD patients[1]. Additionally, exposure to pesticides
or insecticides such as rotenone, a selective inhibitor of METC, is associated with an
elevated risk of PD development[5;6]. Inhibition of mitochondrial function leads to a
decrease in ATP production and increased oxidative stress generation [7-9]. Therefore,
rotenone has been a useful pharmacological agent to study PD in vivo and in vitro.
Rotenone induces neurodegeneration and α-syn accumulation in both in vitro and in vivo
models of PD. Moreover, rotenone-induced oxidative stress has been reported to induce
post-translational modifications of α-syn [10], which are thought to promote its
aggregation and toxicity [11-14].
120
Rotenone-induced neuron death has been associated with
initiation of the
intrinsic apoptotic pathway by promoting cleavage-dependent activation of aspartatespecific effector caspases (caspases-3, 6, and 7)[15]. Moreover, rotenone has been
reported to affect other cellular pathways such as the autophagy lysosomal pathway
(ALP), which is emerging as an important regulator of neuron survival in PD[16-18].
ALP is responsible for regulation of intracellular energy balance by recycling outlived
and/or damaged proteins and organelles. Autophagic vacuoles (AVs), double-membrane
vesicles carrying cargo for degradation via ALP, have been detected in survived SN DA
neurons in human PD brain and several PD animal models[19]. Moreover, autophagy is
believed to be the main route for degradation of α-syn oligomers and aggregates, further
implicating this process in PD pathology[20].
Rotenone-induced cell death is accompanied by AV accumulation, likely an effect
of elevated oxidative stress resulting from inhibition of complex I of mitochondrial
electron transport chain (METC) [16-18].
Autophagy is generally considered a
cytoprotective mechanism, as it promotes degradation of damaged mitochondria and
aggregated α-syn [17;18]. Likewise, autophagy induction via inhibition of mammalian
target of rapamycin (mTOR), which inhibits AV formation [21], has been shown to
attenuate rotenone-induced apoptosis [18]. AV accumulation can result from either
increased autophagy induction (regulated in part via mTOR) or inhibition of autophagy
completion, which requires fusion of AVs with the lysosomes. Lysosomal proteases such
as cathepsins with acidic pH optima carry out degradation of AVs and their cargo. Proper
function of the lysosome is critical because ALP, as a housekeeping and recycling
121
mechanism, can only be effective if lysosomal degradation of AV cargo is
accomplished[22].
Whether autophagy induction and/or inhibition of autophagy completion are
responsible for rotenone-induced AV accumulation and neuron death has not been
investigated. However, a recent study reported a decrease in lysosomal number and
function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of PD.
MPTP is a toxin that similarly to rotenone inhibits complex I of METC [23]. Here we
report that in SH-SY5Y cells and primary telencephalic neuron cultures rotenone induces
neuron death that is accompanied by alterations in lysosomal function. Rotenone induced
a rapid increase in LC3 II levels, indicative of AV accumulation, and an increase in red
acridine orange (AO) staining, a fluorescent compound used for assessment of
intracellular acidic compartments, suggesting an increase in acidic lysosomal or
autophagosomal compartment volume and/or acidity. Rotenone-induced increase in AV
levels was not accompanied by an increase in autophagic flux, suggesting a compromise
of lysosomal function, which was further evidenced by increases in p62, a protein known
to be degraded by the ALP. Our findings indicate the importance of further investigation
of pathways regulating lysosomal pH, volume, and autophagic flux as they may provide
novel therapeutic targets to attenuate protein aggregation and neuron loss associated with
PD.
122
Materials and 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 cells/mm2 in media
containing 2% B-27 supplement (Invitrogen, CA) 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 0 - 72 hours in B-27 and 10µM retinoic acid
supplemented media.
Primary telencephalic cultures. Mice were housed and cared for according to the NIH
Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care
Committee of the University of Alabama at Birmingham. p53−/− mice were purchased
from Taconic (Germantown, NY) and backcrossed for at least 6 generations onto the
C57BL/6J background. Pregnant mice were anesthetized with Nembutal (Lundebeck Inc.,
Deerfield, IL) and euthanized by cervical dislocation. Embryos (E12.5) were removed
and telencephalic cells were dissociated and plated in a chemically defined serum-free
medium containing insulin, transferrin, selenium, progesterone, putrescine, glucose and
glutamine followed by incubation at 37°C in humidified 5% CO2 atmosphere for 48 h, as
123
previously described [24]. Genotyping of mouse pups was performed by PCR using DNA
extracted from tail or limb clips. Cells were then placed in fresh medium containing 2%
FBS with or without rotenone or AraC (Sigma) for an additional 24 - 48 h.
Measurement of Cell Viability and Caspase-3-Like Activity. Cell viability was measured
via Calcein AM fluorogenic conversion assay (Molecular Probes, Eugene, OR). Caspase3-like activity was detected via fluorogenic DEVD cleavage assay and expressed relative
to untreated controls. The assays were performed following protocols previously
described by Nowoslawski L et al. [25].
