Activation of autophagy against Bisphenol-A
1
Activation of autophagic flux against xenoestrogen Bisphenol-A induced hippocampal
neurodegeneration via AMPK/mTOR pathways
Swati Agarwal#,1,2
, Shashi Kant Tiwari#, 1,2
, Brashket Seth1,2
, Anuradha Yadav1,2
, Anshuman Singh1,
Anubha Mudawal1,2
, Lalit Kumar Singh Chauhan1, Shailendra Kumar Gupta
1,2, Vinay Choubey
3, Anurag
Tripathi1, Amit Kumar
1, Ratan Singh Ray
1, Shubha Shukla
4, Devendra Parmar
1, and Rajnish Kumar
Chaturvedi1,2,
*
1CSIR-Indian Institute of Toxicology Research, 80 MG Marg, Lucknow 226001, India
2 Academy of Scientific and Innovative Research (AcSIR), India
3Department of Pharmacology; Centre of Excellence for Translational Medicine; University of Tartu;
Tartu, Estonia. 4Department of Pharmacology; CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension,
Lucknow 226031, India
Running title: Activation of autophagy against Bisphenol-A
# These two authors contributed equally
*To whom correspondence should be address: Dr. Rajnish Kumar Chaturvedi, Developmental Toxicology
Division, Systems Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), 80 MG Marg,
Lucknow 226001, India, Tel: +91-522-2228227, Fax: +91-522-2628227, E-mail: [email protected]
Keywords: Autophagy, Bisphenol-A, neurotoxicity, hippocampus, neural stem cells
Capsule
Background: The effects of xenoestrogen Bisphenol-
A on autophagy, and association with oxidative stress
and apoptosis are still elusive.
Results: Transient activation of autophagy protects
against Bisphenol-A induced neurodegeneration via
AMPK activation and mTOR down-regulation.
Conclusion: Autophagy induction against Bisphenol-
A is an early cell’s tolerance response.
Significance: Autophagy provides an imperative
biological marker for evaluation of neurotoxicity by
xenoestrogen.
Abstract
The human health hazards related to persisting use of
Bisphenol-A (BPA) are well documented. BPA
induced neurotoxicity occurs with the generation of
oxidative stress, neurodegeneration, and cognitive
dysfunctions. However, the cellular and molecular
mechanism(s) of the effects of BPA on autophagy, and
association with oxidative stress and apoptosis are still
elusive. We observed that BPA exposure during early
postnatal period enhanced the expression and the
levels of autophagy genes/proteins. BPA treatment in
presence of bafilomycin A1 increased the levels of
LC3-II and SQSTM1 and also potentiated GFP-LC3
puncta index in GFP-LC3 transfected hippocampal
neural stem cells derived neurons. BPA induced
generation of reactive oxygen species and apoptosis
were mitigated by pharmacological activator of
autophagy (rapamycin). Pharmacological (wortmannin
and bafilomycin A1), and genetic (beclin siRNA)
inhibition of autophagy aggravated BPA neurotoxicity.
Activation of autophagy against BPA resulted
intracellular energy sensor AMPK activation,
increased phosphorylation of raptor and ACC, and
decreased phosphorylation of ULK1 (Ser757), and
silencing of AMPK exacerbated BPA neurotoxicity.
Conversely, BPA exposure down regulates mTOR
pathway by phosphorylation of raptor as a transient
cell’s compensatory mechanism to preserve cellular
energy pool. Moreover, silencing of mTOR enhanced
autophagy, which further alleviated BPA induced ROS
generation and apoptosis. BPA mediated neurotoxicity
also resulted in mitochondrial loss, bioenergetic
deficits, and increased Parkin mitochondrial
translocation, suggesting enhanced mitophagy. These
results suggest implication of autophagy against BPA
mediated neurodegeneration through involvement of
AMPK and mTOR pathways. Hence, autophagy which
arbitrates cell survival and demise during stress
conditions requires further assessment, to be
established as biomarker of xenoestrogen exposure.
http://www.jbc.org/cgi/doi/10.1074/jbc.M115.648998The latest version is at JBC Papers in Press. Published on July 2, 2015 as Manuscript M115.648998
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Activation of autophagy against Bisphenol-A
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Introduction
Bisphenol-A (BPA) is a potent endocrine disruptor as
well as neurotoxicant (1,2). It is released from the
polycarbonate plastics containing various food items
and dental sealants (3). The prevalent usage of BPA
amongst the human population has raised concern
regarding the possible health hazards worldwide.
Compelling evidences from animal studies have
strongly implicated neurotoxic potential of BPA (4-9).
Prenatal low dose BPA exposure impairs the
hippocampal neurogenesis and causes learning and
memory deficits (10-12). BPA increases the levels of
intracellular peroxides and mitochondrial superoxides,
and induces apoptotic cell death in neuronal cells by
the generation of reactive oxygen species (ROS) and
activation of MAP kinases and nuclear factor-kappa B
(13,14). Accumulated evidence supports the toxic
effects of BPA and the resulting pathogenesis of
neurodegenerative as well as other diseases (15-22). BPA exposure resulted in significant increase of
oxidative stress, as evidenced by the increased
malondialdehyde levels, decreased glutathione levels
and superoxide dismutase activity in the brain (15-21).
However, till date there is no information available
regarding the cellular and molecular mechanism(s) of
the effects of BPA on the regulatory dynamics of
autophagy in the brain. Moreover, the interplay
between autophagy, apoptotic cell death, and ROS
generation in BPA mediated neurotoxicity remains
largely unanswered and is not entirely understood.
In order to maintain cellular homeostasis, three
types of cell death mechanism occur namely
autophagy, apoptosis, and necrosis (23,24). Autophagy
is a protective cellular cleanup process involved in the
removal of unwanted and misfolded proteins from the
cells, by delivering them to the lysosomes for their
degradation (25). Autophagy and apoptosis work in a
co-ordinated manner to regulate cell survival and death
(24-26). Several conditions such as starvation,
toxicants exposure and mechanical injury result in the
generation of ROS, and concomitant accumulation of
damaged mitochondria and misfolded proteins inside
the cells. Any alterations in the basal levels of
autophagy may lead to several pathogenic conditions
viz cancer, neurological and neurodegenerative
disorders such as Parkinson’s and Alzheimer’s disease
(25,27,28). Recent studies have found that several
environmental toxicants such as arsenic (29),
cadmium, chromium (30,31), dibenzofuran (32),
paraquat (33), and ethanol (34,35) cause alterations in
the basal levels of autophagy, leading to cellular
toxicity. Autophagy acts as a cardinal process and
interconnects several cell survival pathways viz AMP
kinase (AMPK), mammalian target of rapamycin
(mTOR) and PI3K/Akt (36,37). Stress leads to decline
in the ATP levels and also accretion of cellular AMP.
Thus, AMPK act as an intracellular energy sensor,
which activates under low nutrient or energy deprived
conditions (38). AMPK restrains cell growth and
metabolism through phosphorylation of acetyl CoA
carboxylase (ACC) and raptor (37,39-41) during stress
conditions. AMPK is involved in several functions like
autophagy, apoptosis and cell migration (37,42,43).
The activity of mTOR (a serine/threonine protein
kinase and master regulator of autophagy) is activated
under nutrient enriched conditions, and inhibited under
starvation conditions thereby leading to inhibition and
activation of autophagy respectively (44). Herein, we
studied the effects of BPA exposure on autophagy in
vitro hippocampal neural stem cells (NSC) derived
neurons, and in the hippocampus region (crucial region
for learning and memory regulation) of the rat brain.
We elucidated the molecular mechanism(s) underlying
the AMPK pathway activation and mTOR down-
regulation in response to BPA exposure. The decline
in ATP levels, after BPA exposure activates AMPK to
preserve cellular energy pool by inhibiting the
anabolic processes, while turning on the catabolic
pathways. Moreover, AMPK balances energy levels by
enhancing autophagy, and inhibits mTORC1 by
phosphorylation of raptor (37,39,41). In addition,
autophagy induced against BPA also results with the
phosphorylation of an AMPK substrate ACC.
Contrarily, BPA induced energy depletion leads to the
reduction in the phosphorylation of ULK1 at Ser 757.
Therefore, a concerted coordination is maintained
among the three kinase complexes in order to regulate
the autophagy induction and cell survival during BPA
exposure. Interestingly, inhibition of autophagy
through the genetic and pharmacological approaches
aggravated BPA induced neurotoxicity, and enhanced
ROS generation and apoptosis. Thus, our studies
delineate that autophagy acts as a transient cellular
protective response against BPA induced
neurotoxicity.
Material and Methods
Materials:
BPA [4,4′-(propane-2,2-diyl) diphenol], bafilomycin
A1, wortmannin, rapamycin, anti-SQSTM1 primary
antibody, EGF, bFGF, DHE (2,7-Diamino-10-ethyl-9-
phenyl-9,10-dihydrophenanthridine) and Lipid
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Peroxidation Assay Kit were procured from Sigma-
Aldrich . Primary antibodies such as anti-β-actin, anti-
beclin-1, anti-ACC, anti-p-ACC anti-raptor, anti-p-
raptor, anti-ULK1, anti-p-ULK1, anti-P70S6K, anti-
p-P70S6K, anti-AMPK, anti-p-AMPK, anti-mTOR
anti-p-mTOR were obtained from from Cell Signaling
Technology, and anti-HMGB1, anti-caspase-3, anti-
TOMM20, anti-COX-IV, anti-PINK1, anti-Parkin,
anti-GAPDH, anti-VDAC procured from Abcam.
Secondry antibodies such as Alexa flour-594 goat anti-
rabbit IgG, anti-rabbit IgG peroxidase antibody, anti-
mouse IgG peroxidase antibody, MitoTracker,
LysoTracker, 10-N-nonyl acridine orange were
obtained from Invitrogen (LifeTechnologies, USA).
Neurobasal media, B-27 and N-2 supplement were
obtained from Gibco. GFP-LC3 Plasmid from
Invivogen, anti-LC3A/B pAb from Novus , all siRNAs
from Dharmacon , GSH/GSSG-Glo™ Assay kit and
Cell Titer-Glo Luminescent Cell Viability Assay kit
for ATP measurement were from Promega .
Animals and treatment:
Adult Wistar rats (180–220g) were obtained from
Animal Breeding Colony of the CSIR-Indian Institute
of Toxicology Research. Rats were kept in a 12h
light/dark cycle with ad libitum water and pellet diet
(Hindustan Lever Laboratory Animal Feed, India).
Experimental animals were handled according to the
guidelines laid down by the Institute’s Ethical
Committee for Animal Experiments. Animals were
randomly segregated into the following groups.
(I) Vehicle control group: Received daily single oral
administration of vehicle (corn oil), from PND14-
PND21.
(II) BPA (40µg) group: Received daily single oral
administration of BPA (40µg/kg body weight) from
PND14-PND21.
(III) BPA (400µg) group: Received daily single oral
administration of BPA (400µg/kg body weight) from
PND14-PND21.
The doses of BPA (40 and 400μg/kg body
weight) were selected on the basis of earlier studies,
where BPA induced neurobehavioral and
neurochemical alterations in the rat brain (10-12). Rat
pups were sacrificed at PND21, and the effects of BPA
on autophagy were studied by qRT PCR, western blot
and transmission electron microscopy analysis. In
order to study the effects of BPA on cleaved caspase-3
levels in the hippocampus of the rat brain, a group of
pups were treated with 400μg/kg body weight BPA,
and BPA along with rapamycin (rapamycin; 1 and 2
mg/kg body weight, i.p).
Hippocampal NSC derived neuronal culture and
BPA treatment:
Hippocampal NSC were cultured and differentiated in
neurons as describer earlier (45). Neuronal cells
grown in flasks were treated with different
concentrations of BPA dissolved in DMSO (0, 25, 50
100, 200 and 400µM) for 24h. The non-cytotoxic
concentration of BPA was determined by trypan blue
and propidium iodide (PI) uptake analysis. We found
that BPA was non-cytotoxic at concentrations upto
100µM. In order to study the levels of LC3-II protein,
cells were grown in the presence of non-cytotoxic
concentrations of BPA (0, 25, 50 and 100µM) for 3, 6
and 12h. Further, the cells were treated in the
presence/absence of pharmacological inducer
(rapamycin; 100nM) and inhibitor of autophagy
(wortmannin; 10 µM). To induce starvation conditions
in NSC derived hippocampal neurons, neuronal media
was replaced with HBSS. After respective treatments
cells were analyzed for autophagy by western blotting,
flow cytometry and immunofluorescence studies.
