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Department of Neuroscience, The Ohio State University, Columbus, Ohio, USA
1Correspondence: Department of Neuroscience, The Ohio State University, 4198 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210, USA. E-mail: lin.492{at}osu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxidative stress • oxidative damage • Alzheimer's disease • neurodegeneration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
, Parkinson's disease (6
7
8)
, and amyotrophic lateral sclerosis (ALS) (9
10
11)
. Oxidative stress is defined as an imbalance of the oxidant-antioxidant ratio in favor of the former, leading to cell damage (12)
. The central nervous system is highly vulnerable to oxidative imbalance because of its high rate of oxygen consumption, enrichment of polyunsaturated fatty acids, relative paucity of antioxidant system, and high content of transition redox metals. Depending on the substrates attacked by reactive oxygen species (ROS), oxidative damage has been manifested as lipid peroxidation, protein oxidation, DNA oxidation, and RNA oxidation. In the past, oxidative damage to intracellular molecules was viewed as the result of neurodegeneration. In recent years, numerous investigations have pointed to the functional importance of oxidative imbalance as a crucial event in mediating disease pathogenesis (13
, 14)
. Studies of oxidation of proteins and RNAs in AD brains demonstrated that the increased oxidative modification occurs in some morphologically intact neurons and is not consistently associated with mature senile plaques, suggesting that oxidation of intracellular macromolecules precedes neurodegenerative pathology (15
, 16)
. Furthermore, Pratico and Sung investigated a marker of lipid peroxidation in vivo using urine, plasma, and cerebrospinal fluid (CSF) samples from living patients, in combination with the clinical diagnosis of AD, and found that oxidative damage to lipids is an early event during the evolution of the disease (5)
. In addition, they studied subjects with mild cognitive impairment (MCI), which is predictive with up to 50% accuracy of progression to symptomatic AD within a 4-year period (17)
. Similar to AD subjects, patients with MCI also have an increased level of lipid oxidation compared with cognitively normal elderly controls; however, no significant difference in the level of CSF tau and amyloid ß (Aß)42, markers of AD neuropathology and disease progression (18)
, was observed between MCI subjects and controls. These studies indicate that oxidative damages are present before detection of the typical neuropathological changes of early AD. The ROS-induced damage to intracellular molecules is not a simple consequence of cell death, but may play an important role in AD pathogenesis.
ROS can hydroxylate guanine to produce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8OHdG) in DNA and 8-oxo-7,8-dihydroguanosine (8OHG) in RNA (19)
. Nunomura et al. demonstrated that most of the oxidized nucleoside is associated with cytoplasmic RNA in AD brains (16)
. Increased cytoplasmic RNA oxidation was also found in substantia nigra neurons of Parkinson's disease (7)
. A recent study (20)
has shown that patients carrying a presenilin-1 (PS-1) mutation, which results in a type of familial AD, show a considerable level of neuronal RNA oxidation. Neuronal RNA oxidation is also a prominent feature of familial AD attributable to the amyloid ß protein precursor (AßPP) gene, especially for those cases with a lower percentage area of Aß42 burden. These findings suggest that RNA oxidation is an early event involved in the pathological cascade of AD. To investigate the role of RNA oxidation in AD pathogenesis, we developed a novel immunoprecipitation procedure to isolate oxidatively damaged RNA (21)
, and found that up to 30–70% of messenger RNAs (mRNA) are oxidized in AD frontal cortices (22)
. Identification of oxidized mRNA species revealed that some mRNAs are more susceptible to oxidative damage; thus, RNA oxidation is not random but highly selective. Investigation of the consequence of oxidatively damaged mRNAs revealed that oxidized mRNA cannot be translated properly, leading to reduced protein expression, hence, loss of normal protein function. Furthermore, two recent studies demonstrated that ribosomal RNA (rRNA) in AD is oxidized by bound redox-active iron (23)
and that impairments in protein synthesis occur in the earlier stages of AD, which apparently mediates by alterations in ribosomal nucleic acids as well as the polyribosomal complex itself (24)
. In the present study, we investigated RNA oxidation in the process of neuronal death induced by insults mediated by the production of ROS in cortical primary dissociated cultures. The results indicate that mRNA oxidation may be an important factor initiating the cascade of neurodegeneration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell cultures
Primary rat cortical neuronal cultures were prepared from E16 embryonic forebrain (25)
. The cortices were dissected out of the brains, incubated in activated papain for 30 min at 37°C, triturated by repeated pipetting with a small bore pipette, and plated onto poly-D-lysine-coated (0.1 mg/ml) plastic culture dishes or glass slides. These cultures were maintained in DMEM, 0.5% FBS, and 1 x B-27 supplement. HEK293 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in minimum essential medium with Eagle's salts (MEM; Invitrogen) supplemented with 10% FBS (Invitrogen), 100 µM MEM nonessential amino acids (Invitrogen), and penicillin-streptomycin-glutamine (100 U/ml-100 µg/ml-2 mM; Invitrogen).
