• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Open Neurosci J. Author manuscript; available in PMC Nov 5, 2009.
Published in final edited form as:
Open Neurosci J. Jan 1, 2009; 3: 26–34.
doi:  10.2174/1874082000903010026
PMCID: PMC2773510
NIHMSID: NIHMS114639

Differential Expression of Redox Factor-1 Associated with Beta-Amyloid-Mediated Neurotoxicity

Zhiqun Tan,*§,1 Lei Shi,§†,1 and Steven S. Schreiber1,2,3

Abstract

Redox factor-1 (Ref-1), also known as HAP1, APE or APEX, is a multifunctional protein that regulates gene transcription as well as the response to oxidative stress. By interacting with transcription factors such as AP-1, NF-kappaB and p53, and directly participating in the cleavage of apurininic/apyrimidinic DNA lesions, Ref-1 plays crucial roles in both cell death signaling pathways and DNA repair, respectively. Oxidative stress induced by aggregated beta-amyloid (Aβ) peptide, altered DNA repair and transcriptional activation of cell death pathways have been implicated in the pathophysiology of Alzheimer’s disease (AD). Here we show that varying concentrations of Aβ1–42 differentially regulate Ref-1 expression, Ref-1 function and neuronal survival in vitro. Aβ (5.0 μM) caused a relatively rapid decrease in Ref-1 expression and activity associated with extensive DNA damage and neuronal degeneration. In contrast, Ref-1 induction occurred in cells exposed to Aβ (1.0 μM) without significant neuronal cell death. Aβ-induced attenuation of Ref-1 expression and endonuclease activity, and neuronal cell death were prevented by the anti-oxidant, catalase. Similar differential effects on Ref-1 expression and cell viability were observed in N2A neuroblastoma cells treated with either high or low dose hydrogen peroxide. These findings demonstrate the differential regulation of Ref-1 expression by varying degrees of oxidative stress. Parallels between the Ref-1 response to Aβ and H2O2 suggest similarities between DNA repair pathways activated by different inducers of oxidative stress. In AD brain, colocalization of Ref-1 and Aβ the absence of significant DNA damage are consistent with the cell culture results and suggests that Ref-1 may play a more neuroprotective role under these conditions. Modulation of Ref-1 expression and activity by local variations in Aβ concentration may be an important determinant of neuronal vulnerability to oxidative stress in AD.

INTRODUCTION

Increasing evidence indicates that cellular reduction-oxidation (redox) status regulates a variety of key intracellular processes including gene expression, cell proliferation and cell death [1-3]. Redox factor-1 (Ref-1), also known as APE-1 or APEX or HAP1, is a 37 kDa nuclear protein that participates in the redox regulation of gene transcription as well as the cellular response to oxidative stress [3, 4]. Through cleavage of abasic (apurinic/apyrimidinic) sites by its endonuclease (APE) activity, Ref-1/APE-1 plays a major role in the repair of DNA stand breaks induced by endogenously generated reactive oxygen species (ROS) and exogenous agents such as ionizing radiation and chemotherapeutic drugs [5-7]. In addition, Ref-1 modulates the redox state of conserved cysteine residues within the DNA-binding domain of transcription factors such as AP-1, NF-kappaB and p53, resulting in transcriptional activation of downstream genes such as nitric oxide synthase [8, 9].

Currently the dual functions of Ref-1, i.e., DNA repair versus transcriptional regulation, relevant to the central nervous system (CNS) response to injury are poorly understood. Ref-1 is highly expressed in proliferating cells during embryonic and early postnatal development [10, 11]. In contrast, relatively lower amounts of Ref-1 are found in the adult CNS, particularly in astrocytes, neurons in hippocampus and piriform cortex, as well as cerebellar Purkinje cells [12]. Attenuation or loss of Ref-1 activity has been associated with marked sensitivity to DNA-damaging agents and increased cell death [7, 13, 14]. Downregulation of Ref-1 expression has also been reported in vulnerable neurons following cerebral ischemia [15-17]. However, increased Ref-1 expression has been demonstrated in both surviving and vulnerable neurons following an ischemic or excitotoxic insult in rat brain [18, 19]. In Alzheimer’s disease (AD) brain, Ref-1 protein was found to accumulate in vulnerable neurons as well as senile plaques [20]. As β-amyloid (Aβ)-induced oxidative stress, altered DNA repair and activation of transcription factors such as AP-1 and p53 have been implicated in the pathophysiology of AD [21-24], a role for Ref-1 as a mediator of Aβ-induced neurodegeneration seems plausible.

