down-regulation of apurinic/apyrimidinic endonuclease 1 (ape1) in spinal motor neurones under...

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Down-regulation of apurinic/apyrimidinic endonuclease 1 (APE1) in spinal motor

neurons under oxidative stress1

Tak-Ho Chua,b, Anchen Guoa and Wutian Wua,b,c,d *

a Department of Anatomy; b Research Center of Reproduction, Development and Growth; c

State Key Laboratory of Brain and Cognitive Sciences, Li Ka Shing Faculty of Medicine, the

University of Hong Kong, Pokfulam, Hong Kong SAR, China; d GHM Institute of CNS

regeneration, Jinan University and The University of Hong Kong, Guangzhou, China

Running Title: APE1 in degenerating motor neurons

*Correspondence and request for reprints to:

Wutian Wu, M.D., Ph.D.

Department of Anatomy

Li Ka Shing Faculty of Medicine

The University of Hong Kong

21 Sassoon Road, Hong Kong SAR

China

Phone: (852) 28199187

Fax: (852) 28170857

E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nan.12071

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Abstract

Aim: Apurinic/ apyrimidinic endonuclease 1 (APE1) is an intermediate enzyme in base

excision repair which is important for removing damaged nucleotides under normal and

pathological conditions. Accumulation of damaged bases causes genome instability and

jeopardizes cell survival. Our study is to examine APE1 regulation under oxidative stress in

spinal motor neurons which are vulnerable to oxidative insult.

Methods: We challenged the motor neuron-like cell line NSC-34with hydrogen peroxide and

delineated APE1 function by applying various inhibitors. We also examined the expression of

APE1 in spinal motor neurons after spinal root avulsion in adult rats.

Results: We showed that hydrogen peroxide induced APE1 down-regulation and cell death in

a differentiated motor neuron-like cell line. Inhibiting the two functional domains of APE1,

namely, DNA repair and redox domains potentiated hydrogen peroxide induced cell death.

We further showed that p53 phosphorylation early after hydrogen peroxide treatment might

contribute to the down-regulation of APE1. Our in vivo results similarly showed that APE1

was down-regulated after root avulsion injury in spinal motor neurons. Delay of motor

neuron death suggested that APE1 might not cause immediate cell death but render motor

neurons vulnerable to further oxidative insults.

Conclusion: We conclude that spinal motor neurons down-regulate APE1 upon oxidative

stress. This property renders motor neurons susceptible to continuous challenge of oxidative

stress in pathological conditions.

Keywords: Base excision repair, NSC34, root avulsion

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Introduction

Numerous evidences have shown linkage between oxidative stress and neurodegenerative

diseases and acute injuries [1]. Excessive level of reactive oxygen species (ROS) causes

damage to cellular components, subsequently affects biological functions and leads to cell

death. ROS such as the highly reactive hydroxyl radical reacts with DNA, causing oxidative

DNA damage. Oxidative DNA damage is primarily repaired by an intrinsic mechanism called

DNA base excision repair (BER). BER pathway uses various glycosylases to recognize and

remove specific damaged bases, forming abasic or apurinic/apyrimidinic (AP) sites. The

phosphodiester backbone immediately 5’ to the abasic site is then removed by apurinic/

apyrimidinic endonuclease 1 (APE1), leaving a 3’ hydroxyl group, a 5’-deoxyribose-5-

phosphate (5’-dRP) moiety and a single strand break. The 5’dRP is then removed and the

nick is filled by DNA polymerase beta (Pol β). The strand is eventually sealed by DNA ligase.

The short patch pathway replaces a single nucleotide while a long-patch repair replaces

approximately 2-13 nucleotides when the 5’-dRP is resistant to modification by Pol β. Most

of the repair processes are dependent on the short patch pathway.

In the current study, we focused on the ubiquitously expressed rate-limiting enzyme, APE1,

in the BER pathway. APE1 contains two functional domains: the C-terminal domain is

responsible for its DNA repair function as described above while the N-terminal domain is

responsible for reduction/oxidation (redox) regulation [2]. Redox control of APE1 is

important for gene expression as many of its targets are transcription factors, e.g. nuclear

factor κB (NFκB), activator protein-1 (AP-1) and p53 [3]. Although the exact mechanism of

how APE1 modulates transcription factor activation is not known, it is generally believed that

the redox sensitive cysteine 65 in APE1 interacts with the cysteine residues lying either in the

DNA-binding domains or within the regulatory elements of the transcription factors [4].

Reduction of the cysteine residues of these transcription factors facilitates DNA binding

activity and initiates gene expression [5].

APE1 has attracted much attention in cancer biology as a potential therapeutic target. An

elevated level of APE1 in cancer cells confers resistance to chemotherapeutic drugs and

promotes expression of several transcription factors important for disease promotion and Acc

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progression via as its DNA repair function and redox function respectively [6]. The role of

APE1 in neuronal cells is relatively less well studied. However it is becoming clear that

accumulated nuclear and mitochondrial DNA damage in neurons can lead to

neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases [7]. Thus

maintenance of genome stability by BER pathway is crucial for proper functions for post-

mitotic neurons which survive for life-long.

Previous animal experiments showed that APE1 responded differentially to acute injuries

with regard to age, type of injuries and location of lesions in the central nervous system [8].