Western Blot. Whole cell lysates were obtained as described previously [26]. Briefly,
detached cells and conditioned media were collected and centrifuged (700xg, 5 minutes,
4 οC). The pellet was re-suspended in 1ml of ice-cold PBS and following subsequent
centrifugation (700xg, 5 minutes, 4 οC) the pellet was lysed with buffer containing 1%
SDS and 1% Triton X-100). Protein concentrations were determined using BCA assay
(Pierce). Equal amounts of protein were electrophoresed on SDS-polyacrylamide gel and
subsequently transferred to PVDF membranes (BioRad, CA). Western blots were probed
for LC3 (Abgent), p62 (Abnova), or LAMP1 (1D4B; Hybridoma bank, Johns Hopkins
University School of Medicine, Baltimore, MD). GAPDH (Cell Signaling, Beverly, MA)
or actin (Sigma) were used as loading controls. X-ray films of western blots were
scanned for densitometric analysis using UN-SCAN-IT gel 6.1 software (UN-SCAN-IT,
Orem, UT).
Measurement of AO intensity. To assess changes in lysosomal pH, we utilized the
fluorescent probe acridine orange (AO) (Molecular Probes, Eugene, OR). Cells were
124
seeded on 4- or 8-well Lab-Tek II glass chamber slides (Fisher, Rochester, NY) coated
with 0.1 mg/ml poly-L-lysine (Sigma) and 0.01 mg/ml laminin (BD Biosciences,
Bedford, MA), and were maintained and treated as described above. Prior to fluorescent
probe application, treatment medium was removed and cells were washed in PBS. AO
(5µM) was added to cells in 0% FBS containing MEM/F12 medium for 15 min. At the
end of the incubation period, the fluorescent probe was removed from the cells, the cells
were washed again with PBS, and chambers were removed from chamber slides. Cells on
the slides were coverslipped with fresh 0% FBS containing MEM/F12 medium and were
immediately imaged using Zeiss Axioskop fluorescent microscope. Exposure time was
set based on AO red and green fluorescence (set individually) of the earliest time point
control and applied to the remaining control and treatment conditions of the same
experiment (wells on the same chamber slide). Three images were captured per treatment
conditions (at least 20 cells per image) from different locations on the well. Image
analysis was performed with AxioVision Rel. 4.8 software following image collection.
Cell body regions ware selected and the red AO fluorescent signal intensity over the
selected region (Rel. AO intensity) was computed for each cell. Values of Rel. AO
intensity were polled together from three images of the same condition. The highest value
of Rel. AO intensity of earliest time point control was used to compute the value of 70%
of maximal Rel. AO intensity which was set as the threshold. The percent of cells with
Rel. AO intensity higher that this threshold was than computed per each condition from
the same experiment (wells on the same chamber slide).
Statistics. Significant effects of treatment were analyzed either by one-factor ANOVA
(when effects of treatment or time were assessed) or by two-factor ANOVA (when the
125
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.
Results
Rotenone-induced neuron death is attenuated by p53 deficiency but not by broad caspase
inhibition. In RA differentiated SH-SY5Y cells, rotenone induced concentration and
time-dependent cell death (Fig. 1A, C). A decrease in cell viability was observed starting
at 24h of 10µM rotenone treatment. About a 50% decrease in SH-SY5Y cell viability was
achieved at 48h hours, which progressed to 70% at 72h of rotenone treatment (Fig. 1C).
Rotenone-induced SH-SY5Y cell death was accompanied by concentration-dependent
increase in caspase 3-like activity (Fig. 1B). A fourfold increase in caspase 3-like
enzymatic activity in 10µM rotenone treated SH-SY5Y cells vs. vehicle control was also
detected at 48 and 72h, suggesting that rotenone-induced cell death was accompanied by
caspase activation (Fig 1D).
p53, a transcription factor and a well studied tumor
suppressor protein, has been reported by our lab and others to regulate apoptosis via
transcription-dependent and –independent mechanisms. p53 has also been reported to
regulate autophagy [27]. We observed an increase in nuclear accumulation of p53
following rotenone in SH-SY5Y cells (Fig 2A). Cytosine arabinoside (AraC), a well
studied genotoxic agent, known to induce p53 nuclear accumulation and neuron death
[28], in SH-SY5Y cells also induced increase in nuclear p53 immunoreactivity, and was
used as a positive control (Fig. 2A). To test whether p53 is an important regulator of
rotenone-induced neuron death, we tested the effect of rotenone on survival of primary
126
telencephalic neurons generated from p53 wild type (wt) and p53 deficient (-/-) mouse
embryos. Rotenone induced concentration and time-dependent primary telencephalic
neuron death (Fig 3A, C). Previously our laboratory reported that p53 deficiency in a
gene dose-dependent manner attenuates AraC- induced primary telencephalic neuron
death in vitro [28]. Here we observed similar results; at 48h of 50µM AraC treatment,
p53 deficient neurons were almost completely protected against AraC-induced neuron
death (Fig. 3B). p53 deficiency was also found to significantly attenuate rotenoneinduced primary telencephalic neuron death. However, this protective effect was not as
robust as observed for AraC-induced death (Fig. 3D), suggesting that p53 is not as critical
a regulator of rotenone-induced neuron death as it is for genotoxin-induced death. To
determine if caspase activation is required for rotenone-induced neuron death, we tested
the effect of rotenone on SH-SY5Y cell viability in the presence of a broad caspase
inhibitor Boc-Asp(OMe)-FMK (Boc-FMK) (Fig 2B). Boc-FMK at a concentration that
completely inhibited rotenone-induced caspase-3-like activity (data not shown) failed to
inhibit rotenone-induced cell death (Fig. 2B). This data suggests that although rotenone
can activate one or more apoptotic pathway, caspase activation per se, is not required for
death to occur.