The present study was conducted to determine the effects of different concentrations of Aβ and oxidative stress on Ref-1 expression and neuronal survival, and to examine the potential relationship between Ref-1 induction, Aβ accumulation and neurodegeneration in AD. The results strengthen the idea that Ref-1 induction is neuroprotective while loss of Ref-1 may lead to neurodegeneration. Differential modulation of Ref-1 expression by local variations in the concentration of β-amyloid may contribute to selective neuronal vulnerability in AD.

MATERIALS AND METHODOLOGY

β-amyloid (Aβ1-42) peptide was purchased from U.S. Peptide (Fullerton, CA, USA). Neurobasal medium and B27 supplement were purchased from Invitrogen (Carlsbad, CA). In Situ Cell Death Detection and Cytotoxicity Detection Kits were purchased from Roche Diagnostics (Indianapolis, IN). Rabbit anti-Ref-1 and anti-Aβ1-42 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Quality Controlled Biochemicals (QCB, Hopkinton, MA), respectively. Mouse anti-Ref-1 monoclonal antibody was purchased from BD PharMingen (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG and catalase were purchased from Sigma (St. Louis, MO). Texas Red-or rhodamine-conjugated goat anti-mouse IgG was purchased from Chemicon (Temecula, CA). Biotinylated goat anti-rabbit IgG, ABC and diaminobenzidine (DAB) staining kits were purchased from Vector Laboratories (Burlingame, CA).

Timed pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). Human postmortem brain samples (12 subjects with a histopathological diagnosis of AD and 9 age-matched controls) were obtained from the Alzheimer’s Disease Research Consortium at the University of Southern California and the Institute for Brain Aging and Dementia Tissue Repository at the University of California Irvine. The average age of each group was 81.0 and 79.5 years for the AD cases and controls, respectively.

All procedures were approved by both University of Southern California Keck School of Medicine and University of California Irvine School of Medicine Institutional Review Boards and Animal Utilization and Care Committees. Rats were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Cell Culture

Neuron cultures were prepared from the hippocampus of Sprague-Dawley rats at 18 days of gestation (E18) as previously described [25, 26]. Briefly, hippocampi were dissected in Ca2+/Mg2+-free Hank’s balanced salt solution (HBSS) under a stereomicroscope and incubated in 0.1% trypsin (Irvine Scientific) at 37 °C for 5 min. The tissue was triturated by aspirating 7 to 10 times with a flame-narrowed Pasteur pipette. Cells were plated onto poly-D-lysine-coated 60 mm tissue culture dishes, 8-well chamber slides or 96-well microplates at approximately 104 cells per cm2 in serum-free Neurobasal medium plus B27 supplement for optimum survival of CNS neurons. The medium was exchanged with fresh medium 3 days after initial plating. All cultures were grown in a humidified atmosphere containing 5% CO2 at 37 °C.

Preparation and Exposure of Aggregated β-Amyloid Peptide

1-42 peptide was initially dissolved in 10 mM HCl and then preaggregated in 0.1 M phosphate buffer (pH 7.2) for 3 days prior to use. Aggregation was confirmed by visualization of β-amyloid fibrils using phase contrast microscopy. Seven days after initial plating the culture medium was changed to medium supplemented with either aggregated Aβ1-42 peptide or vehicle. At the specified times cells were either fixed with 4% paraformaldehyde in 1xphosphate buffer (PBS) for immunocytochemistry or harvested for biochemical analysis.

Immunostaining

For immunocytochemistry, fixed cells were incubated with a rabbit anti-human Ref-1 polyclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hr at room temperature, followed by a biotinylated secondary antibody for 30 min and 0.6% H2O2 /2% Triton X-100 in 1 X PBS for 10 min. Immunoreactivity was detected using the ABC method (Vector Laboratories, Burlingame, CA) followed by staining with diaminobenzidine (DAB) (Vector Laboratories).

