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. 2017 Oct;16(4):4521-4528.
doi: 10.3892/mmr.2017.7203. Epub 2017 Aug 10.

Amyloid β-42 induces neuronal apoptosis by targeting mitochondria

Xiao-Jian Han  1 , Yang-Yang Hu  1 , Zhang-Jian Yang  2 , Li-Ping Jiang  2 , Sheng-Lan Shi  1 , Ye-Ru Li  2 , Miao-Yu Guo  1 , Hong-Li Wu  3 , Yu-Ying Wan  3
Affiliations

Amyloid β-42 induces neuronal apoptosis by targeting mitochondria

Xiao-Jian Han et al. Mol Med Rep. 2017 Oct.

Abstract

Alzheimer's disease (AD), with a typical pathological hallmark of amyloid‑beta (Aβ)‑containing plaques and neurofibrillary tangles, is one of the most common types of chronic neurodegenerative diseases. Aβ oligomers serve a crucial role in the pathogenesis of AD, and lead to neuronal loss. However, the precise mechanism of Aβ oligomers in AD remains to be elucidated. The present study demonstrated that 10 µM Aβ‑42 activated the caspase signaling pathway, and induced significant apoptosis in primary cultured mouse cerebral cortical neurons. The results of reverse transcription‑quantitative polymerase chain reaction and western blotting demonstrated that Aβ‑42 (10 µM) also significantly upregulated the transcription and expression of the mitochondrial fission protein dynamin‑related protein 1 (Drp1), and downregulated the transcription and expression of mitochondrial fusion proteins, including mitofusin 1/2 (Mfn1/2) and mitochondrial dynamin like GTPase (OPA‑1). Neurons were transfected with pDsRed2‑Mito for mitochondrial imaging, which revealed that 10 µM Aβ‑42 induced mitochondrial fission in cortical neurons. In addition, 2',7'‑dichlorodihydrofluorescein diacetate and tetramethylrhodamine ethyl ester staining indicated that Aβ‑42 increased the reactive oxygen species (ROS) level and reduced mitochondrial membrane potential in neurons. Inhibition of Drp1 activity by Mdivi‑1 efficiently prevented Aβ‑42‑induced ROS production and disruption of mitochondrial membrane potential. Loss of mitochondrial membrane potential may activate PTEN‑induced putative kinase 1 (Pink1), the prominent sensor for mitochondrial damage, and trigger the process of mitophagy to remove the damaged mitochondria. In the present study, western blotting revealed that the levels of autophagy marker microtubule‑associated proteins 1A/1B light chain 3B (LC3B) and Pink1 were upregulated after Aβ‑42 stimulation. In conclusion, these data indicated that Aβ‑42 induces neuronal apoptosis by targeting mitochondria, including promotion of mitochondrial fission, disruption of mitochondrial membrane potential, increasing intracellular ROS level and activation of the process of mitophagy. Therefore, mitochondria may represent a potential therapeutic target for AD in the future.

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Figures

Figure 1.
Figure 1.
Effect of Aβ-42 at different concentrations on mouse cerebral cortical neurons. Representative fluorescence microscopy images of mouse cerebral cortical neurons (10 days in vitro) exposed to Aβ-42. Scale bar=20 µm. Aβ-42, amyloid β-42.
Figure 2.
Figure 2.
Aβ-42-induced cortical neuronal apoptosis and activated caspase pathway. (A) Representative fluorescence microscopy images and (B) quantification of neuronal apoptosis of mouse cerebral cortical neurons (10 days in vitro) exposed to Aβ-42 and stained with Hoechst 33258 and MAP2. Scale bar=50 µm. (C) Representative western blot images and (D) quantification of caspase 3 protein expression levels. Data are presented as the mean ± standard deviation of at least three independent experiments. *P<0.05 vs. 0 h, **P<0.01 vs. Cont or 0 h. Cont, control; Aβ-42, amyloid β-42.
Figure 3.
Figure 3.
Transcription and expression of mitochondrial fission and fusion proteins after Aβ-42 treatment. mRNA expression levels of (A) Drp1 and (B) Mfn1, Mfn2 and OPA-1. (C) Representative western blot images and (D) quantification of Drp1, Mfn1, Mfn2 and OPN-1 protein expression levels. GAPDH served as an endogenous control. Data are presented as the mean ± standard deviation of at least three independent experiments. *P<0.05, **P<0.01 vs. 0 h. Aβ-42, amyloid β-42; Drp1, dynamin-related protein 1; Mfn, mitofusin; OPA-1, mitochondrial dynamin like GTPase.
Figure 4.
Figure 4.
Effect of Aβ-42 on mitochondrial fission and intracellular ROS production. (A) Mitochondrial morphology in neurons pre- and post-treatment with Aβ-42. Scale bar=5 µm. (B) Effect of Aβ-42 on intracellular ROS production. Scale bar=20 µm. ROS, reactive oxygen species; Aβ-42, amyloid β-42.
Figure 5.
Figure 5.
Aβ-42 disrupts mitochondrial membrane potential and upregulates expression of LC3B and Pink1. (A) Effect of Aβ-42 on mitochondrial membrane potential in neurons, as assessed by tetramethylrhodamine ethyl ester staining. Scale bar=20 µm. (B) Representative western blot images and (C) quantification of protein expression levels of LC3B and Pink1 after Aβ-42 treatment. GAPDH was used as an endogenous control. Data are presented as the mean ± standard deviation of at least three independent experiments. *P<0.05 vs. 0 h. Aβ-42, amyloid β-42; LC3B, microtubule-associated proteins 1A/1B light chain 3B.
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