RNA Oxidation Research Articles

Messenger RNA oxidation is an early event preceding cell death and causes reduced protein expression

We previously reported that up to 50% of messenger RNAs (mRNA) are oxidatively damaged in the affected area of Alzheimer's disease (AD) brains. The role of RNA oxidation in the cell death process is unknown. In the present study, we used cortical primary dissociated cultures to investigate the relationship between RNA oxidation and neuron degeneration induced by various insults, including hydrogen peroxide, glutamate, and amyloid beta peptide. These insults mediate the production of reactive oxygen species and thus induce oxidative stress. The results showed that RNA oxidation was an early event far preceding cell death, not merely a consequence of dying cells. RNA oxidation occurred primarily in a distinct group of neurons that died later. Identification of oxidized RNA species revealed that significant amounts of mRNAs were oxidized and that some mRNA species were more susceptible to oxidative damage, consistent with findings in the AD brain. The level of protein corresponding to the oxidized mRNA species was significantly decreased. Polyribosome analysis indicated that oxidized bases in mRNAs caused ribosome stalling on the transcripts, which led to a decrease of protein expression. These results suggest that RNA oxidation may be directly associated with neuronal deterioration, rather than harmless epiphenomena, during the process of neurodegeneration.

Stress response of a freshwater clam along an abiotic gradient: too much oxygen may limit distribution

SummaryDistribution and abundance patterns are influenced by cellular responses to abiotic stressors.The freshwater clamSphaeriumsp. inhabits swamp/stream systems in Uganda, in which dissolved O2(DO), pH and water transparency form ecotonal gradients along tributary streams. Along a swamp/stream transect, clam abundance was negatively related to DO, pH and transparency; clams were most abundant in the hypoxic/acidic swamp, less abundant in the ecotone, and absent from stream sites (neutral pH and higher DO).We tested the hypothesis that individuals living in low abundance at the distribution edge have increased stress by assessing cellular‐level stress indicators in clams from four sites: dense swamp interior (reference site), swamp margin, ecotone and stream. Compared with swamp interior clams, stream clams had decreased RNA : DNA ratio (decreased protein synthesis and growth) and increased DNA and RNA oxidation (free radical damage). Of seven stress proteins related to general damage and free radical detoxification, expression of nearly all were higher in margin and ecotone clams than in swamp interior or stream clams.This pattern, which includes the first measurement of DNA and RNA oxidation in a natural population, demonstrates that stress increases along the transect, inversely to clam abundance. Additionally, this pattern is consistent with free radical stress, which suggests that inhabiting swamp conditions minimizes oxidative damage in this species.

Oxidized messenger RNA induces translation errors

To investigate the effect of RNA oxidation on normal cellular functions, we studied the translation of nonoxidized and oxidized luciferase mRNA in both rabbit reticulocyte lysate and human HEK293 cells. When HEK293 cells transfected with nonoxidized mRNA encoding the firefly luciferase protein were cultured in the presence of paraquat, there was a paraquat concentration-dependent increase in the formation of luciferase short polypeptides (SPs) concomitant with an increase in 8-oxoguanosine. Short polypeptides were also formed when the mRNA was oxidized in vitro by the Fe-ascorbate-H(2)O(2) metal-catalyzed oxidation system before its transfection into cells. Translation of the in vitro oxidized mRNA in rabbit reticulocyte lysate also led to formation of SPs. The SPs formed by either procedure contained the N-terminal and the C-terminal portions of the tagged luciferase. In addition, the oxidized mRNA was able to associate with ribosomes to form polysomes similar to those formed with nonoxidized mRNA preparations, indicating that the oxidized mRNAs are mostly intact; however, their translation fidelity was significantly reduced. Nevertheless, our results indicate that the SPs were derived from both premature termination of the translation process of the oxidized mRNA and the proteolytic degradation of the modified full-length luciferase resulting from translation errors induced by oxidized mRNA. In light of these findings, the physiological consequences of mRNA oxidation are discussed.