Flow cytometry and trypan blue dye exclusion
assay for cell viability:
Effects of BPA on viability of hippocampal NSC
derived neurons were examined through trypan blue
dye exclusion assay and flow cytometry as described
earlier (46). The results are expressed in terms of
percentage of controls.
Plasmids and siRNA transfection in neuronal cells:
The hippocampal neuronal cells were transiently co-
transfected with plasmids for mammalian GFP-LC3,
pmKate mitochondrial reporter gene, YFP-PARK2,
mito-CFP, and beclin, mTOR, and AMPK siRNA and
cells were treated with BPA. Cells were visualized
using a Nikon Eclipse Ti-S inverted fluorescent
microscope equipped with Nikon Digital Sight Ds-Ri1
CCD camera and NIS Elements BR imaging software
(Nikon, Japan).
Quantification of GFP-LC3 puncta:
For GFP-LC3 puncta counting, a person unknown to
experimental design blindly selected 50 cells from
each group, number of GFP-LC3 puncta were counted
individually in each cell, and averaged as per well
established methods (47,48). The results were
represented as mean±SEM of three independent
experiments.
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Oxidative stress and antioxidant levels:
ROS production in neuronal cells was assessed using
2,7-dichlorohydrofluorescein diacetate (DCFH-DA)
and dihydroethidium (DHE) dyes (49). Briefly,
neuronal cells (1x103
cells) were treated with BPA
(100 µM) for 12h, followed by incubation with DCFH-
DA (10µM) for 30 min at 37ºC. Fluorescence intensity
was monitored by a spectrofluorometer at
excitation/emission wavelengths of 485/530nm,
respectively. To evaluate superoxide generation, DHE
(5µM) was added to control and treated cultures for 30
min, and a ratio of ethidium/DHE was analyzed. DHE,
reacts with superoxide anion, and forms a red
fluorescent product, 2-hydroxyethidium with
maximum excitation and emission peaks at 500 and
580 nm, respectively. Further, following article from
(50), we have assessed lipid peroxidation (LPO),
malondialdehyde (MDA) levels using lipid
peroxidation assay kit as per manufacturer’s
instruction. Antioxidant levels were evaluated by
measuring total glutathione (GSH) levels in control
and treated cultures following manufacturer’s protocol.
The protein levels of superoxide dismutase (SOD) and
catalase were also estimated by immunoblotting.
Gene expression analysis by qRT-PCR:
In order to study the expression of genes involved in
autophagy, qRT-PCR analysis was carried out
following our earlier published method (45).
Levels of protein analysis by western immunoblot:
Hippocampal tissue/cells were lysed with cell lytic MT
MammalianTissue Lysis/Extraction Reagent.
Membranes were blocked with LC3-II (1:1,000),
SQSTM (p62) (1:1000), beclin-1 (1:1000), HMGB1
(1:10000), mTOR (1:1000), p-mTOR (1:1000),
P70S6K (1:1000), p-P70S6K (1:1000), AMPK
(1:1000) p-AMPK (1:1000), p-ACC (1:1000), ACC
(1:1000), p-raptor (1:1000), raptor (1:1000), p-ULK1
(1:1000), ULK1 (1:1000), cleaved-caspase-3 (1:500),
Lamp-2 (1:500), SOD (1:500), catalase (1:500)
TOMM20 (1:1000), COX-IV (1:1000), VDAC (1:
1500), GAPDH (1:2000) and -actin (1:10,000).
Similarly, the levels of PINK-1 and parkin were
studied in cytoplasmic, mitochondrial and total
fractions, using VDAC. Protein bands were quantified
using Scion Image for Windows (NIH, USA).
Two-dimensional gel electrophoresis: For two dimensional polyacrylamide gel
electrophoresis (2D-PAGE) analysis, three sets of
pooled protein samples were prepared from
hippocampal tissue of control and treated rats using
tissue lysis buffer, followed by acetone precipitation.
Protein content was quantified using Bradford reagent.
Samples were then resolved through 2D-PAGE.
Isoelectric focusing (IEF) was carried out using 11-cm
immobilized pH gradient strips (IPG, 4-7 pH gradient,
Bio-Rad). IEF was carried out at 20ºC for 30000 Volt-
hours in a protein i12 IEF Cell (Bio-Rad). Strips were
incubated with gentle shaking in Ready Prep2-D
starter kit equilibration solution-I and equilibration
solution-II for 10 min in each solution. Gels were run
in triplicate, stained in coomassie G-250 (0.2%), and
destained. Images were acquired using GS-800
densitometer (Biorad, USA) and analyzed using PD
quest software followed by densitometric analysis of
individual spot of interest. Spots of interest were
identified by peptide mass fingerprinting through
matrix-assisted laser desorption/ionization-time of
flight (MALDI-TOF) using previously described
protocol (51). Peptide mass fingerprinting (PMF) was
performed using 4800 Proteomic analyzer MALDI-
TOF/TOF mass spectrometer (Applied Biosystems,
Warrington, U.K) in positive reflector. The deduced
peptide sequences were submitted to SwissProt
database (http:// www.expasy.ch) or NCBI non-
redundant database (http://www.ncbi.nlm.nih.gov).
The data presented as mean±standard error mean
(S.E.M) of 3 samples from each group and statistically
analyzed using non-parametric t-test.
Immunofluorescence study:
Immunohistochemical localization of activated
caspase-3 was carried out as described earlier (45,52).
30μm thin serial coronal sections encompassing the
hippocampus were incubated with primary cleaved
caspase-3 antibody (1:500). Slides were analyzed
under a Nikon Eclipse Ti-S inverted fluorescent
microscope. The quantification of cleaved caspase-3
positive cells in the hippocampus region was carried
out following our earlier study.
Conditioned Avoidance Response (CAR):
The learning and memory responses were studied in
control, BPA, rapamycin and BPA+rapamycin treated
rats. Rats received daily single i.p. injection of
rapamycin (0.1 mg/kg body weight) from PND 28-90
served as rapamycin treated group. Similarly, in
BPA+rapamycin group, BPA treated rats (40µg) from
PND 28-90 received daily single i.p. injection of
rapamycin (0.1 mg/kg body weight from PND 28-90.
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We performed assessment of two-way conditioned
avoidance behavior using a shuttle box apparatus
(Columbus Instruments) as described earlier (45,53).
Learning and memory in treated groups were studied
as compared to percent control.
Fluoro-Jade-B labeling for degenerating neurons in
the hippocampus:
Fluoro-Jade B labeling was performed in both control
and treated group according to manufacturer’s
protocol. Fluoro-Jade B+ degenerating neurons were
bilaterally counted in a total of six sections and
averaged for each rat as described earlier (10,45).
Mitophagy study:
Mitophagy was analyzed by studying the
mitochondrial mass using NAO (mitochondrial
specific dye), mitochondrial copy number (ratio of
nuclear encoded gene 18S and mitochondrial encoded
gene COX-II) (54), pmKate mitochondrial reporter
gene (55,56) and its co-localization with GFP-LC3. To
study effects of BPA on mitophagy, neuronal cells
were co-transfected with GFP-LC3, TOMM20 and
pmKate mitochondrial reporter plasmids. Similarly,
neuronal cells were transfected with YFP-PARK2 and
Mito-CFP to study BPA mediated effects on PARK2
mitochondrial translocation. The mitoTracker and
lysostracker co-localization assay was also done using
MitoTracker Green FM and 200nM LysoTracker Red
DND-99 (data not shown). Co-localization was
quantified using ImageJ (National Institutes of Health)
employing the JACoP plugin (57). The statistical test
included in the analysis was Mander's co-localization
coefficient (M), showing the percentage of co-
localization (M=1 regarded as perfect correlation). Data were rendered as the percentage co-localization
obtained by analysis of combining data from at least
three independent experiments.
ATP measurement assay:
Intracellular ATP levels were studied using Cell Titer-
Glo Luminescent Cell Viability ATP determination kit
as per manufacturer’s instructions.
TEM analysis:
To study the effects of BPA on autophagy,
ultrastructure studies were performed by TEM in the
hippocampus region of the rat pups brain as protocol
described earlier (45).
Statistical analysis:
Statistical analysis was carried out by using GraphPad
InStat statistical analysis software (SanDiego, CA,
USA). Homogeneity of variance between all the
experimental groups was ascertained and mean
significant difference in the experimental groups was
determined using one-way analysis of variance
(ANOVA) followed by the Tukey–Kramer post hoc
multiple comparisons test. P-values of 0.05 were
considered to be statistically significant.
Results
Effects of BPA on cell viability and
neurodegeneration in the hippocampus-
We assessed the effects of BPA at various
concentrations (25, 50, 100, 200, and 400μM) on
viability of hippocampal NSC derived neurons by
Trypan blue and PI uptake through flow cytometry
(Fig. 1 A-B). BPA significantly decreased viability
above 100μM concentration (Fig. 1A-B). We found
that 100μM was a non-cytotoxic dose of BPA, and
above it significantly reduced the cell viability and
increased the number of PI+
cells (Fig. 1A-B).
Further, in vivo we studied the effects of BPA on
neuronal cell death in the hippocampus region using
immunohistochemical analysis of activated caspase-3.
Cells labeled with activated caspase-3 undergo
apoptosis following BPA treatment at both 40 and
400μg/kg body weight doses (Fig.1C). Quantitative
analysis revealed that BPA treatment dose dependently
enhanced activated caspase-3+cells in the hippocampus
as compared to control group (Fig. 1C-D). Next, we
determined the effects of BPA treatment on neuronal
degeneration in the hippocampus by labeling sections
with Fluoro-Jade B. We found that BPA significantly
enhanced the number of Fluoro-Jade B+ cells at both
the doses as compared to control (Fig.1E-F). These
results suggest that BPA caused apoptosis and
enhanced neuronal degeneration in the hippocampus.
BPA induced the expression of autophagy marker
genes in the hippocampus region of the rat brain-
As we observed that BPA caused reduced cell viability
and increased neurodegeneration, therefore we
hypothesized that it could be due to alteration in cell
survival process autophagy. We treated Wistar rats
during postnatal day 14-21 (PND14-PND21) with two
doses of BPA (40 and 400µg/kg body weight) and
examined its effects on alteration of the expression of
autophagy marker genes in the hippocampus of the rat
brain. We found significant increase in the mRNA
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expression of LC3, HMGB1, beclin-1, Atg5, Atg12,
Atg3 and Lamp-2 genes, and decrease in the
expression of p62 following BPA exposure at both the
doses studied (Fig. 2A). However, the expression of
Atg7 was not significantly altered by 40µg/kg of BPA
treatment. Similarly, the levels of autophagy proteins
LC3-II, beclin-1, and HMGB1 in the hippocampus
were significantly increased after BPA treatment at
both the doses as compared to control (Fig. 2B-C).
Contrarily, BPA treatment decreased the levels of p62
in the hippocampus region of the rat brain (Fig. 2B-C).
The increased expression and the levels of
autophagosome associated form i.e. LC3-II from
soluble form LC3-I signifying the increased formation
of autophagosome (58).
We next carried out ultrastructural transmission
electron microscopy analysis, in order to assess
whether increased expression of autophagy marker
genes is also associated with enhanced formation of
autophagosome. We observed increased generation of
multilamellar vesicular bodies (59) autophagosomes
(A), autolysosomes (AL) and degradative autophagic
vacuole (AVd) in the hippocampus of BPA treated rats
as compared with control (Fig.2D-E). The presence of
increased degradative autophagic vacuole (AVd)
rather than accumulation of autophagosomes by BPA
treatment suggests that BPA neurotoxicity is relieved
by the generation of intralysosomal degradation
pathway.
Proteomics analysis of autophagy associated
proteins in the hippocampus region of BPA treated
rats-
The effects of BPA on autophagy in the hippocampus
of BPA (40 and 400μg/kg body weight) treated rat
pups were further studied using 2D in tandem with
Mass Spectrometry proteomics approach. Total 50
protein spots on the gel were detected and quantitative
analysis of protein was performed (Fig 2F, Table 1).
We observed alterations in the levels of various
autophagy regulating proteins in BPA treated rats at
both the doses as compared to control (Fig. 2F and
Table 1). The levels of 21 proteins were up-regulated
and 6 were down-regulated by BPA treatment in
comparisons to control (Table 1). Cathepsin D, which
is a lysosomal protein required for complete
autophagic degradation, was significantly up-regulated
by BPA treatment, suggesting autophagic induction
against BPA, rather than accumulation of
autophagosomes. Phosphatidylethanolamine binding
protein, which is involved in LC3-II lipidation, was
also significantly increased due to BPA treatment.
