Some mRNA species are more susceptible to oxidative damage in SOD1^G93A mice.

<p>(A) Semi-quantitative RT-PCR analysis confirmed that the identified mRNA species by DNA microarray were present in the oxidized mRNA pool. MBP, cytochrome c, cytochrome c oxidase Va and ribosome protein S6 mRNAs, which had strong signal intensities on the arrays, were present in the oxidized mRNA pool. MAP2 and PCM1 mRNAs, which had very weak signal intensities on the arrays, were hardly detected in the oxidized mRNA pool. These oxidized mRNA species are significantly decreased in vitamin E treated SOD1^G93A mice (<i>G93A+vitE</i>). (B) Semi-quantitative RT-PCR analysis showed that the oxidized mRNA species are not upregulated in the whole spinal cord of SOD1^G93A mice compared to their non-transgenic littermates. SOD1 mRNA, which includes endogenous mouse SOD1 and transgenic human SOD1, was used as a control. n = 3.</p

RNA oxidation is an early event preceding motor neuron degeneration.

<p>Lumbar spinal cords from indicated age of SOD1^G93A mice (<i>G93A</i>) and non-transgenic littermates (<i>WT</i>) were examined. (A) In the ventral horn, motor neurons with strong 15A3 immunoreactivity had normal nuclear and chromatin morphology by Hoechst 33342 staining; the dying motor neurons showed abnormal nuclear and chromatin morphology but less 15A3 immunoreactivity. <i>Arrows</i> point to the same neuron. Scale bar, 25 µm. (B) Motor neurons with strong 15A3 immunoreactivity had only minor mitochondrial vacuolization as examined by electron microscopy. Scale bar, 0.5 µm. (C) Motor neurons with strong 15A3 immunoreactivity did not have ubiquitinated protein aggregation.</p

Increased RNA oxidation in motor neurons and oligodendrocytes of SOD1^G93A spinal cord at pre-symptomatic stage.

<p>Lumbar spinal cord sections from different ages of SOD1^G93A mice (<i>G93A</i>) and non-transgenic littermates (<i>WT</i>) were immunolabeled with 15A3 antibodies. The intensity of 15A3 immunofluorescence in SOD1^G93A mice was significantly increased at 45 days of age (D), further enhanced at 60 days of age (B, E), and then diminishes during the symptomatic stage (F, G, H), while only a very faint signal was detected in non-transgenic littermates (A, C). 15A3 immunoreactivity was prominent in the ventral horn motor neurons and the white matter oligodendrocytes (K, L), as identified by CC1 immunostaining (M). No increase of 15A3 immunofluorescence was observed in the brain sections (N, O). The immunoreactivity was diminished greatly by the RNase treatment (J) and when the antibody was pre-incubated with 8-OHG (I). Scale bar, 50 µm. (P) Statistical analysis of motor neuron 15A3 immunofluorescence intensity (n = 20). Asterisks indicate statistical significance compared with WT, 60 d (*<i>P</i><0.0001).</p

mRNAs are oxidatively damaged in ALS-affected areas and pre-symptomatic stage of mutant SOD1 mice.

<p>(A) Southern blot analysis of 15A3-immunoprecipitated oxidized mRNAs in ALS patients. mctx, motor cortex; sc, spinal cord; cbm, cerebellum; no Ab, no antibody; Ab block, antibody block (antibodies pre-incubated with 8-OHG). (B) Quantitative analysis of the magnitude of mRNA oxidation by comparing the signal density of oxidized mRNAs (<i>Oxi</i>) to serial dilutions of non-oxidized mRNA (<i>Non-oxi</i>) via Southern blot analysis. An example of analysis of SALS1 motor cortex is shown. The non-oxidized mRNA fraction was diluted 1 to 15. (C) Southern blot analysis of oxidized (<i>O</i>) and non-oxidized (<i>N</i>) mRNA pools prepared from spinal cords of indicated mice (60 day-old, n = 3). The percentages of total spinal cord mRNA that were oxidized in SOD1^G93A (<i>G93A</i>), SOD1^WT (<i>WtSOD1</i>), and non-transgenic (<i>WT</i>) mice are about 30%, 3–5%, and barely detectable, respectively. No mRNA was precipitated in the SOD1^G93A sample when 15A3 antibodies were pre-incubated with 8-OHG (<i>Ab block</i>). (D) Less amount of spinal cord mRNAs were oxidized (∼10% or less) in the end stage of SOD1^G93A mice (n = 3?). (E) RNA oxidation occurred in the pre-symptomatic stage of different mutant SOD1 mice. Oxidized mRNA pools were prepared from the spinal cords of indicated mutant SOD1 mice (n = 3). The ages of tested mice (<i>Age</i>) and the age of onset (<i>Onset</i>) are noted. Right panel shows densitometry analysis of Southern blot results.</p

Vitamin E reduces RNA oxidation and delays the disease onset, but not the survival.

<p>(A) immunofluorescent staining of lumbar spinal cords sections prepared from indicated mice showed that RNA oxidation is significantly decreased in vitamin E treated mice (n = 3 per group). Statistical analysis of immunoreactivity within motor neurons (n = 20) is shown. *<i>P</i><0.01 (B & C) The decline in motor performance by measuring grip strength was significantly delayed (∼14 days, *<i>P</i><0.005) in the vitamin E treated mice. There was no significant difference in the life span between the vitamin E treated and non-treated mice (D). The disease duration is significantly shortened (*<i>P</i><0.01) (E). n = 17 each group.</p

Some proteins corresponding to oxidized mRNA species are decreased.

<p>(A & B) immunofluorescent staining of lumbar spinal cord sections prepared from indicated mice (n = 3) showed that downregulation of protein levels in SOD1^G93A mice was found in cytochrome c oxidase VIb (<i>Cox VIb</i>) and NADH-ubiquinol oxidoreductase subunit 39 kDa (<i>NADH oxi</i>), whose mRNAs were highly oxidized but not EAAT3 protein, whose mRNA was not oxidized. Statistic analysis of immunoreactivity within motor neurons (n = 20) is shown. *<i>P</i><0.0001 (C) Immunoblot analysis showed that MBP, whose mRNA was oxidized, was decreased. Densitometry analysis (standardized by actin intensity) of immunoblot is shown (n = 3). #<i>P</i><0.01.</p