For example, exposure of young and aged rats to hyperoxia revealed up-regulation of APE1

in hippocampus and basal forebrain in young rats while no changes in all brain areas in aged

rats [9]. Transient global cerebral ischaemia caused down-regulation of APE1 protein and

apoptotic cell death in hippocampal CA1 neurons [10-11] but accumulation of APE1 in

ischaemia- resistant dentate gyrus [12]. Similar results were also found in rabbit spinal cord

ischaemia model which showed reduction of APE1 at 1 day and motor neuron loss at 7 days

after ischaemic injury [13]. Reduction of APE1 was also reported in cold-injury and impact

induced brain injuries [14-15].

In our study, we aimed to examine the change of APE1 under oxidative stress in spinal motor

neurons. We used a motor neuron-like cell line NSC-34 as an in vitro model and spinal root

avulsion injury as an in vivo model. NSC-34 is produced by fusing motor neuron-enriched

mouse embryonic spinal cord cells with neuroblastoma cells [16]. These cells possess several

important properties of motor neurons, e.g. they express motor neuron marker choline

acetyltransferase and neurofilament triplet proteins; they can generate axon potentials and

induce acetylcholine receptor clustering when contacted with myotube. Therefore the cell line

is widely used in studying neurotoxicity and motor neuron –related disorders [17-19]. We

further differentiated the fast-proliferating cell line to a slower -growing phenotype. Previous

reports showed that differentiated NSC-34 more closely resemble primary motor neurons by

having longer neurites and glutamate sensitivity than their undifferentiated cells [19]. We

incubated the differentiated NSC-34 cells in hydrogen peroxide to induce oxidative stress and

evaluated change in APE1 level. We then used different inhibitors to block the two functional

domains to determine their roles in cell death. We also tested the inihibitors in another

neuronal cell line PC12 and a non-neuronal cell line HEK293 to evaluate their specificity of

actions in motor neuron. Acc

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Spinal root avulsion involves pre-ganglionic rupture of spinal rootlets. Motor neurons in the

avulsed spinal segment degenerate progressively [20-21]. Previous work showed that the

mechanism of motor neuron death involved apoptosis [22-23]. Further study using immuno-

staining of single-stranded DNA and 8-hydroxy-2-deoxyguanosine (8OHdG), a marker for

oxidative DNA damage showed that DNA damage precedes apoptosis [24]. The staining

intensity of 8OHdG increased progressively from 2 days after injury and peaked at 5 days,

suggesting that oxidative DNA damage is an early event. Therefore root avulsion is a suitable

model for studying oxidative stress induced motor neuronal degeneration. How the DNA

repair enzyme APE1 reacted to the damage is currently unknown. We hypothesize that down-

regulation of APE1 in spinal motor neurons early after injury renders it susceptible to further

oxidative insult, e.g. nitric oxide from nitric oxide synthase at later time course.

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Materials and Methods

NSC-34, PC12 and HEK293 Cell Culture

Motor neuron-like hybrid cell line NSC-34 was used (Cedarlane Laboratories, Burlington,

ON, Canada). The cell line was routinely maintained in Dulbecco's modified Eagle's medium

(DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin

(P/S). Cells were passaged every 3-4 days. To slow down proliferation and induce cell

differentiation, NSC-34 was treated as previously described [17, 19]. Briefly, after confluent,

culture medium was switched to differentiation medium containing 1:1 DMEM plus Ham’s

F12 supplemented with 1% FBS, 1% modified Eagle’s medium non-essential amino acid and

1% P/S. Most cells died within 2-4 days, the flask was gently tapped and medium was

replaced with fresh differentiation medium. Remaining cells were further cultured for 9-10

days. These differentiated cells survived in low serum condition and extended long and

multiple neurites. They were passaged once for subsequent experiments. They are referred as

NSC-34D.

PC12 cells (American Type Culture Collection, ATCC) were maintained in RPMI1640

medium supplemented with 10% horse serum, 5% FBS and 1% P/S. To induce neuronal

differentiation, growth medium was switched to differentiation medium containing

RMPI1640 supplemented with 1% horse serum, 0.5% FBS, 1% P/S and 50ng/ml nerve

growth factor (NGF, Upstate Biotech, Lake Placid, NY). Cells were incubated in the medium

for 6 days. NGF was removed 4 hr prior to drug treatment in differentiation medium.

HEK293 cells (American Type Culture Collection, ATCC) were maintained in DMEM

supplemented with 10% FBS and 1% P/S. Medium was switched to DMEM containing 1%

FBS and 1% P/S 4 hr prior to and during drug treatment. All culture medium and

supplements were obtained from Invitrogen (Carlsbad, CA) unless otherwise specified.

Drug Treatment

NSC-34D cells were seeded in 2% growth factor-reduced Matrigel (BD-Biosciences, Bedford,

MA) coated 6-well plate at 5 x 104 cells per well in differentiation medium. After 6 days, the

cells washed with PBS and pre-incubated with inhibitors including APE1 redox domain

inhibitor (2E)-2-[(4,5-Dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene]-

undecanoic acid (E3330, Sigma, St Louis), DNA repair domain inhibitor 7-Nitroindole-2-

carboxylic acid (CRT0044876, Sigma, St Louis) and alkylating agent methoxyamine Acc

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hydrochloride (MX, Sigma, St Louis) for 2 hrs. They were then co-incubated in 20µM-

300µM of hydrogen peroxide (H2O2) for 1 hr. Cells were then washed twice with PBS and

replaced with inhibitor- containing medium for 24 hours. Cell viability was assessed using

MTT colorimetric assay. Effects of co-treatment with H2O2 and inhibitors were also assessed

using PC12 and HEK293. For experiments using CRT0044876 or E3330, corresponding

amount of DMSO was added to control groups.

