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Published on: October 18, 2016
[Effects of RNA interference targeting P2X7 receptor on microglial phagocytosis of amyloid-β protein]
1Department of Medical Imaging, Tongji Hospital, Tongji University, Shanghai 200065, China.
Objective:
To investigate the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial phagocytosis of amyloid-β (Aβ) protein and to explore its possible mechanism.
Methods:
The small interfering RNA (siRNA) targeting the P2X7R gene was identified. The microglial cells activated by Aβ1-42 were infected with the Lipofectamine-siP2X7R and it was designated as siP2X7R group. Microglia infected with Lipofectamine-siNC was designated as siNC group and non-infected cells as con group. The levels of P2X7R mRNA were detected by real-time PCR and the P2X7R protein was determined by Western blotting. The levels of IL-1β and TNF-α were measured by ELISA. The microglial phagocytosis of Aβ1-42 was observed by ELISA and immunocytochemistry staining.
Results:
Detected by the Real-time PCR, the expression level of P2X7R mRNA of siP2X7R group decreased significantly versus siNC and con groups (P<0.05). The lowered expression of P2X7R protein detected by Western blotting was consistent with Real-time PCR. After RNA interference silencing P2X7R, the levels of IL-1β and TNF-α detected by ELISA in siP2X7R group less than those in con, siNC groups, significantly (P<0.05). In con, siNC and siP2X7R groups respectively, the levels of Aβ1-42 in supernatant were (423±20) pg/ml, (417±16) pg/ml, (296±30) pg/ml and the levels of Aβ1-42 in the microglia were (190±37) pg/ml, (187±39) pg/ml, (322±26) pg/ml. Compared to siNC and con groups, in siP2X7R group the levels of Aβ1-42 in supernatant decreased (P<0.05) and the levels of Aβ1-42 in the microglia increased (P<0.05). Aβ1-42 immunofluorescence staining showed that the red fluorescent products were seen in the cytoplasm of most microglias in siP2X7R group, but in con or siNC groups in only few microglias these products were depicted.
Conclusions:
The silence expression of P2X7R by RNA interference effectively decreases the levels of IL-1β and TNF-α released by microglia and promotes microglia to phagocytose Aβ. P2X7R could be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.
Insights
Silencing the P2X7 receptor (P2X7R) with RNA interference enhances microglial phagocytosis of amyloid-beta (Aβ) protein. This approach reduces inflammatory markers and shows potential for Alzheimer's disease treatment.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Context:
- Alzheimer's disease is characterized by amyloid-beta (Aβ) plaque accumulation.
- Microglia play a crucial role in brain's immune response and Aβ clearance.
- The P2X7 receptor (P2X7R) is implicated in neuroinflammation.
Purpose:
- To investigate the effect of silencing P2X7R using RNA interference on microglial phagocytosis of Aβ.
- To explore the underlying mechanisms, including inflammatory cytokine release.
Summary:
- RNA interference was used to silence P2X7R expression in microglial cells.
- Silencing P2X7R significantly reduced P2X7R mRNA and protein levels.
- This led to decreased levels of IL-1β and TNF-α.
- Microglial phagocytosis of Aβ1-42 was significantly enhanced, with more Aβ internalized by cells.
Impact:
- P2X7R silencing effectively reduces pro-inflammatory mediators released by microglia.
- This strategy promotes microglial phagocytosis of Aβ.
- P2X7R represents a potential therapeutic target for RNA interference-based treatments for Alzheimer's disease.