For the AD studies, tissue blocks containing hippocampus plus surrounding temporal cortex were fixed in 10% neutral-buffered formalin, paraffin-embedded and sectioned at 6 μm. Deparaffinized sections were-rehydrated prior to microwave antigen retrievial. Sections were then incubated with the following combinations of antibodies: mouse anti-Ref-1 (1:100, Chemicon) plus rabbit anti-Aβ1-42 (1:100, QCB), or rabbit anti-Ref-1 and either mouse anti-GFAP (1:200, Roche) or anti-NeuN (1:200, Chemicon) overnight at 4 °C. This was followed by incubation with FITC-conjugated sheep anti-rabbit (1:200, Sigma) and Texas Red-conjugated goat anti-mouse (1:80, Chemicon) secondary antibodies for 30 min. To block endogenous lipofucsin autofluorescence sections were stained with 0.5% Sudan black B in ethanol for 10 min followed by several PBS washes [27]. For dual labeling of DNA damage and Ref-1, sections were re-microwaved after TUNEL staining (see below), incubated in blocking buffer for 30 min at room temperature, washed in PBS and incubated with Ref-1 antibody followed by a biotinylated secondary antibody. After several PBS washes Ref-1 immunoreactivity was detected using streptavidin-conjugated Texas Red (Amersham, Piscataway, NJ).

Evaluation of Neuronal Damage

TdT-mediated dUTP nick end labeling (TUNEL) was employed to detect neuronal DNA damage in both cultured cells and human brain sections. Brain sections were fixed and microwaved as described above, labeled with an In Situ Cell Death Detection (fluorescein) kit (Roche, Indianapolis, IN) according to the manufacturer’s protocol, and analyzed by fluorescence microscopy. For combined labeling of TUNEL and Ref-1, slides were then incubated with mouse anti-Ref-1 monoclonal IgG (1:200, BD PharMingen) followed by incubation with a rhodamine-conjugated goat anti-mouse IgG (Chemicon) and visualized by fluorescence microscopy. For N2A cells, TUNEL was performed using an In Situ Cell Death Detection (POD) kit followed by (aminoethylcarbazole) AEC chromogenic visualization. Cell death in vitro was also assayed by lactate dehydrogenase (LDH) release using a Cytotoxicity Detection Kit (Roche) following manufacturer’s manual.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from cultured cells using TRI-zol (Invitrogen) and reverse transcribed using 1 μg oligo(dT) in 20 μl of reaction buffer (1 mM each dNTPs, 25 mM Tris-Cl (pH 8.3), 25 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, 0.25 mM spermidine, 10 u RNase inhibitor (Roche, Indianapolis, IN), 10 u avian myeloblastosis virus reverse transcriptase (Promega) incubated for 1 hr at 42 °C and 40 min at 52 °C. The reaction was stopped by adding 160 μl of 10 mM Tris-HCl (pH 7.4) and 1mM EDTA. PCR was performed with 10 μl of RT reaction mixture, 50 mM KCl, 10 mM Tris-Cl (pH 9.0), 0.1% Triton X-100, 1 mM MgCl2, 400 nM each primer, 200 μM each dNTP, 5 u Taq DNA polymerase (Boehringer) in a final volume of 50 μl. Ref-1 primers: (forward) 5’-AATGTGGATGGGCTTCGA-3’; (reverse) 5’-GCGCCAACCAACATTCTT-3’ (Integrated DNA Technologies, Coralville, IA). Samples were subjected to 30 cycles (94 °C, 1 min; 58 °C, 1 min; 72 °C, 3 min) in a DNA Thermal Cycler (Perkin Elmer, Norwalk, CT), and 5 μl of the PCR products were analyzed by electrophoresis on a 1% agarose gel. For semi-quantitative PCR, amplification of β-actin was used as a control for densitometric analysis.

AP Endonuclease Activity Assay

AP endonuclease activity was assayed as previously described [28] with certain modification. Briefly, harvested cells were re-suspended and sonicated in AP lysis buffer (70 mM HEPES pH 7.6, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.2% Triton X-100). Total protein concentrations of the supernatants were determined by a Sigma Total Protein Assay kit. To assay AP activity of prepared lysates, QIAGEN column-purified pDsRed1-C3 (4.7 kb) (Clontech) plasmid DNA was depurinated by heating in 10 mM sodium citrate, 0.1 M NaCl pH5.2 at 70 °C for 45 min. APE reaction consisted of 20 ng (total protein) of cell extract, 300 ng of undamaged (control) or depurinated pDsRed1-C3 plasmid (Clontech) in 40 mM HEPES, 5 mM MgCl2, 0.5 mM DTT, 2 mM ATP, 75 mM NaCl, 0.36 mg/ml bovine serum albumine pH7.8 at 30 °C for 10 min in a total volume of 50 μl. The reaction was stopped by addition of EDTA to 20 mM final concentration and by freezing on dry ice. Nicked (APE-cleaved) and closed supercoiled forms of plasmids were resolved by 1.0 % agarose gel electrophoresis in the presence of ethidium bromide.