Cell Viability Assay

Cell viability was examined by MTT colorimetric assay and cell counting of DAPI-stained

nuclei. Tetrazolium salt (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium

bromide, Sigma, St Louis) is converted to a blue formazan product by dehydrogenase in

living cells. It is widely used for evaluation of cytotoxicity in vitro. Briefly, samples in 96-

well plate were incubated with MTT (0.16 mg/ml) for 4 hr and removed. The blue insoluble

precipitate was dissolved in DMSO and optical density was determined at 570nm. Cell

survival profile at different concentrations of H2O2 was also assessed by counting the number

of nuclei of cells grown on coverslip. Briefly, 104 cells were seeded in 2% growth factor-

reduced Matrigel coated coverslips. After H2O2 treatment, cells were washed with 0.01M

PBS and fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature. Cell nuclei

was then stained with 4′ 6-diamidino-2-phenylindole (DAPI) and mounted on slide for

fluorescent microscopy, 5 random fields were chosen per coverslips and photographed at 10X

magnification. Number of DAPI positive nuclei was counted by ImageJ. All experiments

were performed in triplicate and repeated at least 3 times.

Western Blotting

Cell samples were washed with PBS to remove all floating dead cells and then lysed using

ice-cold RIPA buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA,

1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM

beta-glycerophosphate, 1 mM sodium orthovandate, 1 µg/ml leupeptin (Cell Signalling

Technologies, Beverly, MA) and 1mM phenylmethylsulfonyl fluoride (PMSF) for 10 minutes

on ice. For spinal cord tissue samples, C7 segment contralateral and ipsilateral ventral horns

were carefully dissected and homogenized with ultra-sonicator. Total cell lysate protein

content was measured by Bradford method. 10µg of total protein was subjected to 12.5%

SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were

blocked with 5% non-fat dry milk in 0.01M Tris buffer (TB) with 0.1% Tween-20 at 4ºC Acc

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overnight and then incubated with rabbit anti-APE1 (1:2000, Novus Biologicals, Littleton,

CO); rabbit anti-phospho-p53 at Serine 15 (1:500, Novus Biologicals, Littleton, CO) or

mouse anti-p53 (1:500, Abcam, Cambridge, MA) at room temperature for 1 hr. Loading

control was mouse anti-β actin (1:10000, Sigma, St Louis, MO) for cell culture samples and

mouse anti-β-tubulin (Sigma, St Louis, MO, USA) for tissue samples. β-tubulin was used

because it is more suitable for spinal cord injury tissue samples [25]. After three washes in

TB/0.1% Tween-20, the membranes were incubated in peroxidase conjugated secondary

antibodies at room temperature for 1 hr. The membranes were washed extensively and

developed using enhanced chemiluminescence procedure (ECL-plus, GE Healthcare, Little

Chalfont, UK). Blots were quantified by Gel Analyzer in ImageJ (NIH, Bethesda, MD).

Immunocytochemistry

Cells seeding on glass coverslips were fixed with 4% PFA in 0.1 M phosphate buffer (pH 7.4)

for 15 minutes at room temperature. The coverslips were gently washed with 0.01M

phosphate buffered saline (PBS) and then incubated in blocking solution containing 10%

normal serum, 1% bovine serum albumin (BSA) in 0.01M PBS at room temperature for 1 hr.

Primary antibodies including mouse anti-NF-κB (1:1000, Cell signalling, Beverly, MA);

rabbit anti-phospho-p53 at Serine 15 (1:200, Novus Biologicals, Littleton, CO) and mouse

anti-p53 (1:200, Abcam, Cambridge, MA) were then added and incubated at 4°C for 14-16

hrs. After washes, corresponding fluorescent dye conjugated secondary antibodies (1:500,

Molecular Probes, Eugene, OR) were added and incubated for 1 hr. After extensive washes,

nuclei were counterstained with DAPI and then mounted with fluorescent mounting medium

(DAKO, Glostrup, Denmark) for microscopy.

Animals

Animal use for the study was conducted according to section 7 of the Animals (Control of

Experiments) Ordinance (Cap. 340) under Hong Kong law and legislation. All procedures

carried out in the study were approved by the Committee for the Use of Live Animals in

Teaching and Research at the University of Hong Kong. A total of 50 adult male Sprague-

Dawley rats weighing 280 to 320 g were used.

Surgical Procedures

Animals underwent surgical procedures similar to previously described with minor

modifications [26]. Briefly, rats were anesthetized by intraperitoneal injection of ketamine Acc

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(80 mg/kg) and xylazine (8 mg/kg). Under aseptic conditions, dorsal hemilaminectomy at the

sixth cervical vertebra was performed on the right side. The seventh cervical (C7) spinal roots

were identified and the dorsal root was cut to expose the underneath ventral root for clearer

visibility. The ventral root was avulsed with hook made of fire-polished capillary tube. The

success of avulsion was confirmed by examining any remaining rootlets under dissecting

microscope. The avulsed roots together with the spinal nerve were cut and removed to

prevent re-innervation. After the surgical procedures, muscles and skin were sutured in layers.

