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Published on: December 26, 2016
METTL3-mediated TIGAR m6A modification and its role in microglia activation related to Alzheimer's disease
Jing Kang1, Xin Du1, Xiaoting Zhang2
1Department of Clinical Laboratory, The Third Affiliated Hospital of Qiqihar Medical University.
Objective:
This study focused on clarifying whether methyltransferase3 (METTL3) participates in the polarization and activation of microglia in Alzheimer's disease (AD) by mediating the N6-methyladenosine (m6A) modification level of TP53-induced glycolysis and apoptosis regulator (TIGAR).
Methods:
Human microglia HMC3 cells were transfected with overexpression or knockdown lentivirus of METTL3, TIGAR, or TIGAR before being induced by Aβ treatment to establish an in-vitro AD cell model. The expression of TIGAR and METTL3 was measured by real-time quantitative PCR and western blot. Microglial polarization was assessed by detecting the expression of M1 microglia marker CD86 and M2 marker CD206 using immunofluorescence and measuring the protein expression of M1-associated iNOS and IL-1β, and M2-associated Arg-1 and IL-10 using western blot. PAR-CLIP was employed to examine the binding of METTL3 to TIGAR mRNA, and MeRIP was used to measure the m6A level of TIGAR mRNA. The stability of TIGAR mRNA was evaluated by an actinomycin D assay.
Results:
In Aβ-induced HMC3 cells, both METTL3 and TIGAR expressions were reduced. Aβ treatment in HMC3 cells increased M1 polarization and decreased M2 polarization. But this effect was partially reversed by overexpression of either METTL3 or TIGAR. METTL3 binds to TIGAR mRNA and increases its m6A level, thereby promoting TIGAR mRNA stability.
Conclusion:
METTL3 modulates the balance of Aβ-induced polarization and microglia activation in HMC3 cells by upregulating TIGAR, promoting polarization toward an anti-inflammatory profile.
Insights
Methyltransferase-like 3 (METTL3) regulates microglia activation in Alzheimer's disease (AD) by increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) stability. This promotes an anti-inflammatory response, crucial for AD pathogenesis.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Alzheimer's disease (AD) involves complex neuroinflammatory processes.
- Microglia play a critical role in AD pathogenesis, with their polarization state influencing disease progression.
- N6-methyladenosine (m6A) modification is increasingly recognized as a key regulator of cellular processes.
Purpose of the Study:
- To investigate the role of methyltransferase-like 3 (METTL3) in microglia polarization and activation in Alzheimer's disease.
- To determine if METTL3 mediates the m6A modification of TP53-induced glycolysis and apoptosis regulator (TIGAR) in this context.
Main Methods:
- Established an in-vitro Alzheimer's disease (AD) cell model using human microglia (HMC3) cells treated with Aβ.
- Manipulated the expression of METTL3 and TIGAR using lentivirus transfection.
- Assessed microglial polarization (M1/M2 markers), gene/protein expression (qPCR, Western blot), METTL3 binding to TIGAR mRNA (PAR-CLIP), m6A levels (MeRIP), and mRNA stability (actinomycin D assay).
Main Results:
- Aβ treatment reduced METTL3 and TIGAR expression in HMC3 cells, promoting M1 (pro-inflammatory) microglia polarization.
- Overexpression of METTL3 or TIGAR partially reversed Aβ-induced M1 polarization.
- METTL3 was found to bind TIGAR mRNA, increase its m6A modification, and enhance its stability.
Conclusions:
- METTL3 upregulates TIGAR expression by increasing TIGAR mRNA stability via m6A modification.
- METTL3 modulates the balance of Aβ-induced microglia polarization and activation, shifting it towards an anti-inflammatory profile.
- These findings highlight METTL3-TIGAR axis as a potential therapeutic target for Alzheimer's disease.