Western Blot

After treatment cultured cells were rinsed and harvested in buffer containing 20 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1.2% triton X-100, 1 mM Na3VO4, 100 μg/ml phenylmethylsulfonyl fluoride, 2 μg /ml leupeptin, 5 μg /ml aprotinin. Cell lysates were rotated for 1 hr at 4 °C, centrifuged at 10,000 X g for 10 min and 50 μg of protein per lane was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were transferred to a nitrocellulose membrane (Millipore, Bedford, MA) in a Hoefer Transphor Electrophoresis Unit. Following blocking with 10 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.01% Tween-20 and 5% nonfat dry milk, membranes were incubated for 2 hr with anti-Ref-1 antibody (1:500 dilution, Santa Cruz) and anti-rabbit HRP secondary antibody for 1 hr. Proteins were detected by enhanced chemiluminescence and exposure to hyperfilm (Amersham). Protein assays were performed using a Total Protein Assay Kit (Sigma).

RESULTS

Effects of Aβ on Expression and Activity of Ref-1 Protein in Neurons

Previous studies demonstrated dose-dependent neurotoxic effects of Aβ peptides in vitro [29, 30]. High concentrations of pre-aggregated Aβ1-42 (> 5.0 μM) directly cause acute neuronal cell death in cultures, whereas the lower concentrations of Aβ (0.1~1 μM) might sensitize neuronal cells to make them more vulnerable to further stress without causing immediate neuronal degeneration [30]. To examine whether low and high concentrations of Aβ1-42 have differential effects on Ref-1 expression, primary hippocampal neurons maintained in culture for 5-7 days were incubated in medium containing pre-aggregated Aβ1-42 at a concentration of either 1.0 μM or 5.0 μM. In vehicle-treated cells there was a moderate amount of Ref-1 immunoreactivity that was predominantly nuclear (Fig. 1A). Incubation with lower concentrations (0.5~1.0 μM) of Aβ1-42 caused an increase in Ref-1 expression (Figs. 1D & E) that was maintained for up to 48 hr as confirmed by Western blotting (Fig. 1G). However, persistent exposure to 1.0 μM Aβ1-42 for more than 72 hrs was associated with a decline in Ref-1 expression (Fig. 1G). In contrast, there was a marked decrease in Ref-1 expression, by 6 hr following exposure to 5 μM Aβ1-42 (Fig. C). These findings were corroborated by Western blot analysis of Ref-1 expression along with similar changes from RT-PCR analysis of Ref-1 mRNA (Figs. 1D & E). Additionally, an apurinic/apyrimidinic endonuclease (APE) activity assay was performed to measure the major DNA repair function of Ref-1. The results demonstrated changes that paralleled those of Ref-1 protein (Fig. 1F).

Fig. (1)
Differential expression and APE activity of Ref-1 following Aβ treatment in primary neurons. (A-C) Immunohistochemistry using Ref-1 specific antibody reveals nuclear-predominant staining in vehicle control (A), increased staining after incubation ...

Effects of Aβ Concentration on Neuronal Survival

Decreased Ref-1 abundance has been linked with increased neuronal cell death both in vivo and in vitro [14, 15]. As Ref-1 expression varied with the concentration of Aβ, the differential effects of low versus high dose Aβ on neuronal survival were evaluated in cultured neurons by phase contrast microscopy, TUNEL staining and lactose dehydrogenase (LDH) release assay. In vehicle-treated control cultures as well as cultures receiving a relatively low dose of Aβ (1.0 μM), cellular morphology appeared normal (Figs. 2A & B). In contrast, exposure to Aβ (5.0 μM) resulted in the rapid appearance of morphological and biochemical evidence of cell death, i.e., positive TUNEL staining and increased LDH release (Figs. 2C-H). Importantly, persistent exposure to Aβ (1.0 μM) for more than 72 hrs was associated with biochemical evidence of neuronal degeneration (Fig. 2H).

Fig. (2)
Reciprocal relationship between Ref-1 and neuronal cell death due to Aβ treatment. (A-C) Phase contrast microscopy shows normal or relatively normal morphology in vehicle-treated or low dose Aβ1-42-treated neurons at 24 hr (A & ...

