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.