Other proteins detected were heat shock cognate
protein (Hsc71, Hsc70 and Hsc60), and peroxiredoxin-
2, which play a vital role in the identification and
proteasomal degradation of redundant proteins as
well as enhancing their lysosomal activity. Similarly
peroxiredoxin, which is involved in peroxisome
mediated autophagy was also significantly up-
regulated by BPA. Several other metabolic enzymes
such as pyruvate kinase, malate dehydrogenase and its
isoforms, and α and γ enolase and their isoforms were
also significantly up-regulated by BPA (Table 1).
BPA induces autophagy in the hippocampal NSC
derived neurons in vitro
To examine, whether BPA induces autophagy,
neuronal cells were treated with various non-cytotoxic
concentrations of BPA i.e. 0, 25, 50 and 100μM. The
levels of LC3-II were significantly up-regulated in the
hippocampal NSC derived neurons by 100μM
concentration of BPA as compared to control (Fig. 3A-
B). BPA (100μM) significantly enhanced the levels of
LC3-II during 12h of exposure (Fig.3A-B). Significant
increase was found in the levels of LC3-II after 3h of
BPA exposure in neuronal cultures, which further
augmented at 12h (Fig.3C-D). We next studied the
effects of BPA on the formation or accumulation of
GFP-LC3 puncta in GFP-LC3 transfected
hippocampal NSC derived neuronal cells. After
transfection, we examined the formation of GFP-LC3
puncta and puncta index in neuronal cells, which is an
indicator of formation of autophagosomes in the cells.
We observed significantly increased number of GFP-
LC3 puncta in 100μM of BPA treated neuronal
cultures as compared to control (Fig. 3E-F). Further,
we confirmed that increased LC3-II levels observed
are due to the enhanced autophagy rather than
blockade at any step during autophagy. To this end, we
arrested LC3-II mediated autophagosome degradation
using lysosomal protease inhibitor bafilomycin A1 (60)
in BPA treated neuronal cells. BPA treatment in the
presence of bafilomycin A1 potentiated LC3-II
lipidation and GFP-LC3 puncta index in neuronal
cultures in comparison with BPA alone treated
cultures (Fig. 3G-J). Moreover, for starvation neuronal
media was replaced with (Hank’s balanced salt
solution; HBSS) (Fig. 3G-H). We further validated the effects of BPA on
LC3-II lipidation and GFP-LC3 puncta formation in
the presence/absence of pharmacological activator
(rapamycin) and inhibitor (wortmannin), and genetic
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inhibitors (beclin siRNA) of autophagy. Beclin siRNA
significantly reduced beclin protein levels (Fig.4A). BPA along with rapamycin enhanced the levels of
LC3-II protein as well as increased GFP-LC3 puncta
index in neuronal cultures (Fig.4B-E). Moreover, pre-
incubation of neuronal cultures with wortmannin
followed by BPA exposure inhibited LC3-II lipidation,
and also decreased the number of GFP-LC3 puncta.
Further, siRNA mediated knockdown of beclin
suppressed the formation of GFP-LC3 puncta after
BPA treatment (Fig. 4B-E). Thus, these results infer
that rapamycin potentiate, and wortmannin and beclin
siRNA inhibit LC3-II levels and GFP-LC3 puncta
formation after BPA treatment in hippocampal NSC
derived neuronal cultures.
To examine the effects of BPA on autophagic
substrate, the levels of p62 were studied in the cultures
of hippocampal neurons treated with BPA in the
presence/absence of bafilomycin A1. BPA treatment in
the presence of bafilomycin A1, increased the levels of
p62 protein (Fig. 4F-I). Moreover, treatment of BPA
alone in neuronal cultures decreased the levels of p62
protein (Fig. 4F-I). Treatment of BPA along with
rapamycin further decreased the levels of p62 (Fig. 4F-
I). Contrarily, BPA treatment along with rapamycin
enhanced the levels of p62 in the presence of
bafilomycin A1 (Fig. 4F-I). These results suggest that
BPA induces autophagic flux rather than inhibiting
autophagic proteolysis.
HMGB1; an endogenous pro-autophagic
protein, participates in the autophagy process,
increases cell survival, and retards apoptotic cell death
(61,62). Translocation of HMGB1 from nucleus to the
cytosol induces autophagy (61,62). We found that
HMGB1 translocation takes place during autophagy
conditions like starvation and in the presence of
autophagy inducer i.e. rapamycin in neuronal cells
(Fig. 5A-B). Further, we observed that BPA treatment
caused increased HMGB1 cytosolic translocation as
compared to control. Moreover, wortmannin and
beclin siRNA inhibited migration of HMGB1 in the
cytosol after BPA treatment (Fig. 5A-B). Thus, overall
suggesting that BPA also causes cytosolic
translocation of HMGB1 leading to increased
autophagy.
Autophagy compensates against BPA induced
cytotoxicity and apoptosis in neuronal cells and in
the hippocampus of the rat brain-
BPA causes apoptotic cell death in neurons (8), and
autophagy is reported to regulate apoptosis (23,63).
Therefore, to study the role of autophagy in the
regulation of BPA mediated apoptotic cell death, we
treated neuronal cells with rapamycin as well as
wortmannin. BPA treatment significantly enhanced
number of PI positive and cleaved caspase-3 positive
cells in vitro (Fig 6A-B) and in vivo (Fig 6C-D).
Treatment of rapamycin along with BPA significantly
mitigates BPA mediated toxicity and apoptotic cell
death. Conversely, wortmannin further enhanced
cytotoxicity and apoptosis in BPA treated cultures
(Fig. 6A-B). Concurrently, beclin knockdown resulted
in significant reduction of autophagy and exacerbated
BPA mediated apoptotic cell death in neuronal
cultures (Fig.6A-B). Scramble siRNA sequence
showed no significant effect on cell viability (data not
shown). The pre-treatment of catalase decreased BPA
induced cytotoxicity and apoptotic cell death even in
beclin siRNA transfected neuronal cultures after BPA
treatment. These findings suggest that BPA induced
cell death was significantly exacerbated and mitigated
by the inhibition and activation of autophagy
respectively (Fig. 6A-B).
We next studied the role of autophagy in the
regulation of BPA mediated apoptotic cell death in the
hippocampus of the rat brain. BPA significantly
enhanced the number of caspase-3 positive cells in the
dentate gyrus, hilus and molecular layer region of the
hippocampus as compared to control (Fig.6C-D).
Rapamycin (1 and 2mg/kg body weight, i.p) reduced
the number of caspase-3 positive cells in BPA treated
rats. These results suggest that the induction of
autophagy reduces BPA mediated apoptotic cell death
in the hippocampus region of the rat brain.
Autophagy as a protective response against BPA
induced ROS generation- Exposure with BPA causes ROS generation, resulting
neurodegeneration inside the cell (1, 48). Therefore,
we studied the role of autophagy in BPA mediated
ROS generation in neuronal cultures. AMPK and
mTOR siRNA significantly reduced respective protein
levels (Fig.7A). The neuronal cultures were pre-
treated with an antioxidant enzyme catalase, and an
antioxidant N-acetylcysteine followed by BPA
treatment (Fig.7B). BPA significantly enhanced the
levels of LC3-II (Fig.7B). BPA induced increase in
LC3-II lipidation was significantly reduced in neuronal
cells, in which treatment of catalase and NAC was
given prior to BPA, in comparison with BPA alone
treated culture (Fig.7B). Thus, both NAC and catalase
inhibited autophagy by reducing oxidative stress and
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ROS generation in BPA treated neuronal cells. BPA
significantly increased ROS generation in a time
dependent manner in neuronal cells (Fig.7C-E). BPA
mediated ROS generation was decreased by autophagy
inducer rapamycin, and further enhanced in the
presence of bafilomycin A1.
Next, we carried out genetic inhibition of
autophagy by beclin and AMPK siRNA, and activation
of autophagy by mTOR siRNA in neuronal cultures.
siRNA mediated knockdown of beclin and AMPK
resulted in elevation of ROS levels in BPA treated
neuronal cultures, suggesting that inhibition of
autophagy may aggravate BPA induced ROS
generation and oxidative stress (Fig.7C-E). The mTOR
is a cell survival pathway, and inhibition of the mTOR
during stress conditions leads to activation of
autophagy (34). Interestingly, siRNA mediated
knockdown of mTOR caused significant decrease in
ROS generation in BPA treated neuronal cultures
(Fig.7C-E). BPA induced ROS generation was
aggravated in the presence of bafilomycin A1, beclin
and AMPK siRNA, while decreased by rapamycin and
mTOR siRNA (Fig.7C-E).
Further, we examined increase in the levels of
MDA after BPA exposure. Interestingly, BPA
exposure enhanced the levels of MDA in beclin and
AMPK siRNA, and bafilomycin treated neuronal cells,
where autophagy was reduced (Fig.7F). In contrast,
induction of autophagy by mTOR siRNA and
rapamycin alleviated BPA induced increase in LPO.
Similarly, activation of autophagy increased the levels
of total glutathione in BPA treated neuronal cells. On
the other hand, inhibition of autophagy further reduced
the glutathione levels in BPA treated cells (Fig.7G).
BPA induced autophagy is associated with the
activation of AMPK and down-regulation of mTOR
signaling-
The AMPK and mTOR pathways are primarily
involved in regulation of autophagy process in the
cells. Therefore, we studied the role of the
AMPK/mTOR pathways in regulation of BPA
mediated autophagy. We observed dose dependent
decline in the ATP levels in response to BPA exposure
in hippocampal NSC derived neuronal cultures (Fig.
8A). We determined the effects of BPA on neuronal
cells after silencing the AMPK and mTOR genes,
through flow cytometry PI uptake apoptosis analysis
(Fig. 8B-C). BPA exposure increased the number of
apoptotic cells, which was further enhanced after
AMPK knockdown in neuronal cells. Contrarily,
mTOR knockdown in BPA exposed cultures
significantly decreased the numbers of apoptotic cells
(Fig. 8B-C). These results suggest involvement of
AMPK activation in autophagy induction against BPA
induced cell death, while silencing of mTOR enhanced
autophagy leading to decreased apoptosis after BPA
exposure (Fig. 8 B-C). Silencing of AMPK decreased
BPA induced phosphorylation of AMPK at Thr 172 in
neuronal cells (Fig. 8D-E). Moreover, BPA induced
GFP-LC3 puncta and LC3-II levels were also
decreased after AMPK knockdown (Fig. 8D-H).
Furthermore, BPA decreased the
phosphorylation of mTOR at Ser 2481, and
phosphorylation of its downstream target P70S6K at
Thr 389 (Fig. 9A-B). In addition, BPA also decreased
the phosphorylation of ULK at Ser 757 in neuronal
cells (Fig. 9A-B). Interestingly, BPA increased the
phosphorylation of AMPK, ACC at Ser 79 and raptor
at Ser 792 (Fig. 9C-D). AMPK knockdown resulted in
significant down regulation of ACC and raptor
phosphorylation, but up-regulation of ULK1 at Ser 757
in BPA exposed neuronal cultures (Fig. 9C-D).
Corresponding with these findings, BPA mediated
decrease in p-mTOR levels were suppressed by
AMPK knockdown, suggesting involvement of the
mTOR as a downstream effector of the AMPK
pathway in regulation of autophagy induced against
BPA exposure (Fig. 9E-F).
BPA induced mitochondrial loss and mitophagy in
neuronal cells-
Mitochondrial dysfunction is a major source of ROS
generation and cell death, therefore we hypothesized
that BPA mediated cell death could be attributed to
enhanced mitochondrial dysfunction. To study the
effects of BPA on mitochondrial mass in neuronal
cells, we stained neuronal cells with mitochondria
specific dye NAO. Treatment of neuronal cells with
25, 50, and 100μM BPA for 12h caused reduction in
mitochondrial mass dose dependently (Fig. 10A). We
next examined the ratio of mRNA for COX-II
(mitochondrial DNA-encoded gene) and 18S rRNA
(nuclear encoded transcript), to study mitochondrial
DNA content (54). We found significant reduction in
COX-II/18S rRNA ratio after BPA exposure in
neuronal cells, suggesting mitochondrial loss by BPA
treatment (Fig.10B). Mitochondrial dysfunction plays
an important role in the generation of ROS and
dysfunctional mitochondria are removed by a process
known as mitochondrial autophagy (mitophagy) (64).