Rotenone affects ALP function. Previous studies have identified AV accumulation in SN
neurons in human PD brain, suggesting that ALP is involved in the regulation of DA
neuron death [29;30]. ALP has been also reported to regulate rotenone-induced neuron
death [16-18]. To investigate the possible involvement of the ALP in regulation of
rotenone-induced neuronal death, we first tested the effects of rotenone on AV
accumulation and autophagic flux in differentiated SH-SY5Y cells. We observed a
127
significant increase in AVs as early as 6h and through 48h after 10µM rotenone
administration, as demonstrated by an increase in LC3 II levels (best accepted marker of
AVs) in rotenone-treated cells vs. vehicle controls for respective time points (Fig. 4A, B).
AV accumulation may be due to aberrant autophagy induction and/or inhibited AV
degradation, since under baseline conditions, AVs generated in a cell are rapidly
degraded via fusion with lysosomes [19;31]. Therefore, we performed an autophagic flux
assay [31] to test the hypothesis that rotenone promotes autophagy induction. We treated
SH-SY5Y cells with 10µM rotenone for 24 and 48h. At the last 4 h of rotenone
treatment, AV turnover was inhibited with 100nM bafilomycin A1 (BafA1), an inhibitor
of V-ATPase activity that disrupts lysosomal function and AV degradation (Fig. 5A). At
24 and 48h of 10µM rotenone treatment combined with 100nM BafA1 at the last 4h of
treatment (point N-4 at Fig. 5A), we found no significant increase in LC3II levels relative
to 100nM BafA1 treated cells (Fig. 5B, C). This data suggests that rotenone induced
increase in LC3 II levels was not the result of increased AV formation, but rather, from
decreased AV degradation at the lysosome. Autophagic flux is also commonly assessed
by detecting protein levels of p62/A170/SQSMT1, a ubiquiting and LC3 binding protein
which can bind to ubiquitin aggragates
and be degraded via autophagy [31;32].
Therefore, decreases in p62 levels are associated with increased autophagic flux as
observed for example during serum starvation [31]. However, p62 accumulates when
autophagy completion is blocked and/or AV formation is inhibited [31;33;34]. We
observed a time dependent increase in p62 levels in rotenone treated SH-SY5Y cells vs.
vehicle controls. Starting at 12h of rotenone treatment there was about a two fold increase
in p62 levels that was sustained through 48h of rotenone treatment (Fig 6A, B). This data
128
further supports our negative autophagic flux results, suggesting that rotenone inhibits
autophagy completion, likely by causing lysosomal damage.
Rotenone increases AO staining. Lysosomal dysfunction can lead to AV accumulation
and inhibition of autophagic flux. Maintaining acidic pH in the lysosomal lumen is
essential for proper function of lysosomal enzymes, degradation of aggregation-prone
proteins, and cell survival [35]. Therefore, to investigate the effects of rotenone on acidic
compartment pH and volume we used pH sensitive dye AO. AO fluoresces red at low pH
and green at normal intracellular pH. AO is also attracted to the nucleus because of its
abundance in DNA where it also fluoresces green. Vehicle control cells loaded with AO
exhibited only modest levels of red punctate AO staining in the cytoplasm (Fig 7A). We
used 100nM BafA1 as the positive control for AO assay because BafA1 at high
concentrations is known to increase lysosomal pH by inhibiting V-ATPase. SH-SY5Y
cells treated with 100nM BafA1 for 24h did not display any red AO puncta, indicating an
increase in lysosomal pH, as expected (data not shown). At 6h of rotenone treatment,
numerous bright red AO puncta were observed in SH-SY5Y cells (Fig. 7A). AO intensity
in rotenone treated cells was considerably higher than in untreated controls (Fig. 7A, B).
AO puncta also appeared to increase in size and accumulate in tight clusters in the
perinuclear region of the rotenone treated cells. At 24 and 48h of 10µM rotenone
treatment, bright red and punctate AO staining was still observed in many cells (Fig. 7A).
We quantitated our AO results by assessing percentage of cells with red AO fluorescence
intensity higher than threshold red AO intensity in vehicle control cells. Threshold was
set at 70% of maximum red AO fluorescence intensity of 6h vehicle control cells. We
observed a sustained increase in number of cells with red AO fluorescence intensity
129
higher than threshold starting at 6h of 10µM rotenone treatment (Fig. 7B). Increase in
red AO staining intensity can signify either drop in acidic vacuole pH (lysosomes,
autophagolysosomes, and late endosomes) or increase in acidic vacuole volume. We
tested the effect of rotenone on levels of lysosomal membrane associated protein 1
(LAMP1), lysosomal structural protein present on lysosomal membrane and a well
accepted marker of lysosomes. A recent study reported a decrease in LAMP1 levels and
lysosomal number in MPTP mouse models of PD [23]. Therefore, since we observed
rotenone-induced increase in red AO puncta size and staining, signifying increase in
acidic vacuole volume, we expected to see an increase in LAMP1 protein levels as well.
Surprisingly, we did not observe a time-dependent increase in LAMP1 levels in 10µM
rotenone treated SH-SY5Y cells vs. control at either of the time points tested ( 24 and
48h) (Fig. 8A,B). Therefore, our data suggests that rotenone-induced increase in acidic
compartment volume is not coupled to an increase in LAMP1 density in lysosomal
membrane. These observations may indicate an increase in the slightly acidic
autophagolysosome compartment which accumulates in rotenone treated cells due to
defective lysosome degradation or lysosome turnover.
