Mitochondrial cholesterol loading exacerbates amyloid beta peptide-induced inflammation and neurotoxicity

Abstract

The role of cholesterol in Alzheimer's disease (AD) has been linked to the generation of toxic amyloid beta peptides (Abeta). Using genetic mouse models of cholesterol loading, we examined whether mitochondrial cholesterol regulates Abeta neurotoxicity and AD pathology. Isolated mitochondria from brain or cortical neurons of transgenic mice overexpressing SREBP-2 (sterol regulatory element binding protein 2) or NPC1 (Niemann-Pick type C1) knock-out mice exhibited mitochondrial cholesterol accumulation, mitochondrial glutathione (mGSH) depletion and increased susceptibility to Abeta1-42-induced oxidative stress and release of apoptogenic proteins. Similar findings were observed in pharmacologically GSH-restricted rat brain mitochondria, while selective mGSH depletion sensitized human neuronal and glial cell lines to Abeta1-42-mediated cell death. Intracerebroventricular human Abeta delivery colocalized with mitochondria resulting in oxidative stress, neuroinflammation and neuronal damage that were enhanced in Tg-SREBP-2 mice and prevented upon mGSH recovery by GSH ethyl ester coinfusion, with a similar protection observed by intraperitoneal administration of GSH ethyl ester. Finally, APP/PS1 (amyloid precursor protein/presenilin 1) mice, a transgenic AD mouse model, exhibited mitochondrial cholesterol loading and mGSH depletion. Thus, mitochondrial cholesterol accumulation emerges as a novel pathogenic factor in AD by modulating Abeta toxicity via mGSH regulation; strategies boosting the particular pool of mGSH may be of relevance to slow down disease progression.

Figure 1.

Increased susceptibility to Aβ1–42 exposure of brain mitochondria and cortical neurons from SREBP-2 transgenic mice. A, Total cholesterol levels and GSH levels of brain homogenate (Homo.) and isolated mitochondria (Mit.) from WT and SREBP-2 transgenic (Tg) mice; *p < 0.05 (n = 6–8 per genotype). B, C, WT and transgenic (TgSREBP-2) mitochondria (1 mg/ml) were exposed to Aβ1–42 (1 μm) for 2 h. B, Hydrogen peroxide generation determined by DCF fluorescence (A.U.: arbitrary units). *p < 0.05, **p < 0.01 (n = 6–8 per genotype). C, After Aβ treatment mitochondria were pelleted and the resulting supernatants (Spnt) and pellets were analyzed for the presence of apoptogenic proteins. Shown are representative immunoblottings of cytochrome c (Cyt C) and Smac/DIABLO (n = 4). D, E, Primary neurons isolated from WT and transgenic (TgSREBP-2) cerebral cortices at 6 DIV. D, Representative confocal images of cortical neurons stained with filipin (red) and mouse anti-cytochrome c antibody followed by the appropriated FITC-conjugated secondary antibody (Cyt C, green). Scale bar: 10 μm. E, Cell death of cortical neurons after exposure to increasing doses of Aβ1–42 as indicated for 48 h. *p < 0.05 versus untreated cells (0 nm), # p < 0.01 versus WT cells (n = 4–6). Values are mean ± SD; mean differences were compared by unpaired Student's t test.

Figure 2.

Isolated brain mitochondria from NPC1 knock-out mice show an increased sensitivity to Aβ1–42. A, Total cholesterol levels (white bars) and GSH levels (black bars) of isolated mitochondria from NPC1^+/+ and NPC1^−/− brains. *p < 0.05 (n = 3–4 per genotype). B, Hydrogen peroxide generation determined by DCF fluorescence of mitochondria (1 mg/ml) incubated with Aβ1–42 (1 μm) for 2 h. *p < 0.05, **p < 0.01 (n = 3–4 per genotype). C, After 2 h treatment with Aβ1–42 (1 μm) mitochondria were pelleted and the resulting supernatants (Spnt) and pellets were analyzed by Western blotting for the presence of cytochrome c (Cyt C) and Smac/DIABLO. Representative immunoblottings are shown. Values are mean ± SD; mean differences were compared by unpaired Student's t test. prot., Protein; A.U., arbitrary units.

Figure 3.

