Early oxidative stress and DNA damage in Aβ-burdened hippocampal neurons in an Alzheimer's-like transgenic rat model

Abstract

Oxidative stress is a key contributor to AD pathology. However, the earliest role of pre-plaque neuronal oxidative stress, remains elusive. Using laser microdissected hippocampal neurons extracted from McGill-R-Thy1-APP transgenic rats we found that intraneuronal amyloid beta (iAβ)-burdened neurons had increased expression of genes related to oxidative stress and DNA damage responses including Ercc2, Fancc, Sod2, Gsr, and Idh1. DNA damage was further evidenced by increased neuronal levels of XPD (Ercc2) and γH2AX foci, indicative of DNA double stranded breaks (DSBs), and by increased expression of Ercc6, Rad51, and Fen1, and decreased Sirt6 in hippocampal homogenates. We also found increased expression of synaptic plasticity genes (Grin2b (NR2B), CamkIIα, Bdnf, c-fos, and Homer1A) and increased protein levels of TOP2β. Our findings indicate that early accumulation of iAβ, prior to Aβ plaques, is accompanied by incipient oxidative stress and DSBs that may arise directly from oxidative stress or from maladaptive synaptic plasticity.

Fig. 1. Laser captured iAβ-burdened neurons have increased expression of genes related to oxidative stress and DNA damage response.

a Representative image of McSA1 (Aβ) immunoreactivity in Tg hippocampus. b High magnification McSA1 immunoreactivity in subiculum (I) and CA1 (II) in Tg rats as from A. c Schematic depicting laser capture microdissection of CA1 and subiculum neurons from Tg Aβ-burdened neurons and Wt neurons not burdened with Aβ. d Differentially expressed genes including Ercc2, Fancc, Sod2, Gsr (GR), and Idh1 in Aβ-burdened Tg hippocampal neurons as compared to Wt neurons. Fold changes were normalized to Wt expression. Scale bars represent 500 µm in A, 50 µm in (b). n = 5–9 (Wt), n = 6–8 (Tg). Error bars indicate SD. two-tailed t-test, *p < 0.05, **p < 0.01.

Fig. 2. Protein levels of XPD (gene product of Ercc2) and Fancc in hippocampal neurons.

a Quantification of XPD immunoreactivity in subiculum and CA1 neurons of Wt and Tg rats normalized to Wt fluorescence intensity. b Representative images of XPD immunoreactivity (red) in Wt and Tg subiculum neurons with NeuN in green. c Higher magnification images of XPD immunoreactivity (red) in subiculum neurons with NeuN in green. d Quantification of Fancc immunoreactivity in CA1 and subiculum neurons of Wt and Tg rats normalized to Wt fluorescence intensity. e Quantification of Fancc immunoreactivity in the nuclei of CA1 and subiculum neurons normalized to Wt fluorescence intensity. f Representative images of Fancc immunoreactivity (red) in Wt and Tg CA1 neurons with NeuN in green, and DAPI in cyan. n = 9 (Wt), n = 7–8 (Tg). Error bars represent SD. Scale bars represent 50 µm in B and 10 µm in C. ns = non-significant, two-tailed t-test, *p < 0.05.

Fig. 3. Protein levels of GR and SOD2 in hippocampal neurons.

a Quantification of GR immunoreactivity in CA1 and subiculum neurons of Wt and Tg rats normalized to Wt fluorescence intensity. n = 9 (Wt), n = 7 (Tg). b Representative images of GR immunoreactivity (red) in Wt and Tg CA1 neurons with NeuN in cyan. c Enzyme activity of GR in 3-month (n = 6 (Wt), n = 6 (Tg)) and 5-month-old (n = 12 (Wt), n = 15 (Tg)) Wt and Tg cortical homogenates. d Quantification of SOD2 immunoreactivity in CA1 and subiculum neurons of Wt and Tg rats that had no parvalbumin (PV-) immunoreactivity. Values were normalized to Wt fluorescence intensity. e Quantification of SOD2 immunoreactivity in CA1 and subiculum of Wt and Tg PV+ neurons normalized to Wt fluorescence intensity. n = 9 (Wt), n = 7 (Tg). f Representative CA1 images of a Tg rat showing SOD2 immunoreactivity in red (I), PV+ neurons in green indicated by asterisks (*) (II), merged with NeuN in cyan (III). A higher magnification image of SOD2 (red) and NeuN (cyan) in CA1 (IV). Error bars represent SD. Scale bars represents 50 µm in B, F (I-III) and 10 µm in F (IV). ns = non-significant, two-tailed t-tests.

Fig. 4. Damage in iAβ-burdened neurons.

a Quantification of neurons with γH2AX-positive foci in CA1 and subiculum. b Representative image of γH2AX-positive foci in green (arrowhead) in the Tg subiculum (NeuN in magenta). c Quantification of 4HNE immunoreactivity in CA1 and subiculum of Wt and Tg neurons normalized to Wt fluorescence intensity. d Representative images of 4HNE immunoreactivity (red) in Wt and Tg CA1 neurons with an inset showing NeuN in green. e Quantification of 8-oxo-dG immunoreactivity in CA1 and subiculum of Wt and Tg neurons normalized to Wt fluorescence intensity. The left-most panels show the cytoplasmic or nuclear masks used to distinguish cytoplasmic versus nuclear immunoreactivity for quantification. f Representative image of 8-oxo-dG immunoreactivity in Wt and Tg CA1 neurons (green) with inset showing NeuN (magenta). n = 9 (Wt), n = 7 (Tg). Error bars represent SD. Scale bars represent 50 µm. ns = non-significant, two-tailed t-tests, *p < 0.05.

Fig. 5. Increased transcript levels of ERGs and TOP2β protein levels in hippocampal homogenates.

a Differentially expressed synaptic plasticity genes including Grin2b, CamkIIα, BdnfIV, c-fos and Homer1A in Tg hippocampal homogenates as compared to Wt. b Representative images (left) and quantification (right) of TOP2β immunoreactivity normalized to GAPDH levels in Wt as compared to Tg hippocampal homogenates as determined by Western blotting. Fold changes were normalized to Wt expression. n = 10–13 (Wt), n = 12–15 (Tg). Error bars indicate SD. Two-tailed t-test, *p < 0.05.