Immunofluorescence
The cultures were plated onto poly-D-lysine-coated glass coverslips and fixed with 2% paraformaldihyde supplemented with 1.7% sucrose in phosphate buffer for 30 min at room temperature. After being permeabilized with 0.1% saponin in 1 x PBS, the cultures were blocked with 1% bovine serum albumin (BSA) in 1 x PBS with 0.1% saponin for 1 h at room temperature, then incubated with primary antibodies overnight at room temperature. After rinsing, the coverslips were incubated with secondary antibody solution (Alexa Fluor 488 goat-anti-mouse or rabbit IgG (1:1000), and Alexa Fluor 594 goat anti-rabbit or mouse IgG (1:1000) in PBS with BSA) for 60 min at room temperature. Nuclear staining was accomplished by adding Hoechst 33342 fluorescence dye into secondary antibody solution (0.25 µg/ml). The coverslips were then rinsed and mounted onto glass slides with ImmuMount (Shandon Lipshaw, Pittsburgh, PA, USA).
Western blot
Protein extracts were generated from primary cultures, resolved by SDS-PAGE, and transferred onto PVDF membranes. After being blocked by 5% nonfat milk in PBS with 0.1% Tween-20, the membrane was incubated with the following primary antibodies in 1% milk overnight at 4°C (rabbit anti-hSOD1 pAb or goat anti-ß-actin mAb), followed by peroxidase-conjugated goat anti-rabbit IgG (1:1200; ICN Biomedicals, Aurora, OH, USA) for 60 min at room temperature. The immunoreactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate according to the manufacturer's directions.
Isolation of mRNAs
Total RNAs were isolated from cultures using TRIZOL (Invitrogen) according to the manufacturer's directions. mRNAs were extracted from total RNAs using the Oligo-tex mRNA Purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer's directions. The concentration of mRNA was measured with the OD value at 260 nm UV light by Beckman DU640 spectrophotometry (Beckman Instruments Inc., Palo Alto, CA, USA).
Immunoprecipitation of oxidized mRNAs
mRNA (1.5 µg) was incubated with 2.5 µg of anti-8OHG antibody 15A3 at room temperature for 1 h, then 20 µl of immobilized protein L gel beads (Pierce) was added and incubated at 4°C for another 15 h. The beads were washed three times by PBS with 0.04% (v/v) Nonidet P-40 (Roche Applied Science, Indianapolis, IN, USA). After precipitation of beads by centrifuge, the oxidized mRNA:antibody:protein L complexes were separated from nonoxidized mRNAs, which remained in the supernatant. Nonoxidized mRNAs were precipitated with ethanol and dissolved in 10 µl DEPC-treated H2O. The following items were added into the precipitant of oxidized mRNAs in the order shown: 300 µl of PBS with 0.04% Nonidet P-40, 30 µl of 10% (w/v) sodium dodecyl sulfate (SDS), and 300 µl of PCI (phenol: chloroform: isoamyl alcohol; 25:24:1). The mixture was incubated at 37°C for 15 min (vortexing every 5 min) and separated to aqueous phase and organic phase by spinning at 14,000 rpm for 5 min. The aqueous layer containing oxidized mRNAs was collected and mixed with 40 µl of 3 M sodium acetate, pH 5.2, 2 µl of 5 mg/ml glycogen plus 1 ml of 95% (v/v) ethanol. The sample was frozen at –80°C for 1 h and centrifuged for 20 min. The pellet was washed by 75% ethanol and air-dried. It was resuspended in 10 µl DEPC-treated H2O.
RT-PCR
mRNA from cultures or immunoprecipitated mRNAs was reversely transcribed with AMV reverse transcriptase (Invitrogen) and gene-specific primers, including Cu/Zn SOD1 F (5'-atggcgacgaaggccgtgtgcgt) and R (5'-ctggcaaaatacaggtcattgaaacagaca) or R (SP6 sequence; 5'-catttaggtgacactatag), Glutamate dehydrogenase 1 F (5'-agttccaagacaggatatcgggt) and R (5'-actcagcttgtacatgatccgcta), ß-actin F (5'-cgggacctgacagactacctcat-3') and R (5'-accgactgctgtcaccttcacc-3'), MAP2 F (5'-ttggctcacttgacaatgctcacc-3') and R (5'-aatatgacacctgctcagagccca-3'). PCRs were performed in the presence of 3 mM MgCl2, 0.2 mM dNTP, 0.25 µM primers, and 2 U TaqDNA polymerase (Invitrogen) in 1x PCR buffer. A series of cycles (20, 25, 30 cycles) was performed (95°C for 30 s, specific annealing temperature to each set of primers for 45 s, and 72°C for 1 min). PCR products were visualized as single bands on 1% agarose gels stained with ethidium bromide.
