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Tp47 induces cell death involving autophagy and mTOR in human microglial HMO6 cells
Wen-Na Liu1, Xiao-Yong Jiang2, JunRen2
1Center of Clinical Laboratory, Zhongshan Hospital, School of Medical, Xiamen University, Xiamen, Fujian Province, China.
Background:
Tp47 can induce immune cells to produce numerous inflammatory factors, some of which can trigger autophagy. Increased autophagy has a dual effect on cell survival. However, whether Tp47 induces autophagy in microglia is unknown.
Objective:
To evaluate the potential role of Tp47 in microglia.
Methods:
After treatment with Tp47, autophagy-related proteins were assessed in HMO6 human microglial cells by flow cytometry, Western blotting and immunofluorescence. Cell death was assessed by flow cytometry and trypan blue staining. Changes in mTOR pathway proteins were explored by using Western blotting.
Results:
After treatment with Tp47, a gradual increase in total LC3 expression was observed as a dose- and time-dependent accumulation of its active form, LC3-II (P < 0.05), but P62 expression was downregulated (P < 0.05). Moreover, microglial mortality gradually increased in a dose- and time-dependent manner. 3-Methyladenine (3-MA), a specific inhibitor of PI3KC3, reversed autophagy and cell death. The mortality rate of HMO6 microglial cells treated with Tp47 was approximately 13.7 ± 2%, and the basal expression of p-mTOR, p-p70s6k and p-S6 in these cells was significantly downregulated by Tp47. Moreover, the mortality rate of microglia was significantly reduced after mTOR agonist intervention.
Conclusion:
In human microglial HMO6 cells, Tp47 induces autophagy- and mTOR pathway-dependent cell death.
Insights
Tp47 protein induces autophagy and cell death in human microglia. This process involves the mTOR pathway and can be reversed by inhibiting autophagy or activating mTOR signaling.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Tp47 protein can trigger inflammatory responses and autophagy in immune cells.
- Autophagy's role in cell survival is complex and context-dependent.
- The effect of Tp47 on microglia, key immune cells in the brain, was previously unknown.
Purpose of the Study:
- To investigate the role of Tp47 in human microglial cells.
- To determine if Tp47 induces autophagy in microglia.
- To elucidate the molecular mechanisms underlying Tp47's effects on microglia.
Main Methods:
- HMO6 human microglial cells were treated with Tp47.
- Autophagy markers (LC3, P62) and mTOR pathway proteins were analyzed using flow cytometry, Western blotting, and immunofluorescence.
- Cell death was quantified via flow cytometry and trypan blue staining.
- The effects of autophagy inhibitor 3-Methyladenine (3-MA) and an mTOR agonist were assessed.
Main Results:
- Tp47 treatment led to a dose- and time-dependent increase in autophagy markers (LC3-II accumulation) and a decrease in P62.
- Microglial cell death increased with Tp47 exposure.
- Inhibition of autophagy by 3-MA reduced Tp47-induced cell death.
- Tp47 downregulated key proteins in the mTOR pathway (p-mTOR, p-p70s6k, p-S6).
- Activating the mTOR pathway with an agonist significantly reduced microglial mortality.
Conclusions:
- Tp47 induces autophagy in human microglial cells.
- Tp47-induced cell death in microglia is dependent on both autophagy and the mTOR signaling pathway.
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