Relationship between Ref-1 and Aβ in Alzheimer’s Disease

As Ref-1 is regulated by Aβ in a concentration-dependent manner in vitro, the accumulation of Ref-1 in AD [20] suggested that there may be a relationship between Ref-1 and Aβ in AD. Double-labeling of Ref-1 and either a neuronal (NeuN) or astrocytic (glial fibrillary acidic protein) marker demonstrated that Ref-1-immunoreactive cells were predominantly neurons. In addition, Ref-1 immunoreactivity colocalized with intracellular Aβ (Figs.3G-I), and both diffuse and dense-core senile plaques (Figs. 3J-O). Dual staining of Ref-1 and TUNEL demonstrated that a small number of Ref-1-immunoreactive cells in the AD hippocampus were TUNEL-positive (Figs. 3P-R). Conversely, the majority of Ref-1-positive cells in the AD hippocampus and all of the Ref-1-positive cells in control brains were TUNEL-negative (Figs. 3P-U).

Fig. (3)
Pattern of Ref-1 expression in the AD brain. Brain sections were dual stained for Ref-1 and other markers as indicated at the top of the figure. (A-F) Ref-1 immunoreactivity (A,D) and co-labeling with either (B) a neuronal marker, NeuN, or (E) an astrocytic ...

Differential Regulation of Ref-1 Expression and Modulation of Neuronal Survival by Oxidative Stress

Aβ-induced production of hydrogen peroxide (H2O2), may contribute to neurodegeneration in AD [29, 31]. We therefore sought to determine the effects of H2O2 on Ref-1 expression. N2A neuroblastoma cells, which express a relatively high basal level of Ref-1, were treated with H2O2 (0.1 mM ~ 10 mM) and both Ref-1 expression and cell death were evaluated by Western blotting and TUNEL, respectively. Several hours following treatment with 10 mM H2O2 a striking downregulation of Ref-1 expression and activity occurred (Fig. 4D) in association with increased cell death (Fig. 4C). In contrast, cells treated with 0.1 mM H2O2 exhibited a transient increase in Ref-1 expression (Fig. 4E) without any significant morphological changes (Figs. 4A, B). To further explore the connection between Aβ, H2O2 and Ref-1, Aβ-treated neurons were incubated with catalase (CAT), which catalyzes the breakdown of H2O2. Co-treatment with CAT significantly prevented Aβ-induced downregulation of Ref-1 protein and loss of APE activity (Fig. 4I). In contrast there were no significant changes in either Ref-1 expression or activity in control cells after CAT treatment (data not shown). In association with the preservation of Ref-1 expression and activity, CAT efficiently protected against Aβ-induced neuronal cell death as demonstrated by cell morphology (Figs. 4F-H).

Fig. (4)
Dose-dependent effects of hydrogen peroxide on Ref-1 expression and cell survival. (A) TUNEL staining reveals no DNA damage in untreated N2A cells. (B) A small number of cells treated with low dose H2O2 (0.1 mM, 24 hr) are TUNEL-positive. (C) In contrast, ...

DISCUSSION

The importance of Ref-1 during the response to oxidative stress has previously been demonstrated in non-neural cells [5, 7, 32]. Ref-1 expression is induced within several hours of exposure to ROS-generating agents [5, 7]. In contrast, Ref-1 depletion increases vulnerability to DNA-damaging agents as well as hypoxia and hyperoxia [7]. Among its major functions Ref-1 protein is an endonuclease that participates in the base excision repair of DNA strand breaks induced by ROS [6, 33]. We surmised that Ref-1 might play an important role in the CNS response to ROS-generating agents such as H2O2 and Aβ. Accordingly, we found that Ref-1 expression and activity are differentially regulated by exposure to varying concentrations of ROS generators. The results imply an indirect relationship between the level of Ref-1 expression/activity and neuronal damage. Specifically, high concentrations of ROS-generators may impair Ref-1-mediated DNA repair as evidenced by downregulation of Ref-1 expression and widespread DNA damage. In contrast, exposure to lower concentrations of ROS-generators induce moderate DNA damage and activation of the Ref-1 pathway. In addition to functioning in DNA damage repair, Ref-1 protein may activate p53, NFkB and other transcription factors that regulate cell death [4, 9]. Increased Ref-1 expression might simultaneously activate the transcriptional activity of p53, a tumor suppressor that plays important roles in apoptosis and neuronal degeneration [34, 35], resulting in delayed cell death. This is supported by our finding that massive cell death eventually occurred despite an induction of Ref-1 protein in cells exposed to low toxin concentrations. Taken together, our results suggest that upregulation of Ref-1 by mild to moderate degrees of oxidative damage may facilitate transient cell survival through its DNA repair function, while severe oxidative damage may lead to decreased Ref-1 production which promotes rapid cell death. This hypothesis is supported by varying levels of Ref-1 expression in association with either neuronal survival or degeneration following transient cerebral ischemia [15-18]. Additionally, a direct causal relationship between Ref-1 induction and neuronal survival was recently demonstrated in vitro [14].