LDH release assay
Cytotoxicity was assessed by measuring the amount of lactate dehydrogenase (LDH) released from the cells after treatment. Aliquots of culture media were removed from wells at various time points after treatment. Culture media (50 µl/reaction) was mixed with an equal volume of substrate mixture [25 mM sodium lactate, 3.76 mM ß-NAD+ (nicotinamide adenine dinucleotide), 603.4 µM MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), 100 µM MPMS (1-methoxyphenazine methosulfate), and 0.1% Triton X-100 in 200 mM Tris–HCl, pH 8.0] and incubated for 30 min at 37°C. The reaction was stopped by adding 100 µl stop solution (20% SDS in 50% dimethylformamide). Absorbance readings were obtained with a GENios microplate reader (Tecan Co., San Jose, CA, USA; 
ex=570 nm).
DCF assay
Media were removed from the dissociated cultures and replaced with phenol red-free DMEM containing 100 µM H2DCFDA. Cultures were placed back into the incubator for 30 min, followed by washing with PBS. Cultures were treated with 50 µM hydrogen peroxide at 37°C for 1 h, then examined by fluorescence microscope.
In vitro transcription and oxidation
PCR templates were amplified from pcDNA3-SOD1 and pcDNA3-luciferase plasmid DNAs using T7+ and SP6-(dT)30 primers (T7+: 5'-ggatcctaatacgactcactatagg; SP6-(dT)30: 5'-(dt)30catttaggtgacactatag). After purification, the amplified cDNAs were transcribed to RNA containing poly(A) tail and CAP using m7G(5')ppp(5')G RNA capping analog (Invitrogen) by T7 RNA polymerase (U.S. Biological, Swampscott, MA, USA). For oxidation, capped poly(A) RNAs were incubated with H2O2 and cytochrome c (Sigma, St. Louis, MO, USA) at different dosages at 37°C for 1 h. RNA was purified by RNeasy mini kit (Qiagen).
In vitro translation
In vitro translation using the Rabbit Reticulocyte Lysate system (Promega, Madison, WI, USA) was performed according to the manufacturer's direction on in vitro generated luciferase RNAs, both nonoxidized and oxidized, for 1.5 h at 30°C. Biotinylated luciferase was created by incorporating biotinylated lysine residues into nascent proteins during translation using the TranscendTM Non-Radioactive Translation Detection Systems (Promega). Horseradish peroxidase Avidin D (Vector Laboratories, Burlingame, CA, USA) was used to visualize the biotinylated proteins, followed by chemiluminescent detection.
RNA transfection
Cells were seeded to 80–90% confluence in 6-well tissue culture plates. Two micrograms of in vitro synthesized and/or oxidized mRNAs were diluted in serum-free OPTI-MEM (Invitrogen) containing 6 µl DMRIE-c reagent (Invitrogen). After incubation with an RNA mixture for 6 h at 37°C, cells were cultured in normal complete cell media for another 16 h, then harvested for protein detection by SDS-PAGE.
Immunoprecipitation
Rabbit reticulocyte lysates were diluted in 1 x PBS containing complete protease inhibitor cocktail (Roche Applied Science) with 1% Triton X-100 and incubated with immobilized NeutrAvidin binding protein (Pierce) at 4°C for 16 h. The immunobead-bound protein complexes were washed three times with PBS buffer plus 0.1% Triton X-100 and resolved in SDS-PAGE.
Polyribosome analysis
Sucrose gradients (15 to 60%) were prepared by layering 1.5 ml of 15% sucrose on top of an equal volume of 60% sucrose in Beckman polyallomer centrifuge tubes (Beckman). Gradients were formed by placing tubes in a horizontal position for 5 h (26)
. Fifty microliters of translational products in rabbit reticulocyte lysates diluted in 300 µl of buffer (100 mM KCl, 2 mM DTT, 2 mM magnesium acetate, 20 mM Tris-HCl, pH 7.5) was layered onto a sucrose gradient and spun in a Beckman SW40 rotor at 30,000 rpm for 3 h at 4°C. Ten fractions were collected per tube, starting from the top, and mixed thoroughly with an equal volume of phenol-chloroform-isoamyl alcohol. The resulting mixtures were centrifuged at 14,000 rpm for 5 min and the aqueous phases after centrifugation were precipitated with 5 µg of glycogen, 2 volumes of 95% ethanol, and 0.1 volume of 3M acetic acid (pH 5.2). Precipitated RNAs were resuspended in 5 µl of DEPC-treated ddH2O for Northern blot analysis.