Discussion
The data presented in this report indicate that rotenone induces neuron death
accompanied by activation of caspases and p53 nuclear translocation (Fig. 1 and 2). p53
deficient neurons demonstrated a significant but modest reduction in rotenone-induced
death (Fig. 3). p53 has been implicated in induction of autophagy, which in respect to
130
rotenone-induced cell death, has been reported to be a cell protective process possibly by
promoting degradation of damaged mitochondria [18;36;37]. Therefore, p53 knockdown
might have resulted in inhibition of both cell death promoting pro-apoptotic pathways
and pro-survival autophagic pathways. Overall, it appears that multiple cell death
pathways are activated by rotenone, as indicated by the fact that broad caspase inhibition
failed to rescue rotenone-induced cell death (Fig. 2B). Therefore, our data suggest that
inactivating caspases is not sufficient to protect neurons from rotenone-induced toxicity
and other cellular targets of rotenone should be explored for elucidation of better
therapeutic targets to protect against rotenone toxicity.
It is becoming more apparent that autophagy plays a critical role in regulation of
neuron survival, particularly in the context of neurodegenerative diseases such as PD.
AV accumulation has been reported following rotenone exposure and with other
inhibitors of mitochondrial function such as MPP+ and thenoyl trifluoroacetone (TTFA)
(complex II METC inhibitor) [16;38]. We also observed a time dependent increase in
AV accumulation in SH-SY5Y cells with rotenone administration (Fig. 3). AV
accumulation was suggested to result from increased oxidative stress induced by
mitochondrial dysfunction because addition of antioxidants can attenuate rotenone and
TTFA induced AV accumulation and cell death [16]. Likewise, autophagy is believed to
be a cytoprotective mechanism in the case of mitochondrial dysfunction. In agreement
with this, induction of autophagy with the mTOR inhibitor rapamycin was also reported
to attenuate rotenone-induced neuron death, likely by promoting degradation of damaged
mitochondria [18;36]. Consistent with these reports, we observed a significant increase in
AV accumulation after rotenone treatment (Fig. 4). Rotenone-induced AV accumulaiton
131
was accompanied by an increase in red AO fluorescence intensity (Fig. 7). This data
suggest that the ALP is highly sensitive to rotenone toxicity; activation of the ALP may
be a pro-survival response to help relieve the increased oxidative stress burden associated
with rotenone toxicity, at least initially.
Prolonged oxidative stress and/or autophagy induction is implicated in
development of lysosomal dysfunction [22]. Indeed, although LC3 II levels were
elevated, we were unable to detect a statistically significant increase in autophagic flux at
24 and 48h of rotenone treatment (Fig. 5), suggesting that ALP function has been
negatively affected. Consistent with this hypothesis, prolonged rotenone exposure was
associated with an increase in p62 levels (Fig. 6). p62 has been implicated in cell
signaling and regulation of cell death and survival [39] because, as a scaffolding protein,
it has multiple protein-interaction domains [40]. p62 levels have also been reported to
increase following proteasome inhibition and increased oxidative stress; both of these
processes have been implicated in rotenone toxicity [41;42]. Therefore, although other
factors might have contributed to the increase in p62 protein levels, p62 accumulation is
in agreement with our autophagic flux data suggesting that the increase in p62 levels
following rotenone treatment is in part due to inhibited lysosomal function. These
findings are also in agreement with recent publications demonstrating inhibition of
autophagic flux and lysosomal membrane permeabilization in MPP+ treated neurons
[23;43].
LAMP1 is a structural protein localized to lysosomal membrane. It is highly
glycosylated, which is believed to protect the lysosome from self-digestion [44].
132
Knockdown of lamp1 has been reported to sensitize cancer cells to siramesine and photooxidation-induced lysosomal destabilization and cell death[45]. Overexpression of the
transcription factor EB (TFEB) which was reported to regulate lysosome biogenesis, has
been demonstrated to induce an increase in LAMP1 levels and protect against MPP+induced neuron death [23]. We observed a rapid increase in size of AO positive puncta
(puncta of red AO fluorescence) and red AO fluorescence intensity (Fig. 7). However, we
did not detect a significant increase in LAMP1 levels following rotenone treatment (Fig.
8). These data suggest that although rotenone causes an increase in volume of acidic
compartments, the expanding surface of these acidic compartments was relatively devoid
of LAMP1. This may compromise lysosomal membrane integrity and promote induction
of lysosomal membrane permeability (LMP), a process resulting in release of lysosomal
proteases into the cytosol an cell death induction [22].
We hypothesize that prolonged oxidative stress induced by rotenone-mediated
mitochondrial dysfunction results in damage to the lysosomal compartment and decrease
in autophagic flux. Rotenone-mediated damage to the lysosomal membrane can be
induced either by inhibition of expression of LAMP1 and other lysosomal proteins, direct
oxidative modifications of lysosomal membrane structural proteins which hinder their
normal function, or deregulation of mechanisms keeping the acidic compartments from
self-digestion. Likewise, the increase in red AO intensity with rotenone treatment, may
also suggest increase in acidic compartment acidity. Prolonged decrease in acidic
compartment pH may result in digestion of lysosomal membrane and LMP induction.
