Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 35 (2), 302-10

Oxidative Damage to the Promoter Region of SQSTM1/p62 Is Common to Neurodegenerative Disease

Affiliations

Oxidative Damage to the Promoter Region of SQSTM1/p62 Is Common to Neurodegenerative Disease

Yifeng Du et al. Neurobiol Dis.

Abstract

Recently we reported that declined SQSTM1/p62 expression in Alzheimer disease brain was age-correlated with oxidative damage to the p62 promoter. The objective of this study was to examine whether oxidative damage to the p62 promoter is common to DNA recovered from brain of individuals with neurodegenerative disease. Increased 8-OHdG staining was observed in brain sections from Alzheimer's disease (AD), Parkinson disease (PD), Huntington disease (HD), Frontotemporal dementia (FTD), and Pick's disease compared to control subjects. In parallel, the p62 promoter exhibited elevated oxidative damage in samples from various diseases compared to normal brain, and damage was negatively correlated with p62 expression in FTD samples. Oxidative damage to the p62 promoter induced by H2O2 treatment decreased its transcriptional activity. In keeping with this observation, the transcriptional activity of a Sp-1 element deletion mutant displayed reduced stimulus-induced activity. These findings reveal that oxidative damage to the p62 promoter decreased its transcriptional activity and might therefore account for decreased expression of p62. Altogether these results suggest that pharmacological means to increase p62 expression may be beneficial in delaying the onset of neurodegeneration.