Northern blot
DIG-labeled 18S cRNA probes were synthesized by in vitro transcription using 1 µg of DNA template (linearized pcDNA3–18S rRNA sequence), DIG-11-UTP (350 µM; Roche Applied Science), and SP6 RNA polymerase (100 U; U.S. Biochemicals, Cleveland, OH, USA). In vitro transcription proceeded for 2.5 h at 37°C, after which the template was digested with deoxyribonuclease I (5 U; Invitrogen) for 15 min at 37°C. DIG-labeled DNA probes were generated from luciferase PCR products using Digoxogenin High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science). mRNAs were fractionated by electrophoresis through a 1% agarose/2.2 M formaldehyde gel, then transferred to positively charged nylon membranes by overnight capillary transfer with 10 x SSC. The RNA was then fixed to the membrane by UV cross-linking as above, followed by baking for 30 min at 80°C. The membranes were incubated with modified Church buffer (10% SDS, 1 mM EDTA, 2% blocking reagent (Roche Applied Science) in 250 mM Na2HPO4, pH 7.2 (27)
, for 3 h at 68°C, then with DIG-labeled cRNA probe or DNA probe in modified Church buffer overnight at 68°C or 55°C, respectively. The membranes were subsequently washed three times for 20 min each at 68°C with washing buffer (1% SDS and 1 mM EDTA in 25 mM Na2HPO4, pH 7.2). Detection of DIG-labeled probe hybridization was accomplished using Digoxogenin High Prime DNA Labeling and Detection Starter Kit II according to the manufacturer's direction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80–90% neurons as determined by MAP-2 (a neuron marker) and GFAP (an astrocyte marker) immunostaining (data not shown). Different insults, including H2O2, glutamate, and Aß 25–35, were used to generate oxidative stress. These insults have been implicated in the pathogenesis of AD and mediate the production of ROS (28
29
30)
. The 7-day-old cultures were treated with 50 µM H2O2 or 50 µM glutamate (in DMEM) for 30 min or with 20 µM Aß25–35 for 48 h. The toxic effects of each insult were visualized as morphological changes by MAP-2 immunostaining. In all three conditions, neurons underwent degeneration as shown by beadlike structures on neurites (31)
at 24 h postexposure to H2O2, 10 h postexposure to glutamate, or 24 h exposure to Aß25–35 (Fig. 1
).
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To investigate whether and when RNA oxidation occurs under these insults, we performed immunostaining using the 15A3 antibody, which recognizes both 8-OHG and 8-OHdG, markers of oxidative damage to RNA and DNA, respectively. In H2O2-treated cultures, the intensity of 15A3 immunofluorescence was significantly increased 1 h post-treatment, further enhanced at 4 h, and diminished at 10 h; a very faint signal was detected in untreated (DMEM) cultures (Fig. 2
A, B). Immunoreactivity was prominent in the cytoplasm and was diminished greatly by RNase treatment, but only slightly by DNase I treatment, indicating cytoplasmic RNA is the major site of nucleic acid oxidative damage (Fig. 2A
). Moreover, the immunoreactivity was diminished completely when the antibody was preincubated with 8-OHG (Fig. 2A
, Ab block), indicating that the observed immunoreactivity was specific to oxidized base 8-OHG in RNA. Only some of the treated cells showed 15A3 immunoreactivities, indicating that some cell types were more susceptible to RNA oxidation. We measured LDH activity in the culture media taken at various time points to monitor H2O2-mediated cytotoxicity. LDH release increased with time in both treated and untreated cultures (Fig. 2D
). At 4 h post-treatment, the amount of LDH release was low, and the treated cultures had about double the amount of LDH release as did the untreated cultures. By 6 h post-treatment, LDH release had increased significantly in the treated cultures, and the increase grew exponentially with time. H2O2-induced cell death occurs via an apoptotic cell suicide pathway (32)
. We performed nuclear staining using fluorescent nuclear dye Hoescht 33342 to identify apoptotic cells. Nuclear and chromatin morphology of H2O2-treated cells was comparable to untreated cells within 15 h post-treatment (Fig. 2C
). Abnormal nuclear and chromatin morphology was observed in the treated cells at 24 h post-treatment, as neurons began to degenerate (Fig. 1)
.