Further studies using ratiometric imaging, a technique that is emerging as a more reliable
method for acidic compartment pH assessment [46], would be necessary to test whether
133
rotenone-induced increase in red AO fluorescence intensity is indeed due to the decrease
in acidic compartment pH following rotenone treatment.
Autophagy is a key player in regulation of neuronal cell survival following
mitochondrial
dysfunction
and
oxidative
stress
which
are
associated
with
neurodegenerative diseases like PD. However, the effects of autophagy can be
maintained as long as the lysosomes are capable of degrading AV cargo. Here we report
that rotenone induces rapid and prolonged increase in AV levels and red AO fluorescence
intensity, likely initially cytoprotective mechanisms that could help degrade damaged
cellular constituents such as mitochondria. We hypothesize that at later time points of
rotenone treatment, oxidative burden results in decreased lysosomal degradative capacity
and ultimately, cell death. Our data suggests that better understanding of the pathways
regulating ALP function following rotenone-induced neuronal injury might provide novel
targets for treatment of neurodegenerative diseases associated with protein accumulation
and oxidative stress such as PD.
134
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Fig. 1: Rotenone induced SH-SY5Y cell death and caspase 3-like activity is
concentration and time dependent. Rotenone induces concentration-dependent increase
in cell death (A) and caspase 3-like activity (B) at 24h of treatment. 10µM rotenone
induced time dependent increase in SH-SY5Y cell death (C) that was accompanied by
increase in caspase 3-like activity (D) at 48 and 72h of treatment. * p<0.05 vs. 0µM
rotenone (A, B); * p<0.05 vs. 0h rotenone (C, D).
139
Fig. 2: Rotenone-induced SH-SY5Y cell death is accompanied by increased nuclear
p53 accumulation but is not dependent on caspase activation. Rotenone (10µM)
induced an increase in nuclear p53 accumulation at 24h, as demonstrated by increased
nuclear p53 immunoreactivity in rotenone treated SH-SY5Y cells vs. control. 50µM
AraC (24h) was used as a positive control for p53 nuclear accumulation (A). Complete
inhibition of rotenone-induced caspase 3-like activity (not shown) with general caspase
inhibitor Boc-FMK (Boc-Asp-FMK) does not attenuate rotenone-induced cell death (B).
*p<0.05 compared to CTL. Scale bar equals to 20µm.
140
Fig. 3: Rotenone induced concentration and time dependent primary telencephalic
neuron death that was attenuated by p53 deficiency. At 24h rotenone induced a
concentration-dependent increase in primary telencephalic neuron death (A) with a
further increase at 48h (C). p53 deficiency significantly attenuated primary telencephalic
neuron death induced by 10nM rotenone (D). p53 deficiency significantly attenuated
AraC-induced primary telencephalic neuron death. 50µM AraC was used as a positive
control (B). * p<0.05 vs. 0nM rotenone (A, C); *p<0.05 vs. wt p53 primary telencephalic
neurons (B, D).
141
Fig. 4: Rotenone induced a time-dependent increase in AV accumulation. 10µM
rotenone induced a time-dependent and sustained increase in AV accumulation in SHSY5Y cells as demonstrated by increases in LC3 II levels over GAPDH, loading
control, vs. vehicle controls. Controls were generated for each time point examined (A).
Levels of LC3 II over GAPDH were quantitated from 4 separate experiments (B).
* p<0.05 vs. vehicle controls for the respective time point.
142
Fig. 5: Rotenone inhibits autophagic flux. To test the effects of rotenone on autophagic
flux, autophagic flux assay was performed by adding 100nM BafA1 at the last 4 hours of
rotenone treatment (point N-4) (A). For each time point (24 or 48h) a separate 100nM
BafA1 lysate was collected to account for any time-dependent starvation-induced
differences in AV accumulation (B, C). 100nM BafA1 alone and in combination with
10µM rotenone induced a significant increase in AV accumulation, as demonstrated by
the increase in LC3 II levels over GAPDH, loading control, vs. vehicle CTL (B).
Western blot quantitation of three separate experiments did not indicate significantly
higher LC3 II/GAPDH levels in 10µM rotenone plus 100nM BafA1 vs. 100nM BafA1
treated SH-SY5Y cells at either 24 or 48h (C). * p <0.05 vs. CTL.
143
Fig. 6: Rotenone induces time-dependent p62 accumulation. 10µM rotenone induced a
time-dependent and sustained increase in p62 accumulation in SH-SY5Y cells (A). p62
levels were significantly higher starting at 12h of rotenone treatment as compared to the
vehicle controls generated for each time point examined (A, B). Levels of p62 over
GAPDH were quantitated from four separate experiments (B). * p<0.05 vs. vehicle
controls for the respective time point.
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Fig. 7: Rotenone-induced SH-SY5Y cell death is accompanied by increases in
volume of acidic compartments. Rotenone induced a rapid and sustained increase in red
AO staining, suggesting an increase in the volume of acidic compartments. 10µM
rotenone also induced a change in the subcellular localization of red AO puncta - from
diffuse in vehicle CTL to aggregated in the perinuclear region of rotenone treated SHSY5Y cells (A). To quantitate rotenone-induced changes in AO intensity, a threshold of
AO intensity was established that represented AO fluorescence intensity value equal to
70% of maximum AO intensity in 6h vehicle CTL. Quantitation of red AO staining
intensity indicated that the number of cells with red AO fluorescence intensity higher
than threshold increased in rotenone treated SH-SY5Y cells vs. vehicle CTL (B).