Accumulating evidence has implicated ROS in the pathophysiology of AD and other neurodegenerative diseases [24, 36, 37]. Oxidative damage has been reported in transgenic mouse models as well as human AD [23, 38-40], while other reports have demonstrated increased ROS production in neurons exposed to either H2O2 or Aβ [41, 42]. Thus, Aβ-mediated generation of H2O2 and ROS may contribute to oxidative damage and neuronal loss in AD. In this regard, prior reports of DNA damage, abnormal DNA repair and neuronal apoptosis [23, 43, 44] suggest that Ref-1 may regulate the cellular response to ROS in AD. Our finding that CAT blocked Aβ-induced downregulation of Ref-1 protein strengthens this notion. We previously found that Ref-1 accumulates in vulnerable neurons and senile plaques in the AD brain [20]. The present study corroborates and extends these observations by demonstrating both neuronal and astrocytic accumulation of Ref-1, and colocalization of Ref-1 with Aβ in senile plaques and a subpopulation of cells in the AD hippocampus. While these findings support a connection between Ref-1 and oxidative stress in AD, the nature of the relationship between Ref-1 and Aβ remains to be determined. Specifically, regulation of Ref-1 by exogenous administration of Aβ, as we have shown here, may be quite distinct from endogenous Aβ in AD.

In addition to its DNA repair function, Ref-1 modulates the redox state and DNA binding of key transcription factors including Fos, Jun, NF-kappaB and p53, which have been implicated in AD pathogenesis [43, 45, 46]. Ref-1 may therefore have dual roles in the CNS response to oxidative stress. Specifically, Ref-1 DNA repair function may play an important role in neuroprotection, whereas redox regulation of gene expression may contribute to neuronal cell death. Thus, alterations in the balance between Ref-1 DNA repair and redox functions may be a critical determinant of neuronal survival following oxidative stress. Future studies will need to clarify the relative importance of each of the functions of Ref-1 protein in either the recovery or death of damaged CNS neurons.

In conclusion, we have shown that Ref-1 is differentially regulated by varying levels of oxidative stress in neurons. Importantly, reduction of Ref-1 expression and activity by high concentrations of ROS-generators were associated with neuronal death, while lower concentrations of ROS-generators transiently induced Ref-1 expression in association with prolonged neuronal survival. Our results therefore support a neuroprotective role for Ref-1 following oxidative stress. Additional work is needed to elucidate the relative importance of the dual functions of Ref-1 in the neuronal injury response, which could lead to new strategies to optimize neurorecovery.

Acknowledgments

This study was supported by NIH AG-26637 to Z.T. and an Alzheimer’s Association IIRG grant to S.S.S. The brain tissue used in this project was provided by the Institute for Brain Aging and Dementia Tissue Repository at UCI and Alzheimer’s Disease Research Consortium at the University of Southern California.

ABBREVIATIONS

Ref-1
Redox factor-1
β-amyloid
AD
Alzheimer’s disease
ROS
reactive oxygen species
CNS
central nervous system
APE
apurinic/apyrimidinic endonuclease
DAB
diaminobenzidine
FITC
fluorescein isothiocyanate
DAPI
4’,6-Diamidino-2-phenylindole
HBSS
Hank’s balanced salt solution
PBS
phosphate buffer
TUNEL
TdT-mediated dUTP nick end labeling
RT-PCR
reverse transcription-polymerase chain reaction
AEC
aminoethylcarbazole
GFAP
glial fibrillary acidic protein
EDTA
ethylenediaminetetra-acetate
NeuN
neuronal nuclear antigen
LDH
lactose dehydrogenase

Footnotes

AUTHORS’ CONTRIBUTION ZT conceived of the study, participated in its design, carried out immunohistochemistry experiments, and drafted the manuscript. LS carried out the cell culture, Western blotting and AP assays. SSS participated in the study design and helped to draft the manuscript. All authors read and approved the final manuscript.