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Similar phenomena were also observed in glutamate- or Aß 25–35-treated cultures. A significant increase of 15A3 immunofluoresence was detected as early as 2 h after glutamate treatment (Fig. 3
A) or after 12 h exposure to 20 µM or 50 nM of Aß 25–35 (Fig. 3B
). LDH release was significantly increased after RNA oxidation occurred (Fig. 3A, B
). These results indicate that oxidation of RNA is not a consequence of dying cells; it is an early event that far precedes cell death and may even contribute to it.
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RNA oxidation occurs primarily in a distinct group of neurons that die later
The above immunostaining results (Figs. 2
, 3)
indicated that only some treated cells were susceptible to RNA oxidation. To identify the cell types, we performed double-labeling of 15A3 with a neuron-specific marker or with an astrocyte-specific marker on the H2O2-treated cultures. An antibody against neuronal excitatory amino acid transporter 3 (EAAT3) was used to label neurons and an anti-GFAP antibody was used to identify astrocytes. The results showed that RNA oxidation was primarily located in a distinct group of neurons (Fig. 4
A), and these neurons appeared to die later. We further examined reactive oxygen species levels in each cell type by dichlorofluorescence (DCF) assay. After being oxidized by reactive oxygen species, the nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH) become DCF and emit fluorescence. As shown in Fig. 4B
, neurons exhibited a strong DCF fluorescence (solid arrow) whereas astrocytes showed a faint signal (open arrow).
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Poly(A)+ mRNAs are significantly oxidized in H2O2-treated cultures
We previously developed an immunoprecipitation procedure to isolate oxidatively damaged RNA (21)
and found that up to 50% of poly(A)+ mRNAs are oxidized in AD brains (22)
. We used this established procedure to investigate whether mRNAs are oxidized in H2O2-treated cultures. The cultures were treated with 50 µM H2O2 for 30 min, then harvested 1, 2, or 4 h after treatment. Poly(A)+ mRNAs were isolated from each sample and oxidized mRNAs were separated from nonoxidized mRNAs by immunoprecipitation with 15A3 antibodies. Both oxidized and nonoxidized mRNA pools were then reversely transcribed to cDNAs. DIG-labeled dUTPs were incorporated into cDNAs so as to facilitate analysis by Southern blot. A significant time-dependent increase of the level of oxidized mRNAs was observed in treated samples compared with untreated controls (Fig. 5
A). About 60–70% of mRNAs were oxidized at 4 h post-treatment (Fig. 5B
).
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In our previous study (21)
, we cloned and identified oxidized mRNA species isolated from AD frontal cortex and found that some mRNA species were more susceptible to oxidative damage; thus, RNA oxidation is not random, but highly selective. We investigated whether those mRNA species found to be oxidized in AD, including Cu2+/Zn2+ superoxide dismutase (SOD1), glutamate dehydrogenase 1 (GDH), cytochrome c oxidase-Va, and c-Ha-ras, are oxidatively damaged and whether selective RNA oxidation occurs in the cultured neurons under oxidative stress. RT-PCR analysis was performed and revealed that these mRNA species were found in the oxidized mRNA pool prepared from H2O2-treated cultures (Fig. 5C
shows SOD1 and GDH). ß-Actin and MAP-2 mRNAs are highly abundant mRNA species, and only small amounts of ß-actin and MAP-2 mRNAs are oxidized in AD brains. These two mRNA species were not detected in an oxidized mRNA pool (Fig. 5C
). These results indicate that primary neuronal culture may have a similar mechanism to selectively oxidize mRNAs to the AD brain.
Oxidative modification of mRNA leads to reduced protein expression
We previously expressed the oxidized luciferase RNA as well as green fluorescence protein RNAs in HEK293 cells and found that the oxidized RNA cannot be translated properly, leading to reduced protein expression and, consequently, loss of normal protein function (21)
. We investigated whether proteins corresponding to the oxidized mRNAs in H2O2-treated cultures show abnormal expression. SOD1 mRNA and ß-actin mRNA were chosen for this study because SOD1 mRNA is highly oxidized in H2O2-treated cultures as well as in AD brains, but ß-actin mRNA is not. The cultures were treated with H2O2 for 30 min, then harvested 3, 6, and 12 h post-treatment. Immunoblot analysis revealed that the SOD1 protein level had dramatically decreased at 12 h post-treatment whereas the ß-actin protein remained constant (Fig. 6
A). The decrease of the SOD1 protein level was not due to increased degradation of SOD1 protein by oxidative stress because it still occurred in the presence of 30 µM of proteasome inhibitor MG132 (Fig. 6A
). Moreover, it was not due to reduced mRNA expression as determined by quantitative RT-PCR analysis (Fig. 6B
). It could be due to oxidative damage to SOD1 mRNA.
