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: rajnish@iitr.res.in

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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Activation of autophagy against Bisphenol-A

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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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Activation of autophagy against Bisphenol-A

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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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Activation of autophagy against Bisphenol-A

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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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Activation of autophagy against Bisphenol-A

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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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Activation of autophagy against Bisphenol-A

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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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Activation of autophagy against Bisphenol-A

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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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Activation of autophagy against Bisphenol-A

32

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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Activation of autophagy against Bisphenol-A

35

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

36

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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