The animals were kept warm and allowed to recover from anaesthesia before returning to

their cages. They were allowed to survive for up to 4 weeks.

Perfusion and Tissue Processing

At the end of the survival period, the animals were given a lethal dose of sodium

pentobarbital and perfused intracardially with 0.9% normal saline followed by perfusion with

200 to 300 mL of fixative containing 4% PFA in 0.1 M phosphate buffer (pH 7.4). The spinal

cord was harvested and post-fixed with fresh fixative overnight and subsequently placed in

30% phosphate buffered sucrose. After the samples had sunk, the cords were cut into 40µm

cross sections on a sliding microtome. The sections were collected and stored in 0.01 M PBS

(pH 7.4) at 4ºC for later use.

Counting of Surviving Motor neurons

To assess motor neuron survival, every alternate section from all samples was counterstained

with 1% neutral red (Sigma), which stains the Nissl substance. Motor neurons were counted

under light microscope by a blinded observer following the method previously described [27].

Briefly, motor neurons were identified based on their large somata, location of cell bodies in

ventral horn and granular appearance of Nissl bodies. Motor neurons in the contralateral and

ipsilateral sides of the C7 segment were counted. Only those with visible nuclei were counted.

Immunohistochemistry

Floating sections were washed with PBS and then incubated in blocking solution containing

10% normal goat serum and 1% BSA for 1 hour at room temperature. The sections were

subsequently incubated in primary antibodies rabbit anti-APE1 (1:1000, Novus Biologicals,

Littleton, CO) and mouse anti-SMI-32 (1:1000, Sternberger Monoclonals, Lutherville, MD)

at 4°C for 14-16 hrs. After washes with PBS, sections were incubated in corresponding

fluorescent dye conjugated secondary antibodies (1:500, Molecular Probes, Eugene, OR) for Acc

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1 hr at room temperature. After thorough washes, sections were mounted onto gelatine-coated

glass slide and mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark) for

microscopy.

Measurement of APE1 Nuclear Staining

Every tenth section was pooled from serial sections and immunostained with anti-APE1

(conjugated with Alexa Fluor 568 secondary antibodies) and SMI-32 (conjugated with Alexa

Fluor 488 secondary antibodies) as described above. At least 4 sections were collected for

each sample. All nuclei of motor neurons, as immuno-positive for SMI-32 in the cell bodies,

were photographed under 40X lens with Carl Zeiss fluorescence microscope. At least 100

motor neurons were analysed for each side in each time point. Area, mean gray value and

integrated optical density were measured by ImageJ (NIH, Bethesda, MD).

Data Analysis

Results are expressed as mean ± SEM. One-way analysis of variance and the Tukey multiple

comparisons test were used to determine the statistical significance of differences among the

means. A value of p < 0.05 was considered significant.

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Results

Hydrogen peroxide causes down-regulation of APE1 and motor neuron death

To examine the change in APE1 level in motor neurons upon ROS insult, we challenged

NSC-34D with increasing doses of H2O2 for 1 hour. Western blot analysis of samples

collected at 24 hours after treatment showed that NSC-34D down-regulated APE1 starting at

100µM H2O2 (Fig 1A & B). Cell survival assay by counting DAPI-stained nuclei showed a

dose-dependent cell death starting at 100µM H2O2 (Fig 1C). Similar results were obtained by

MTT assay which measures metabolic activities of living cell. A significantly lowered

activity was detected starting at 50µM (Fig 1D), perhaps due to higher sensitivity of the assay.

We then examined the time course of APE1 level under 100 µM H2O2, a significant decrease

in APE1 started at 4 hours after treatment. It remained down-regulated at 24 hours after

treatment (Fig 2A & C). Previous report showed that sub-lethal dose of ROS induced

activation of APE1 in Hela cells, lung primary fibroblast and CHO cells [28], we then treated

the cells with 20µM H2O2, results showed a slight but insignificant decrease of APE1 at any

time point after treatment (Fig 2A & B). Taken together, these results suggested that APE1 is

correlated with concentration dependent cell death and NSC-34D responded differently from

other cell types.

Inhibiting DNA repair and redox functions of APE1 increased NSC-34D cell death

Since APE1 contains dual functional domains, silencing APE1 protein cannot provide a clear

indication on the role of each domain. We therefore used small molecules to inhibit individual

domain. CRT0044876 was identified as a potent APE1 inhibitor at low micromolar

concentrations. CRT0044876 was not cytotoxic to NSC-34D as high as 25µM (Fig 3A).

Survival curve for co-incubation of 10µM of CRT0044876 with increasing doses of H2O2

was no different from that of DMSO containing H2O2 control (Fig 3D). Previous reports on

lack of effect of this inhibitor suggested that CRT0044876 might not be a suitable APE1-

DNA repair domain inhibitor [29-30]. We then tried to block DNA repair process by

methoxyamine (MX), a drug binds covalently with AP sites, subsequently preventing

recognition by APE1. Inherent cytotoxicity assay showed that NSC-34D tolerated well to

concentration of MX lower than 50µM (Fig 3B). We then co-incubated 10µM of MX with

increasing doses of H2O2, results showed that MX significantly potentiates the cytotoxicity of Acc

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H2O2 starting at 50µM (Fig 3D). These results showed that inhibiting DNA repair increased

NSC-34D cell death.