Mitophagy involves selective degradation of damaged
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mitochondria by autophagosomes, by passing them to
lysosomes (64,65) (data not shown). Co-localization of
pmKate mitochondrial reporter gene (55,56) or
pDsRed2 with GFP-LC3 is an indicator of mitophagy
process in the cells (66). We observed enhanced co-
localization of pmkate mitochondrial reporter gene;
red with GFP-LC3; green (Manders co-localization
coefficient; M=0.19) after BPA exposure as compared
to control (M=0.05). In BPA+rapamycin and
BPA+beclin siRNA treated cultures co-localization
coefficient were M=0.27 and 0.10 respectively (Fig.
10C-D). We observed that increased autophagic
activity, in response to BPA was involved with
increase in the number of mitochondria co-localized
with autophagosomes in neuronal cells i.e LC3
positive mitochondria. Additionally, we observed that
rapamycin treatment enhanced the number of GFP-
LC3-positive autophagosomes as well as the number
of mitochondria that co-localized with
autophagosomes (Fig.10C-D). Contrarily, we also
found that knock down the expression of beclin
inhibits the number of mitochondria co-localized with
autophagosomes (Fig. 10C-D). We have also studied
the increase in autophagosome number was due to an
increase in autophagosome formation rather than
proteolytic inhibition (data not shown).
BPA enhances mitophagy by increasing the levels
of PINK1 and parkin, and PARK2 mitochondrial
translocation-
Next, we studied the effect of BPA on the expression
and levels of parkin and PINK, which are involved in
mitochondrial quality control (67). Several evidences
underpin that damaged mitochondria leads to
neurodegenerative disorders (68). It is observed that
PINK1 and Parkin recruit on the damaged
mitochondria and promote their separation from
mitochondrial circuitry by degradation via mitophagy
(67-69). BPA treatment enhanced the expression of
PINK and Parkin (PARK2) genes, and their protein
levels in mitochondrial fractions dose dependently
(Fig. 11A-C). To study the effects of BPA on PARK2
mitochondria translocation, neuronal cells were
transfected with YFP-PARK2 and Mito-CFP plasmids.
BPA treatment caused increased PARK2 translocation
to mitochondria, suggesting enhanced mitophagy (Fig.
11D-E). We observed enhanced co-localization of
Park2; green with mito-CFP; red (M=0.29) after BPA
exposure as compared to control (M=0.04). In BPA+
beclin siRNA and BPA+PINK1 siRNA treated
cultures co-localization coefficient were M=0.14 and
0.08 respectively. BPA treatment also increased Parkin
levels in mitochondrial fraction, leading to decreased
levels in cytosol (Fig.11F). Further, we also examined
the effects of BPA on mitophagy in neuronal cells
after AMPK silencing. BPA enhanced co-localization
of GFP-LC3 plasmid with TOMM20 protein. We
observed enhanced co-localization of TOMM20
protein; red with GFP-LC3; green (M=0.31) after BPA
exposure as compared to control (M=0.08). In AMPK
siRNA and BPA+AMPK siRNA treated cultures co-
localization coefficient were M=0.04 and 0.12
respectively. We observed BPA induced damaged and
fragmented mitochondria were taken up by
autophagosomes for degradation (Fig.11G-H). In
addition BPA decreased the levels of mitochondrial
proteins TOMM20 and COX-IV, and p62, depicting
enhanced mitophagy, which was significantly blocked
by AMPK knockdown in neuronal cells (Fig.11 I-J).
These results suggest that mitophagy was induced in
response to BPA exposure, through activation of the
AMPK pathway.
Time dependent responses of BPA on autophagy,
apoptosis and antioxidant levels in neuronal cells-
We studied a time course response of BPA on the
levels of autophagy proteins p62 and LC3-II,
antioxidant enzymes catalase and SOD, and apoptotic
protein cleaved-caspase-3. NSC derived neuronal
cultures were exposed with BPA upto 72h. The levels
of LC3-II by BPA exposure were significantly
increased only upto 12h (Fig.12A-B). Further, BPA
decreased the levels of p62 upto 12h, which were
increased at 24-72h (Fig.12A-B). Additionally, LC3-II
levels were also significantly decreased in the presence
of bafilomycin only after 12h, signifying the
impairment in autophagic flux at later time points
(Fig.12C-D). BPA exposure also up-regulated the
levels of cleaved-caspase-3, and decreased the levels
of Lamp-2 after 12h (Fig.12A-B), suggesting the
impairment of autophagy-lysosome pathway and up-
regulation of apoptosis. Similarly, BPA also caused
time dependent decrease in the levels of catalase and
SOD (Fig.12A-B). These results suggest that, prolong
BPA exposure caused impairment of autophagic flux,
which resulted in BPA mediated apoptosis. Further,
we also found that long term exposure of BPA
enhanced hippocampal neurodegeneration (Fig. 12E-
F) leading to impaired learning and memory ability in
the rats (Fig. 12G). Interestingly, activation of
autophagy by administration of rapamycin caused
reduced neurodegeneration and also restored learning
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and memory deficits induced after BPA treatment (Fig.
12E-G).
On the basis of our experimental studies, we
proposed a schematic model illustrating the plausible
mechanism(s) of BPA mediated alterations on
regulatory dynamics of autophagy (Fig.13). BPA
induced loss in the ATP levels were compensated with
rise in the phosphorylation of AMPK, suggesting that
autophagy generated against BPA induced toxicity was
accompanied by the up-regulation of AMPK.
Furthermore, AMPK activation resulted in
phosphorylation of AMPK substrates such as ACC and
raptor to preserve cellular ATP pool during BPA
exposure. BPA induced autophagy by decreased
phosphorylation of mTOR, and its downstream target
molecules including P70S6K and also of ULK1 at
Ser757. Interestingly, silencing the expression of
AMPK enhances the phosphorylation of ULK1 at Ser
757 and mTOR supporting the involvement of AMPK
chiefly in autophagy generated against BPA.
Inhibition of autophagy aggravated BPA induced
neurotoxicity by enhancing ROS generation and
apoptotic cell death. Activation of autophagy by
rapamycin and mTOR siRNA reduced the number of
BPA induced apoptotic cells in vitro and in vivo.
Mitophagy was also increased against BPA
neurotoxicity, which reduced the accrual of damaged
mitochondria by mitochondrial translocation of
PARK2. Beclin and PINK1 siRNA blocked BPA
mediated enhanced PARK2 mitochondrial
translocation, thus mitophagy. Thus, BPA induced
toxicity was mitigated through the generation of
autophagic response.
Discussions
Several clinical and experimental studies suggest that
exposure of xenoestrogen BPA causes cognitive and
behavioral alterations in the human and animals
(1,2,4,7,10,11,70,71). BPA causes ROS generation,
which decreases the process of generation of new
neurons (neurogenesis) and myelination in the
hippocampus, as well as alters cognitive functions
(10,11,72). BPA exerts neurotoxicity through the
induction of apoptosis in neurons (14). However,
whether BPA induced ROS generation and apoptosis
in the brain are associated with other form of cell death
mechanism i.e. autophagy, is still elusive. Autophagy
is found to be decreased in the pathogenesis of several
neurodegenerative and neurological disorders (25). A
recent study has reported the role of autophagy in BPA
induced glucose metabolic disorders in the pancreas of
high fat diet fed rats (73). Herein, for the first time, we
explored the role of autophagy in BPA mediated
neurotoxicity. We examined the underlying molecular
mechanism(s) of BPA induced autophagy in the
hippocampus region of the rat brain and hippocampal
NSC derived neurons in culture. We reported that
autophagy flux was generated as a cellular protective
response against BPA neurotoxicity through the
dynamic interplay amongst the AMPK and mTOR
pathways. Strikingly, autophagy by increasing the
cellular tolerance might acts as a secondary protective
response against the oxidative stress (74).
BPA induces autophagic flux- Autophagy acts as a
cardinal process in sustaining the protein machinery by
the process of membrane trafficking, involving
degradation and recycling excess of defective
macromolecules inside the cells (75). Autophagy is
greatly enhanced during developmental stages, and
also gets activated in response to environmental
stressors to maintain the cellular homeostasis (31,76).
The conversion of LC3-I to LC3-II and the formation
of autophagosomes are hallmark features of autophagy
(58). However, an increase in the LC3-II levels may
also be due to the decrease in autophagic
proteolysis.(58)
We found that BPA enhanced LC3-II
lipidation both in vivo as well as in vitro. The
expression and levels of p62, were decreased by BPA
treatment time dependently. Further, we found that the
mRNA expression of autophagy genes such as LC3,
HMGB1, beclin-1, Atg5, Atg12, Atg3 and Lamp-2
were significantly increased following BPA treatment.
Similarly, the protein levels of LC3-II, HMGB1 and
beclin-1 were also increased by BPA. Subsequently,
using electron microscopy we found that BPA
significantly enhanced generation of multilamellar
vesicular bodies and single membrane organelles
autolysosomes in the hippocampus, suggesting the
induction of autophagy. In addition, with the help of
the 2D analysis in tandem with mass spectrometry, we
found alterations in the proteomics profile of several
autophagy related proteins in the hippocampus region
of BPA treated rat as compared to control. Next, we
found that BPA enhanced the formation of GFP-LC3
puncta and up-regulated LC3-II levels in the presence
of bafilomycin A1 (a persuasive inhibitor of lysosome
V-ATPases), and caused accumulation of
autophagosomes by impairing the fusion between
autophagosome and lysosome (60). These results
suggest that BPA induced autophagic flux rather than
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inhibiting autophagic proteolysis. Similarly, other
toxicants such as ethanol (34) and cadmium(30) were
also found to increase GFP-LC3 puncta and decreased
levels of p62.
Further, we studied the detailed mechanistic
aspect pertaining to autophagy generated against BPA
neurotoxicity. We found that the exposure of
rapamycin (an autophagy inducer) enhances BPA
mediated increase in the levels of LC3-II and number
of GFP-LC3 puncta in neuronal cells. Conversely,
wortmannin (an autophagic inhibitor), and beclin
siRNA restrains BPA induced GFP-LC3 puncta and
LC3-II levels. Considering the fact that autophagy is
enhanced in serum deprived conditions, the
experiments were performed both in HBSS i.e.
starvation and nutrient rich conditions to avoid any
discrepancy.
Autophagy as cell's self compensatory response
against BPA induced cytotoxicity as well as
oxidative damage-
Several studies reported that BPA exposure results in
enhanced oxidative stress, apoptotic cell death, and
neurotoxicity (1,5,14,77). Numerous compounds
including NAC, melatonin, green tea etc. are reported
to ameliorate BPA induced neurotoxicity as well as
cognitive deficits, suggesting that BPA exposure may
deplete the levels of antioxidants inside the cells
(1,15,78). We also found that antioxidant enzymes like
catalase and an antioxidant NAC inhibited BPA
induced LC3-II up-regulation, signifying that
autophagy is generated against BPA induced depletion
of antioxidant enzyme.
The paradox regarding the autophagy mediated
cell death still exists, whether autophagy is protective
or deleterious. Autophagy is also responsible for cell
death, and it has been reported that run-away
autophagy that is excessive autophagy results in
breakdown of crucial cell organelles as well as
biomolecules (26,79). Thus, it is very important that
autophagy should take place at a particular threshold,
and beyond that it may prove to be deleterious (26,80).
Overall, our results suggest that autophagy is
generated as a transitory cellular tolerance against
BPA mediated toxicity to mitigate BPA induced
oxidative stress and cell death. Moreover, exposure
with antioxidants curtails BPA induced neurotoxicity
by decreasing oxidative stress and ROS generation in
neuronal cells. However, our study suggested that
activation of autophagy either pharmacologically or
genetically alleviated BPA induced ROS generation,
by decreasing the levels of peroxides and superoxides
(13), as well as by decreasing the levels of MDA
which were increased after BPA exposure (15-21). In
addition, BPA induced decrease in total glutathione
levels were also ameliorated by activation of
autophagy. Further, autophagy activation also
decreased caspase-3 activation and neurodegeneration
in the hippocampal neurons and also caused inhibition
of cognitive impairments. Conversely, inhibition of
mTOR enhanced autophagy in BPA treated neuronal
cultures; thereby decreasing BPA induced ROS
generation and apoptotic cell death.
