Beta-amyloid-induced neuronal apoptosis involves c-Jun N-terminal kinase-dependent downregulation of Bcl-w

Abstract

beta-Amyloid protein (Abeta) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimer's disease (AD). Abeta directly induces neuronal apoptosis, suggesting an important role of Abeta neurotoxicity in AD neurodegeneration. However, the mechanism(s) of Abeta-induced neuronal apoptosis remain incompletely defined. In this study, we report that Abeta-induced neuronal death is preceded by selective alterations in expression of the Bcl-2 family of apoptosis-related genes. Specifically, we observe that Abeta significantly reduces expression of antiapoptotic Bcl-w and Bcl-x(L), mildly affects expression of bim, Bcl-2, and bax, but does not alter expression of bak, bad, bik, bid, or BNIP3.Abeta-induced downregulation of Bcl-w appears to contribute to the mechanism of apoptosis, because Abeta-induced neuronal death was significantly increased by Bcl-w suppression but significantly reduced by Bcl-w overexpression. Downstream of Bcl-w, Abeta-induced neuronal apoptosis is characterized by mitochondrial release of second mitochondrion-derived activator of caspase (Smac), an important precursor event to cell death. We observed that Smac release was potentiated by suppression of Bcl-w and reduced by overexpression of Bcl-w. Next, we investigated the upstream mediator of Abeta-induced Bcl-w downregulation and Smac release. We observed that Abeta rapidly activates c-Jun N-terminal kinase (JNK). Pharmacological inhibition of JNK effectively inhibited all measures of Abeta apoptosis: Bcl-w downregulation, Smac release, and neuronal death. Together, these results suggest that the mechanism of Abeta-induced neuronal apoptosis sequentially involves JNK activation, Bcl-w downregulation, and release of mitochondrial Smac, followed by cell death. Complete elucidation of the mechanism of Abeta-induced apoptosis promises to accelerate development of neuroprotective interventions for the treatment of AD.

Figure 1.

Bcl-wexpression is downregulated during Aβ-induced neuronal death. Primary neuron cultures were exposed to 25 μm aggregated Aβ peptide for the indicated times and then assayed for cell viability or Bcl-w expression. A, Aβ induces time-dependent neuronal death. Data show mean ± SEM cell viability from a representative experiment (n = 6). Significance is defined as ^*p < 0.05 and ^**p < 0.01 in comparison to vehicle-treated control group (Ctrl). B, Aβ induces time-dependent decrease in mRNA levels of Bcl-w. The mRNA expression of Bcl-2 family members (Bcl-2, Bcl-x_L, Bcl-w, bax, bak, bad, bik, bid, BNIP3, and bim_EL) was detected by RT-PCR followed by agarose gel electrophoresis. β-Actin served as internal control. C, Full-length Aβ peptides (25 μm) Aβ_1-40 and Aβ_1-42 also reduce mRNA levels of Bcl-w as detected by RT-PCR. D, Aβ induces time-dependent decrease in protein levels of Bcl-w. Protein expression of Bcl-w (top) was analyzed by Western blot. β-Tubulin (bottom) was used as a control. The picture shown is a representative of duplicated experiments. E, Relative amounts of Bcl-w were determined by densitometric scanning of Western blots from three independent experiments. Data is represented as a mean ± SEM percentage of control values. ^*p < 0.05 and ^**p < 0.01 relative to vehicle-treated control group.

Figure 2.

Overexpression of Bcl-w protects against Aβ-induced neuronal death. A, Primary neuron cultures were infected AAV-Bcl-w, AAV-GFP, or reagents without vector (Vehicle) for 3 d. Bcl-w expression was detected by immunocytochemistry using anti-human Bcl-w antibody. In comparison to negative control without first antibody, only weak Bcl-w expression was detected in vehicle-treated and AAV-GFP-infected cultures. Almost all of neurons exhibited strong expression of Bcl-w in AAV-Bcl-w-infected cultures. B, Infection with AAV-Bcl-w yielded strong expression of human Bcl-w mRNA (top), as detected by RT-PCR. β-Actin served as internal control (bottom). C, Bcl-w protein expression was significantly increased in cultures infected with AAV-Bcl-w (top), as analyzed by Western blot using an antibody that recognizes both rodent and human Bcl-w. β-Tubulin was used as a control (bottom). The picture shown is a representative of duplicated experiments. D, Relative amounts of Bcl-w were determined by densitometric scanning of Western blots from three independent experiments. Data is represented as a mean ± SEM percentage of vehicle (1 d) control values. Significance is defined as ^**p < 0.01 relative to respective vehicle-treated control group. E, Infection with AAV-Bcl-w significantly protects neuron cultures from cell death induced by 48 h exposure to 25 μm Aβ_25-35. Data show mean ± SEM cell viability from a representative experiment (n = 6). Significance is defined as ^**p < 0.01 in comparison to AAV-GFP condition.

Figure 3.