Next we used APE1 redox domain inhibitor E3330 to block redox regulation in NSC-34D.

Inherent cytotoxicity assay showed that E3330 at lower than 10µM did not affect NSC-34D

cell survival (Fig 3C). Co-incubation of 10µM of E3330 with increasing doses of H2O2

resulted in increased cell death at 50µM and 100µM but not higher concentration of H2O2

(Fig 3D). To test whether blocking redox function in other cell types also affect cell survival,

we used human embryonic kidney cell line HEK293 and differentiated neuronal cell line PC-

12 and repeated the experiments. They have different cytotoxicity profiles of E3330 with

NSC-34D, but 10µM is non-cytotoxic to all cell types (Fig 4). Results showed that the

survival curve for co-incubation of 10µM E3330 with increasing doses of H2O2 did not differ

from that of DMSO containing H2O2 control in HEK293 and PC12 cells. These results

suggested that maintaining redox regulation by APE1 is important for survival in NSC-34D

but not in other cell types.

APE1 down-regulation was p65 independent

Pharmacological action of E3330 was shown to block the interaction between APE1 and

nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [31], thus inhibiting

reduction of NF-κB and nuclear translocation for gene transcription. Therefore we examined

p65, a subunit of NF-κB complex, translocation by immunohistochemistry. Results showed

that p65 did not accumulate in nuclei neither in control (Fig 5A) nor in 100µM of H2O2 (Fig

5B) at all time points examined. Addition of E3330 in 100µM H2O2 also did not cause or

prevent p65 nuclear translocation but neurite retraction and cell rounding were observed (Fig

5C). Cytoplasmic shrinkage caused a higher staining intensity but western blot analysis did

not show up-regulation of p65 (data not shown). These results showed that oxidative stress

did not cause NF-κB translocation in NSC-34D and it was not responsible for APE1-

mediated survival.

p53 phosphorylation preceded APE1 down-regulation

It is well known that DNA damage triggers p53 activation. However whether p53 regulates

APE1 or the vice versa remains elusive [32-33]. Western blot showed that p53 was

phosphorylated (pSer15) immediately upon H2O2 addition (0 hour) and peaked at 2 hours

after treatment. The level returned to baseline level at 4 hours after treatment whereas total

p53 remained relatively constant (Fig 6A). Immunohistochemical staining of phospho-p53 Acc

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revealed the same pattern. Time point studies showed that pSer15 staining appeared in nuclei

as early as 30 minutes after addition of H2O2 whereas total p53 staining intensity remained

constant (Fig 6B). These results suggested that p53 phosphorylation preceded APE1 down-

regulation.

Progressive down-regulation of APE1 after spinal root avulsion

Spinal root avulsion causes progressive degeneration of motor neurons across several weeks.

We used western blot to examine APE1 level at multiple time points after injury. Semi-

quantitative results showed that APE1 was down-regulated at 3 days after injury but up-

regulated afterward (n=6 in each time point, Fig 7A). Since APE1 is ubiquitously expressed,

other cells such as inflammatory cells might contribute to the increased amount of APE1. We

then used SMI-32 immunohistochemistry to specifically look for changes in motor neurons

(n=5 in each time point, Fig 7B), nuclear size and APE1 optical density were measured.

Results showed that the size of motor neuron nuclei was significantly smaller at 14 and 28

days after injury (Fig 7C), suggesting nuclear shrinkage. Mean gray value of APE1 staining

showed that APE1 was significantly lowered and progressively down-regulated in ipsilateral

side as compared to contralateral side at all time points examined. Integrated optical density

which takes into account the nuclear size, showed significant change starting from 7 days

after injury (Fig 7C). Our results indicated that spinal motor neurons also down-regulated

APE1 under oxidative stress in its pathophysiological context.

We then examined the effect of APE1 decrease on motor neuron survival by counting the

number of surviving motor neurons at various time points (n=5 at each time point, Fig 8).

Spinal root avulsion caused minimal cell lost (5.44±5.13%) at one week after injury. A

progressively lost of 23±6.55% and 48.2±1.19% of cells happened at 2 weeks and 4 weeks

after injury. The results are consistent with our previous publications [21]. The results

suggested that down-regulation of APE1 preceded cell death evident at 2 weeks after injury.

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Discussion

Our results showed that oxidative stress caused down-regulation of APE1 and cell death in a

motor neuron-like cell line. Inhibiting APE1 DNA repair or redox regulation functions

potentiated oxidative stress induced cell death, suggesting that both functions are important

for cell survival under oxidative challenge. We further showed that early phosphorylation of

p53 suggested its up-steam regulatory role on APE1. Our in vivo data demonstrated similar

results. Down-regulation of APE1 in motor neurons preceded cell death in spinal root

avulsion injury. Our results imply that oxidative stress induced down-regulation of APE1 in

spinal motor neurons renders them susceptible to further oxidative challenge such as nitric

oxide from nitric oxide synthase expressed at later time course. It represents one of the

pathways leading to motor neuron death.

Neurons are prone to accumulating DNA damage. First, the neuron is one of the most

metabolically active types of cell, while most of them are retained for the whole lifespan.