Autophagy induced against BPA involved dynamic
interplay among the AMPK and mTORC pathways
AMPK is attributed as one of the fundamental
controller of cellular metabolism in eukaryotes, which
activates during cellular ATP depletion (38). Inhibition
of AMPK significantly inhibits autophagy and also
decreases viability of astrocytes during oxygen and
glucose deprived conditions (81). Autophagy induction
is lethal in neurons lacking AMPK, while AMPK
induces autophagy by inhibiting the mTOR pathway
(82). During stress conditions AMPK activates the
catabolic pathways, and inhibits energy utilizing
processes inside the cell (37,42). On the contrary,
mTOR acts as a crucial target of many essential
pathways such as insulin, growth factors and nutrient
transporters, and also involved in regulation of
autophagy (83). The mTOR pathway is activated in the
presence of excess nutrients, neurotrophic factors, and
neurotransmitters, which increases protein synthesis
and inhibits autophagy (83). BPA exposure results in
neuronal migration loss, increased neurodegeneration
and decreased synaptogenesis (8,84). In our study we
observed that BPA treatment depleted the energy
content inside the cells by lowering the ATP levels.
Thus, to ablate BPA mediated low energy levels inside
the cell autophagy was enhanced and accompanied by
rise in the levels of AMPK. The activation of AMPK
pathway results in increased phosphorylation of raptor
at Ser 792 to preserve cellular energy pool.
Correspondingly, we also found that BPA down-
regulated phosphorylation of mTOR as well as its
downstream components like P70S6K. In addition,
BPA also induced the phosphorylation of ACC at
Ser79. Further, to acknowledge the pivotal role of
AMPK in BPA mediated autophagy, we carried out
siRNA mediated knockdown of AMPK. We observed
that silencing the expression of AMPK decreased BPA
induced rise in the levels of LC3-II as well as GFP-
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LC3 puncta, and also suppressed phosphorylation of
raptor and ACC. Interestingly, AMPK knockdown
resulted in increased phosphorylation of mTOR and
also ULK1 (Ser 757), which was decreased after BPA
treatment, suggesting the involvement of mTOR as an
effector downstream module of AMPK pathway in
BPA mediated autophagy. Most importantly, it was
observed that BPA decreased the phosphorylation of
ULK1 at Ser 757. During nutrient rich conditions,
mTORC1 gets activated and disrupts ULK1-AMPK
interaction, leading to the inhibition of autophagy
(37,41,85,86). At the time of starvation or stress
conditions AMPK activation inhibits mTORC1 by
phosphorylation of TSC and raptor, which reduces
ULK1 Ser 757 phosphorylation (37,41). Therefore,
unphosphorylated ULK1 at Ser 757 is able to interact
with and activated by AMPK (41,87). Thus, a
balanced coordination among these kinases helps in
regulation of cellular decision regarding autophagy
induction and cell proliferation (37,85). We observed
that BPA exposure decreased the phosphorylation of
ULK1 Ser 757, which results the inability of ULK1 to
interact with mTOR. Thus, over all it can be concluded
that autophagy is induced against BPA neurotoxicity
through the involvement of the AMPK and mTOR
pathways.
BPA mediated induction of mitophagy blocked by
AMP inhibition
Mitochondria are indispensable organelles in
regulation of the cellular homeostasis. The exclusion
of defective mitochondria by delivering them to
lysosomes for their degradation i.e through autophagy
is known as "mitophagy" (64). We observed that
mitophagy is enhanced against BPA neurotoxicity,
which comprises mitochondrial loss leading with
bioenergetic deficits. BPA enhanced the co-
localization of GFP-LC3 with pmKate mitochondrial
reporter gene, which was further increased in the
presence of rapamycin, suggesting increased
mitophagy. Contrarily, beclin1 knockdown decreased
BPA induced mitophagy, thereby decreasing the co-
localization of GFP-LC3 with pmKate. Recent studies
revealed a vital role of PINK1 and Parkin in regulation
of mitophagy. Intriguingly, we also found that
enhanced mitophagy against BPA was corroborated
with the rise in the expression and levels of PINK1 and
Parkin (PARK2). We found that BPA induced PARK2
mitochondrial translocation, as observed by enhanced
YFP-Parkin and mito-CFP co-localization. Several
recent studies suggest vital involvement of AMPK in
the regulation of mitophagy process (88). We
observed that AMPK knockdown resulted in defective
mitophagy, and increased levels of TOMM 20, COX-
IV and p62 from basal level. Strikingly, exposure of
BPA decreased the levels of TOMM 20, COX-IV and
p62 even after AMPK knockdown, unambiguously
depicting that BPA induced mitochondrial loss
resulting mitophagy. Next, we also observed that BPA
induced the co-localization of TOMM20 and GFP-
LC3, which was reduced by the AMPK knockdown.
These results suggest vital involvement of the AMPK
pathway in mitophagy induced against BPA. Hence,
we may conclude that mitophagy is enhanced against
BPA neurotoxicity to ablate damaged mitochondria,
and to prevent the oxidative stress and maintain the
mitochondrial quality control.
Inhibition of autophagy enhances BPA induced
apoptosis, while induction of autophagy is
neuroprotective- A co-ordinate relationship exist between autophagy
and apoptosis (89), and if any one pathway is blocked
the other may take over the charge. In current study we
found that early BPA exposure activates autophagy as
a cellular tolerance response, but prolong exposure
leads to impaired autophagic flux and apoptotic cell
death. The levels of p62 were found to be increased,
while levels of Lamp-2 were significantly decreased at
later time points, suggesting impaired autophagy-
lysosome pathway and cognitive dysfunctions (90,91).
The decrease in the levels of Lamp-2 results in
accumulation of p62 aggregates and
neurodegeneration (91). Thus, we demonstrated that
impaired autophagic flux by BPA enhances apoptosis,
by elevating the levels of cleaved-caspase-3. Several
studies report that autophagy impairment increases
caspase-3 activity and cell death (92,93). Interestingly,
long term exposure of BPA caused hippocampal
neurodegeneration and reduced learning and memory
ability in the rats. Activation of autophagy reduced
BPA mediated neurodegeneration and also restored
learning and memory deficits.
In conclusion, these results suggest that,
autophagy acts as transitory survival pathway under
stress conditions, but at the same time when it occurs
excessively or when it’s impaired it may be deleterious
and also lead to activation of apoptosis mediated
cellular demise. Lastly, the autophagic responses
against the widespread usages of environmental
xenoestrogen require further assessments.
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Activation of autophagy against Bisphenol-A
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Acknowledgments:
This work was supported by the Council of Scientific
and Industrial Research (CSIR) - Network grant
InDEPTH (BSC0111) to RKC. We are thankful to the
Director, CSIR-IITR, for his constant support during
this study. SA and BS are recipients of Senior
Research Fellowship and Junior Research Fellowship
respectively from CSIR, New Delhi. SKT and AY are
recipients of Senior Research Fellowship and Junior
Research Fellowship respectively from University
Grants Commission, New Delhi. CSIR-IITR
Manuscript Communication number-
Conflict of Interest: The authors declare no competing
financial interest.
Author contribution: SA, SKT and RKC conceived
and coordinated the study, performed experiments, analyzed data, and wrote the paper. SA, SKT, BS, AY, RSR, SKG, AT, AK and SS designed, performed and analyzed the experiments shown in Figures 1 and 3-9. AS, AM and DP designed, performed and analyzed the experiments shown in Figure 2. SA, SKT, and VC performed and analyzed the experiments shown in Figures 10-12. All authors reviewed the results and approved the final version of the manuscript.
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Figure Legends:
Figure 1: BPA reduces viability of neural stem cells (NSC) derived neurons and induces apoptosis
and neurodegeneration in the hippocampus. (A-B) Primary hippocampal NSCs derived neurons were treated with BPA for 24h. Graph showing
percent cell viability by trypan blue assay as compared to control, and values of PI+ cells expressed in
terms of % PI+ cells as compared to control. The values are expressed as mean±SEM (n=3 independent
experiments). (C) Representative immunofluorescent photomicrograph showing cells labeled with
activated caspase-3 (red, apoptotic cell marker) counterstained with nuclear stain DAPI (Blue) in the
hippocampus. Arrows indicate activated caspase-3+ cells. Scale bar=100μm. (D) Quantitative analysis of
activated caspase-3+ co-labeled cells in the hippocampus (E) Fluoro-Jade B stained degenerating neurons
in the hippocampus. Arrows indicate the degenerating neurons. (F) Quantification analysis of Fluoro-Jade
B+ degenerating neurons in the hippocampus. Values are expressed as mean±SEM (n=6 rats per group).
*p<0.05 vs control. Scale bar=20μm
Figure 2: BPA enhances autophagy in the hippocampus region of the rat brain. (A) Effect of BPA (40 and 400μg/kg body weight, oral) on the expression of autophagy genes in the
hippocampus region of the rat brain was studied by qRT-PCR. β-actin served as housekeeping gene for
normalization. The data expressed as mean+SEM (n=6 rats/group). *p<0.05 vs control. (B) Western blot
analysis of levels of autophagy proteins in the hippocampus (C) Quantification of relative protein density
after normalization with β-actin. (D) Transmission electron microscopic examination of the hippocampus
region. Several multilamellar bodies observed by TEM were indicated by single arrow. Increased number
of curving phagophores (C) and autolysosmes (AL) were found in BPA treated rats. Double membrane
organelles (autophagosomes; A) were scarcely found in 40μg and rare in 400μg BPA treated pups. (E)
Quantification of TEM images. (F) Effects of BPA on proteomic profile of autophagy related proteins in
the hippocampus of the rat brain. Separation of proteins found to be involved in autophagy by two-
dimensional gel electrophoresis. Altered proteins by BPA treatment are labelled with arrows. *p<0.05 vs
control. The data expressed as mean+SEM (n=3 rats/group)
Figure 3: BPA induces autophagy in the hippocampal NSC derived neuronal cells.
(A-D) Neuronal cultures were treated with various concentrations of BPA (25, 50 and 100 μM). At
100µM concentration the time (0, 3, 6 and 12h) dependent analysis of LC3-II protein levels was
performed by immunoblotting. Relative protein levels were quantified after normalization of LC3-II with
β-actin. The data represented as mean ±SEM (n=3 independent experiments) *p<0.05. (E-F) Neuronal
cells were transfected with GFP-LC3 plasmid, following treatment with various concentrations of BPA.
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After 12h BPA treatment, GFP-LC3 puncta were observed and counted by fluorescence microscopy
analysis and expressed as GFP-LC3 puncta/cells. Scale bar=20µm. (G-J) Neuronal cultures were pre-
incubated with bafilomycin A1 (10nM) and treated with BPA (100μM) for 12h. The experiments were
performed both in the presence and absence of serum (HBSS conditions), and the levels of LC3-II protein
in neuronal cells after BPA treatment were studied. Relative protein density was quantified after
normalization of LC3-II with β-actin. Further, neuronal cells were also transfected with GFP-LC3 plasmid
and treated with BPA, Bafilomycin A1 and BPA+Bafilomycin to study the autophagic flux. Scale
bar=20µm *p<0.05.
Figure 4: BPA induces generation of autophagic flux in the hippocampal neuronal cultures.
(A-C) Beclin siRNA decreased the beclin protein levels. Neuronal cells were pre-incubated with
(rapamycin; 100nM), and (wortmannin; 10μM) and treated with BPA (100μM) for 12h. The levels of
LC3-II protein (LC3-II lipidation) were studied in neuronal cells after BPA treatment. Relative protein
densities were quantified after normalization with β-actin. (D-E) Treatment of BPA (100μM) was given in
the presence and absence of pharmacological activator (rapamycin; 100nM), inhibitor (wortmannin;
10μM), and beclin siRNA co-transfection in GFP-LC3 transfected neuronal cells. GFP-LC3 puncta were
observed and quantified under a fluorescence microscope. The data represented as mean ± SEM (n=3
independent experiments). Scale bar=20µm, *p<0.05. (F-I) The levels of p62 were studied in the presence
of bafilomycin A1 and rapamycin followed by BPA treatment, both in control and HBSS conditions.
Figure 5 : Autophagy generated against BPA promotes extranuclear HMGB1 translocation: (A-B) Rat hippocampal NSC derived neurons were treated with rapamycin (100nM), wortmannin (10μM), and
beclin siRNA followed by BPA (100µM) for 12h and then were immunostained with HMGB1. The figure
depicts translocation of HMGB1 from the nucleus to the cytosol. The mean nuclear and cytosolic HMGB1
intensity per cell were observed by imaging cytometric analysis. The data represented as mean ±SEM
(n=3 independent experiments). Scale bar=20µm *p<0.05.