Oxidative stress alters neuronal RNA- and protein-synthesis: Implications for neural viability

Recent studies have demonstrated that impaired protein synthesis occurs in several neurodegenerative conditions associated with oxidative stress. Studies have also demonstrated that administration of oxidative stressors is sufficient to impair different and discrete regulatory aspects of protein synthesis in neural cells, with the majority of these studies focused on the effects of oxidative stressors towards initiation factors. Currently, little is known with regards to oxidative stress effects on the rates of RNA- and protein-synthesis, or the relationship between oxidant-induced impairments in RNA-/protein-synthesis to subsequent neuron death. In the present study, we demonstrate that administration of an oxidative stressor (hydrogen peroxide) induces a significant and time-dependent decrease in both RNA- and protein-synthesis in primary neurons and neural SH-SY5Y cells. Increases in RNA oxidation and disruption of ribosome complexes were selectively observed following the longer durations of oxidant exposure. Significant correlations between the loss of RNA- and protein-synthesis and the amount of oxidant-induced neuron death were also observed. Interestingly, the addition of a protein synthesis inhibitor (cycloheximide) did not significantly alter the amount of neuron death induced by the oxidative stressor. These data demonstrate that oxidant exposure promotes a time-dependent decrease in both RNA- and protein-synthesis in neurons, and demonstrate a role for elevations in RNA oxidation and ribosome dysfunction as potential mediators of impaired protein synthesis. These data also suggest that there is a complex relationship between the ability of oxidative stressors to modulate RNA- and protein-synthesis, and the ability of oxidative stressors to ultimately induce neuron death.

Effect of olive oils on biomarkers of oxidative DNA stress in Northern and Southern Europeans

High consumption of olive oil in the Mediterranean diet has been suggested to protect DNA against oxidative damage and to reduce cancer incidence. We investigated the impact of the phenolic compounds in olive oil, and the oil proper, on DNA and RNA oxidation in North, Central, and South European populations. In a multicenter, double-blind, randomized, controlled crossover intervention trial, the effect of olive oil phenolic content on urinary oxidation products of guanine (8-oxo-guanine, 8-oxo-guanosine and 8-oxo-deoxyguanosine) was investigated. Twenty-five milliliters of three olive oils with low, medium, and high phenolic content were administered to healthy males (n=182) daily for 3 wk. At study baseline the urinary excretion of 8-oxo-guanosine (RNA oxidation) and 8-oxo-deoxyguanosine (DNA oxidation) was higher in the Northern regions of Europe compared with Central and Southern European regions (P=0.035). Urinary excretion of the 8 hydroxylated forms of guanine, guanosine, deoxyguanosine and their nonoxidized forms were not different when comparing olive oils with low, medium, and high phenolic content given for 2 wk. Testing the effect of oil from urinary 8-oxo-deoxyguanosine changes from baseline to post-treatment showed a reduction of DNA oxidation by 13% (P=0.008). These findings support the idea that ingestion of olive oil is beneficial and can reduce the rate of oxidation of DNA. This effect is not due to the phenolic content in the olive oil. The higher DNA and RNA oxidation in Northern European regions compared with that in Central and Southern regions supports the contention that olive oil consumption may explain some of the North-South differences in cancer incidences in Europe.

RNA damage and surveillance under oxidative stress

RNA damage has been recently reported to increase under oxidative stress and in patients with many degenerative diseases, which has drawn attention to the consequences of RNA oxidation at the molecular and cellular levels. Under similar conditions the levels of oxidative damage in RNA are usually higher than those in DNA, which may impair protein synthesis or other RNA function. Therefore, accumulation of RNA damage must be prevented and cells have developed specific mechanisms to remove oxidatively-damaged RNA and to block incorporation of oxidized nucleotides during RNA synthesis. Removal of oxidized RNA may be mediated by specific proteins that recognize oxidative lesions and direct the RNA degradation machinery to eliminate the damaged RNAs. During RNA synthesis, oxidized ribonucleotides are hydrolyzed or discriminated from normal ribonucleotides during transcription, preventing their incorporation into RNA. Collective evidence suggests that RNA oxidative damage is a challenging and persistent problem normally controlled through RNA surveillance mechanisms, making them critical to maintaining cellular health and preventing disease.