Even under normal condition, a small amount of electrons leaking from mitochondrial

electron transport chain reacts with oxygen to form the ROS precursor superoxide anion [34-

35]. Second, neurons repair mitochondrial DNA (mtDNA) damage less efficiently than glial

cells in the CNS [36]. Post-mortem studies showed that oxidized DNA increased

progressively with normal aging but the amount of oxidized mtDNA is 10-fold more than

nuclear DNA in neurons [7]. Third, as neurons are terminally differentiated, as they do not

require DNA replication mechanism which possesses backup repair pathways to ensure

genome stability, less efficient repair in post-mitotic cells leads to build up of DNA adducts

[37]. Fourth, under pathological conditions, excessive ROS can be generated from

overproduction from pro-oxidant enzymes; by-product of enzymatic activities; inactivation of

anti-oxidant defense or oxidation of neurotransmitters [38-41]. ROS can also be derived from

infiltrating cells such as microglia during inflammation [42]. Excessive levels of ROS cause

damage to all parts of the cell and eventually affects cellular functions. Although recent

studies showed that astrocytes can play a supportive role by releasing glutathione to buffer

ROS exposed to neurons [43], inefficient intrinsic defense and repair mechanisms render

neurons vulnerable.

Oxidative DNA damage is primarily repaired by the BER repair pathway. Over-expression of Acc

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the rate limiting enzyme APE1 in hippocampal and sensory cells enhanced cell survival under

hydrogen peroxide treatment, suggesting that the role of APE1 is neuroprotective [44]. It was

shown that adenoviral transfer of APE1 or injection of APE1 peptide into ischaemic mice

brain can decrease DNA and tissue damage [45-46]. Conversely, a link between APE1

reduction and neurotoxicity was demonstrated by RNA silencing experiment, reduction of

APE1 level caused enhanced cell death in differentiated neuronal cell line SH-SY5Y, dorsal

root ganglion cells and hippocampal neurons co-treated with oxidative and alkylating agents

[44, 47]. One would expect that the cell might up-regulate APE1 to speed up DNA repair

upon oxidative stress, but interestingly, APE1 expression in neurons is generally down-

regulated upon oxidative injuries as mentioned in the introduction section and also shown in

our studies. It either implies another poor intrinsic defense mechanism of neuron or that the

enzyme is down- regulated by other upstream molecules.

To delineate which functional domain is responsible for the protective effect, we used small

molecules to inhibit individual functions. It is not surprising to find that inhibiting DNA

repair by MX caused enhanced cell death in increasing dose of hydrogen peroxide. However,

it is intriguing to find that inhibiting the redox function domain by E3330 also affected motor

neuron survival. Our results showed that it is specific to the motor neuron-like cell line but

not to another neuronal cell line PC-12 or non-neuronal cell line HEK293. Previous

experiments by others also showed that the redox functional domain is important for cell

survival under oxidative stress for primary hippocampal and dorsal root ganglion cells but not

in differentiated neuroblastoma cell SH-SY5Y [47]. Therefore it seems that redox regulation

by APE1 for survival under oxidative stress is cell type specific. Apparently, inhibiting the

redox domain of APE1 in other cells types elicits different responses. For example, E3330

inhibits pancreatic cancer growth and migration [48], induces embryonic stem cell to

differentiate into hematopoietic lineage [49] and inhibits inflammatory cytokine release by

activated macrophage [50].

Functional studies showed that E3330 mediates its effect by preventing interaction of APE1

with several transcription factors including NFκB [31]. NFκB is an important modulator in

neuronal survival, differentiation and axon morphology [51]. Its effect of activation on

neuronal survival still remains controversial. Glutamate toxicity induced NFκB activation in

another motor neuron-like cell line VSC 4.1, suppression of its activation potentiated

glutamate induced cell death [52]. However, it was also shown that NFκB is involved in Acc

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chronic glutamate induced motor neuron death in spinal cord organotypic culture [53]. We

therefore examined whether NFκB is involved in oxidative stress induced cell death with our

cell line. Our results showed that the NFκB subunit, p65, was localized in the cytoplasm.

Neither control condition nor hydrogen peroxide treatment initiated translocation of p65 into

the nucleus. Therefore we believe that NFκB is not involved in the process and APE1 is

likely to affect other transcription factors to mediate cell survival.

From our time course studies in APE1 down-regulation, reduction of APE1 protein level was

evident at 4 hours after hydrogen peroxide treatment. Delayed decrease of APE1 suggested

that multiple steps are involved in regulating APE1 level. It is well known that DNA damage

triggers p53 activation. A previous report showed that introduction of wild type but not

mutant p53 into p53-null cells down-regulates endogenous APE1 level and inhibits its gene

expression [33]. The authors further showed that p53 interferes with specificity protein-1

binding to APE1 promoter and subsequent hinders recruitment of transcription machinery.

However it was also shown that silencing APE1 expression caused an up-regulation of

phosphorylated p53 in cisplatin-treated sensory neurons [32]. We then examined the temporal

relationship of APE1 and p53 phosphorylation in our cell line model. Although we did not

show a mechanistic linkage between APE1 and p53, our results showed that phosphorylation

of p53 preceded down-regulation of APE1, suggesting that phospho- p53 might mediate the

down-regulation of APE1.