Figure 6: Autophagy compensates against BPA induced cytotoxicity and apoptosis in neuronal cells
and in the hippocampus of the rat brain. (A-B) Neuronal cells were pre-incubated with rapamycin, wortmannin, catalase, and beclin siRNA and
treated with BPA (100μM) for 12h. Propidium idodide (PI) staining was done through flow cytometry in
order to further study the number of PI+ cells (apoptotic cells). The data represented as mean ±SEM (n=3
independent experiments) *p<0.05. (C-D) Rat pups were orally gavaged with BPA (400μg/kg body
weight) and/or i.p rapamycin (1 and 2mg/kg body weight) during PND14-21. Arrows indicate activated
caspase-3+ cells (apoptotic marker) in the dentate gyrus region of the hippocampus. Scale bar: 100μm.
Values are expressed as mean ± SEM (n= 6 rats per group). *p<0.05 vs control.
Figure 7: Autophagy as a protective response against BPA induced ROS generation. (A-B) AMPK and mTOR siRNA reduced the respective protein levels. Neuronal cultures were treated
with BPA (100μM) in the presence/absence of catalase (10,000 U/ml) and N-acetyl-cysteine (NAC;
10mM). The LC3-II lipidation was determined by immunoblotting. Relative LC3-II protein levels were
quantified after normalization with β-actin. The data represented as mean ±SEM (n=3 independent
experiments) *p<0.05. (C-D) Relative ROS levels were determined using DCFH-DA dye time
dependently from 3-12h (E) Levels of intracellular superoxides were monitored using DHE for 12h. (F-G)
Effects of BPA on lipid peroxidation and total glutathione levels were monitored in comparison with
control groups.
Figure 8: Effects of BPA on ATP levels and the AMPK/mTOR pathways involved in autophagy.
(A) Neuronal cells were treated with different concentrations of BPA (100µM) for 12h, and ATP levels
were monitored through ATP measurement kit using a luminometer. The data represented as mean ±SEM
(n=3 independent experiments) *p<0.05. ( B-C) Effects of BPA on cytotoxicity after knockdown the
expression of AMPK and mTOR in neuronal cells were studied by flow cytometry using PI. (D-F)
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Activation of autophagy against Bisphenol-A
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Neuronal cells were transfected with AMPK siRNA and were treated with BPA. Effects of BPA on the
levels of LC3-II and on the phosphorylation levels of AMPK, were analyzed by immunoblotting. Relative
protein levels were quantified after normalization with β-actin. (G-H) Neuronal cells were co-transfected
with GFP-LC3 plasmid and AMPK siRNA, and effects of BPA on the expression of GFP-LC3 puncta
were studied. Scale bar=10µm.
Figure 9: AMPK is chiefly involved in autophagy generated against BPA. (A-B) Neuronal Cells were incubated with BPA (100μM) for 12h and the proteins levels were analyzed.
The data represented as mean ±SEM (n=3 independent experiments) *p<0.05. (C-D) Neuronal cells were
transfected with AMPK siRNA followed by exposure with 100µM BPA. The transfected cells were
incubated in the absence or presence of BPA for 12h, and the levels of proteins were analyzed by
immunobloting. Relative protein levels were quantified after normalization with ACC, raptor and ULK1.
The mTOR pathway is downstream of AMPK pathway in BPA treated neuronal cells. The p-mTOR and
mTOR levels were analyzed after transfection with AMPK siRNA.
Figure 10: BPA induced mitochondrial loss and mitophagy in neuronal cells.
(A) BPA elicits significant mitochondrial loss in neuronal cells. The neuronal cells were treated with BPA
for 12h. Mitochondrial specific dye NAO staining was used to analyze mitochondrial mass. (B)
Mitochondrial copy number was determined by evaluating the ratio of COX-II and 18S in neuronal cells
after BPA exposure (C-D) Neuronal cells were transfected with GFP-LC3 and pmKate mitochondrial
resistance plasmid and were treated with rapamycin, BPA, and cells were also transfected with beclin
siRNA. Scale bar=10µm *p < 0.05. We have calculated the co-localization data with Manders coefficient;
in which we found that % of red colocalized with green, (Fig.10C) Control cells depicted co-localization
of pmKate mitochondrial reporter gene (red) with GFP-LC3 , (Manders co-localization coefficient; M=
0.05) BPA exposure enhanced the colocalization (M=0.19), further BPA+rapamycin (M=0.28) and BPA
treatment in beclin knock down cultures results (M= 0.11). The data represented as mean ±SEM (n=3
independent experiments).
Figure 11: BPA enhances mitophagy by increasing the levels of PINK1 and Parkin, and PARK2
mitochondrial translocation and AMPK is involved in mitophagy generated against BPA exposure (A-C) Neuronal cells were exposed with BPA for 12h, and the expression and levels of PINK and Parkin
were studied. (D-E) Neuronal cells were co-transfected with YFP-PARK2 and mito-CFP plasmids, along
with PINK1 and beclin siRNA, followed by treatment with BPA. The data represented as mean ±SEM
(n=3 independent experiments). Scale bar=10µm *p<0.05 (F) The levels of PARK2 were studied in total
cell lysates, mitochondrial, and cytosolic fractions. BPA increased the levels of PARK2 in the
mitochondrial fraction and decreased in the cytosol. (G-H) Neuronal cells were co-tranfected with GFP-
LC3 plasmid and AMPK siRNA, and were exposed with BPA for 12h, TOMM20 immunocytochemical
analysis was done along with GFP-LC3 to observe the number of damaged mitochondria undergoing
mitophagy. Scale bar=10µm (I-J) Knockdown the expression of AMPK resulted in increased levels of
TOMM20, p62 and COX-IV in neuronal cells, which was further decreased after BPA exposure.
Figure 12: Effects time dependent responses of BPA on autophagy, apoptosis, antioxidant levels in
neuronal cells and conditioned avoidance response in rats
(A) Neuronal cells were exposed with BPA at various time points from 3-72 h and the levels of LC3-II,
p62, Lamp-2, Cleaved-caspase-3, Catalase and SOD were determined by immunoblotting. (B) To study
the prolong effects of BPA on the autophagic flux in the hippocampal neuronal cultures, neuronal cultures
were exposed with BPA at 12, 24 and 48h in the presence of bafilomycin. Relative protein levels were
quantified after normalization with β-actin. The data represented as mean ±SEM (n=3 independent
experiments) *p<0.05. (C) Rat pups were orally gavaged with BPA (40μg/kg body weight) from PND14-
90 and/or i.p rapamycin (0.1mg/kg body weight) during PND21-90 Arrows indicate Fluoro-Jade B+
degenerating neuronal population in the hippocampus.. Scale bar=20μm, values are expressed as mean ±
SEM (n= 6 rats per group). *p<0.05 vs control. (D) The cognitive ability (learning and memory) of the
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control, BPA, rapamycin and BPA+rapamycin, treated rats was measured following assessment of two-
way conditioned avoidance behavior. Rapamycin significantly decreased BPA induced learning and
memory deficits.
Figure 13: Proposed schematic model for the role of autophagy against BPA induced neurotoxicity
in the hippocampus region through modulation of the AMPK and mTOR pathways: BPA induced
neurotoxicity may be alleviated by the generation of autophagy. BPA exposure resulted with increased
oxidative stress, ROS generation, mitochondrial damage, ATP depletion and apoptotic cell death.
Autophagy was generated against BPA induced neurotoxicity by the up-regulation of genes involved in
autophagy process and down-regulation of autophagic substrate p62 and mTOR pathway. ATP depletion
was alleviated by increased phosphorylation of AMPK, which further up-regulated phosphorylation levels
of ACC and raptor to preserve cellular energy pool. Pharmacological and genetic inhibition of autophagy
by wortmannin, bafilomycin A1, and beclin and AMPK siRNA aggravated BPA induced neurotoxicity.
Pharmacological activator of autophagy (rapamycin) and mTOR siRNA ameliorated BPA induced ROS
generation and apoptotic cell death. Further, siRNA mediated knockdown of mTOR ameliorated BPA
induced ROS generation and apoptotic cell death, suggesting that mTOR inhibition leads to activation of
autophagy. Likewise, mitophagy was also enhanced against BPA induced neurotoxicity to mitigate the
accumulation of damaged mitochondria and to prevent oxidative stress. The mitochondrial translocation of
Park2 was enhanced after BPA exposure.
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Table 1: Effects of BPA on proteomic profile of autophagy related proteins in the hippocampus
SPOT
No.
Identification by
MALDI-TOF/Peptide
Mass finger printing
SWISS
PROT/NCBI
Acession No.
Optical Density Units (ODU) MASS
OBSERVED
(KDa)
pI
(OBSER
VED)
(KDa)
Control BPA
(40μg/kg b.wt)
BPA
(400μg/kg
b.wt)
1 Calmodulin P97756 2.4x 103±828.2 3.0x 10
4±1081.4 4.2873 x
104±974.4
10.4 4.3
2 Calponin-3 P37397 1.3 x 103±106.5 4.1 x 10
3±141.9 4.129 x
103±202.08
10.7 6.2
3 Profilin-2 Q9EPC6 35 ±3.4 1.6 x 103±67.11 2.7 x 10
3±102.5 7.5 6.9
4 Phosphatidyl ethanolamine
binding protein(1)
P31044 8.0 x 103±82.05 1.2 x 10
4±168.4 1.8 x 10
4±114.6 20.5 5.8
5 Phosphatidyl ethanolamine
binding protein(68)
P31044 2.1 x
104±781.03
2.7 x 104±499.3 3.6 x
104±174.43*
20.5 5.8
6 Cathepsin D P24268 6.0 x
103±45.177
8.4 x 103±63 9.1 x
103±36.29*
27.2 5.7
7 Peroxiredoxin-2 P35704 1.1 x
104±294.50
1.6 x 104±266 2.2 x
104±795.97*
20.3 5.9
8 PyruvateKinase P11980 8.5 x 103±96.82 1.2 x 10
4±190.3 1.3 x
104±129.57*
43.2 6.5
9 Malate dehydrogenase-1 P04636 4.9 x 103±14.4 6.0 x 10
3±181.1 6.5 x
103±59.46*
40.3 6.2
10 Malate dehydrogenase
(cytosolic)
O88989 9.2 x
103±329.07
1.2 x 104±391.2 1.4 x
104±180.15
41.4 6.7
11 α-enolase(1) P04764 2.9 x
104±1679.5
3.7 x 104±940.9 4.1 x
104±561.99*
51.0 6.1
12 α-enolase(68) P04764 3.4 x
104±417.44
3.9 x 104±555.3 3.6 x 10
4±173.4 51.0 6.3
13 γ-enolase(1) P07323 1.7 x
104±264.02
2.4 x 104±531 3.8 x
104±181.6*
51.1 5.2
14 γ-enolase(68) P07323 2.6 x
104±308.50
3.7 x 104±136.2 5.7 x
104±88.63*
51.1 5.3
15 γ-enolase(3) P07323 4.6 x
104±364.25
5.5 x 104±436 5.9 x
104±1161*
51.1 5.5
16 Heat shock cognate-71 P63018 6.7 x 103±96.76 9.9 x 10
3±244 1.0 x
104±730.9*
85.2 6.1
17 Heat shock protein-60 P63039 5.2 x
103±364.86
8.1 x 103±107.8 7.2 x 10
3±157.7 62.5 5.9
18 Heat shock protein-60 P63039 8.9 x 103±107.6 1.2 x 10
4±755.4 1.5 x 10
4±26* 62.6 6.2
19 Heat shock protein-70 Q6LA95 4.8 x 103±65.07 7.2 x 10
3±19.29 1.0 x
104±156.75*
85.1 6.6
20 Protein disulphide
isomerase A3
P11598 2.4 x
104±806.09
2.3 x 104±671.55 2.3 x
104±378.45
79.4 6.8
21 Phenylalanine t-RNA
ligase α-subunit
Q505J8 2.4 x
104±1250.9
2.2 x 104±396.89 2.3 x
104±953.97
70.1 5.9
22 Protein kinase C & casein
kinase substrate in neurons
Q9Z0W5 2.2 x
104±1226.7
2.0 x 104±897.7 2.0 x
104±420.15
70.1 5.8
23 Protein kinase C and
casein kinase substrate in
neurons
Q9Z0W5 2.2 x
104±1368.6
2.0 x 104±521 2.1 x
104±654.04
70.1 5.7
24 RAS Protein activator like-
3
Q8C2K5 2.3 x
104±375.03
2.1 x 104±1004.8 2.1 x
104±478.16
70.1 5.5
25 β-tubulin 2A chain P85108 5.4 x 4.9 x 104± 338 4.4 x 64.1 6.2
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Activation of autophagy against Bisphenol-A
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* Protein spots which are involved in the process of autophagy were found to be statistically significant (p< 0.05), using
non parametric t-test.
