In the present study, we found that mRNA oxidation is a common feature in ALS patients and mutant SOD1 transgenic mice and also an early event preceding motor neuron degeneration. This study suggests that mRNA oxidation may contribute to motor neuron deterioration in ALS.
We used 15A3 antibodies to separate oxidized mRNAs from non-oxidized mRNAs and subsequently quantified and identified oxidized mRNAs. The specificity of 15A3 antibody is very important. This was validated by including the no antibody control and the 8-OHG-blocked antibody control in each experiment. In addition, we analyzed the 15A3-precipitated mRNAs by HPLC-ECD (high-performance liquid chromatography coupled with electrochemical detection) and confirmed that the mRNAs recognized by 15A3 contained high levels of 8-OHG. This indicates that the isolated/detected mRNAs were oxidized mRNAs.
In the ALS postmortem tissues study (), we found that mRNAs are oxidatively damaged to a variable extent in ALS patients. Interpretation of human postmortem tissue pathological changes is difficult, since postmortem tissue represents the very end stage of the neurological disease and reflects the cells that remain but not necessarily those that are at risk. The mutant SOD1 mice studies provide the valuable information of the role of RNA oxidation in ALS. From the results of the SOD1G93A mice study, we speculate that significantly increased mRNA oxidation may occur in the motor neurons and oligodendrocytes of ALS-affected areas at the prodromal stage. This may contribute to neuronal deterioration and in combination with other toxicities eventually leading to motor neuron degeneration.
Previous studies in SOD1G93A
mice showed that significant increases in lipid, protein and DNA oxidation occurs during symptomatic stage 
. We found that mRNA oxidation occurs as early as 45 days, progressively increases with age, until it peaks at 60–70 days of age, and then diminishes when the motor neurons begin to degenerate (). Those motor neurons showing RNA oxidation appear to be still healthy as judged by the nuclear and chromatin morphology and mitochondrial morphology (). These results indicate that mRNA oxidation is an early event.
RNA oxidation primarily occurs in motor neurons and oligodendrocytes at pre-symptomatic stage (). This indicates that both cell types are more vulnerable to RNA oxidation. Like neurons, oligodendrocytes are highly vulnerable to injury by oxidative stress 
. Oligodendrocytes, compared to other cells, have a high lipid content, high iron content, and low supplies of cellular antioxidant 
Increased mRNA oxidation also occurs in the pre-symptomatic stage of mice expressing other mutant SOD1 (). Mutant SOD1G37R
, like SOD1G93A
, retains full dismutase activity 
. Both SOD1G85R
lack dismutase activity and are unstable 
. Mutant SOD1 H46R/H48Q
possesses little or no SOD1 activity 
. The protein levels of mutant SOD1 in these mice are different: SOD1G93A
mice express 17-, 5-, 0.9- and 0.45-fold human mutant SOD1 of mouse wild type SOD1, respectively 
. This suggests that increased RNA oxidation is a common feature in these ALS mouse models and has nothing to do with SOD1 activity or mutant SOD1 expression level. What could be the possible mechanism underlying ROS formation in these mutant SOD1 mice? We would like to propose one possible mechanism. Ferri et al.
have demonstrated that one common property of different FALS-mutant SOD1s with widely differing biophysical properties is the association with mitochondria to a much greater extent than wild-type SOD1 
. Mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and impair mitochondrial function, which could lead to increased ROS formation.
Identification of oxidized mRNA species by DNA microarray revealed that some mRNA species are more susceptible to oxidative damage, which was also observed in our previous studies 
. The phenomenon of selective RNA oxidation was not related to the abundance of mRNA species or the up-regulation of mRNA expression. No common motifs, sequences or structures were found in oxidized mRNA species at this time. Several possible mechanisms are currently under investigation.