Mitochondrial GSH regulates Aβ-induced oxidative stress. Depletion of mitochondrial GSH levels was assessed in vitro by incubation of isolated mitochondria (1 mg/ml) with 5 μm ethacrynic acid (EA) for 15 min or in vivo by inhibiting the GSH synthesis with buthionine sulfoximine treatment (BSO, 3 mmol/kg). A, GSH levels after EA or BSO treatment. *p < 0.05 (n = 4–6). B, C, Mitochondria from rat brain pretreated with EA or BSO were exposed to Aβ 1–42 (5 μm) or the inactive form Aβ 42–1 (5 μm) for 2 h. B, Hydrogen peroxide generation determined by DCF fluorescence (A.U.: arbitrary units; NT: untreated mitochondria). *p < 0.05 (n = 4–6). C, After Aβ treatment mitochondria were pelleted and the resulting supernatants (spnt) and pellets were analyzed for the presence of cytochrome c (Cyt C) and Smac/DIABLO. Shown are representative immunoblottings of three independent experiments. D, Effect of blocking the electron transport chain at complex I, II, or III on the generation of hydrogen peroxide by Aβ. After EA treatment mitochondria were incubated with rotenone and TTFA (Rot/TTFA, 20 and 15 μm, respectively), antimycin A (Antimyc. A, 5 μm), and/or Aβ (5 μm) during 60 min. The blockers of the respiratory complexes were added 15 min before exposure to Aβ. Hydrogen peroxide generation was assessed by DCF fluorescence. *p < 0.05 versus untreated mitochondria; ^# p < 0.01 versus Aβ-treated mitochondria. (n = 3). Values are mean ± SD; mean differences were compared by Dunnett's test. prot., Protein; Ctrl., control.

Figure 4.

Selective depletion of mitochondrial GSH in human neuroblastoma SH-SY5Y cells enhances the apoptotic cell death induced by Aβ. Mitochondrial GSH depletion was assessed by incubating cells with (S)-3-hydroxy-4-pentenoate (HP, 5 mm) for 10 min. A, GSH levels. *p = 0.002 (n = 4). Cyt., Cytosol; Mit., mitochondria. B, Cell viability after 24 h incubation with increasing concentrations of Aβ1–42. *p < 0.01 versus HP-treated cells at 0 μm, **p = 0.02 versus control (Ctrl.) cells at 0 μm; n.s., not significant (n = 4). C, Chromatin morphology of cells exposed to Aβ1–42 (5 μm, 24 h) analyzed by Hoechst-33258 staining. Original magnification, 200× (n = 4). D, Caspase-3 activity of cell extracts from cells exposed to Aβ1–42 (5 μm, 24 h) with or without the pancaspase inhibitor qVD-OPH (20 μm). *p < 0.05 versus HP-treated cells. (n = 3). E, F, After preincubation with the antioxidant agents N-acetyl-cysteine (NAC, 3 mm) or butylated hydroxyanisole (BHA, 20 μm) and/or the caspase inhibitor qVD-OPH (20 μm) for 2 h, mitochondrial GSH was depleted by HP treatment and cells were exposed to Aβ 1–42 (5 μm). (n = 3–4). E, Cell death analyzed by PI staining at 24 h exposure to Aβ1–42. *p < 0.01. F, Hydrogen peroxide generation after 2 h incubation with Aβ1–42. *p < 0.05 (A.U.: arbitrary units). Values are mean ± SD; mean differences were compared by Dunnett's test (B, E, and F) or unpaired Student's t test.

Figure 5.

Increased neuroinflammation in SREBP-2 transgenic mice after Aβ1–42 infusion. WT and SREBP-2 transgenic (TgSREBP-2) mice were subjected to continuous intracerebroventricular infusion of vehicle or human Aβ1–42 solution (1.2 μg/d) for 28 d; then, half of the brain was processed for immunohistochemistry and the other hemisphere used for biochemical analysis. n = 6–8 mice per group. A, Aβ1–42 immunohistochemical staining. Representative photomicrographs of hippocampus from WT and transgenic mice after infusion are shown. Scale bar: 100 μm. B, Confocal analysis of Aβ internalization. Shown are representative images of Aβ1–24 (green) and cytochrome c (red) immunofluorescence from hippocampal sections of TgSREBP-2 brains after the infusion period. Merged image indicates partial colocalization of Aβ with the mitochondrial marker cytochrome c (Cyt C, yellow). Nuclei were stained with Hoechst-33258 (shown only in merged image; blue). Scale bar: 10 μm. C, Activation of microglia and astrocytes analyzed by F4/80 and GFAP immunostaining, respectively. Shown are representative photomicrographs of F4/80 and GFAP immunoreactivity as indicated, in sections of hippocampus from infused WT and SREBP-2 transgenic (Tg) mice. Scale bar: 100 μm. D, TNF-α and IL-1β mRNA expression analyzed by real-time PCR from WT and SREBP-2 transgenic (Tg) brain after vehicle or Aβ infusion. Shown are relative values after normalization against 18 s expression and are representative of three experiments ± SD, *p < 0.01 versus vehicle-infused TgSREBP-2 mice. Mean differences were compared by unpaired Student's t test.