Decreased RNA, and Increased RNA Oxidation, in Ribosomes from Early Alzheimer’s Disease

All cells rely on efficient protein synthesis in order to maintain cellular homeostasis. Recent studies from our laboratory indicate that declines in protein synthesis and ribosome function occur in the earliest stage of Alzheimer's disease (AD). Additional studies indicate a potential role for ribosomal RNA oxidation as a potential mediator of decreased protein synthesis in AD. The ribosome is a complex of proteins and nucleic acids that mediates all protein synthesis. At present it is unclear if significant alterations in ribosomal RNA occurs within the ribosome complex during the progression of AD. In this study we examined the amount of ribosomal RNA in the different ribosomal fractions generated from control subjects, individuals with mild cognitive impairment (MCI), and individuals with AD. Studies were conducted in the inferior parietal lobule of each subject. Together, these data demonstrate that during the progression of AD there is a gross decline in the amount of ribosomal RNA within the ribosome complex. Additionally, these studies provide evidence for gross elevations in RNA oxidation within the ribosome complex of MCI and AD. Together, these data strongly suggest a role for RNA alterations within the ribosome as a mediator of decreased protein synthesis in both MCI and AD.

Hepatic Oxidative Stress During Aging: Effects of 8% Long-Term Calorie Restriction and Lifelong Exercise

Hepatic aging may involve alterations in redox status, resulting in enhanced oxidant production and changes in specific signaling pathways that lead to a pro-inflammatory response. The authors investigated whether mild calorie restriction and long-term voluntary exercise could attenuate these changes. Four groups of male Fischer 344 rats were compared: young (6 mo), old (24 mo), old calorie restricted (8% CR, 24 mo) and old CR with daily voluntary wheel running (Exercise; 8% CR, 24 mo). Levels of endogenous reactive oxygen species (ROS), nitric oxide (NO*), and peroxynitrite (ONOO-) were significantly higher in the old ad libitum fed group compared to the young group. Sulfhydryl (-SH) content was significantly reduced and glutathione (GSH) content tended to be lower in the old animals. Old rats had significantly increased nuclear presence of NF-kappaB and in connection, increased levels of regulatory cytosolic phosphorylated I-kappaBalpha and decreased dephosphorylated I-kappaBalpha, suggesting an increased inflammatory response. Interestingly, a significant increase in liver RNA oxidation (8-oxo-7,8-dihydroguanosine) in the old ad libitum fed rats was detected and DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine) tended to be increased. The age-associated increase in oxidative stress and upregulation of pro-inflammatory proteins was attenuated in the livers from both the CR and the exercise + CR groups.

Reduced production of full length cDNA from oxidatively damaged RNA

Macromolecular damage caused by oxidative stress may account for the pathogenesis of numerous diseases. It has recently reported that the levels of oxidized RNA are increased in patients of various age-related disorders. It is unclear how RNA oxidation affects its biological function, and how cells are protected against RNA damage. Understanding of the mechanisms has been hampered by the lack of efficient and sensitive methodologies detecting RNA damage. Using total RNA from E. coli, we have shown that full-length cDNA for 16S rRNA is made less efficiently from oxidized RNA than from controls. When RNA is oxidized in vitro, the amount of full-length cDNA is reduced depending on the dosage of the oxidant. RNA from E. coli cultures with oxidant treatment also demonstrates significant reduction in the amount of full-length cDNA than the RNA from cultures without oxidant treatment. Interestingly, the level of reduction depends on the distance of cDNA chain extension. Using semi-quantitative or real-time PCR, about 30% to 40% reduction can be detected from each kilobases of cDNA when cells were treated with 0.5 mM H2O2. The data suggest that the deficiency of reverse-transcription is caused by damages that are randomly distributed in RNA templates. PCR-based detection makes this method sensitive and specific to the RNA species of interest.

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.

Oxidative Damage to RNA in Neurodegenerative Diseases

Since 1999, oxidative damage to RNA molecules has been described in several neurological diseases including Alzheimer's disease, Parkinson's disease, Down syndrome, dementia with Lewy bodies, prion disease, subacute sclerosing panencephalitis, and xeroderma pigmentosum. An early involvement of RNA oxidation of vulnerable neuronal population in the neurodegenerative diseases has been demonstrated, which is strongly supported by a recent observation of increased RNA oxidation in brains of subjects with mild cognitive impairment. Until recently, little is known about consequences and cellular handling of the RNA damage. However, increasing body of evidence suggests detrimental effects of the RNA damage in protein synthesis and the existence of several coping mechanisms including direct repair and avoiding the incorporation of the damaged ribonucleotides into translational machinery. Further investigations toward understanding of the consequences and cellular handling mechanisms of the oxidative RNA damage may provide significant insights into the pathogenesis and therapeutic strategies of the neurodegenerative diseases.

