Phorbol myristate acetate induces cellular senescence in rat microglia in vitro
Dan Cao1, Xiao-Hong Li2, Xiao-Guang Luo2
1Department of Geriatrics, The Fourth People's Hospital of Shenyang, Shenyang, Liaoning 110031, P.R. China.
Abstract:
The present study aimed to establish a cellular model to test the hypothesis that oncogene-induced senescence (OIS) is triggered by aging-related activation of microglia. Primary microglia were incubated with phorbol 12-myristate 13-acetate (PMA), and β-galactosidase (β-Gal) staining was applied to subsequent assessment of cellular senescence. Moreover, flow cytometry was employed for examinations of cell cycle arrest and senescence-associated proteins, p53 and p21 were measured by western blotting. Furthermore, examination of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were carried out with microglia supernatants undergoing age-related degenerative diseases in the nervous system, using ELISA. PC12 cells were co-cultured with microglia activated by aging-related alteration(s) to evaluate whether apoptosis was increased in PC12 cells. Cellular senescence-associated β-Gal staining showed that microglial β-Gal expression gradually increased with prolonged PMA stimulation. Microglia in the group receiving 72 h of PMA stimulation displayed the highest percentage of cells arrested in G0/G1, the highest amount of senescence-associated expression of p53 and p21, and the most prominent secretion of TNF-α and IL-1β. In comparison with controls, an increase of apoptotic PC12 cells was detected, which were co-cultured with aging microglia. Taken together, microglia tend to undergo senescence after PMA treatment, suggesting that microglial senescence is associated with inactivation of certain oncogenes.
Insights
Microglia undergo senescence when exposed to PMA, a process linked to aging and neurodegenerative diseases. This microglial senescence may inactivate oncogenes, impacting brain health.
Area of Science:
- Neuroscience
- Cellular Biology
- Immunology
Background:
- Microglia, the immune cells of the central nervous system, play a critical role in neuroinflammation and neurodegenerative diseases.
- Oncogene-induced senescence (OIS) is a cellular process implicated in aging and cancer, but its role in microglia remains unclear.
- Aging-associated microglial activation is a hallmark of neurodegenerative conditions.
Purpose of the Study:
- To establish a cellular model for investigating the link between aging-related microglial activation and oncogene-induced senescence (OIS).
- To determine if OIS in microglia is triggered by aging-related activation.
- To assess the impact of senescent microglia on neuronal apoptosis.
Main Methods:
- Primary microglia were treated with phorbol 12-myristate 13-acetate (PMA) to induce senescence.
- Cellular senescence was assessed using β-galactosidase (β-Gal) staining.
- Cell cycle arrest, senescence-associated proteins (p53, p21), and inflammatory cytokines (TNF-α, IL-1β) were analyzed via flow cytometry, western blotting, and ELISA.
- PC12 cells were co-cultured with activated microglia to evaluate apoptosis.
Main Results:
- PMA stimulation led to increased β-Gal expression in microglia, indicating senescence.
- Microglia exposed to 72h PMA showed G0/G1 cell cycle arrest, elevated p53 and p21 expression, and increased secretion of TNF-α and IL-1β.
- Co-culture with aging microglia resulted in increased apoptosis of PC12 cells compared to controls.
Conclusions:
- Microglia undergo senescence following PMA treatment, supporting the hypothesis that OIS is linked to aging-related microglial activation.
- Senescent microglia secrete pro-inflammatory cytokines and can induce apoptosis in neurons.
- Microglial senescence may be associated with the inactivation of specific oncogenes, contributing to neurodegenerative processes.