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To further test this possibility, we synthesized SOD1 poly(A)+ RNAs in vitro, then oxidized these SOD1 RNAs with the 3.2 µM H2O2 and 0.2 µM cytochrome c. More than 80% of RNAs were oxidized, as determined by immunoprecipitation (not shown). SOD1 RNAs treated with 0.2 µM cytochrome c alone were used as a nonoxidized RNA control. These oxidized and nonoxidized SOD1 RNAs were then expressed in HEK293 cells by transfection. The transfected cells were harvested at 16 h post-transfection to determine protein expression level by immunoblot analysis. The result showed a significant decrease of SOD1 protein expression in the cells transfected with oxidized SOD1 RNAs compared with cells transfected with nonoxidized SOD1 RNAs (Fig. 6C
). To examine the possibility that the decline of protein level is the result of rapid degradation of the oxidized RNAs after entering the cells, we examined levels of oxidized SOD1 RNAs at different time points after transfection by quantitative RT-PCR. The oxidized RNA levels remained similar to the nonoxidized RNA levels for as long as 72 h (Fig. 6D
). The transfected SOD1 RNAs were distinguished from endogenous SOD1 mRNAs by incorporating the SP6 sequence at 3' untranslated region. The result was also confirmed by Northern blot analysis (not shown). These results indicate that oxidative modification of mRNA leads to reduced protein expression.
Oxidative modification of mRNA causes an abnormal increase of polyribosome association during translation
How does oxidative modification of mRNA affect the downstream translational process? We tested two possible mechanisms. First, we investigated whether oxidative modification of mRNA would cause premature termination of translation, and thus generation of truncated protein. We tested this possibility by examining the products translated from oxidatively damaged luciferase RNA in a rabbit reticulocyte lysate. In vitro synthesized luciferase RNAs were oxidized with 3.2 µM H2O2 and 0.2 µM cytochrome c, then subjected to translation in rabbit reticulocyte lysates. The translated products were examined by immunoblot analysis using polyclonal antibodies against the purified luciferase protein. The results showed there was no extra band of a smaller size than the full-length luciferase protein band (Fig. 7
A, upper panel, lysate). Two batches of polyclonal luciferase antibodies from different vendors and different acrylamide concentrations of gel were tested with consistent results. To further confirm this result, we added the biotin-labeled lysines to the translation reaction to label the translated products, which were subsequently detected by HRP-conjugated Avidin D. There are 36 lysine residues relatively evenly distributed in the 550 amino acid luciferase sequence. As shown in Fig. 7A
(lower panel, lysate), no truncated product was identified. Furthermore, we considered that truncated proteins, if produced, may be present in an amount too low to be detected in the total lysate. We used Avidin D conjugated with agar to pull down and concentrate biotin-labeled translated products, then examined them by immunoblot analysis using antiluciferase antibodies (Fig. 7A
, upper panel, IP) or HRP-conjugated Avidin D (Fig. 7A
, lower panel, IP). No truncated luciferase protein was detected. These results indicate that oxidized RNA either does not produce truncated protein or produces such a small amount of truncated protein with different sizes that it cannot be detected by the methods applied.
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Second, we determined whether oxidative modification of the transcript would reduce translation efficiency. We performed polyribosome analysis to compare the amount of ribosome loading onto the oxidized transcript vs. the nonoxidized transcript. A decreased amount of ribosome loading onto the oxidized transcript would suggest that oxidized bases on transcript inhibit the translation process; on the other hand, an increased amount of ribosome loading onto the oxidized transcript would suggest that oxidized bases on transcript cause ribosome stalling on the transcript or slow the translation process. Luciferase RNAs were used in this study. In vitro synthesized luciferase RNAs were treated with either 3.2 µM H2O2 and 0.2 µM cytochrome c as oxidized RNA or with 0.2 µM cytochrome c alone as nonoxidized RNA. Both nonoxidized and oxidized luciferase RNAs were subjected to translation in rabbit reticulocyte lysates for 10 min and put on ice to halt the translation process. The lysates were then loaded onto a 15–60% sucrose gradient and fractionated by centrifugation. The fractions were collected from the top of the gradient (fractions 1–10). The free monoribosomes were expected to remain at the top of the gradient (<20% sucrose) while RNA-associated polyribosomes were expected to be found in the heavier fractions (>35% sucrose) (33)
. We extracted RNAs from each fraction and determined the distribution of luciferase RNA and ribosome by Northern blot analysis using luciferase or 18S rRNA antisense RNA probes. As shown in Fig. 7B