Proteasome Regulation of Oxidative Stress in Aging and Age-Related Diseases of the CNS

Proteasome-mediated protein degradation is responsible for a large percentage of bulk protein turnover, particularly the degradation of short-lived and oxidized proteins. Increasing evidence suggests that proteasome inhibition occurs during the aging of the central nervous system (CNS), and in a variety of age-related disorders of the CNS. The focus of this review is to discuss the role of the proteasome as a regulator of oxidative stress, with preservation of proteasome function playing an important role in preventing oxidative stress, and proteasome inhibition playing an important role as a mediator of oxidative stress. In particular, this review will describe experimental evidence that proteasome inhibition is sufficient to induce mitochondrial dysfunction, increase reactive oxygen species generation, elevate RNA and DNA oxidation, and promote protein oxidation. Taken together, these data indicate that the proteasome is an important regulator of oxidative damage in the CNS, and suggest that proteasome inhibition may serve as an important switch for the induction of oxidative stress in the CNS. Additionally we discuss the likelihood that the 20S proteasome and 26S proteasome may play different roles in regulating oxidative stress and neurotoxicity in the aging CNS, and in age-related disorders of the CNS.

Ribosome Dysfunction Is an Early Event in Alzheimer's Disease

Alzheimer's disease (AD) is a progressive and devastating disorder that is often preceded by mild cognitive impairment (MCI). In the present study, we report that in multiple cortical areas of MCI and AD subjects, there is a significant impairment in ribosome function that is not observed in the cerebellum of the same subjects. The impairment in ribosome function is associated with a decreased rate and capacity for protein synthesis, decreased ribosomal RNA and tRNA levels, and increased RNA oxidation. No alteration in the level of initiation factors was observed in the brain regions exhibiting impairments in protein synthesis. Together, these data indicate for the first time that impairments in protein synthesis may be one of the earliest neurochemical alterations in AD and directly demonstrate that the polyribosome complex is adversely affected early in the development of AD. These data have important implications for AD studies involving proteomics and studies analyzing proteolysis in AD, indicate that oxidative damage may contribute to decreased protein synthesis, and suggest a role for alterations in protein synthesis as a novel contributor to the onset and development of AD.

Quantification of oxidized RNAs in Alzheimer's disease

Cytoplasmic RNA oxidation is a prominent feature of vulnerable neurons in the brains of Alzheimer's disease (AD) patients. We previously demonstrated that messenger RNAs (mRNA) are oxidized in AD brains. However, the magnitude of the mRNA oxidation is unclear. In the present study, we separated oxidized mRNAs from non-oxidized mRNAs by immunoprecipitation and then quantitatively analyzed both mRNA fractions. Surprisingly, 30–70% of the mRNAs isolated from AD frontal cortices were oxidized while the abundance of oxidized mRNAs was low in age-matched normal controls. Furthermore, we previously showed that some mRNA species are more susceptible to oxidative damage. Here, we quantified the degree of oxidation for individual mRNA species that were previously found to be oxidized in AD. Quantitative analysis revealed that the relative amounts of oxidized transcripts reach 50–70% for some mRNA species. These high abundances implicate the potential contribution of mRNA oxidation to the pathogenesis of AD.

Ribosomal RNA in Alzheimer Disease Is Oxidized by Bound Redox-active Iron

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

Proteasome inhibition increases DNA and RNA oxidation in astrocyte and neuron cultures

Increased levels of nucleic acid oxidation have been described as part of normal brain aging and have been demonstrated to occur in multiple neurological disorders. The basis for increased nucleic acid oxidation in each of these conditions is presently unknown. Proteasome inhibition occurs in a host of neurodegenerative conditions and likely contributes to increased levels of oxidative damage and neurotoxicity. In the present study we demonstrate for the first time the ability of proteasome inhibition to increase the level of nucleic acid oxidation in primary neuron and astrocyte cultures. Administration of proteasome inhibitors (MG262, MG115) at concentrations that do not induce neuron death in the first 24 h of treatment, dramatically increase the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8OHG) immunoreactivity in both cell types. Neurons underwent larger increases in nucleic acid oxidation compared to astrocyte cultures. While both DNA and RNA oxidation were observed following proteasome inhibition, RNA appeared to undergo a greater degree of oxidation than DNA. Both 18S and 28S ribosomal RNA were dramatically decreased following proteasome inhibition. Interestingly, an accumulation of unprocessed and/or cross-linked RNA species was observed following proteasome inhibition. Taken together, these data indicate the ability of proteasome inhibition to increase the levels of nucleic acid oxidation in both neurons and astrocytes, and suggest that proteasome inhibition may have deleterious effects on transcription and translation in both neurons and glia.