104±622.51 10
4±1282.47
26 Translationally controlled
tumor protein
P14701 6.4 x
104±208.45
6.7 x 104±598 7.03 x
104±678.07
70.0 6.9
27 BTB/POZ domain
containing protein-7
D3ZCW2 5.7 x
104±1407.7
5.2 x 104± 857 5.5 x
104±361.21
71.2 6.2
28 α-tubulin chain B P05213 6.0 x 104±505.8 5.5 x 10
4±623 6.1 x
104±466.86
65.5 6.6
29 Protein disulfide isomerase
A3
P11598 6.4 x
104±2008.8
6.0 x 104±1200 6.4 x
104±1001.6
78.0 6.4
30 Guanine deaminase Q9WTT6 5.9 x 104±428.5 5.4 x 10
4±516 5.7 x
104±738.05
48.3 6.3
31 Brain creatine kinase P07335 4.3 x
104±1095.4
3.4 x 104±765 3.9 x
104±1646.2
47.4 6.0
32 Brain creatine kinase P07335 5.1 x 104±225.9 4.2 x 10
4±232.2 4.5 x 10
4±951.2 47.5 6.1
33 Brain creatine kinase P07335 6.1 x
104±422.28
5.6 x 104±608.9 5.8 x 10
4±1789 41.0 6.5
34 Annexin-III P14669 6.0 x 104±451.7 6.0 x 10
4±1032.4 6.3 x 10
4±375.3 39.5 6.6
35 Isocitrate dehydrogenase Q99NA5 5.8 x 104±498.4 5.3 x 10
4±708.8 5.7 x 10
4±804.6 40.5 6.1
36 N(G), N(G)
Dimethylarginine
dimethylaminehydrolase-1
O08557 5.7 x 104±817.7 5.1 x 10
4±1306 5.4 x 10
4±700.9 41.7 6.2
37 Secrenin-1 Q6AY84 6.4 x 104±210.9 6.0 x 10
4±630.5 5.9 x
104±459.35
62.8 4.9
38 Cystatin S related
anchoring protein
P19313 6.0 x 104±422.2 5.5 x 10
4±401.6 6.5 x 10
4±60.39 52.7 4.4
39 Glial fibrillary acidic
protein
P47819 6.3 x 104±1222 5.9 x 10
4±794.7 6.1 x
104±1043.9
44.3 4.8
40 Dihydropyrimidinase
related protein 2
P47942 5.7 x
104±1078.9
5.1 x 104±965 4.5 x 10
4±475.6 32.6 4.8
41 Lactoylglutathione lyases Q6P7Q4 6.5 x
104±1315.1
6.2 x 104±455.8 6.1 x 10
4±715.3 25.1 5.5
42 Translationally controlled
tumor protein
P14701 6.6 x
104±610.14
6.3 x 104±863.6 6.1 x
104±468.64
21.9 4.9
43 ADP-ribosylation factor
GTPase activating protein-
2
Q3MID3 6.5 x
104±1493.4
6.1 x 104±479.9 5.3 x 10
4±412.6 20.6 5.6
44 Heme binding protein-1 B4F7C7 6.7 x 104±778.8 6.4 x 10
4±662.4 6.2 x 10
4±659 23.6 5.7
45 Ionosine triosephosphate
pyrophosphatase
D3ZW55 6.8 x 104±176.9 6.5 x 10
4±929.9 6.5 x 10
4±274.5 24.9 6.1
46 FMRF amide like
neuropeptide PF2
P41873 6.9 x
104±1197.6
6.6 x 104±832.1 6.6 x
104±541.19
23.8 6.8
47 UMP-CMP Kinase Q4KM73 7.2 x
104±1872.9
7.1 x 104±202.8 6.9 x
104±308.11
12.4 6.8
48 Cytoplasmic tyrosine
protein kinase 1
O35346 7.0 x 104±423.4 7.0 x 10
4±807.9 6.8 x 10
4±367 17.2 6.4
49 Glial maturation factor B Q63228 6.8 x
104±1344.9
6.7 x 104±702.28 6.2 x 10
4±1646 12.8 5.6
50 Interleukin-9 D4A8I9 6.5 x 104±752.8 6.5 x 10
4±491.5 6.9 x
104±560.41
12.3 5.4
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Activation of autophagy against Bisphenol-A
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(A)
Fig: 1
(B)
(C)0 25 50 100 200 400
BPA (µm) conc.
0
20
40
60
100
120
80
% C
ell
Via
bil
ity
*
*
Control
BPA 40µg
Hilus
GCL
ML
DG
BPA 400µg
0
50
100
150
200
250
Nu
mb
er
of
acti
vate
d
cle
aved
casp
ase-3
+cell
s
*
*
(E)
Nu
mb
er
of
Flu
oro
-Jad
e B
+cell
s
0
40
80
120
160
200
*
*
Fluoro-JadeB DAPI Merged
(D) (F)
BPA 40µg
Control
Hilus
GCLML
DG
Cleaved-caspase-3/DAPI
BPA 400µg
% P
rop
idiu
mIo
did
e+
cell
s
0 25 50 100 200 400
BPA (µm) conc.
0
15
30
45
50
*
*
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Activation of autophagy against Bisphenol-A
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3
3
1
112
2
88
9 9 910 1010
111111
1213
14
1513
14
151314
15
2122
2324
2223
24
2122
2324
21
25 2525 2626 26
27 2727
28 2828
12 12
2929
29
30
3031 32
31 3231
32
34
33 33 33
34
3536
3536 35 36
37373738 38
38
3939 39
40 40 40
4848
4949 5050
50
Phosphatidylethanolamine
binding protein-2
Cathepsin-D
HSP-70HSC-71
HSP-60HSP-60
Cathepsin-D Cathepsin-D
HSC-71HSC-71HSP-70 HSP-70 HSP-60
Control 4μg Treated 40 μg Treated
Peroxiredoxin
-IIPeroxiredoxin
-II
Peroxiredoxin
-II
Phosphatidylethanolamine
binding protein-2Phosphatidylethanolamine
binding protein-2
1
24948
33
43 434342 42
42
34 34
30 30
20 20 20
8
41 414144 44 44
45 45 454646 46
4747
47
Control BPA (40µg/kg b.wt) BPA (400µg/kg b.wt
1μM
BPA400μg
C
ALAVd
AVdA
BPA40μg
AL
AVd
C
A
A
β-actin
Beclin-1
HMGB1
ControlBPA
(40μg)BPA
(400μg)
p62
LC3-I/
LC3-II
Fig: 2 (B)
(C)
(E)
Control
(D)
(F)
12
Rela
tive m
RN
A
exp
ressio
n
0
2
4
6
8
10
(A)
*
**
** *
*
*
*
*
*
**
*
*
*
*
0
0.51.0
1.52.02.5
3.03.5
Rela
tive p
rote
in l
evels
LC3II Beclin-1 p62 HMGB1
*
* *
*
**
*
*
0
5
10
15
Autophagic
vacuoles
Autolysosomes
(Degradative Av)
TE
M Q
uan
tifi
cati
on
*
*
*
*
Control BPA 40μg BPA 400μg
0
0.5
1
1.5
2
2.5
3
1 2 3 4
Control BPA 40μg BPA 400μg
0
0.5
1
1.5
2
2.5
Control BPA 40ug BPA 400ug
Control
BPA 40ug
BPA 400ug
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Fig: 3
(C) 0h 3h 6h 12hLC3-I/
LC3-II
β-actin
(A)0 25 50 100
LC3-I/
LC3-II
β-actin
BPA
conc.(μM)
(D)(B)
LC
3II
/β-a
cti
nra
tio
0 250
1
2
3
10050
*
BPA conc. (µM)
0
1
2
3
0h 3h 6h 12h
**
*
(F)
(G)
0
5
10
15
20
25
30
0 25 50 100
GF
P-L
C3 P
un
cta
/ C
ell
*
*
BPA conc. (µM)
(H)
β-actin
LC3-I
LC3-II
4
0
1
2
3
**
*
*
*
0
10
20
30
40
GF
P-L
C3 P
un
cta
/ C
ell
*
*
*
(J)
LC
3II
/β-a
cti
nra
tio
LC
3II
/β-a
cti
nra
tio
(I)
(E)
Control BPA
BafilomycinBPA+
bafilomycin
25 µM BPA0 µM BPA
100 µM BPA50 µM BPA
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Activation of autophagy against Bisphenol-A
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0
10
20
30
40
GF
P-L
C3 P
un
cta
/ C
ell
*
*
* ** *
*
Fig: 4(B)(A)
(D) (E)
0
0.5
1.0
1.5
2.0
2.5
3.0
Rela
tive p
rote
in l
evels
*
*
*
**
**
+
--
+
-+
+
+-
-
++
-
-+
+
++
-
--
Rapamycin
Bafilomycin
BPA
p62
β-actin
2.0
0.0
0.5
1.0
1.5
Rela
tive p
rote
in l
evels
*
*
*
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5
Rela
tive p
rote
in l
evels
**
*
*
*
*
(H) (I)
+-
+
--
+
-
-+
-
-
+
--
+
+
-
--
+
+-
-
++
-+
-
--
+-
-
--
Control
HBSS
50μM BPA
100μM BPABafilomycin
p62
β-actin
(F) (G)
Rapamcin+ BPARapamycinControl BPA
BeclinsiRNA+BPABeclin siRNAWortmannin
Wortmannin+BPA
Beclin-1
β-actin
0
0.5
1.0
1.5
*
(C)
β-actin
LC3-I
LC3-II
Rela
tive p
rote
in l
evels
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Fig: 5
(A)
0
20
40
60
80
100
HM
GB
1%
*
* *
* *
(B)
control HBSS
Rapamycin Wortmannin
BPA BecnsiRNA
BPA+BecnsiRNABPA+Wortmannin
BPA
control
Rapamycin Wortmannin
Beclin siRNA
HBSS
BPA+Beclin siRNABPA+Wortmannin
HMGB1/DAPI
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Control
Co
un
t
PE-Texas Red-A
5.6 0.240
BPA 100µM
Co
un
t
PE-Texas Red-A
24.73 1.5
Rapamycin
Co
un
t
PE-Texas Red-A
10.4 0.54
Co
un
t
PE-Texas Red-A
BPA +rapamycin
13.0 0.31
Co
un
t 42.33. 0.48
BPA+wortmanninWortmannin
Co
un
t
PE-Texas Red-A
15.2 0.64
PE-Texas Red-A
Co
un
t
PE-Texas Red-A
8.7 1.7
Beclin siRNA
Co
un
t
PE-Texas Red-A
Beclin siRNA +BPA
31.7 1.44
Co
un
t
PE-Texas Red-A
Beclin siRNA+BPA+
catalase
17.23 0.93
Co
un
t
PE-Texas Red-A
Catalase
6.0 0.12
Co
un
t
PE-Texas Red-A
11.2 0.66
BPA+ catalase
0
10
20
30
40
50
% P
rop
idiu
mIo
did
e+
cell
s
*
* **
*
* *
*
*
*
Fig: 6
(A) (C)
(B)
(D)
DG
GCLML
Control
Hilus
DG
GCL
BPA
ML
BPA+Rapamycin 1mg/kg.b.wt
DG
GCL
ML
Hilus
GCL
ML
DGHilus
Cleaved-caspase-3/DAPI
BPA+Rapamycin 2mg/kg.b.wt
# o
f acti
vate
d c
asp
ase
-3
po
sit
ive c
ell
s/s
ecti
on
0
40
80
120
160
200
Granular
layer
Molecular
layer
Hilus
*
*
**
**
***
0
20
40
60
80
100
120
140
160
180
200
Granular Cell layer
Molecular layer
Hilus
Control
BPA
BPA+Rapamycin(1mg/kg b wt)
BPA+Rapamycin (2mg/kg b wt)
0
20
40
60
80
100
120
140
160
180
200
Granular Cell layer
Molecular layer
Hilus
Control
BPA
BPA+Rapamycin(1mg/kg b wt)
BPA+Rapamycin (2mg/kg b wt)
0
20
40
60
80
100
120
140
160
180
200
Granular Cell layer
