Evidence supporting oxidative stress in a moderately affected area of the brain in Alzheimer's disease

Abstract

The pathogenesis of Alzheimer's disease (AD) remains to be elucidated. Oxidative damage and excessive beta-amyloid oligomers are components of disease progression but it is unclear how these factors are temporally related. At post mortem, the superior temporal gyrus (STG) of AD cases contains plaques, but displays few tangles and only moderate neuronal loss. The STG at post mortem may represent a brain region that is in the early stages of AD or alternately a region resistant to AD pathogenesis. We evaluated expression profiles and activity of endogenous anti-oxidants, oxidative damage and caspase activity in the STG of apolipoprotein ε4-matched human AD cases and controls. Total superoxide dismutase (SOD) activity was increased, whereas total glutathione peroxidase (GPX), catalase (CAT) and peroxiredoxin (Prx) activities, were decreased in the AD-STG, suggesting that hydrogen peroxide accumulates in this brain region. Transcripts of the transcription factor NFE2L2 and inducible HMOX1, were also increased in the AD-STG, and this corresponded to increased Nuclear factor erythroid 2-related factor (NRF-2) and total heme-oxygenase (HO) activity. The protein oxidation marker 4-hydroxynonenal (4-HNE), remained unchanged in the AD-STG. Similarly, caspase activity was unaltered, suggesting that subtle redox imbalances in early to moderate stages of AD do not impact STG viability.

Figure 1

Pathology in the AD-STG. (A) (i–ii) Photomicrographs of Garvey silver staining from the STG of (i) a control showing no pathology and (ii) an AD case with numerous plaques and a single intracellular NFT (indicated by →). Scale bar in main images represents 20 μm. Scale bar to the insets represents 40 μm. (iii–iv) Photomicrographs of AT8 immunostaining from the STG of (iii) a control and (iv) an AD case with numerous tau-positive neurons including an intracellular NFT (indicated by →). CP = cored plaque; DP = diffuse plaque; IT = intracellular neurofibrillary tangle. (B) AT8 (tau) immunoblotting of whole brain homogenates showing hyperphosphorylated tau (positive bands in the range 45–79 kDa) in the AD samples (for complete image refer to Supplementary Fig. S1).

Figure 2

SOD and GPX protein expression and activity in the STG of AD cases and controls. Sample supernatants displaying (A) Quantification of SOD1, SOD2 and GPX1 protein expression by direct (B) Total SOD activity, reflected by the inhibition of pyrogallol auto-oxidation as a % of a control lacking the presence of SOD, and (C) Total GPX activity, assessed by monitoring the consumption of NADPH. Data represent mean ± SD, n = 19 for control and n = 20 individual AD cases; *p < 0.05.

Figure 3

Determinations of H₂O₂-consuming activity in the AD- and control-STG. (A) Total H₂O₂ consuming activity in the control and AD STG was assessed by treating supernatants with 0.1% v/v H₂O₂ (vehicle). Additionally, H₂O₂ consuming activity was assessed following pre-treatment with the catalase inhibitor, aminotriazole (AT) and sodium azide in both subject groups. (B) H₂O₂ consumption attributed to catalase (difference between vehicle and AT consumption rates) displayed as a fold change relative to the control group (n = 5 controls and n = 5 AD subjects) (C) A modified FOX assay displaying the peroxiredoxin specific consumption of cumen H₂O₂ (n = 19 control and n = 19 individual AD cases). Data represent mean ± SD; *p < 0.05, **p < 0.01 & ***p < 0.0001.

Figure 4

NRF-2 protein expression and distribution in the AD- and control-STG. Photomicrographs of NRF2 protein staining of the STG grey matter in a (A) control and (B) AD case. (C) Image semi-quantification of NRF2 positively stained pixels. Data represent mean ± SD. Within image panels → indicates cytosolic staining and ►  indicates nuclear staining, n = 14 for control and n = 16 individual AD subjects. (D) Representative blots for cystosolic fraction from control and AD samples (23 μg protein loading) displaying NRF2 band (95 kDa, refer to Supplementary Fig. S2 for complete gel image; protein band at ~50 kDA was not included in the assessment) and corresponding total protein bands imaged prior to protein transfer. (E) Densitometry of NRF-2 immunoblot bands using cytosolic STG fractions, normalized to total protein. Note, multiple blots were processed in parallel with a constant exposure time (125 sec) to allow semi-quantitiation over the complete control and AD sample sets. Data represent mean ± SD, n = 19 controls and n = 19 individual AD cases. *p < 0.05 & **p < 0.01.

Figure 5

Total HO activity in AD and control STG. Microsomal fractions of STG homogenates exposed to hemin and biliverdin reductase allowed for the HO dependent conversion of heme-to-bilirubin. Data reflect bilirubin levels, as determined by HPLC analysis. Data represent mean ± SD, n = 19 for control and n = 21 individual AD cases. *p < 0.05.

Figure 6

4-HNE immune-reactivity in AD- and control-STG. Photomicrographs of 4-HNE immunostaining of the STG grey matter in a (A) control and (B) AD case (C) Image quantification of 4-HNE⁺ stained pixels. Data represent mean ± SD, n = 19 for control and n = 19 AD cases.

Figure 7

Caspase 3/7 activity in AD and control-STG. Supernatants were treated using the Caspase-Glo 3/7 assay kit (Promega). Caspase 3/7 activity was determined for each homogenate after 60 min of monitoring the readout. Data represent mean ± SD, n = 19 for control and n = 20 individual AD cases.

Figure 8

Correlational outcomes of HO, SOD, CAT and Prx activity with AD pathology. Total HO activity in the AD STG was correlated with both (A) plaque areal fraction (p = 0.04, r² = 0.21) and (B) total NTFs (p = 0.03, r² = 0.24) In addition, (C) total NFTs were correlated with total SOD activity (p = 0.03, r² = 0.23) while the number of AT8- tau positive cells were correlated with (D) total CAT activity (p = 0.05, r² = 0.77) and (E) total Prx activity (p = 0.02, r² = 0.26).