Publisher's Disclaimer: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

References

1. Liu H, Colavitti R, Rovira II, Finkel T. Redox-dependent transcriptional regulation. Circ Res. 2005;97:967–74. [PubMed]
2. Kwon YW, Masutani H, Nakamura H, Ishii Y, Yodoi J. Redox regulation of cell growth and cell death. Biol Chem. 2003;384:991–6. [PubMed]
3. Kelley MR, Parsons SH. Redox regulation of the DNA repair function of the human AP endonuclease Ape1/ref-1. Antioxid Redox Signal. 2001;3:671–83. [PubMed]
4. Evans AR, Limp-Foster M, Kelley MR. Going APE over ref-1. Mutat Res. 2000;461:83–108. [PubMed]
5. Ramana CV, Boldogh I, Izumi T, Mitra S. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals. Proc Natl Acad Sci USA. 1998;95:5061–66. [PMC free article] [PubMed]
6. Rothwell DG, Barzilay G, Gorman M, Morera S, Freemont P, Hickson ID. The structure and functions of the HAP1/Ref-1 protein. Oncol Res. 1997;9:275–80. [PubMed]
7. Walker LJ, Craig RB, Harris AL, Hickson ID. A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress. Nucleic Acids Res. 1994;22:4884–89. [PMC free article] [PubMed]
8. Forrester K, Ambs S, Lupold SE, et al. Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53. Proc Natl Acad Sci USA. 1996;93:2442–7. [PMC free article] [PubMed]
9. Jayaraman L, Murthy KG, Zhu C, Curran T, Xanthoudakis S, Prives C. Identification of redox/repair protein Ref-1 as a potent activator of p53. Genes Dev. 1997;11:558–70. [PubMed]
10. Ono Y, Watanabe M, Inoue Y, et al. Developmental expression of APEX nuclease, a multifunctional DNA repair enzyme, in mouse brains. Brain Res Dev Brain Res. 1995;86:1–6. [PubMed]
11. Xanthoudakis S, Smeyne RJ, Wallace JD, Curran T. The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. Proc Natl Acad Sci USA. 1996;93:8919–23. [PMC free article] [PubMed]
12. Dragunow M. Ref-1 expression in adult mammalian neurons and astrocytes. Neurosci Lett. 1995;191:189–92. [PubMed]
13. Walton M, Lawlor P, Sirimanne E, Williams C, Gluckman P, Dragunow M. Loss of Ref-1 protein expression precedes DNA fragmentation in apoptotic neurons. Brain Res Mol Brain Res. 1997;44:167–70. [PubMed]
14. Vasko MR, Guo C, Kelley MR. The multifunctional DNA repair/redox enzyme Ape1/Ref-1 promotes survival of neurons after oxidative stress. DNA Repair (Amst) 2005;4:367–79. [PubMed]
15. Kawase M, Fujimura M, Morita-Fujimura Y, Chan PH. Reduction of apurinic/apyrimidinic endonuclease expression after transient global cerebral ischemia in rats: implication of the failure of DNA repair in neuronal apoptosis. Stroke. 1999;30:441–8. discussion 449. [PubMed]
16. Fujimura M, Morita-Fujimura Y, Kawase M, Chan PH. Early decrease of apurinic/apyrimidinic endonuclease expression after transient focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 1999;19:495–501. [PubMed]
17. Gillardon F, Bottiger B, Hossmann KA. Expression of nuclear redox factor ref-1 in the rat hippocampus following global ischemia induced by cardiac arrest. Brain Res Mol Brain Res. 1997;52:194–200. [PubMed]
18. Edwards M, Kent TA, Rea HC, et al. APE/Ref-1 responses to ischemia in rat brain. Neuroreport. 1998;9:4015–18. [PubMed]
19. Tan Z, Sankar R, Tu W, et al. Immunohistochemical study of p53-associated proteins in rat brain following lithium-piloCarpine status epilepticus. Brain Res. 2002;929:129–38. [PubMed]
20. Tan Z, Sun N, Schreiber SS. Immunohistochemical localization of redox factor-1 (Ref-1) in Alzheimer’s hippocampus. Neuroreport. 1998;9:2749–52. [PubMed]
21. Nunomura A, Castellani RJ, Zhu X, Moreira PI, Perry G, Smith MA. Involvement of oxidative stress in Alzheimer disease. J Neuropathol Exp Neurol. 2006;65:631–41. [PubMed]
22. Markesbery WR, Lovell MA. DNA oxidation in Alzheimer’s disease. Antioxid Redox Signal. 2006;8:2039–45. [PubMed]