Our in vivo results are in general consistent with the in vitro results which showed down-

regulation of APE1 in motor neurons. Spinal root avulsion causes motor neuron degeneration,

and oxidative stress is one of the possible pathways. Inflammatory responses such as

activation of microglia and astrocytes after root avulsion are sources of oxidative stress, and

accumulation of metabolically active mitochondria within the injured motor neurons also

accounts for the generation of reactive oxygen species [23]. A previous report showed that

oxidative stress induced single strand breaks before apoptosis in motor neurons that is p53

dependent after sciatic nerve avulsion [54]. More spinal motor neurons survived in mice

deficient in p53 than that in wild type mice. It is interesting to test whether absence of p53

activation in p53 knock-out mice maintains APE1 level and promotes cell survival. We did

not examine the upstream and downstream molecules in our animal study. However we noted

in our earlier report which we showed decreased phosphorylation of c-Jun in adult spinal

motor neurons after spinal root avulsion [55]. Since c-Jun is one of the downstream targets of Acc

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APE1, it indirectly supports our recent findings that APE1 is down-regulated early after

injury. Our results also showed that APE1 down-regulation at 3 days after injury preceded

motor neuron death, about 5% motor neurons were lost at 7 days after injury while about one-

fourth was lost at 2 weeks after injury. The delay in motor neuron death implies that: 1)

oxidative stress -induced damage slowly accumulates in motor neurons and 2) APE1 down-

regulation renders motor neurons susceptible to second wave ROS attack, e.g. by nitric oxide

released from de novo expression of nitric oxide synthase (NOS) in motor neurons [21, 56].

Our previous experiments showed that neuronal NOS appears at 7 days and peaks at 2 weeks

after root avulsion injury, correlating to the time of motor neuron death. In addition, our

preliminary results in distal nerve axotomy model also showed down-regulation of APE1 in

spinal motor neurons without motor neuron death (data not shown). It suggested that down-

regulation of APE1 might not be the cause of motor neuron death but render the motor

neurons more vulnerable to further genotoxic insults.

It is noteworthy that oxidative stress is not the only factor contributing to motor neuron

degeneration after root avulsion, other possible factors include deprivation of neurotrophic

support from the spinal root and loss of synaptic input, as in our study the dorsal root was

also transected. Further studies are warranted to establish a direct link between oxidative

stress and down-regulation of APE1 in vivo, for example by peripheral nerve transection or

by selective motor root avulsion and quantitative measurement of oxidative stress level in

motor neuron.

We conclude that motor neurons down-regulate APE1 under oxidative stress. Down-

regulation of APE1 is likely to cause less efficient DNA repair and compromised redox

regulation. Although we showed correlation of reduced APE1 level with cell death in cell

culture model, delayed motor neuron death after APE1 protein reduction in spinal root

avulsion model suggested that it might not be the direct cause. It is likely that compromised

APE1 function renders motor neurons susceptible to continuous oxidative stress as in the

pathological conditions.

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Acknowledgments

All authors (CTH, GA and WWT) contributed to designing, performing the experiments and

writing the manuscript.

This study was supported by the University of Hong Kong and National Basic Research

Program of China (973 program, 2011CB504402).

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References

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Figure Legends

Fig 1

To determine the response of APE1 level and cell survival under oxidative stress in NSC-34D,

cells were challenged with increasing doses of hydrogen peroxide (H2O2) for 1 hour and

examined at 24 hour. Western blot analysis showed that APE1 was significantly down-

regulated at concentration at or above 100µM H2O2 (A&B, *, p<0.05; **, p<0.01 vs control).

Significantly number of cells was lost at these concentrations as determined by counting

DAPI-stained nuclei on coverslips. (C, ***, p<0.0001 vs control). MTT colorimetric assay

which measures enzymatic activities in cells showed that NSC-34D was affected even at

50µM H2O2 (D, ***, p>0.0001 vs control). Results showed that oxidative stress induced by

H2O2 caused down-regulation of APE and affected cell survival.

Fig 2

To determine the temporal change of APE1 level, NSC-34D was challenged with low dose

(20µM) and high dose (200µM) of hydrogen peroxide (H2O2). Western blot analysis showed

that APE1 was significantly down-regulated at 4 hour after 1-hour treatment with 200µM

H2O2 and remained down-regulated till 24 hour (A, lower panel and C, **, p<0.01;

***p<0.001 vs control). Previous report showed that sub-lethal dose of H2O2 activated APE1,

we therefore treated cells with 20µM H2O2 which we showed no difference in cell viability

with MTT (data not shown). Our results showed that 20µM H2O2 also caused slight but

insignificant down-regulation of APE1 at all time points examined (A, upper panel and B). It

suggested that even low dose of oxidative stress caused down-regulation of APE1 but not an

increase in motor neurons.

Fig 3

APE1 is a multiple function-enzyme with dual domains. To examine the role of each domain

on cell survival, we used small molecule inhibitors to inhibit individual functions.

CRT0044876 is an inhibitor for DNA repair function domain of APE1, cell viability was not Acc

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affected at concentration at or below 25µM as determined by MTT assay (A). Methoxyamine

(MX) convalently binds to abasic sites and acts as inhibitor of base excision repair pathway,

cell viability was not affected at concentration at or below 25mM as determined by MTT

assay (B). E3330 is a quinone analogue specifically inhibits redox function of APE1, cell

viability was not affected at concentration at or below 10µM as determined by MTT assay

(C). Co-treatment of hydrogen peroxide (H2O2) with inhibitors in NSC-34D showed that

inhibiting DNA repair process with MX and inhibiting redox function of APE1 with E3330

potentiated H2O2 induced cell death at or above concentration of 50µM H2O2 (D, *, p<0.05;

**, p<0.01 and ***, p<0.001 vs control). No difference in cell viability was found when cells

were co-treated with CRT0044876, it suggested that this inhibitor might not be specific to

APE1 as mentioned in other studies.

