#These authors contributed equally to this work
Amyotrophic lateral syndrome (ALS) is a progressive degenerative disorder characterized by motor neuron death and axon degeneration. Mitochondrial dysfunction plays a key role in the pathogenesis of ALS, the mechanism of which remains poorly understood. The B-cell lymphoma-2 (Bcl-2) family of proteins that control and mediate mitochondrial function and apoptosis, including the pro-apoptotic members Bcl2-Associated X (Bax), are involved in ALS development. The death receptor 6 (DR6) regulates motor neuron death in ALS, and DR6 antibodies can prevent axon degeneration and motor neuron damage by blocking DR6. Previous studies demonstrated that PSAP localized to mitochondria and was required for DR6-induced apoptosis. In this study, SOD1G93A was transfected into the motor neuron cell line NSC-34 to serve as an ALS cell model
Amyotrophic lateral syndrome (ALS) is a progressive neuronal degenerative disease that causes damage to motor neurons.
ALS can be divided into familial (fALS) and sporadic ALS (sALS) (
Mitochondrial dysfunction is critical in the pathogenesis of neurodegenerative disorders, such as ALS and Alzheimer’s disease (
Elevated DR6 protein levels have been detected in SOD1G93A mice and patients with ALS. DR6 antibodies can inhibit motor neuron death via blocking DR6, highlighting the role of DR6 in ALS (
The present study demonstrated that SOD1G93A overexpression in NSC-34 cells led to the induction of apoptosis. The knockdown of PSAP, Bax, or Bak abolished PARP cleavage and caspase 3 activation, induced by SOD1G93A, indicating that PSAP, Bax, and Bak mediated cell death following SOD1G93A overexpression. Furthermore, the immunoprecipitation (IP) analysis showed that PSAP interacted with Bax and Bak on SOD1 overexpression. In addition, PSAP was required for SOD1G93A-induced apoptosis, which was dependent on Bax–Bak complex formation.
Lipofectamine 2000 was purchased from Thermo Fisher (CA, USA). A mitochondria isolation kit was procured from Transgen Biotech (Beijing, China). Complete protease inhibitor cocktail tablets, Cell Counting Kit-8 (CCK-) 8, and anti-Cyt C antibody were purchased from Bimake (TX, USA). Anti-myc, Bak, β-actin, and Cox I antibodies, were purchased from Santa Cruz Biotechnology (TX, USA). An anti-PARP antibody was purchased from Cell Signaling Technology (MA, USA). An anti-Bax antibody was purchased from R&D Systems (MN, USA). Both anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from Bioss (Beijing, China). pcDNA3.1/LacZ-Myc-Flag, pcDNA3.1/SOD1-Myc-Flag, and pcDNA3.1/SOD1G93A-Myc-Flag plasmids were amplified as described previously (
NSC-34 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone) supplemented with fetal bovine serum (10%), streptomycin (100 μg/mL), and penicillin (100 units/mL) at 37°C and in the presence of 5% CO2. The cells were transfected with pcDNA3-LacZ, wtSOD1, or SOD1G93A constructs using Lipofectamine 2000 following the manufacturer’s protocols.
The cell survival activity was measured by the CCK-8 method. Briefly, NSC34 cells were seeded into 24-well plates at a density of 105 cells/mL. The cells were transfected with LacZ, wtSOD1, or SOD1G93A plasmids for 24 h. After 24 h-transfection, the CCK-8 reagent was added and cultured for 0.5–5 h at 37°C. The absorbance was finally measured at 450 nm following the manufacturer’s protocols.
A caspase-3 activity kit was purchased from Bioworld (Nanjing, China). NSC34 cells were seeded into six-well plates at a density of 5 × 105 cells/mL. The cells were transfected with LacZ, wtSOD1, or SOD1G93A plasmids for 24 h. Activated caspase-3 cleaved Ac-DEVD-pNA to generate pNA. The absorbance was measured at 405 nm to indicate caspase-3 activity.
siRNA duplexes to PSAP, Bax, and Bak, and scrambled siRNA and RNAi-mate reagent were purchased from Gene Pharma (Shanghai, China). The cells were treated with each siRNA for 48 h and transfected with LacZ, wtSOD1, or SOD1G93A plasmids using Lipofectamine 2000 reagent. The cells were harvested and lysed for further analysis 24 h after transfection.
The cytosolic and mitochondrial fractions were extracted using the mitochondria isolation kit following the manufacturer’s protocols to explore Cyt C release and Bax translocation.
Cell apoptosis was determined using an Annexin V/propidium iodide (PI) kit and analyzed by flow cytometry. The cells were harvested after 24 h transfection, washed twice with an ice-cold phosphate-buffered solution (PBS), and stained with Annexin V/PI solution.
IP and immunoblotting were performed as previously described (
For Western blot analysis, cells were harvested and lysed by sonication for 25 s on ice in lysis buffer. Denatured protein (20 μg) ofeach sample was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk powder in Tris-buffered saline tween (TBST) for 45 min. The protein blot was then incubated with appropriate primary antibodies and HRP-conjugated secondary antibody separately. For immunoprecipitation (IP), cells were harvested and lysed by sonication for 20 s on ice in IP lysis buffer, and samples were centrifuged at 14,000 ×
The data were analyzed by using SPSS 22.0 statistical analysis software, and groups of data are presented as mean ± standard deviation (SD).Statistical significance was performed on the data by analysis of variance for significance test. **
NSC-34 cells were transfected with flag-tagged LacZ or wtSOD1, SOD1G93A to investigate whether SOD1G93A promoted apoptosis.
As shown in
Since mitochondrial damage promotes motor neuron cell death, the study next examined whether SOD1G93A induced the release of mitochondria-related molecules into the cytoplasm. As shown in
SOD1G93A triggered Cyt C release and Bax translocation regulated by the Bcl-2 family of proteins, which are known regulators of the mitochondrial apoptotic pathway. The study therefore determined the effects of Bax and Bak on SOD1G93A-induced apoptosis. Prior to SOD1G93A overexpression, the cells were silenced with Bax, Bak-specific siRNAs, or scrambled controls. The cell viability was then assessed following the transfection of LacZ, wtSOD1, and SOD1G93A.
PSAP specifically localized to the outer membrane of the mitochondria and was associated with Bax or Bak. In addition, PSAP was required for DR6-induced apoptosis (
The possible interaction of Bax, Bak, and PSAP upon SOD1G93A overexpression was explored to further investigate the mechanism by which Bax, Bak, and PSAP mediated SOD1G93A-induced apoptosis. As shown in
This study demonstrated that a specific mitochondrial protein PSAP played an important role in SOD1G93A-induced apoptosis through Bax-Bak interaction. These findings provided new insights into the role of PSAP in motor neuron death and axon degeneration in ALS, highlighting the potential of PSAP as a novel therapeutic target for ALS treatment and prevention.
ALS is a neurodegenerative disease characterized by motor neuron death and axon degeneration that rapidly progresses after onset (
PSAP, also known as mitochondrial carrier homolog 1 (MTCH1) due to its homology with a mitochondrial carrier protein, was identified as a specific mitochondrial resident protein and shown to induce apoptosis via Apaf-1 and Smac (
Oxidative stress, excitotoxicity, inflammation, loss of growth factors, and apoptosis induced ALS. Interestingly, these processes were either directly or indirectly related to mitochondrial function. Mitochondria are crucial for ATP production through oxidative phosphorylation and are critical for neuronal function. Mitochondrial dysfunction results in the loss of ATP production and reactive oxygen species (ROS) generation, promoting cell death. Mitochondrial damage therefore leads to neurodegenerative diseases (
IP was performed to determine the relationship between SOD1, PSAP, Bax, and Bak so as to further explore the mechanism of PSAP regulating SOD1G93A-induced apoptosis. A higher number of SOD1 -Bax complexes were evident after the overexpression of wtSOD1, while SOD1 -Bax complex formation decreased in response to SOD1G93A overexpression. Bax translocation from the cytosol into the mitochondria was also observed in SOD1G93A but not wtSOD1-overexpressing cells. Generally, SOD1 localized to the cytosol, and SOD1 -Bax complexes were abundant following wtSOD1 transfection. Higher levels of Bax translocated into the mitochondria; SOD1G93A transfection explained the decreased SOD1-Bax interaction observed in cells expressing this mutant. The overexpression of SOD1 could disrupt Bax-Bak, PSAP-Bax, and PSAP-Bak complex formation in wtSOD1-transfected cells. In contrast, only slight differences in PSAP-Bax or PSAP-Bak complex formation were observed on SOD1G93A overexpression. However, SOD1G93A dramatically increased the number of Bax-Bak complexes, thereby promoting apoptosis. Furthermore, the knockdown of PSAP inhibited SOD1G93A-induced Bax-Bak complex formation. These data suggested that PSAP regulated the formation of the Bax-Bak complex, either directly or indirectly, and mediated its effects on apoptotic induction through MOMP.
In summary, the results indicated that the mitochondrial protein PSAP was required for SOD1G93A-induced apoptosis mediated through mitochondrial apoptosis-induced channels. PSAP is pivotal for Bax-Bak interaction, leading to caspase activation and apoptosis. Inhibiting these activities of PSAP therefore represents a potential approach for treating ALS.