Trolox and 17β-Estradiol Protect against Amyloid β-Peptide Neurotoxicity by a Mechanism That Involves Modulation of the Wnt Signaling Pathway

Abstract

Oxidative stress is a key mechanism in amyloid ␤-peptide (A␤)-mediated neurotoxicity; therefore, the protective roles of 17␤-estradiol (E 2 ) and antioxidants (Trolox and vitamin C) were assayed on hippocampal neurons. Our results show the following: 1) E 2 and Trolox attenuated the neurotoxicity mediated by A␤ and H 2 O 2 as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays, quantification of apoptotic cells, and morphological studies of the integrity of the neurite network. 2) Vitamin C failed to protect neurons from A␤ toxicity. 3) A␤-mediated endoperoxide production, reported to induce cell damage, was decreased in the presence of E 2 and Trolox. 4) Two key Wnt signaling components were affected by E 2 and Trolox; in fact, the enzyme glycogen synthase kinase 3␤ was inhibited by both E 2 and Trolox, and both compounds were able to stabilize cytoplasmic ␤-catenin. 5) E 2 activated the expression of the Wnt-5a and Wnt-7a ligands, and at the same time, E 2 , through the ␣-estrogen receptor, was able to prevent the excitotoxic A␤-induced rise in bulk-free Ca 2؉as an alternative pathway to increase cell viability. 6) Finally, the Wnt-7a ligand protected against cytoplasmic calcium disturbances induced by A␤ treatment. Our results suggest that control of oxidative stress, regulation of cytoplasmic calcium, and activation of Wnt signaling may prevent A␤ neurotoxicity. Alzheimer disease (AD)1 is a neurodegenerative disease characterized by neuronal cell death, dystrophic neurites, neurofibrillary tangles, and senile plaques (1). Senile plaques are composed by the amyloid ␤-peptide (A␤), a 40 -42-amino acid peptide that originates from the proteolytic cleavage of the amyloid precursor protein (2). There is also evidence relating the etiopathology of AD with oxidative stress induced by A␤ in the brain of AD patients (3-6). A␤ increases the production of intraneuronal reactive oxygen species (ROS) and stimulates hydrogen peroxide (H 2 O 2 ) levels through metal ion reduction (7,8). Free radicals peroxidize membrane lipids (9) and oxidize proteins (10). In vitro experiments also support the observation that the neurotoxic effect of A␤ is mediated by free radical mechanisms (5, 11, 12) and alteration of Ca 2ϩ homeostasis (13). Furthermore, several studies have reported neuroprotection by antioxidants against A␤-mediated cytotoxicity (14 -16). Also, 17␤-estradiol (E 2 ; estrogen) treatment apparently has beneficial effects on AD (17, 18). In addition, E 2 prevents A␤-induced cell death by activation of the ␣-ER (19) and preserves neuronal viability and function in cortical neurons exposed to glutamate toxicity (20). Also, there is evidence that E 2 prevents morphological neurodegenerative changes in hippocampal neurons caused by A␤ deposits (21).On the other hand, neurofibrillary tangles are intracellular aggregates of paired helical filaments produced by hyperphosphorylation of the microtubule-associated protein tau (23). It has been proposed that glycogen synthase kinase-3␤ (GSK-3␤...

Hyperforin prevents β-amyloid neurotoxicity and spatial memory impairments by disaggregation of Alzheimer's amyloid-β-deposits

“…This result is important, since Ab oligomers (including stable dimers and trimers) can interfere with LTP and thus synaptic plasticity 45,47 generating the neurotoxicity observed in triple transgenic mice, 58 and ROS are involved in Ab neurotoxicity. 26,41,59 Therefore, our results suggest that the protection from the Ab neurotoxicity was partially mediated by the ROS reduction.…”

“…24,25 Image analysis The ROS fluorescence was imaged with a LSM 5 Pascal Zeiss confocal microscope, and analyzed by Sigman Scan Pro software. 26 The fluorescence intensity quantification was carried out as the difference between the final fluorescence emitted by the neurons in the presence of the respective treatment and the background fluorescence of the cells without treatment of 10 pictures of three independent experiments. The number of neurons in vivo experiments was calculated with the Image analysis (Image-Pro Express) in  40 magnification coronal brain sections stained with Nissl.…”

“…Primary neuronal hippocampal cell cultures and cell viability assay Hippocampal neurons were dissociated by trypsinization as described previously 26 and seeded at a density of 120 Â 10 3 cells per well of 48-well microtiter plates precoated with (50 mg/ml) polylysine, in 100 ml of Neurobasal medium supplemented with B-27 and maintained at 371C with an atmosphere 8% CO 2 . After 2 h, the culture media was changed.…”

“…Absorption values at 550-650 nm were determined using an automatic microtiter plate reader (Uniskan, Helsinki, Finland) and MTT reduction results were expressed as a percentage of control (untreated) cells. 26 …”

Rosiglitazone Treatment Prevents Mitochondrial Dysfunction in Mutant Huntingtin-expressing Cells

“…The fluorescence changes determined by Fluo-3 represent the cytoplasmic calcium changes (30,31), and Rhod-2 fluorescence indicate calcium changes in the mitochondria (32,33,34). To estimate Rhod-2 fluorescence pattern in live mitochondria, we used Mitotracker Green TM dye (MitoGreen) (33).…”