Fig 4

To determine whether blocking redox function domain in other cell line also potentiated

oxidative stress induced cell death, E3330 was tested in human embryonic kidney cell line

HEK293 and differentiated neuronal cell line PC12. Cell viability was not affected by E3330

at concentration at or below 25µM in HEK293 as determined by MTT assay (A). Co-

treatment of 10µM E3330 with increasing doses of hydrogen peroxide showed that E3330 did

not potentiate oxidative stress induced cell death (B). Cell viability was not affected by

E3330 as high as 25µM in PC12 as determined by MTT assay (C). Co-treatment of 10µM

E3330 with increasing doses of hydrogen peroxide showed that E3330 did not potentiate

oxidative stress induced cell death (D). Results showed that potentiation of oxidiative stress

in E3330 are specific to motor neuron cell line NSC-34D.

Fig 5

E3330 was previously shown to inhibit interaction between APE1 and several transcription

factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). As

NF-κB pathway is involved in neuronal cell survival, growth and morphological change, we

investigated whether NF-κB is involved in APE1-mediated cell survival. Representative

photomicrograph showed immunohistochemistry staining of subunit of NK-κB, p65, is

localized mainly in the cytoplasm after treatments (A). Neither treatment with hydrogen Acc

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peroxide (B) or co-treatment of hydrogen peroxide with E3330 (C) caused nuclear

translocation. Co-treatment with E3330 caused neurite retraction and cell rounding, thus a

higher staining intensity was observed due to cytoplasmic shrinkage. Our results suggested

that NF-κB might not participate in APE1-mediated cell survival. Scale bar= 50µm.

Fig 6

DNA damage triggers p53 activation, we examined whether p53 activation is temporally

correlated with APE1 down-regulation. Western blot analysis showed that p53 is

phosphorylated at Serine 15 (pSer15) immediately after hydrogen peroxide treatment (0h).

Phosphorylated p53 remained up-regulated till 2 hour after treatment and remained at basal

level afterwards. Total p53 remained constant throughout the experiments (A). Representative

photomicrograph showed that p53 is localized in the nucleus. At control condition, only a few

weakly stained phosphos-p53 positive nuclei were found. At 2 hours after hydrogen peroxide

treatment, many cells were phospho-53 positive (B). Our results showed that p53 is activated

early after oxidative stress. It suggested that p53 might act upstream and negatively regulate

APE1 expression. Scale bar= 100µm.

Fig 7

Spinal root avulsion model was used to study the response of APE1 in motor neurons in their

pathophysiological context. Previous reports showed that spinal root avulsion caused motor

neuron degeneration due to oxidative stress. Western blot analysis from avulsed spinal cord

ventral horn tissue showed that APE1 level was down-regulated at 3 days after injury but

increased steadily afterwards (A, representative gel photo shows protein samples from

contralateral and ipsilateral ventral horns of one animal for each time point, n=6 in each time

point for data analysis). As other neighboring cells other than motor neurons also express

APE1, we decided to use immunohistochemistry to examine level change in individual motor

neurons (B). Spinal cord samples were cut into 40µm thick sections. Non-phosphorylated

heavy weight neurofilament marker SMI-32 was used to identity motor neuron cell bodies

(arrows), sections were co-stained with anti-APE1 (n=5 in each time point).

Immunohistochemical staining showed that APE1 mainly localized in motor neuron nuclei,

although slight staining was also observed in cytoplasm under high magnifications. Acc

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Representative photomicrograph showed difference in APE1 staining intensity at

contralateral (left) and ipsilateral (right) ventral horn motor neurons at 2 weeks after avulsion

injury. Marked reduction of APE1 staining was evident. Pictures were taken under 40x lens

and the optical density and size of nuclei was measured. Quantitative data were collected for

each motor neuron which is represented by a dot in the chart (C). Results showed that the

mean gray value of ipsilateral ventral horn motor neurons was significant lower than that in

contralateral motor neurons at 3 days after injury and remained lower throughout the

observation period. Measurement of nuclear size showed a significant reduction of nuclear

area at 2 weeks and 4 weeks after injury. Integrated optical density (IOD) which is the

product of mean gray value and measured area showed a significant reduction of IOD at 1

week after injury. A bar chart showing the percentage of IOD of APE1 in ipsilateral motor

neurons relative to contralateral motor neurons demonstrates the progressive reduction of

staining intensity of APE1 level. The results are in accordance with cell culture data showing

reduction of APE1 under oxidative stress. Scale bar=50µm.

Fig 8

To examine the temporal correlation of APE1 down-regulation and motor neuron cell death

after root avulsion injury. Spinal cord sections were counter-stained with neutral red and

surviving motor neurons were counted under light microscope (n=5 in each time point).

Quantitative results of motor neuron survival relative to contralateral ventral horn showed

that a significant 23% of motor neurons were lost at 14 days after injury. It showed a delay in

APE1 level decrease and motor neuron cell death. MNs: motor neurons.

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down-regulation of apurinic/apyrimidinic endonuclease 1 (ape1) in spinal motor neurones under...

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