23. Beal MF. Oxidative damage as an early marker of Alzheimer’s disease and mild cognitive impairment. Neurobiol Aging. 2005;26:585–6. [PubMed]
24. Mattson MP. Pathways towards and away from Alzheimer’s disease. Nature. 2004;430:631–9. [PMC free article] [PubMed]
25. Tan Z, Sun X, Hou FS, et al. Mutant ubiquitin found in Alzheimer’s disease causes neuritic beading of mitochondria in association with neuronal degeneration. Cell Death Differ. 2007;14:1721–32. [PMC free article] [PubMed]
26. Kaech S, Banker G. Culturing hippocampal neurons. Nat Protoc. 2006;1:2406–15. [PubMed]
27. Schnell SA, Staines WA, Wessendorf MW. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem. 1999;47:719–30. [PubMed]
28. Redaelli A, Magrassi R, Bonassi S, Abbondandolo A, Frosina G. AP endonuclease activity in humans: development of a simple assay and analysis of ten normal individuals. Teratog Carcinog Mutagen. 1998;18:17–26. [PubMed]
29. Behl C, Davis JB, Lesley R, Schubert D. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell. 1994;77:817–27. [PubMed]
30. Qin L, Liu Y, Cooper C, Liu B, Wilson B, Hong JS. Microglia enhance beta-amyloid peptide-induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species. J Neurochem. 2002;83:973–83. [PubMed]
31. Behl C. Amyloid beta-protein toxicity and oxidative stress in Alzheimer’s disease. Cell Tissue Res. 1997;290:471–80. [PubMed]
32. Seki S, Ikeda S, Watanabe S, et al. A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization. Biochim Biophys Acta. 1991;1079:57–64. [PubMed]
33. Fritz G, Grosch S, Tomicic M, Kaina B. APE/Ref-1 and the mammalian response to genotoxic stress. Toxicology. 2003;193:67–78. [PubMed]
34. Vousden KH. p53: death star. Cell. 2000;103:691–4. [PubMed]
35. Morrison RS, Kinoshita Y. The role of p53 in neuronal cell death. Cell Death Differ. 2000;7:868–79. [PubMed]
36. Onyango IG, Khan SM. Oxidative stress, mitochondrial dysfunction, and stress signaling in Alzheimer’s disease. Curr Alzheimer Res. 2006;3:339–49. [PubMed]
37. Lee HG, Castellani RJ, Zhu X, Perry G, Smith MA. Amyloid-beta in Alzheimer’s disease: the horse or the cart? Pathogenic or protective? Int J Exp Pathol. 2005;86:133–8. [PMC free article] [PubMed]
38. Smith MA, Richey HPL, Sayre LM, Beckman JS, Perry G. Widespread peroxynitrite-mediated damage in Alzheimer’s disease. J Neurosci. 1997;17:2653–7. [PubMed]
39. Smith MA, Hirai K, Hsiao K, et al. Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem. 1998;70:2212–5. [PubMed]
40. Pappolla MA, Chyan YJ, Omar RA, et al. Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenic mouse model of Alzheimer’s disease: a chronic oxidative paradigm for testing antioxidant therapies in vivo. Am J Pathol. 1998;152:871–7. [PMC free article] [PubMed]
41. Schubert D, Behl C, Lesley R, et al. Amyloid peptides are toxic via a common oxidative mechanism. Proc Natl Acad Sci USA. 1995;92:1989–93. [PMC free article] [PubMed]
42. Valencia A, Moran J. Reactive oxygen species induce different cell death mechanisms in cultured neurons. Free Radic Biol Med. 2004;36:1112–25. [PubMed]
43. Anderson AJ, Su JH, Cotman CW. DNA damage and apoptosis in Alzheimer’s disease: colocalization with c-Jun immunoreactivity, relationship to brain area, and effect of postmortem delay. J Neurosci. 1996;16:1710–9. [PubMed]
44. Fishel ML, Vasko MR, Kelley MR. DNA repair in neurons: so if they don’t divide what’s to repair? Mutat Res. 2006 [PubMed]
45. de la Monte SM, Sohn YK, Wands JR. Correlates of p53- and Fas (CD95)-mediated apoptosis in Alzheimer’s disease. J Neurol Sci. 1997;152:73–83. [PubMed]
46. Kamasani U, Prendergast GC. Genetic response to DNA damage: proapoptotic targets of RhoB include modules for p53 response and susceptibility to Alzheimer’s disease. Cancer Biol Ther. 2005;4:282–8. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...