1. HMGB1 reverses mitochondrial DNA damage caused by mutant Atxn1 The mitochondrial DNA amplification assay with cerebellar tissue revealed that mitochondrial DNA damage was enhanced in mutant Atxn1‐KI mice but reversed in the double‐transgenic mice.
HMGB1 reverses mitochondrial DNA damage caused by mutant Atxn1 The mitochondrial DNA amplification assay with cerebellar tissue revealed that mitochondrial DNA damage was enhanced in mutant Atxn1‐KI mice but reversed in the double‐transgenic mice. The number of long cDNA fragments decreases when multiple DNA breaks occur between the primers, while short fragments are more easily amplified. The ratio of the long fragment to the short fragment was decreased in mutant Atxn1‐KI mice (right panel). The data are presented as mean ± SD. Statistical analysis involved Student's t‐test. The chloramphenicol (CAP) resistance assay of HeLa cells transfected with a control vector (pDsRed), Atxn1‐86Q expression vector (pDsRed‐Atxn1‐86Q) or Atxn1‐86Q expression vector + HMGB1 expression vector (pCI‐HMGB1). The results showed an increase in mitochondrial DNA damage in mutant Atxn1‐expressing HeLa cells. The increase was abrogated by co‐expression of HMGB1. The data are presented as mean ± SD. Statistical analysis involved Student's t‐test; *P < 0.05. Expression levels of Atxn1 and HMGB1 in transiently transfected HeLa cells used for the CAP assay in (B). Atxn1‐86Q was detected with an anti‐1C2 antibody. Analysis of mitochondrial genomic DNA using a next‐generation sequencer. The cumulative percentages of read counts are plotted against the read length (left panel). The read lengths in the mitochondrial genome are also presented with box plots (right panel). Both analyses indicate that the read length was disproportionally shifted to the shorter fraction in Atxn1‐KI mice. The shortening of the read length was reversed in Atxn1‐KI;HMGB1 mice. The shift of distribution was analysed statistically using Friedman's test (P = 2.19 × 10−31) followed by Wilcoxon rank‐sum test. P‐values were corrected using the Bonferroni method. The P‐value was 3.24 × 10−7 in the comparison between Atxn1‐KI mice and background mice (wild‐type) and 1.84 × 10−3 between Atxn1‐KI;HMGB1 mice and Atxn1‐KI mice. Frequency of mutation in the mitochondrial genome of the three genotypes of mice. The changes of mutation frequency were assessed statistically by Fisher's exact test and the post hoc Bonferroni correction. Mutation frequency was increased in Atxn1‐KI mice compared to background mice (wild‐type; P = 3.24 × 10−7) but normalized in Atxn1‐KI;HMGB1 mice compared to Atxn1‐KI mice (P = 1.84 × 10−3). Source data are available online for this figure.
Hikaru Ito et al. EMBO Mol Med. 2015;7:78-101
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