*p<0.05 vs. vehicle CTL for respective time point. Scale bar equals to 10µm.
145
Fig. 8: Rotenone does not alter LAMP1 levels. LAMP1 levels were measured at 24
and 48h of 10µM rotenone and vehicle CTL treated SH-SY5Y cells (A). Quantitation of
six independent experiments showed no significant difference in LAMP1 levels in
rotenone treated vs. vehicle CTL SH-SY5Y cells (B).
146
CONCLUSIONS AND DISCUSSION
PD is a complex disorder affecting multiple intracellular processes. Oxidative
stress, mitochondrial dysfunction, and protein aggregation have been proposed to be
among the main effectors of PD etiology. There is an increasing body of evidence
indicating that the ALP also plays a critical role in regulation of cell death and cell
survival in PD in part by mediating degradation of damaged mitochondria and
aggregation-prone proteins such as α-syn. Therefore, manipulation of this pathway has
emerged as a potential new approach to PD treatment. However, much remains to be
understood about the interplay between different components of this pathway and their
individual contributions to cell survival.
The overall goal of my thesis work was to characterize the alterations in the ALP
in a mitochondrial dysfunction-induced in vitro model of PD and assess how stimulation
of ALP function may promote neuron survival in PD. In particular, as described in
Chapter 3, I investigated the effects of the mitochondrial toxin rotenone on autophagic
flux and ALP-associated proteins. In Chapter 2, I characterized the mechanism of low
dose bafilomycin A1 (BafA1) mediated neuroprotection, which we hypothesize
attenuates lysosome dysfunction induced inhibition of autophagic flux. Alterations in the
ALP, as evidenced by accumulation of AVs, have been reported in several models of PD.
However, as the findings presented in Chapter 2 and 3 suggest, it is critical to
147
characterize alterations in what pathway is responsible for AV accumulation. AVs can
accumulate due to either increased autophagy induction or hindered autophagic flux.
Therefore, in Chapter 3, I investigated the effects of rotenone on these two mechanisms
regulating AV turnover.
The lysosome is the organelle responsible for degradation of AVs and also
implicated in other cellular functions such as membrane recycling and cell survival
regulation [80;90]. The lysosome is a central link in both the ALP and the endocytic
pathway, but the mechanisms regulating its function are not well characterized. The
digestive capacity of the lysosome depends on activity of digestive enzymes in its lumen.
Maintenance of low lysosomal pH is critical for proper functioning of these enzymes
[90;92]. However, the regulation of lysosomal pH is not well understood especially in the
context of neurodegenerative diseases such as PD. Several factors are involved in
lysosomal pH maintenance. One important component is V-ATPase, a lysosomal
membrane localized multimeric enzyme complex responsible for active pumping of H+
into lysosomal lumen. V-ATPase activity can be regulated in part by variation of its
subunit composition [114]. Other lysosomal trans-membrane proteins acting as ion
channels also have been shown to be important in lysosomal pH regulation [99-101].
Among these are chloride- proton transporters of the CLC family and the cystic fibrosis
transmembrane conductance regulator chloride channel (CFTR). These channels are
thought to regulate lysosomal pH in part by preventing positive charge buildup on the
luminal side of the lysosomal membrane which is generated by V-ATPase activity [102].
148
Proper lysosomal pH is also critical for maintenance of redox balance of the
lysosomes [90]. Metals such as Fe and Zn are found in high concentrations in the
lysosomes being products of metal-containing protein degradation [90;115]. Once
liberated in the lysosomal lumen, metals are thought to be maintained in an inactive state
by metal-to-metal complex formation or coupling with lysosomal proteins [59;116;117].
Lysosomal pH has been shown to affect the percentage of free (active) vs. bound
(inactive) metals in the lysosomal lumen and, therefore, is thought to regulate oxidative
balance of the lysosome [118]. For instance, some anti-malarial lysosomotropic agents
such as CQ are believed to promote malaria parasite plasmodium fulcrum death by
inhibiting Fe complex formation in the parasite’s vacuole (analogue to mammalian
lysosomes) which then can promote induction of oxidative stress in the parasite [119].
As discussed in Chapters 1, 2, and 3 and in the proceeding discussions, lysosome
dysfunction can contribute to neuron death via several components; 1) accumulation of
undigested proteins and AVs, 2) oxidative stress induction, and 3) release of lysosomal
enzymes into the cytosol upon lysosomal membrane permeabilization (LMP). The
contribution of each of these components to neuron death particularly in PD may be
challenging to dissect. However, further research of these components will provide a
better understanding of the pathology and etiology of PD and may lead to the discovery
of novel targets for theraputic intervention.
Regulation of autophagic flux by low dose BafA1
The mechanism of low dose BafA1 cytoprotection against CQ and α-synuclein
overexpression-induced neuron death is yet to be fully characterized. As indicated in
149
Chapter 2, restoration of lysosomal function may in part be responsible for its
cytoprotective effects. Low dose BafA1-induced restoration of lysosomal function may
be mediated by modulation of ion transport across the lysosomal membrane and
stabilization of normal lysosomal pH. Several elements are involved in regulation of
lysosomal pH and function. They include V-ATPase, Clc-7, and CFTR ion channels
[102;114]. Therefore, in order to further assess the mechanisms of lysosomal function
regulation by low dose BafA1, its effects on each of these systems would need to be
analyzed.
