Phorbol myristate acetate induces cellular senescence in rat microglia in vitro

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.

Activated microglia release β−galactosidase that promotes inflammatory neurodegeneration

“…Thus, it is possible that part of the β-galactosidase in senescent cells could end up on the cell surface. This is at least consistent with our findings that: (i) PMA and LPS induce β-galactosidase release [as both PMA and LPS are reported to induce microglial senescence (Yu et al, 2012;Cao et al, 2020)], and (ii) microglia in the brains of aged mice appear to have βgalactosidase on their cell surface. Note, however, that we do not know whether PMA or LPS induced microglial senescence in our cultures, and much more work would be required to confirm that senescent microglia expose β-galactosidase.…”

“…Phorbol 12-myristate 13-acetate (PMA) is a protein kinase C activator that has been reported to induce β-galactosidase and senescence of microglia (Cao et al, 2020). So, we tested whether PMA treatment of microglia could induce β-galactosidase release, and found that BV-2 microglia cultured with PMA had significantly more β-galactosidase activity on the cell surface (Figures 1C, D) and released significantly more β-galactosidase activity into the culture medium (Figures 1E, F).…”

Cellular Senescence in Brain Aging

“…Besides astrocytes, also senescent microglia might influence the pro-inflammatory state in the brain. Experiments performed on microglia derived from rat brain cultured in vitro indicated that those cells can undergo stress-induced senescence, which is accompanied by SASP (Cao et al, 2020). Moreover, it was demonstrated in the elegant studies performed by Sierra et al (2007) that microglia derived from old mice are characterized by higher granularity (flow cytometry analysis) and significantly increased expression of TNFα, IL-1β, IL-6.…”

Targeting senescent cells for a healthier longevity: the roadmap for an era of global aging

“…Besides astrocytes, microglia can also become senescent and influence the pro-inflammatory state of the brain. For example, microglial cells derived from rat brain and cultured in vitro undergo stress-induced senescence, which is accompanied by the SASP [ 75 ]. Microglia of aged mice is characterized by higher granularity and increased expression of TNF-α, IL-1β, IL-6, cytokines that reflect development of the SASP [ 76 ].…”