Mitochondrial failures in Alzheimer's disease

Mitochondrial dysfunction and free radical-induced oxidative damage have been implicated in the pathogenesis of several different neurodegenerative diseases such as Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and Alzheimer's disease (AD). The defective adenosine triphosphate (ATP) production and increased oxygen radicals may induce mitochondria-dependent cell death because damaged mitochondria are unable to maintain the energy demands of the cell. The role of vascular hypoperfusion-induced mitochondria failure in the pathogenesis of AD now has been widely accepted. However, the exact cellular mechanisms behind vascular lesions and their relation to oxidative stress markers identified by RNA oxidation, lipid peroxidation, or mitochondrial DNA (mtDNA) deletion remain unknown. Future studies comparing the spectrum of mitochondrial damage and the relationship to oxidative stress-induced damage during the aging process or, more importantly, during the maturation of AD pathology are warranted.

Delaying the Mitochondrial Decay of Aging with Acetylcarnitine

Oxidative mitochondrial decay is a major contributor to aging. Some of this decay can be reversed in old rats by feeding them normal mitochondrial metabolites, acetylcarnitine (ALC) and lipoic acid (LA), at high levels. Feeding the substrate ALC with LA, a mitochondrial antioxidant, restores the velocity of the reaction (K(m)) for ALC transferase and mitochondrial function. The principle appears to be that, with age, increased oxidative damage to protein causes a deformation of structure of key enzymes with a consequent lessening of affinity (K(m)) for the enzyme substrate. The effect of age on the enzyme-binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product that increases with age). In old rats (vs. young rats), mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O(2) uptake are lower; oxidants/O(2), neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher. Ambulatory activity and cognition decline with age. Feeding old rats ALC with LA for a few weeks restores mitochondrial function; lowers oxidants, neuron RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (as assayed with the Skinner box and Morris water maze). A recent meta-analysis of 21 double-blind clinical trials of ALC in the treatment of mild cognitive impairment and mild Alzheimer's disease showed significant efficacy vs. placebo. A meta-analysis of 4 clinical trials of LA for treatment of neuropathic deficits in diabetes showed significant efficacy vs. placebo.

Neuronal RNA oxidation is a prominent feature of familial Alzheimer's disease

An in situ approach was used to identify the oxidized RNA nucleoside 8-hydroxyguanosine (8OHG) in the frontal cortex of familial Alzheimer's disease (FAD) with a mutation in presenilin-1 (PS-1) or amyloid β protein precursor (AβPP) gene ( n = 13, age 47–81 years). Neurons with marked 8OHG immunoreaction in the cytoplasm were widely distributed in the superior/middle frontal gyrus of FAD. Relative intensity measurements of neuronal 8OHG immunoreactivity showed that there was a significant increase in FAD compared with controls ( n = 15, age 59–81 years), while there was no difference in relative 8OHG between the PS-1 and the AβPP FAD. Interestingly, a presymptomatic case carrying a PS-1 mutation showed a considerable level of relative 8OHG, and the increased levels of neuronal 8OHG in FAD were more prominent in cases with a lower percentage area of Aβ42 burden. These results suggest that oxidative stress is an early event involved in the pathological cascade of FAD.

Factors contributing to the outcome of oxidative damage to nucleic acids.

Oxidative damage to DNA appears to be a factor in cancer, yet explanations for why highly elevated levels of such lesions do not always result in cancer remain elusive. Much of the genome is non-coding and lesions in these regions might be expected to have little biological effect, an inference supported by observations that there is preferential repair of coding sequences. RNA has an important coding function in protein synthesis, and yet the consequences of RNA oxidation are largely unknown. Some non-coding nucleic acid is functional, e.g. promoters, and damage to these sequences may well have biological consequences. Similarly, oxidative damage to DNA may promote microsatellite instability, inhibit methylation and accelerate telomere shortening. DNA repair appears pivotal to the maintenance of genome integrity, and genetic alterations in repair capacity, due to single nucleotide polymorphisms or mutation, may account for inter-individual differences in cancer susceptibility. This review will survey these aspects of oxidative damage to nucleic acids and their implication for disease.