Download Amyloid-β Peptide Induces Mitochondrial Oxidative Stress Damage

Amyloid-β Peptide Induces Mitochondrial Oxidative Stress Damage
in SH-SY5Y Neuroblastoma Cells
Han-Chang Hang1, 2, Chang-Jun Lin1, Zhao-Feng Jiang1, 2,*
1
College of Arts and Sciences, Beijing Union University, Beijing 100191, China
Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University
Beijing 100191, China
2
Dr. Huang Han-Chang
Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University
Beijing 100191, China.
197#, Beitucheng West Road, Haidian District, Beiijing, China
E-mail: [email protected]
Tel: 010-62004534
Corresponding author:
Prof. Zhao-Feng Jiang
Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University
Beijing 100191, China.
197#, Beitucheng West Road, Haidian District, Beiijing, China
E-mail: [email protected]
Tel.: +86 10 62004534;
Fax: +86 10 62388926.
Abstract
Background:
Alzheimer’s disease (AD) is one of the most common forms of neurodegenerative disease.
Amyloid-β (Aβ) is considered as a centre molecule and plays a key role in AD pathological
development. The accumulating aggregation of Aβ in neuritic plaques incurs neuronal oxidative
damage, neurofibrillary tangles, and loss of hippocampal neurite, synapse and neuron. The elevated
oxidative stress is thought to be links the age-related neurodegeneration. The elevated oxidative stress
is also thought to be a key even on the Aβ-induced neurotoxicity. However, the pathways of
Aβ-induced oxidative damages are under controversies. The processing of energy metabolism is a key
way to generate reactive oxygen species (ROS), which are the free radicals damaging bio-molecules.
Cell energy is mainly generated in mitochondria, which plays an important role in cell energy
metabolism. In this study, we investigated Aβ-induced oxidative damages on neurons and sub-cellular
structures, especially on mitochondria, based on our work and on the recent research progress
worldwide on the relations between oxidative stress and Alzheimer’s disease.
Methodology:
Cortical neurons or SH-SY5Y cells were cultured at 37℃ for 7days. After removed the culture
medium and the cultured cortical neurons were added fresh culture medium containing 5, 10 μM Aβ,
incubated at 37℃ in a humidified incubator. Morphological changes of cells were detected using a
microscope at differential interference contrast (DIC) model, and the chromatin condensation of
cultured cells was detected by the staining assay of nucleus with Hoechst 33342 dye (a molecular
probe). The hyperphosphoration of Tau and GSK-3beta activity were detected by Westblot analysis.
Cell viability were assayed by MTT, Hoechst 33342 assay and lactate dehydrogenase (LDH) assay, and
the ROS levels were detected by ROS assay based on the ROS-mediated conversion of nonfluorescent
2',7'-DCFH-DA into fluorescent DCFH. The free intracellular Ca 2+ in cells was measured using the
fluorescence Ca2+ indicator, fura-2/AM. Mitochondrial membrane potential was detected with JC-1
dye. Normal mitochondria (Red for J-aggregates) were recorded at Ex/Em 525/590 nm and depolarized
mitochondria (Green for monomer) were recorded at Ex/Em 490/530 nm with 300 ms integration time.
Cellular ATP levels were measured using a firefly luciferasebased ATP assay kit (Beyotime, China).
Results:
The results indicate that AD-like symptoms are observed in cultured cortical neurons and SH-SY5Y
cells. Aβ results in neuronal damages including cell viability and morphology, such as chromatin
condensation, cell bodies, dendrites, and interaction between cells. Aβ induces hyperphosphorylation of
Tau protein. GSK3-beta, as an important glycogen synthase, is involved in energy metabolism.
GSK-3beta is involved into the pathway of tau hyperphosphorylation. Cell damage induced by Aβ may
involve abnormity of cellular energy metabolism. To further understand the damage to energy
metabolism, we detected the effects of Aβ on the mitochondria. The results indicated that Aβ results in
the depolarization of mitochondrial membrane potential and the decrease of ATP generation but
increase of intracellular ROS. As the same time, cellular oxidative stress is increased when cells were
treated with Aβ; intracellular Ca2+ is increased less than 1 h after Aβ treatment, indicating that Ca2+
signaling is involved into the pathway of Aβ-induced neuronal damages.
Conclusions:
Aβ-induced tau hyperphosphorylation and neuronal damage are involved in mitochondrial
dysfunction. Ca2+ influx is involved into cellular oxidative damage.
Keywords:
protein.
Alzheimer’s disease, Amyloid-β, Mitochondrial damage, Oxidative stress, Tau