BackgroundAccumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration.Methodology/Principal FindingsWe demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons.Conclusion/SignificanceThese findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.

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.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5-untranslated regions (5-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions. Excitatory amino acid transporters (EAATs)2 in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Five sodium-dependent glutamate transporter subtypes have been identified and characterized: GLAST-1 (EAAT1) (1, 2), GLT-1 (EAAT2) (3, 4), EAAC1 (EAAT3) (5, 6), EAAT4 (7), and EAAT5 (8). EAAT2 is expressed mainly in glial cells throughout the brain and is responsible for up to 90% of all glutamate transport in adult tissue (9, 10).A malfunction in the glutamate transport system can lead to accumulation of excessive glutamate in the synapse, which is harmful to neurons and could result in neurodegeneration (11,12). Over the past 15 years, it has been demonstrated that glutamate-mediated toxicity may play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and Alzheimer disease (AD). Rothstein and colleagues (13) reported a significant increase in levels of glutamate in the cerebrospinal fluid of ALS patients and defective glutamate transport in the affected areas of ALS (14). Subsequent studies demonstrated a dramatic and selective loss of EAAT2 protein in the affected areas of ALS (15). Defective glutamate transport and loss of EAAT2 protein were also reported in the affected areas of AD patients (16,17). This phenomenon also occurs in several rod...

In the past decade, RNA oxidation has caught the attention of many researchers, working to uncover its role in the pathogenesis of neurodegenerative diseases. It has been well documented that RNA oxidation is involved in a wide variety of neurological diseases and is an early event in the process of neurodegeneration. The analysis of oxidized RNA species revealed that at least messenger RNA (mRNA) and ribosomal RNA (rRNA) are damaged in several neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). The magnitude of the RNA oxidation, at least in mRNA, is significantly high at the early stage of the disease. Oxidative damage to mRNA is not random but selective and many oxidized mRNAs are related to the pathogenesis of the disease. Several studies have suggested that oxidative modification of RNA affects the translational process and consequently produces less protein and/or defective protein. Furthermore, several proteins have been identified to be involved in handling of damaged RNA. Although a growing body of studies suggests that oxidative damage to RNA may be associated with neuron deterioration, further investigation and solid evidence are needed. In addition, further uncovering of the consequences and cellular handling of the oxidatively damaged RNA should be important focuses in this area and may provide significant insights into the pathogenesis of neurodegenerative diseases.

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