An increase in V-ATPase activity induced by low dose BafA1 may be a
mechanism responsible for the increase in autophagic flux described in Chapter 2.
BafA1, at high concentrations, is classically known as a selective inhibitor of V-ATPase
causing an increase in lysosomal pH, which inhibits lysosomal function and results in AV
accumulation [90]. However, as presented in Chapter 2, our data suggests that the effects
of low dose BafA1 on autophagic flux are independent of its ability to inhibit V-ATPase
activity, as was demonstrated by a lack of AV accumulation and Lysotracker Red, a
fluorescent dye accumulating in acidic cellular compartments such as lysosomes, staining
changes in low dose BafA1 treated cells compared to untreated controls [120;121].
Nevertheless, it is possible that at the low concentrations used in our experiments, BafA1
may act as an allosteric regulator of V-ATPase activity. In this scenario, BafA1 binding
to V-ATPase would promote a change in V-ATPase’s three dimensional structure such
that its ability to translocate protons across the lysosomal compartment would be
enhanced, which would promote a drop in luminal pH. In order to test the hypothesis that
150
low dose BafA1 promotes autophagic flux by directly affecting V-ATPase activity,
several approaches can be undertaken.
Investigating the effects of low dose BafA1 on ATPase activity and proton
pumping ability of V-ATPase isolated from SH-SY5Y cells would be one of the most
direct tests. However, it would be technically challenging because large quantities of SHSY5Y cells would be required for isolation of V-ATPase which then would need to be
inserted into the artificial lipid membrane of liposomes. This experimental setting may
also prove to be unsuitable for detection of any possible allosteric effects of low dose
BafA1 on V-ATPase activity because three dimensional structure of V-ATPase may be
affected by the lipid composition of the membrane as well as the neighboring proteins.
Bowman E. J. et al reported that at 1nM concentration BafA1 did not inhibit ATPase
activity of V-ATPases isolated from vacuolar membranes of Neurospora crassa or
bovine chromaffin granule membranes [122]. The primary structure of V-ATPase is more
than 75% conserved between different species extending from archaebacteria to humans
[123]. Therefore, the findings by Bowman E.J. et al are likely to hold true for SH-SY5Y,
a human neuronal cell line.
Testing the effects of BafA1 derivatives on V-ATPase activity can be a less
technically challenging approach of investigating the effect of low dose BafA1 on VATPase function. BafA1 consists of a 16-membered macrolactone and a tetrahydropyran
rings. Substitutions in one or more structural elements of BafA1 led to the discovery of
derivatives with great variability in efficacy to inhibit V-ATPase activity [124]. For
instance, BafA1 modifications associated with either disruption of its macrolactone or
151
deletion of the tetrahydropyran rings generates compounds that do not inhibit V-ATPase
activity [124]. However, the differences in binding of these compounds to V-ATPase
have not been directly investigated. Comparing their effects on attenuation of CQinduced cell death and autophagic flux inhibition to that of low dose BafA1 may shed
light on the overall importance of BafA1 interaction with V-ATPase activity in its
cytoprotection.
Low dose BafA1 may also modulate lysosomal pH acting as a direct ion
transporter across the lysosomal membrane. However, our data that low dose BafA1
pretreatment is sufficient for attenuation of CQ-induced neuron death is inconsistent with
BafA1 acting directly as an ion transporter. On the contrary, these findings suggest that
low dose BafA1 induces some long-lasting changes in the ALP rendering neurons more
resistant to insults such as CQ.
Low dose BafA1 may regulate lysosomal flux by affecting other ion
transporters regulating lysosomal pH and function such as Clc-7 or CFTR. Both CFTR
and Clc-7 have been implicated in regulation of lysosomal function and cell viability
[100-102]. This has been demonstrated by accumulation of lipofuscin loaded lysosomes,
dysfunction of lysosomal enzymes such as acid ceramidase, and cell death of several cell
types including neurons when CFTR or Clc-7 are mutated or knocked down in vitro or in
vivo [101]. However, recent publications suggest that these ion transporters may not be
critical regulators of lysosomal pH because cells deficient in either CFTR or Clc-7 have
no significant change in lysosomal pH as compared to wild type cells [102;125].
Although these observations may appear contradictory, they suggest that 1) ion transport
152
across the lysosomal membrane is an important contributor to lysosomal function and cell
viability, and 2) lysosomal pH is not the only determinant of lysosomal function.
The effects of CQ, α-syn, and BafA1 (high and low concentration) on the function
of lysosomal ion transporters such as CFTR or Clc-7 to our knowledge has not been
tested. Although the mechanisms of CFTR and Clc-7’s regulation of lysosomal function
are not fully understood, they are clearly important as regulators of the ALP and
lysosomal function. Therefore, investigating the effects of lysosomotropic agents such as
CQ and selective inhibitors of V-ATPase such as high dose BafA1 may facilitate our
understanding of the relationships between ion transporters and ALP function. It can be
hypothesized that inhibition of lysosomal function, such as with CQ treatment, may affect
protein and transcription levels of Clc-7 or CFTR or inhibit their activity. In this case,
cytoprotective effects of low dose BafA1 may be accompanied by normalizing the
activity or expression levels of Clc-7 or CFTR. The requirement for Clc-7 or CFTR to the
effects of low dose BafA1 on autophagic flux and cell viability can be further addressed
by inhibiting Clc-7 or CFTR’s function pharmacologically or via knockdown. Abrogation
of low dose BafA1 effects on autophagic flux and cell viability in these conditions would
indicate that these ion transporters are critical for low dose BafA1-induced
cytoprotection.