, for the nonoxidized RNA sample, 18S rRNA was localized to fractions 4–9 and showed two peaks at fraction 5 and 8. Luciferase RNA was localized to fractions 6–9 and showed one peak at fraction 8. This indicated that fractions 4–5 contained free monoribosomes and fractions 6–9 contained RNA-associated polyribosomes. The predominant RNA-associated polyribosomes were in fraction 8. For the oxidized RNA sample, there were two obvious changes compared with nonoxidized RNA sample: 1) the predominant RNA-associated polyribosomes were localized to the heavier fractions, fractions 9–10, and 2) the amounts of free monoribosomes (fractions 4–5) were significantly reduced and the amounts of RNA-associated polyribosomes (fractions 6–10) were significantly increased. Figure 7C
shows the quantitative analysis of this result by measuring signal intensities using densitometry. We repeated this experiment four times and observed consistent results. Moreover, we noted that more RNA-associated polyribosomes were localized to the bottom fraction 10 when the RNAs were oxidized with higher concentration of oxidants (8 µM H2O2 and 0.5 µM cytochrome c) (not shown). This indicates that more ribosomes load onto the transcript when it had more oxidized bases. Taking these results together suggests that oxidized bases on transcript may cause ribosome stalling on the transcript or slow the translation process.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The conclusion that mRNA oxidation is an early event preceding cell death was made based on the following. 1) In the case of H2O2 treatment, oxidized mRNA was detectable as early as 1 h post-treatment by immunofluorescent staining (Fig. 2A
) and immunoprecipitation (Fig. 5A
). The oxidized mRNA level increased with time for up to 4 h and declined at 10 h post-treatment. 2) The increase of LDH release started at 6 h post-treatment (Fig. 2B
); at this time point, >60% of mRNAs were oxidized (Fig. 5B
). 3) Abnormal nuclear and chromatin morphology, markers of apoptotic cells, were not evidenced within the first 15 h (Fig. 2C
) but were observed 24 h post-treatment, when the neurons started to degenerate (Fig. 1)
. Similar phenomena also occurred in glutamate- or Aß 25–35-treated cultures (Fig. 3)
. Nunomura et al. have demonstrated that, in AD brains, increased RNA oxidation is restricted to healthy-looking vulnerable neurons (16)
. It is not dependent on proximity to senile plaques and is decreased in neurons containing neurofibrillary tangles. They suggested that RNA oxidation is an early event preceding cell death. The results of the present study support their observation.
We speculate that in the case of H2O2 treatment, the initial oxidative modification of mRNA may be the result of the attacks from intracellular ROS generated directly from the diffused H2O2 during treatment. Cells possess a certain level of antioxidant capacity and dispose oxidants via several pathways. Once this capacity is overwhelmed, a chain reaction producing ROS may be turned on in a positive feedback manner. This may explain the continuous rise of oxidized mRNA levels within 4 h after exposure to H2O2. Oxidative damage to mRNA, among damage incurred to other cellular macromolecules, resulted in an LDH release at 6 h. By 10 h post-treatment, the oxidized mRNA level had declined, probably because the apoptotic pathway was initiated at this time and mRNA began to degrade. It has been shown that mRNA degradation precedes abnormal nuclear and chromatin morphology during the apoptotic process (34)
. Neurons eventually degenerated at 24 h.
In situ detection of oxidized RNAs in AD brains reveals that RNA oxidation is primarily present in vulnerable neurons (16)
. This raises the important question of whether neurons are more susceptible to RNA oxidation or whether oxidized RNAs accumulate over decades in neurons because they are nondividing cells. In the present study, neurons and astrocytes were under the same level of oxidative stress, and oxidized RNAs were present predominantly in a distinct group of neurons, not in astrocytes (Fig. 4)
. This indicates that neurons are more susceptible to RNA oxidation. It has been shown that astrocytes are the primary site of the production of ROS that causes delayed neuronal death, but it has no effect on astrocytes themselves (35)
. The resistance of astrocytes to ROS has been ascribed to their greater antioxidant capacity compared with neurons (36)
. The concentration of reduced glutathione, the major intracellular antioxidant, has been found to be markedly higher in astrocytes than in neurons (37)
.
We found that 60–70% of mRNAs were oxidized 4 h post-H2O2 treatment (Fig. 5B
) and that some mRNA species were more susceptible to oxidative damage (Fig. 5C
). These results support our previous studies in AD brain (21)
. The mechanisms underlying the selectivity of mRNA oxidation are currently under investigation. No common motifs, sequences, or structures were found in oxidized mRNA species at this time. Several possible mechanisms are considered, including turnover rate of oxidized mRNAs, spatial conformation of mRNA-protein complex, mRNA stability, and intracellular localization of mRNA species.