Figure 6.

Aβ-infused TgSREBP-2 mice display enhanced oxidative stress and neuronal damage which is prevented by GSH ethyl ester (GSHee) coinfusion. WT and TgSREBP-2 mice were subjected to continuous infusion of vehicle or human Aβ1–42 solution (1.2 μg/d) for 28 d. n = 6–8 mice per group. A, Lipid peroxidation estimated as production of malondialdehyde (MDA). *p < 0.03. B, Representative immunoblotting showing presence of carbonyl proteins in WT and TgSREBP-2 (Tg) brains after infusion. C, Representative immunoblotting showing synaptophysin (Synapt.) protein levels after infusion. Densitometric values of the bands representing synaptophysin immunoreactivity were normalized with the values of the corresponding β-actin bands (O.D.: normalized optical density). D, Representative images of degenerated neurons in hippocampal regions by Fluoro-Jade B staining. Scale bar: 50 μm. E, Representative images of apoptotic cells in hippocampus by terminal deoxynucleotidyl transferase-mediated nick-end labeling. Scale bar: 25 μm. F, Lipid peroxidation in infused TgSREBP-2 brains estimated as malondialdehyde levels (MDA). *p < 0.01 (n = 6). G, TNF-α and IL-1β mRNA expression analyzed by real-time PCR from infused TgSREBP-2 brains. Relative values were normalized against 18 s expression. *p < 0.05, **p < 0.01 (n = 6). Values are mean ± SD; mean differences were compared by unpaired Student's t test.

Figure 7.

GSH ethyl ester (GSHee) intraperitoneal therapy protects against Aβ-induced neurodegeneration in Tg-SREBP-2 mice. WT and TgSREBP-2 mice were subjected to continuous infusion of human Aβ1–42 solution (1.2 μg/d) for 28 d. GSH ethyl ester (1.25 mmol/kg/d) or vehicle alone were administered intraperitoneally over the last 2 week of the infusion period. n = 4–6 mice per group. A, Mitochondrial GSH levels. *p = 0.01. prot., Protein. B, Lipid peroxidation estimated as production of malondialdehyde (MDA). *p < 0.01. C, Representative immunoblotting showing presence of carbonyl proteins. D, Activation of astrocytes analyzed by GFAP immunostaining. Shown are representative photomicrographs GFAP immunoreactivity in hippocampal sections. Scale bar: 100 μm. E, TNF-α expression analyzed by real-time PCR. Relative values were normalized against 18 s expression. *p < 0.05. F, Representative images of apoptotic cells in hippocampus by terminal deoxynucleotidyl transferase-mediated nick-end labeling. Scale bar: 25 μm. Values are mean ± SD; mean differences were compared by unpaired Student's t test.

Figure 8.

Brain mitochondria from 10-month-old AD transgenic mice exhibit increased cholesterol and depleted GSH levels. A, Total cholesterol levels. B, GSH levels of isolated brain mitochondria from WT mice and AD transgenic mice (Tg-APP/PS1) at the indicated ages. *p < 0.05 (n = 3–4). C, D, SREBP-2 expression in brain extracts from WT mice and Tg-APP/PS1 transgenic mice at the indicated ages. C, SREBP-2 mRNA expression analyzed by real-time PCR. Relative values were normalized against 18 s expression. *p < 0.05 (n = 3–4). D, Representative immunoblotting showing SREBP-2 protein levels. β-Actin levels were analyzed as a loading control. Values are mean ± SD; mean differences were compared by unpaired Student's t test. prot., Protein.