A very striking finding in this study is that many identified known oxidized mRNAs are related to ALS: (1) mRNAs corresponding to genes linked to familial ALS or ALS-like human motor neuron disease, including SOD1, dynactin 1, and vesicle-associated membrane protein 1 (VAMP) mRNAs. A missense mutation in the p150 subunit of the dynactin
) gene has been described in a human kindred with a slowly progressive, autosomal dominant form of lower motor neuron disease 
. A dominant missense mutation in the VAMP-B
gene (ALS8) has been linked to an atypical ALS that is accompanied by an unusual tremor 
. VAMP-B interacts with VAMP-A involving in vesicular trafficking 
. (2) mRNAs encoding neurofilament subunits. The genes encoding three neurofilament subunits have long been suspected as causative for ALS because of their link with motor neuron pathology in mice and humans 
. (3) mRNAs encoding proteins involved in protein folding and degradation. Reduction of proteasome 26S function and protein chaperone activities have been found in SOD1G93A
transgenic mice 
. Impaired function of protein folding and degradation pathway can lead to protein aggregation, one of hallmarks of ALS. (4) mRNAs encoding proteins involved in the mitochondrial electron transport chain (ETC). Dysfunction of mitochondrial ETC has been previously found in SOD1G93A
mice and ALS patients 
. It is possible that mRNA oxidation is responsible for the observed dysfunction and also the mitochondria vacuolization (). (5) mRNAs encoding proteins involved in glycolysis and tricarboxylic acid (TCA) cycle. Depleted ATP levels and reduction of glucose use have been reported in spinal cords of SOD1G93A
mice at pre-symptomatic stage 
. Abnormal glycolysis and TCA process combined with mitochondrial electron transport chain dysfunction could result in ATP synthesis impairment. (6) mRNAs encoding metallothioneins. Metallothioneins, known to bind copper ions and decrease oxidative toxicity, have been suggested to have important roles in the pathophysiology of ALS 
. (7) mRNAs encoding proteins involved in protein transport. Defective axonal transport has been found to cause late-onset progressive degeneration in transgenic mice 
. Dominant point mutations in dynein causing motor neuron disorders have been found in both ALS patients and mouse models 
. (8) mRNAs encoding proteins involved as structural constituents of myelin sheath, including MBP, proteolipid protein (PLP) and myelin-associated oligodendrocytic basic protein (MOBP). MBP was reported to be related to axon degeneration 
. MBP protein level is significantly decreased in ALS spinal cords 
. We show that MBP protein is significantly decreased in the presymptomatic stage of SOD1G93A
spinal cords (). mRNA oxidation may be responsible for loss of MBP protein. Myelin sheaths contribute to the structure and stability of the axons 
. Abnormally expressed myelin proteins may affect axon stability and contribute to axon degeneration.
We examined protein expression levels for the oxidized mRNA species and found that some proteins corresponding to oxidized mRNA species are decreased (). Our previous studies demonstrated that oxidized bases in mRNAs can cause ribosome stalling on the transcripts, leading to decreased protein expression 
. A recent study 
demonstrates that oxidized mRNA induces translation errors, producing short polypeptides because of premature termination or translation error-induced degradation.
Does mRNA oxidation contribute to the pathogenesis of the disease? We approached this question by treating SOD1G93A
mice with vitamin E. As mentioned above, RNA oxidation occurs at an early age of mice, but significant protein, lipid and DNA oxidations do not occur until active disease progression stage 
; thus, the protective effects by vitamin E in the early symptomatic stage may be primarily due to reduced mRNA oxidation. Four observations strongly support that mRNA oxidation does contribute to the disease. First, the protein expression levels for the oxidized mRNA species are decreased (). Second, the vitamin E treated mice still had normal motor performance and were still active at the age of 100 days while the non-treated mice were already sick (). The treated mice developed onset rapidly between 110 and 120 days and died within a similar age range as the non-treated mice. This is probably because reduced mRNA oxidation by vitamin E partially diminishes toxicity in motor neurons, but toxicities from other non-neuronal cells are still present. Third, reduced mRNA oxidation by vitamin E significantly reduces mitochondria vacuolization in motor neurons (). Finally, many identified known oxidized transcripts are related to ALS. RNA oxidation may account for many neuropathological changes reported previously.
Dietary supplement with vitamin E is widely used in the clinical as an antioxidant for ALS patients. Although in previous studies vitamin E did not prolong survival of ALS patients 
, Ascherio et al.
found that those individuals who took vitamin E supplements for 10 years had less than 50% risk of death from ALS than that of vitamin E nonusers 
. These studies support our observations in SOD1G93A
mice that RNA oxidation is an early event and contributes to motor neuron degeneration. Thus, blocking RNA oxidation at prodromal stage may prevent/slow the disease progression; however, at the disease stage, the antioxidant treatment may be already too late so there is no significant beneficial effect.