SOD1 mRNA is oxidized in the AD brain (21)
and also in cultured neurons under oxidative stress (Fig. 5C
). We observed that the SOD1 protein level was dramatically decreased at 12 h post-H2O2 treatment (Fig. 6A
) and that this decrease was not due to reduced mRNA expression (Fig. 6B
). It could be due to oxidative damage to SOD1 mRNA. We expressed oxidized SOD1 mRNA in HEK293 cells and found that oxidative modification of SOD1 mRNA led to a decrease of normal SOD1 protein expression (Fig. 6C
).
We then investigated how oxidative modification of mRNA affects the downstream translational process. We asked whether oxidative modification of mRNA would cause premature termination of translation. Based on the methods applied in this study, we concluded that oxidized RNA may not produce truncated protein (Fig. 7A
). However, it is possible that oxidized mRNAs did generate truncated proteins with different sizes that are present in a very small quantity individually and our methods were not sensitive enough to detect them. We also considered whether oxidized mRNAs produce missense mutated proteins. We compared protein levels and activities of luciferase translated from oxidized luciferase RNAs. Quantitative analysis revealed that the luciferase activity was highly correlated with the luciferase protein level (not shown); this suggests that oxidized mRNAs may not produce missense mutated proteins that possess lower luciferase activity. However, it is still possible that oxidized mRNAs produce missense mutated proteins and the majority of mutation sites are outside of the enzymatically functional site of the luciferase protein.
We also asked whether oxidative modification on transcript would reduce translation efficiency. Based on our polyribosome analysis, we concluded that oxidized bases on the transcript may cause ribosome stalling or slow the translation process (Fig. 7B, C
). Eukaryotic translation can be subdivided into three sequential phases of initiation, elongation, and termination. An oxidized base may impair any of these phases, depending on its location on the transcript. Oxidation on the ribosome small subunit binding site and/or the 5'-untranslated region (UTR) may reduce the affinity of ribosomes for the transcript and migration rate of ribosome small subunits along the 5' UTR during the initiation phase. Oxidation within the coding region may affect codon-anti-codon pairing between mRNA and t-RNA during the elongation phase; further, the elongation rate may decrease or halt if the ribosome complex cannot pass through the oxidized sites efficiently. Oxidation of bases encoding stop codons may interfere in the normal termination procedure. Oxidation on 5'- or 3'-UTRs may affect the translational regulation controlled by these regions. It is likely that the presence of oxidation lesions on coding regions or stop codons contributes to the ribosome stalling observed by polyribosome association experiments (Fig. 7B, C
). Furthermore, oxidized mRNAs may mediate their deleterious effects by stalling ribosomes on their transcripts, thereby consuming the intracellular ribosome pool, resulting in a decrease of protein production.
Cells may protect mRNA from oxidation via three pathways. First, the intracellular antioxidant system in which Cu2+/Zn2+ SOD1, catalase, and glutathione peroxidase are the major enzymes modulates the neutralization of ROS. Studies have shown that antioxidant enzyme activities are reduced in AD brains (38)
. Neurons in AD-affected regions were under the attack of increased ROS. mRNA may be a major target because of its widespread subcellular distribution, relative abundance, single-strand nature, and lack of protection from histone proteins. Second, the discovery of RNA repair enzymes rectified the conventional thought that damaged RNA is irreparable. mRNA repair mechanisms have long been underestimated because any given message may be present in a cell in several to thousands of copies and are replaceable. Recently, an RNA repair enzyme, hABH3, which repairs the methylated mRNAs, has been reported (39)
. Although no repair enzyme for oxidized mRNA has been found yet, there is a possibility that such an enzyme is present in cells, as mRNA oxidation occurs no less frequently than does methylation. The alteration of such an enzyme, if it exists, could be studied in the AD brain to better understand mRNA oxidation/repair mechanisms. Finally, oxidized mRNAs may be recognized by an RNA degradation system, and thereby be removed from the cytoplasm.
Studies have shown that RNA alone can be pathogenic, altering cellular functions in several human diseases (40)
. Oxidized mRNAs may mediate a toxic gain-of-function by attracting and sequestering ribosomes, RNA binding proteins, RNA degradation system proteins, or RNA repair enzymes from their normal functions. It is also possible that different oxidized mRNA species may behave differently and have different effects depending on their natures. Further investigation should be performed to better understand the involvement of mRNA oxidation in neurodegeneration.
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ACKNOWLEDGMENTS |
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Received for publication January 23, 2007. Accepted for publication April 5, 2007.
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REFERENCES |
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