Figures

Fig. 1
Fig. 1
Representative immunohisitochemical staining for 8-OHdG in brain. Sections were treated with DNase I or incubated without primary antibody did not display any immunoreactivity for 8-OHdG. Little or no immunoreactivity was observed in sections taken from normal brain. DNA damage is present in neurodegenerative diseased brains than in normal brains. Variable but reproducible staining to 8-OHdG was observed in sections of AD, FTD, HD, Pick’s and PD. The staining was mostly cytoplasmic with some nuclear deposits (Inset-arrow).
Fig. 2
Fig. 2
Profile of oxidative damage in 3 amplicons within p62 promoter of normal and various samples from neurodegenerative diseased brains. A. Three amplicons, amplicon 1 (−1085 ~ −954), amplicon 2 (−407 ~ −321) and amplicon 3 (−1608 ~ −1484), were designed within the human p62 promoter. Amplicon 2 is the closest amplicon to the transcription initiation site and amplicon 3 is the furthest one. GC content of each amplicon and transcription factor binding sites within the p62 promoter were shown. B. Oxidative damage to each amplicon in control and various neurodegenerative diseased brains was assessed. Neurodegenerative diseased samples (N shown in the bar) include Alzheimer’s Disease (AD), Pick’s, Frontotemporal Dementia (FTD), Huntington Disease (HD) and Parkinson Disease (PD). Oxidative damage is expressed as Mean % ± S.D. One-way ANOVAs indicated significant overall differences among the 6 groups for each amplicon (amplicon 1, F = 6.21, p <0.001; amplicon 2, F = 4.03, p < 0.01; amplicon 3, F = 4.98, p < 0.01). Individual comparisons between control and disease associated damage levels were conducted using one-tailed t tests with the group-wise p-value adjusted for multiple tests by the Stepdown Sidak approach. Damage to each amplicon from all 5 disease groups was significantly higher than control subjects (* indicates adjusted p < 0.05). C. DNA immunoprecipitation of amplicon 2 within the p62 promoter with a polyclonal antibody to 8-OHdG (Chemicon) in normal and various neurodegenerative diseased brain samples. Input DNA for each sample is shown. Relative density of each band was quantified. The PCR products of amplicon 2 from each of the neurodegenerative diseased samples were higher than control samples. ANOVA indicated overall statistical significance among the 8-OHdG levels in the 5 diseased and 1 control groups (F = 5.69. p = 0.028). Significant differences between individual diseases and the control are indicated by ** (adjusted p < 0.001).
Fig. 2
Fig. 2
Profile of oxidative damage in 3 amplicons within p62 promoter of normal and various samples from neurodegenerative diseased brains. A. Three amplicons, amplicon 1 (−1085 ~ −954), amplicon 2 (−407 ~ −321) and amplicon 3 (−1608 ~ −1484), were designed within the human p62 promoter. Amplicon 2 is the closest amplicon to the transcription initiation site and amplicon 3 is the furthest one. GC content of each amplicon and transcription factor binding sites within the p62 promoter were shown. B. Oxidative damage to each amplicon in control and various neurodegenerative diseased brains was assessed. Neurodegenerative diseased samples (N shown in the bar) include Alzheimer’s Disease (AD), Pick’s, Frontotemporal Dementia (FTD), Huntington Disease (HD) and Parkinson Disease (PD). Oxidative damage is expressed as Mean % ± S.D. One-way ANOVAs indicated significant overall differences among the 6 groups for each amplicon (amplicon 1, F = 6.21, p <0.001; amplicon 2, F = 4.03, p < 0.01; amplicon 3, F = 4.98, p < 0.01). Individual comparisons between control and disease associated damage levels were conducted using one-tailed t tests with the group-wise p-value adjusted for multiple tests by the Stepdown Sidak approach. Damage to each amplicon from all 5 disease groups was significantly higher than control subjects (* indicates adjusted p < 0.05). C. DNA immunoprecipitation of amplicon 2 within the p62 promoter with a polyclonal antibody to 8-OHdG (Chemicon) in normal and various neurodegenerative diseased brain samples. Input DNA for each sample is shown. Relative density of each band was quantified. The PCR products of amplicon 2 from each of the neurodegenerative diseased samples were higher than control samples. ANOVA indicated overall statistical significance among the 8-OHdG levels in the 5 diseased and 1 control groups (F = 5.69. p = 0.028). Significant differences between individual diseases and the control are indicated by ** (adjusted p < 0.001).
Fig. 3
Fig. 3
Correlation between p62 expression and oxidative damage in the p62 promoter in FTD. A. p62 expression in 5 human FTD brain tissues. An equal concentration of whole lysate protein (50 µg) from 5 human FTD brains were Western blotted with p62 and tubulin. P62 relative expression was expressed as the ratio of the relative intensity of p62 to that of tubulin. B. Correlation analysis between p62 expression and the average oxidative DNA damage of 3 amplicons. p62 relative expression is negatively correlated with DNA damage within the p62 promoter (r = −0.85; p = 0.067).
Fig. 4
Fig. 4
Diagram of primers used for p62 promoter amplification and sequencing. Two specific primers, PWF and PWR were used to amplify p62 promoter from genomic DNA. The location of five sequencing primers, PWF1-S, PWF2-S, PWF3-S, PWF4S, and PWR1S were shown.
Fig. 5
Fig. 5
Oxidative stress reduces p62 promoter activity. A. p62 promoter activity decreased upon in vitro H2O2 treatment. p62 promoter-pGL3 was incubated in the absence or presence of 100 µM H2O2 for one hour at 4°C in vitro. Non-treated and H2O2-treated p62 promoter-pGL3 were transfected into HEK cells along with pRL-TK and PDEF. Cells were lysed and analyzed by Dual-Luciferase Reporter Assay after 48 h. Relative promoter activity was expressed as Mean ± S.E.M. (N = 3). B. Oxidative DNA damage to 3 amplicons within the p62 promoter upon in vitro H2O2 treatment. Non-treated p62 promoter-pGL3 construct and in vitro 100 µM H2O2-treated p62 promoter-pGL3 were subjected to oxidative DNA damage assay for three amplicons. The 10 X dilution of the p62 promoter-pGL3 construct was used to generate standard curve. * p < 0.05, significant difference.
Fig. 6
Fig. 6
Induction of the wild type and Sp-1 deleted p62 promoter upon H2O2 treatment. WT p62 promoter-pGL3 and Sp-1 deletion mutant were transfected into HEK cells along with pRL-TK and PDEF. The cells were treated with 600 µM H2O2 for 16 h, post-transfection, lysed and subjected to dual-luciferase assay. The p62 promoter-pGL3 activity was normalized by pRL-TK activity. The induction was expressed as the ratio of the relative promoter activity of 600-µM-treatment to that of non-treatment (Mean ± S.E.M. (N = 6); ** p < 0.001).

Similar articles

See all similar articles

Cited by 27 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback