PPARγ coactivator-1α (PGC-1α) protects neuroblastoma cells against amyloid-beta (Aβ) induced cell death and neuroinflammation via NF-κB pathway
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Alzheimer’s disease is characterized by the accumulation of amyloid beta (Aβ) and the formation of neurofibrillary tangles. Aβ is the main constituent of senile plaques and is largely involved in neuronal death and neuroinflammation. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is one of the main transcriptional coactivator and has been related to many fields such as energy metabolism, cardiovascular disease, neurodegenerative disorders, and so on.
Treatment with Aβ1–42 reduced the expression of PGC-1α in both protein and RNA levels of neuroblastoma N2a cells. Aβ1–42 induced a robust activation of cleaved caspase-3 while PGC-1α suppressed this activation and protected N2a cells from Aβ-induced cell death. Overexpression of PGC-1α significantly reduced the level of main proinflammatory cytokines. In addition, PGC-1α inhibited the transportation of NF-κB p65 from cytoplasm to nucleus and IκBα degradation induced by Aβ1–42.
Our results have demonstrated that PGC-1α can protect neuroblastoma cells against Aβ-induced neuronal death and neuroinflammation. Moreover, this neuroprotective effect of PGC-1α is regulated through NF-κB pathway. Taken together, our work provides evidence that PGC-1α could be beneficial in targeting Aβ neurotoxicity.
KeywordsPGC-1α Aβ Neuronal death Neuroinflammation NF-κB pathway
peroxisome proliferator-activated receptor gamma coactivator-1 alpha
nuclear factor-kappa B
Cell Counting Kit-8
tumor necrosis factor-alpha
Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative disorders and is characterized by neuronal degeneration and death in the central nervous system [1, 2]. At present, AD affects about 40 million people and has become a healthcare challenge worldwide. Two predominant pathological features of AD brains are extracellular aggregation of amyloid-beta (Aβ) and formation of intracellular neurofibrillary tangles . As a major factor in the development of AD, Aβ is the main constituent of senile plaques and is largely involved in neuronal death and neuroinflammation [4, 5, 6, 7]. However, the molecular mechanisms underlying Aβ induced neurotoxicity and neuroinflammation still remain unclear [8, 9, 10, 11, 12].
It has been reported that Aβ-induced neuroinflammation contributes to the progress of neurodegeneration and neuronal death in Alzheimer’s disease . Under the inflammatory condition, glia cells and some immune cells are activated directly or indirectly by increased levels of potent proinflammatory cytokines such as IL-1α, IL-1β, IL-6 and TNF-α, etc [14, 15]. Moreover, some signaling pathways are initiated during this process including nuclear factor-kappa B (NF-κB) pathway. The transcription factor NF-κB is crucial for many biological processes such as inflammation, differentiation, apoptosis, neuronal survival, and so on. In the central nervous system, NF-κB signaling pathway has drawn attention because of its functions in synaptic plasticity, neuronal survival, and neurotransmission [16, 17, 18, 19]. For example, the activation of NF-κB was detected in AD brains and is a key step in Aβ-induced neurotoxicity . Therefore, the regulation of the NF-κB pathway could be a perspective target in the treatment of AD.
The peroxisome proliferator-activated receptor gamma (PPARγ) is an isoform of PPARs that regulates numerous biological processes like metabolism of glucose and lipids, inflammation, cellular differentiation, and proliferation . This nuclear receptor is regulated by other genes called transcriptional coactivators. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is one of the main transcriptional coactivators and has been involved in many processes such as energy metabolism, cardiovascular disease, neurodegenerative disorders, and so on. Several studies have reported that overexpression of PGC-1α can reduce the production of Aβ [22, 23]. Thus, whether PGC-1α can protect neurons from Aβ-induced cytotoxicity has not been well understood.
In this study, we investigate first, whether Aβ treatment can induce the change of PGC-1α expression in neuroblastoma cell line; second, whether PGC-1α can protect neuroblastoma cells from Aβ-induced neuronal death and neuroinflammation; and third, whether the neuroprotective characteristic of PGC-1α to Aβ is mediated by the NF-κB pathway. Notably, we determined that PGC-1α protects the neuroblastoma cells from Aβ neurotoxicity by inhibiting the NF-κB pathway. These results provide us with a novel and potential therapeutic strategy for AD.
Antibodies and reagents
The following antibodies were used in this study: anti-PGC-1α (1:1000, #4259), anti-cleaved caspase-3 (1:1000, #9664), anti-NF-κB p65 (1:1000, #8242), anti-phospho-IκBα (1:1000, #2859), anti-Lamin B1 (1:1000, #13435), anti-IκBα (1:1000, #9242), and β-actin (1:3000, #3700) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Aβ1–42 fragments were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). A stock solution of the peptides were dissolved in ultrapure water at 1 mM. Aβ1–42 was then diluted with the experimental solution to 25 μM and vortexed thoroughly. This Aβ1–42 solution was used within 5 min of preparation. With this preparation procedure, the Aβ1–42 was largely present as stable oligomers.
Cell culture and transfection
We used mouse neuroblastoma N2a cells from American Type Culture collection (Manassas, VA, USA) in our experiments. Cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) with 10% Fetal Bovine Serum (Biomeda, Fostercity, CA, USA) and 1% penicillin at 37 °C in a humidified incubator with 5% CO2. All other cell culture regents were obtained from Gibco (Carlsbad, CA, USA). Transfection with empty plasmid or pcDNA4 myc PGC-1α (PGC-1α plasmid) (Addgene, Cambridge, MA, USA) were performed according to the manufacturer’s instruction with Lipofectamine 2000 from Invitrogen (Carlsbad, CA, USA). Cells were used for the following experiments 48 h after transfection.
Western blot analysis
After different treatments, cells were lysed by sonication in CelLytic MT buffer (Sigma, St. Louis, MO, USA) and proteins were extracted. 50 μg protein samples were separated on a denaturing 10% SDS-PAGE gel, and transferred to polyvinylidene fluoride membranes. The membrane was then blocked in PBST with 5% non-fat milk at room temperature for 1 h, followed by incubation with specific primary antibodies at 4 °C overnight. Blots were incubated with appropriate secondary antibodies at room temperature for 1 h the next day. The signals were detected by ECL detection kit. Blots were analyzed by ImageJ software.
Quantitative real-time PCR
mRNA levels of PGC-1α were determined by quantitative real-time polymerase chain reaction (qRT PCR). Total RNA was extracted from 1 × 107 with TRIzol (Invitrogen, Carlsbad, CA, USA) and cDNA from different samples were amplified with specific primers for human PGC-1α [5′-GGC AGA AGG CAA TTG AAG AG-3′ (forward) and 5′-TCA AAA CGG TCC CTC AGT TC-3′ (reverse)] and GAPDH [5′-ATCAGCAATGCCTCCTGCAC-3′ (forward) and 5′-CGTCAAAGGTGGAGGAGTGG-3′ (reverse)] Data were normalized to GAPDH expression and the control group was set as 1.
TUNEL assay was performed to measure the apoptotic neuroblastoma cells after treatment with an in situ cell death detection kit (Roche Diagnostics, Indianapolis, IN, USA). According to the manufacturer’s instruction, cells were TUNEL-stained, followed by counterstaining with DAPI. TUNEL positive cells were detected and counted with a confocal microscope.
Cell viability assay
1 × 105 N2a cells were plated in each well of a 96-well plate with 100 μl medium. After treatment, cell viability was assessed by the Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer’s instructions. We prepare control wells without cells and subtract the background absorbance of the control wells.
Enzyme-linked immunosorbent assay (ELISA)
After treatment, levels of IL-1β and TNF-α in the culture supernatant were measured by IL-1β and TNF-α ELISA kits (BD Bioscience, San Diego, CA, USA) according to the manufacturer’s instructions.
All data was analyzed using unpaired Student’s t tests or two-way ANOVA with Bonferroni post-tests (GraphPad Software) and represented as the mean ± SEM of at least three independent experiments. p values were calculated with the appropriate statistical tests using GraphPad Prism software 7.0. A significant difference was considered to be present at p < 0.05.
Treatment with Aβ1–42 reduces the expression of PGC-1α in neuroblastoma N2a cells
PGC-1α attenuates Aβ1–42 induced N2a cell death
PGC-1α prevents neuroinflammation induced by Aβ1–42
PGC-1α inhibits Aβ1–42 induced N2a cell death via NF-κB pathway
AD is a prevalent neurodegenerative diseases with cognitive function and memory impairment in the elderly. Several hypotheses of AD pathogenesis have been proposed in current AD studies such as Aβ hypothesis, tau hypothesis and the cholinergic hypothesis . Recently Aβ hypotheses are most commonly used in this field. Molecular mechanisms of Aβ-induced neurotoxicity have been investigated for the last two decades to understand the pathology process of AD. However, current therapeutics has very minimal impact to target the underlying cause of this disease [25, 26, 27]. In this study, our aim is to determine a coactivator of PPARγ, PGC-1α, and to test whether this coactivator can protect neuroblastoma cells from Aβ-induced neurotoxicity.
We first measured the levels of PGC-1α after the treatment with Aβ1–42. Both protein and mRNA levels of PGC-1α are reduced with Aβ challenge. This data is consistent with the conclusion of some studies based on animal model and clinical trial. These studies showed that PGC-1α expression decrease in AD brains as the results of Aβ pathology .
We then test how PGC-1α affects the Aβ-induced neuronal death and neuroinflammation. After treating with Aβ1–42 for 6 h, PGC-1α prevents neuroblastoma cells from death and largely reduces the level of IL-1β and TNF-α. Inflammation in the brain is a complicated network and is affected by many proinflammatory cytokines, including IL-6, IL-1β, TNF-α, IL-17, and so on [7, 28, 29]. Among these cytokines, IL-1β and TNF-α are key regulators involved in AD pathogenesis. Some studies have shown that their levels and activities were increased in the AD brains. Moreover, both of them are implicated to have protective effects to Aβ [30, 31, 32, 33, 34].
The transcription factor NF-κB plays numerous roles in the nervous system, including its function in synaptic processes, neuroprotection, neural stem cell proliferation, neurotransmission, and so on. In addition, its involvement in brain inflammation is very important for us to understand the protective role of PGC-1α. In AD brain, many mediators can activate the NF-κB pathway, including Aβ accumulation, reactive oxygen species, and numerous proinflammatory cytokines [35, 36, 37]. During this activation, the IκBα will be phosphorylated and then degraded. NF-κB will be activated and enters into the nucleus where it induces the gene transcription for some inflammatory mediators. In this study, we determined that PGC-1α inhibits the activation and translocation of NF-κB from cytoplasm to nucleus. Meanwhile, the degradation of IκBα is also inhibited by PGC-1α. These results implicate that NF-κB pathway is essential in the protective processes of PGC-1α against Aβ.
Our work demonstrates the neuroprotective function of PGC-1α to Aβ-induced neurotoxicity and the biological mechanisms underlying this process. These results provide us with promising evidence that PGC-1α could have a therapeutic effect to combat the neuronal death and neuroinflammation associated with AD.
YZ performed experiments, analyzed data and contributed to write the manuscript. CC performed the qRT PCR and ELISA experiments. YJ contributed to analyze the data. SW performed all the cell culture work. XW contributed to the study design. KW designed this study, supervised the experiments and contributed to write the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
This research was supported by grants from the National Natural Science Foundation of China (Nos. 81673066 and 81473684). The funding agency had no role in the design of the study and collection, analysis, and interpretation of data in writing the manuscript.
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- 22.Katsouri L, Lim YM, Blondrath K, Eleftheriadou I, Lombardero L, et al. PPARgamma-coactivator-1alpha gene transfer reduces neuronal loss and amyloid-beta generation by reducing beta-secretase in an Alzheimer’s disease model. Proc Natl Acad Sci USA. 2016;113:12292–7.CrossRefPubMedPubMedCentralGoogle Scholar
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