Suppression of endogenous Bcl-w expression exacerbates Aβ-induced neuronal death. Primary neuron cultures were transfected for 1 or 3 d with siRNA that either specifically targeted Bcl-w [siBcl-w(73), siBcl-w(362)] or served as scrambled or mismatched controls. A, The specific Bcl-w siRNAs but not the control siRNAs decreased endogenous Bcl-w mRNA expression as detected by RT-PCR using rat-specific primers (top). β-Actin served as internal control (bottom). B, Protein levels of Bcl-w were similarly affected by the siRNA treatments, as determined by Western blot with Bcl-w antibody (top).β-Tubulin served as a control (bottom). The picture shown is a representative of duplicated experiments. C, Relative amounts of Bcl-w were determined by densitometric scanning of Western blots from three independent experiments. Data is represented as a mean ± SEM percentage of ncBcl-w (1 d) control values. Significance is defined as ^**p < 0.01 relative to respective ncBcl-w control group. D, Neuron cultures were exposed for 48 h to 25 μm Aβ_25-35 24 h after transfection with siRNA. Data show mean ± SEM cell viability from a representative experiment (n = 6). Significance is defined as ^**p < 0.01 in comparison to ncBcl-w condition.

Figure 4.

Aβ-induced apoptosis involves Smac release from mitochondria to cytosol in neuron cultures. A, Primary neuron cultures were exposed to vehicle (Ctrl) or 25 μm Aβ_25-35 for 6-48 h. Levels of Smac in mitochondrial (Mt) and cytosolic (Cyt) extracts were analyzed by Western blot. B, Neuron cultures were pretreated for 1 h with 0 or 40 μm of the caspase 9 inhibitor Z-LEHD-FMK followed by exposure to 25 μm Aβ_25-35 for 48 h. Data show mean ± SEM cell viability from a representative experiment (n = 3). Significance is defined as ^**p < 0.01 in comparison to Aβ condition.

Figure 5.

Bcl-w inhibits Aβ-induced Smac release. A, Overexpression of Bcl-w inhibits Aβ-induced Smac release. Neuron cultures were infected with AAV-Bcl-w or AAV-GFP or vehicle for 24 h, followed by exposure to 25 μm Aβ_25-35 for 48 h. Smac levels in mitochondrial (Mt) and cytosolic (Cyt) extracts were analyzed by Western blot. B, Suppression of Bcl-w expression increases Aβ-induced Smac release. Neuron cultures were transfected with siRNA for 24 h, followed by exposure to 25 μm Aβ _25-35 for 48 h. Smac levels in mitochondrial and cytosolic extracts were analyzed by Western blot. ncBcl-w, Scrambled siRNA as a negative control; mmBcl-w, mismatched siRNA control; siBcl-w (73), siRNA targeting Bcl-w nt 73-93; siBcl-w (362), siRNA targeting Bcl-w nt 362-382.

Figure 6.

Aβ-induced Bcl-w downregulation and Smac release are dependent on JNK activation. A, JNK is activated (phosphorylated) in a time-dependent manner by Aβ. Neuron cultures were treated with 25 μm Aβ_25-35 for the indicated times and then probed by Western blot with phospho-JNK (p-JNK) or pan JNK antibodies. The JNK inhibitor SP600125 (100 nm) decreases both basal levels (B) and Aβ-induced increases (C) in phosphorylated JNK, as determined by Western blot with p-JNK and pan JNK antibodies. D, Inhibition of JNK with SP600125 inhibits Aβ-induced Bcl-w downregulation. Neuron cultures were pretreated with 100 nm SP600125 for 90 min, followed by exposure to 25 μm Aβ_25-35 for the indicated times, and were then analyzed for Bcl-w mRNA expression by RT-PCR. β-Actin served as internal control. E, Western Blot confirmed that SP600125 inhibits Aβ-induced Bcl-w downregulation (top). Neuron cultures were pretreated with 100 nm SP600125 for 90 min, followed by exposure to 25 μm Aβ_25-35 for 48 h, and were then analyzed for Bcl-w expression by Western blot. As a control, Aβ-induced Bcl-x_L downregulation was also inhibited by SP600125 (middle). β-Tubulin was used as a control (bottom). F, Relative amounts of Bcl-w were determined by densitometric scanning of Western blots from three independent experiments and are represented as a mean ± SEM percentage of control values. ^**p < 0.01 relative to the Aβ condition. G, JNK inhibition attenuates Aβ-induced Smac release. Neuron cultures were pretreated with 100 nm of SP600125 for 90 min, followed by exposure to 25 μm Aβ_25-35 for 48 h, and were then analyzed for Smac content in mitochondrial (Mt) or cytosolic (Cyt) extracts Western blot. H, JNK inhibition blocks Aβ-induced neuronal death. Neuron cultures were pretreated with increasing concentrations (0-1 μm) of SP600125 for 90 min, followed by exposure to 25 μm Aβ_25-35 for 48 h, and were then assayed for cell viability. Data show mean ± SEM cell viability from a representative experiment (n = 6). Significance is defined as ^**p < 0.01 in comparison to Aβ_25-35 condition. Ctrl, Control.

Figure 7.

Bcl-w does not affect Aβ-induced JNK activation. Neuron cultures were exposed to 25 μm Aβ_25-35 for the indicated times 24 h after AAV-Bcl-w infection. Western blots of whole-cell extracts were probed with a phospho-JNK (p-JNK) antibody (top) or JNK antibody (bottom). Results parallel those in noninfected cultures (Fig. 6 A).