Molecular layer
Hilus
Control
BPA
BPA+Rapamycin(1mg/kg b wt)
BPA+Rapamycin (2mg/kg b wt)
0
20
40
60
80
100
120
140
160
180
200
Granular Cell layer
Molecular layer
Hilus
Control
BPA
BPA+Rapamycin(1mg/kg b wt)
BPA+Rapamycin (2mg/kg b wt)
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Fig: 7
(A)M
DA
F
orm
ati
on
rela
tive f
old
ch
an
ge
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5 Control
BPA
*
*
**
*
*
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Fo
ld c
han
ge c
om
pare
d t
o c
on
tro
l
Eth
idiu
m/D
ihyd
roeth
idiu
m
flu
ore
scen
ce
Control
BPA
*
*
*
*
*
*
(C)
(D) (E)
(F) (G)
0
0.5
1.0
1.5
2.0
2.5
3.0
Rela
tive
RO
Sco
ncen
trati
on
Control
BPA
*
*
**
*
*
0
20
40
60
80
100
120
To
tal G
luta
thio
ne l
evel
( %
co
ntr
ol) *
*
**
*
*
(B)
β-actin
AMPK
mTOR
β-actin
Rela
tive R
OS
Co
ncen
trati
on
s
0
0.5
1.0
1.5
2.0
2.5
3.0
BPA
BPA+Beclin siRNA
BPA+Rapamycin
BPA+Bafilomycin
BPA+mTOR siRNA
BPA+AMPK siRNA
0h 3h 6h 12h
*
*
*
**
*
*
*
*
*
Rela
tive R
OS
co
ncen
trati
on
Rela
tive R
OS
Co
ncen
trati
on
s
0
0.5
1.0
1.5
2.0
2.5
3.0
BPA
BPA+Beclin siRNA
BPA+Rapamycin
BPA+Bafilomycin
BPA+mTOR siRNA
BPA+AMPK siRNA
0h 3h 6h 12h
**
*
**
*
*
*
*
*
LC3-I
LC3-II
β-actin
-
-
-
-
+
-
-
-
+
+
-
-
+
+
-
+
-
+
BPA
Catalase
NAC
Control
BPA
0
0.5
1.0
1.5
2.0
2.5
LC
3II
/β-a
cti
nra
tio
*
* *
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Activation of autophagy against Bisphenol-A
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120
0
20
40
60
80
100
AT
P (%
Co
ntr
ol)
*
**
*
BPA conc. (µM)
Fig: 8
(A)
Control
siRNA
AMPK
siRNA
BPA
p-AMPK (Thr 172)
AMPK
LC3I/LC3II
β-actin
- + - +
Control
Co
un
t
PE-Texas Red-A
5.6 0.24
Co
un
t
PE-Texas Red-A
mTOR siRNA + BPA
10.73 0.6
AMPK siRNA
Co
un
t
PE-Texas Red-A
12.6 0.4
AMPK siRNA+BPA
Co
un
t
PE-Texas Red-A
43.5 0.23
BPA 100µM
PE-Texas Red-A
24.73 1.5
Co
un
t
PE-Texas Red-A
mTOR siRNA
7.2 1.02
Co
un
t
(B)
(C) (D)
BPA
AMPKsiRNA AMPK siRNA+ BPA
Control
GFP-LC3
GF
P-L
C3 P
un
cta
/Cell
0
5
10
15
20
25
30
*
*
- - + +AMPK siRNA0
0.5
1.0
1.5
2.0
2.5
Rela
tive p
rote
in
Rati
o o
f L
C3II
/β-a
cti
n
*
*
AMPK siRNA0
0.5
1.0
1.5
2.0
2.5
- - + +
Rela
tive p
rote
in
Rati
o o
f p
-AM
PK
/AM
PK
*
*
*
0
0.5
1
1.5
2
2.5
Control BPA Control BPA
Control BPA
Control
BPA
(G) 0
0.5
1
1.5
2
2.5
Control BPA Control BPA
Control BPA
Control
BPA
(F)
(H)
0
10
20
30
40
50
% P
rop
idiu
mIo
did
e+
cell
s
(E)
*
*
*
* *
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Fig: 9
(C) (E)
p-mTOR (Ser 2481)
mTOR
BPA
Control
siRNA
AMPK
siRNA
- + - +
Rela
tive p
rote
in
rati
o o
f p
-rap
tor/
rap
tor
0
0.5
1.0
1.5
2.0
2.5
- - + +
*
*
Rela
tive p
rote
in
rati
o o
f p
-UL
K1/U
LK
1
0
0.5
1.0
1.5
2.0
- - + +
(D)
(F)
0
0.5
1
1.5
2
2.5
Control BPA Control BPA
Control BPA
Control
BPA
*
*
*
p-ACC (Ser79)
ACC
p-raptor (Ser792)
raptor
p-ULK1 (Ser757)
ULK1
Control
siRNA
AMPK
siRNA
BPA- + - +
0
0.5
1.0
1.5
2.0
2.5
Rela
tive p
rote
in
rati
o o
f p
-AC
C/A
CC
- - + +AMPK
siRNA
*
*
**
Rela
tive p
rote
in r
ati
o o
f
p-m
TO
R/m
TO
R
0
0.5
1.0
1.5
2.0
2.5
- - + +AMPK
siRNA
0
0.5
1
1.5
2
2.5
Control BPA Control BPA
Control BPA
Control
BPA
*
*
P70S6K
p-P70S6K
(Thr 389)
mTOR
p-mTOR
(Ser 2481)
ULK1
p-ULK
(Ser 757)
Control BPA
(A) (B)
0
0.5
1.0
1.5
Rela
tive p
rote
in levels
p-mTOR/
mTOR
p-P70S6K/
P70S6K
p-ULK1/
ULK1
* * *
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Activation of autophagy against Bisphenol-A
33
120
Mit
oc
ho
nd
ria
l m
as
s %
co
ntr
ol
0
20
40
60
80
100
Control 25 50 100
BPA (conc µM)
*
*
Control 25 50 100
BPA (conc µM)
0
0.5
1.0
1.5
*
*
MtD
NA
co
py
nu
mb
er
(CO
X-I
I/1
8S
ra
tio
)
GFP-LC3
pmKate mt
Reporter gene
GFP-LC3
/pmKate mtReporter gene
Fig:10
(B)(A)
(C)
(D)
% r
ed
co
-lo
cali
sed
wit
h g
reen
0
10
20
30
40
*
*
*
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Activation of autophagy against Bisphenol-A
34
TOMM20
Control AMPK siRNA
β-actin
COX IV
p62
BPA- + - +
Rela
tive p
rote
in L
evels
AMPK siRNA0
0.5
1.0
1.5
2.0
2.5
- - + + - - + + - + - +
*
*
*
*
*
*
*
*
*
TOMM 20 p62 COX IV
Co
ntr
ol
BP
AB
PA
+
Be
clin
siR
NA
BP
A+
PIN
K s
iRN
A
YFP-PARK2 Mito CFP MergeYFP-PARK2 Mito-CFP Merged
0 25 50 100
BPA
conc.(μM)
Parkin
PINK
β- actin
Parkin
VDAC
Parkin
GAPDH
Parkin
β-actin
Mitochondria
Lysate
Cytoplasm
PINK1 Parkin
Rela
tive P
rote
in l
evel
0
1
2
3
0 25 50 100
BPA Conc.(μM)
% g
reen
co
-lo
cali
sed
wit
h r
ed
0
10
20
30
40
Rela
tive m
RN
A e
xp
ressio
n
PINK Parkin0
0.51.01.52.02.53.0
Control BPA 25μM BPA 50μM BPA 100μM
Control BPA 25μM BPA 50μM BPA 100μMControl BPA 25μM BPA 50μM BPA 100μM
Control BPA 25μM BPA 50μM BPA 100μM
Fig:11
% r
ed
co
-lo
cali
sed
wit
h g
reen
0
10
20
30
40
(A) (B) (C) (F)
(D)(G)
(F) (H)(E)
(J)(I)
Control BPA
TOMM20 GFP-LC3 Merged
Control
BPA
AMPKsiRNA
BPA+
AMPKsiRNA
**
**
*
*
*
*
*
*
*
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Fig: 12
(A) (C)
+
-
-
-
+
-
-
-
+
-
+
+
+
-
-
-
+
-
-
-
+
-
+
+
12h
LC3I/LC3II
β-actin
+
-
-
-
+
-
-
-
+
-
+
+
24h 48h
Control
BPA
Bafilomycin
0
20
40
60
80
100
Learning Memory
Control BPA
Rapamycin BPA+Rapamycin
0 3 6 12 24 48 72
p62
β-actin
Catalase
SOD
LC3I/LC3II
Cleaved-caspase-3
Lamp-2
(E)
(G)
(B)
(F)
No
. o
f F
luo
ro-J
ad
eB
+
neu
ron
s
0
40
20
60
80
100 *
*
Rela
tive l
evels
of
LC
3II
/β-a
cti
n
Co
ntr
ol
BP
AB
afi
lom
ycin
Bafi
lom
ycin
+ B
PA
Co
ntr
ol
BP
AB
afi
lom
ycin
Bafi
lom
ycin
+ B
PA
12h 24h
0
0.5
1.0
1.5
2.0
2.5
3.0
Co
ntr
ol
BP
AB
afi
lom
ycin
Bafi
lom
ycin
+ B
PA
48h
*
**
*
* *
*
**
0
0.5
1.0
1.5
2.0
0 3 6 12 24 48 72
Rela
tive l
evels
of p
62/β
-acti
n
Rela
tive l
evels
of L
C3II
/β-a
cti
n
0
0.5
1.0
1.5
2.0
0 3 6 12 24 48 72
(D)
**
*
** *
*
*
* *
0 3 6 12 24 48 72
Rela
tive l
evels
of C
C3/β
-acti
n
0
1
2
3
0 3 6 12 24 48 72
Rela
tive l
evels
of L
am
p-2
/β-a
cti
n
0
0.5
1.0
1.5
2.0
*
*
**
**
*
*
0 3 6 12 24 48 72 0
0.5
1.0
1.5
Rela
tive l
evels
of C
ata
lase/β
-acti
n
0 3 6 12 24 48 72 0
0.5
1.0
1.5
Rela
tive l
evels
of S
OD
/β-a
cti
n
** * *
*
**
** * *
Learning Memory% C
on
dit
ion
ed
Avo
idan
ce
Resp
on
se
0
20
406080
100
0
20
40
60
80
100
Learning Memory
Control BPA
Rapamycin BPA+Rapamycin
120
*
*
*
*
Control
BPA 40µg/kg. b.wt
BPA + Rapamycin
0.1 mg/kg b.wt
Fluoro-Jade B
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Activation of autophagy against Bisphenol-A
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Transient activation of autophagy againsts Bisphenol-A mediated
neurodegeneration via AMPK/mTOR pathways
Brain Affecting Hippocampus (memory and learning process)
ROS
Mitochondria
damage
Cleaved
caspase-3
Energy
depletion
NEUROPROTECTION
AMPK
raptor ACC
ULK1
Ser 757mTOR
ATP
decline
NEUROPROTECTION
P
P P
+ Bafilomycin
+ Beclin siRNA
(Negative modulators
of autophagy)
+ m
TO
Rs
iRN
A
+A
MP
K s
iRN
A
(mTOR inactive)
= BPA
= inhibition
= activation
Cell growth &
proliferation
Autophagyinduces
against BPA
(Active)
(inactive)
Mitophagy
P
Fig: 13
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Rajnish Kumar ChaturvediAnuarg Tripathi, Amit Kumar, Ratan Singh Ray, Shubha Shukla, Devendra Parmar and
Anubha Mudawal, Lalit Kumar Singh Chauhan, Shailendra Kumar Gupta, Vinay Choubey, Swati Agarwal, Shashi Kant Tiwari, Brashket Seth, Anuradha Yadav, Anshuman Singh,
neurodegeneration via AMPK/mTOR pathwaysActivation of autophagic flux against xenoestrogen Bisphenol-A induced hippocampal
published online July 2, 2015J. Biol. Chem.
10.1074/jbc.M115.648998Access the most updated version of this article at doi:
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