Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β-amyloid peptide

Abstract

Alzheimer's disease (AD) is an aging-related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP-ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up-regulated the lysoso-mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3-methylade-nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ-induced apoptosis.

Fig 1

Western blotting of low MW Aβ peptide. Sixteen percent tris-tricine SDS-PAGE showing the molecular weight of low MW Aβ (A) of indicated amount loaded, and (B) after incubation in culture media of indicated time. 6E10 antibody was used for the detection of Aβ peptide.

Fig 2

Low MW Aβ induced cleavage of caspase 3 and PARP. Western blotting of cleaved caspase 3 (Asp 175) and PARP in primary cortical neurons treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 4 to 30 hrs. β-actin was used as an internal control. Figure shows the representative result from three independent experiments.

Fig 3

Low MW Aβ did not significantly affect AIF gene expression. AIF mRNA level in neurons treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 4 to 30 hrs was measured by Q-PCR with 18S ribosomal RNA as internal control. Relative gene expression level was analysed with Pfaffl method. Bar chart plots the means ± S.E.M. of a triplicate from three independent experiments. Empty bars indicate the control at each time-point.

Fig 4

Low MW Aβ did not induce mito-chondrial release and nuclear entry of AIF. Neurons transfected with Mito-GFP were treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 8 or 24 hrs. The neurons were stained with anti-AIF antibody. Representative Z-stack confocal images from two independent experiments are shown. Scale bar, 10 μM.

Fig 5

Low MW Aβ induced the formation of autophagic vesicles. Primary cortical neurons transfected with LC3-DsRed were treated with low MW Aβ_1–42 of 10 μM or DMSO (vehicle) for indicated time. Representative Z-stack confocal images from three independent experiments are shown. Scale bar, 10 μM.

Fig 12

Autophagy protects neurons from Aβ-induced apoptosis. (A) Neurons trans-fected with LC3-DsRed were treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 18 hrs. Neurons were then stained with anti-cleaved caspase 3 (Asp 175) antibody. Representative Z-stack con-focal images from two independent experiments are shown. Scale bar, 10 μM. (B) Percentage of LC3-trans-fected cells showing positive immunoreactivity for cleaved caspase 3. Bar chart plots the means ± S.E.M. of three independent experiments. *P < 0.05, represents significant difference from the reference value. Empty bars indicate the control at each time-point.

Fig 10

Low MW Aβ induced the lysosomal machinery. Neurons were treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 24 hrs. (A) Neurons were stained with anti-LAMP2 antibody. Scale bar, 20 μM. (B) Neurons transfected with LC3-DsRed prior treatment were stained with 200 nM LysoTracker Green®. Representative live cell Z-stack confocal images from two independent experiments are shown. Scale bar, 10 μM.

Fig 6

Low MW Aβ did not significantly affect BECN1 gene expression. mRNA levels of BECN1 in neurons treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 4 to 30 hrs were measured by Q-PCR with 18S ribosomal RNA as internal control. Relative gene expression level was analysed with Pfaffl method. Bar chart plots the means ± S.E.M. of a triplicate from three independent experiments. Empty bars indicate the control at each time-point.

Fig 7

Low MW Aβ did not significantly induce BECN1. (A) Neurons were treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 4 to 8 hrs. The neurons were stained with anti-BECN1 antibody. Representative Z-stack con-focal images from two independent experiments are shown. Scale bar, 20 μM. (B) Relative intensity of BECN1 staining. Intensity of signals were measured by Image J. Bar chart plots the means ± S.E.M. of at least three images. *P < 0.05, represents significant difference from the reference value. Empty bars indicate the control at each time-point.

Fig 8

Low MW Aβ did not significantly affect ATG5 gene expression. mRNA levels of ATG5 in neurons treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 4 to 30 hrs were measured by Q-PCR with 18S ribosomal RNA as internal control. Relative gene expression level was analysed with Pfaffl method. Bar chart plots the means ± S.E.M. of a triplicate from three independent experiments. Empty bars indicate the control at each time-point.

Fig 9

Low MW Aβ induced ATG12. Neurons were treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 24 hrs. (A) Neurons were stained with anti-ATG12 antibody. Representative confocal images from two independent experiments are shown. Scale bar, 20 μM. (B) Relative intensity of ATG12 staining. Intensity of signals were measured by Image J. Bar chart plots the means ± S.E.M. of three images. (C) Neurons trans-fected with LC3-DsRed prior treatment were stained with anti-ATG12 antibody. Representative Z-stack confocal images are shown. Scale bar, 10 μM.

Fig 11

Low MW Aβ reduced phosphorylation of DAPK. Western blotting of p-DAPK (S308) in neurons treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) for 4 to 30 hrs. β-actin was used as an internal control. Figure shows the representative result from two independent experiments.

Fig 13

Inhibition of autophagy advanced Aβ-induced apopto-sis. Western blotting of (A) LC3, and (B) cleaved caspase 3 (Asp 175) in neurons treated with low MW Aβ_1–42 of indicated concentrations or DMSO (vehicle) with or without 3-MA of 10 mM for 24 hrs. β-actin was used as an internal control. Figure shows the representative result from three independent experiments.