Oxidative Stress implications in the lysosomal function regulation
Oxidative stress signaling has been implicated in regulation of many intracellular
pathways including the ALP. Prolonged oxidative stress is believed to cause cell death
due to alterations in structure and function of intracellular components. However, initially
153
oxidative stress may be a signaling mechanism to help cells overcome stress [90]. There
is increasing evidence of a complex cross talk between mitochondria and the ALP. It is
thought that damaged mitochondria are degraded by selective auotophagy, a process
termed “mitophagy” [126]. The mechanisms that would indicate which population of
mitochondria is damaged and in turn removed from the cell by mitophagy have yet not
been elucidated. Alternatively, lysosomes have been implicated in oxidative stress
induction because of the high metal content within the lysosomal lumen [90]. Lysosomeinduced oxidative stress has been reported to affect mitochondrial function, exacerbating
oxidative stress signaling and promotion of cell death [90].
One of the cytotoxic effects of CQ is increased oxidative stress, which
accompanies lysosomal function inhibition. Therefore, attenuation of CQ-induced neuron
death by low dose BafA1 described in chapter 2 may be mediated by inhibition of CQinduced oxidative stress. This hypothesis can be tested by assessing the changes in
fluorescence of oxidative stress sensitive fluorescent probes such as dichlorofluorescein
diacetate (DCFDA) or by measuring changes in levels of hydroxynonenal (HNE) protein
modifications in CQ vs. CQ plus low dose BafA1 treated cells. Increased oxidative stress
induced by CQ would be accompanied by elevated levels of DCFDA fluorescence or
HNE. If co-administration or pre-treatment with low dose BafA1 were associated with a
decrease in oxidative stress levels as compared to CQ alone, the next questions that arise
would be what protein(s) and mechanism(s) are responsible for attenuation of CQinduced oxidative stress by low dose BafA1. Examining the changes in redox signaling
pathways induced by low dose BafA1 alone vs. CQ may help address this question. The
154
signaling cross-talk between lysosomes, mitochondria, and cytosolic components
involved in oxidative stress regulation are not fully mapped out, but are likely complex.
Therefore, it is possible that attenuation of CQ-induced oxidative stress and cell death by
low dose BafA1 may be achieved by affecting components other then the ALP.
Another project discussed in Chapter 3 of my dissertation investigated the effects
of mitochondrial dysfunction on lysosomal function. As described in greater detail in
Chapter 3, rotenone-induced death was associated with AV accumulation and an
increase in AO red fluorescence intensity suggesting increase in lysosomal volume and/or
acidification of ALP or late endosomal compartments. These observations suggest that
rotenone directly or indirectly affects pathways regulating lysosomal turnover and pH.
Rotenone is known to induce oxidative stress by inhibiting complex I of the METC
[127]. Therefore, it is likely that rotenone’s effects are mediated by increased oxidative
stress which can modulate structure and function of proteins involved in lysosome pH or
the volume regulation of lysosomes. Indeed, oxidative stress induced by H2O2 has been
reported to promote alteration in structure and function of lysosomal membrane localized
proteins such as Hsp70, a chaperone protein suggested to play a role in regulation of
lysosomal membrane composition and stability [128;129].
Candidate proteins responsible for rotenone-induced effects on the ALP include
lysosomal V-ATPase or other ion channels involved in lysosomal function regulation
such as Cl- / H+ transporters, Clc-7, and CFTR. Rotenone may affect expression and/or
activity of these protein complexes to promote degradation of proteins and organelles
damaged by oxidative stress, at least initially. This interpretation is consistent with the
155
observation of increased AV accumulation induced by rotenone, as described in chapter 3
and reported by others. Testing whether V-ATPase, Clc-7 and CFTR are also affected in
other pharmacological or genetic models of PD that have been associated with lysosomal
dysfunction and in post-mortem SN tissue of PD patients would allow us to determine if
these lysosomal protein complexes are implicated in PD pathology.
In summary, projects presented in this dissertation raise a question on the
similarities in mechanisms of cytotoxicity between classical PD-associated toxins such as
rotenone and known lysosomotropic agents like CQ. Although they have different
mechanisms of action, both rotenone and CQ affect the ALP and lysosome function and
cause neuronal death. This finding further adds to the growing body of evidence
indicating that lysosomal dysfunction plays a role in regulation of DA neuron death in
PD. Lysosomotropic agents such as CQ have been extensively studied as antimalarial
agents. Therefore, knowledge accumulated from studies on CQ may prove to be highly
beneficial for PD research as well.
156
Fig. 2: Proposed model of the ALP regulation by CQ, rotenone, and low dose
BafA1. CQ and rotenone alter lysosomal function, causing inhibition of autophagic flux
which results in accumulation of numerous autophagosomes in the cell. Low dose BafA1
restores lysosomal function, as demonstrated by attenuation of CQ-induced autophagic
flux inhibition (A). V-ATPase and Cl- transporters, Clc-7 and CFTR, regulate lysosomal
pH and function. CQ, rotenone, and low dose BafA1 may exert their effects on the ALP
by modulating function of these ion transporters (B). Further investigations of these ion
channels in different PD models will contribute to better understanding of the ALP
regulation and may yield novel therapeutic targets for treatment of PD.
157
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