Akt and mTOR mediate programmed necrosis in neurons

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Necroptosis is a regulated form of necrosis implicated in neuronal death following stroke and brain trauma.
• While necroptosis initiation is understood, the downstream signaling events remain largely unexplored.
• The Akt pathway is known to inhibit apoptosis, and its downstream effector, mTOR, regulates protein synthesis.

Purpose of the Study:

• To investigate the role of Akt and mTOR signaling in regulating necroptosis in neuronal cells.
• To determine if Akt/mTOR activation is a downstream event in necroptosis signaling.

Main Methods:

• Induced necroptosis in HT22 hippocampal cells using TNFα and zVAD.
• Assessed Akt/mTOR pathway activation via phosphorylation of AKT, GSK-3β, mTOR, and S6.
• Utilized Akt inhibitor viii, rapamycin, and siRNA for AKT1/2 and mTOR knockdown.
• Administered necrostatin-1 to confirm pathway involvement.
• Measured cell death, mitochondrial reactive oxygen species (ROS) production, and protein complex assembly (RIPK1-RIPK3-pAkt).

Main Results:

• TNFα/zVAD treatment induced rapid necroptosis, preceded by RIPK1-RIPK3-pAkt complex formation and Akt/mTOR pathway activation.
• Inhibition of Akt (using Akt inhibitor viii) and mTOR (using rapamycin) significantly reduced necroptosis (>50%) and ROS production, without affecting RIPK1-RIPK3 assembly.
• siRNA-mediated knockdown of AKT1/2 and mTOR confirmed these inhibitory effects.
• Necrostatin-1 inhibited necroptosis, Akt/mTOR phosphorylation, ROS generation, and RIPK1-RIPK3-pAkt complex assembly.

Conclusions:

• Akt and mTOR signaling pathways are activated downstream of RIPK1 in the context of necroptosis.
• Targeting Akt/mTOR represents a potential therapeutic strategy to mitigate necroptotic neuronal death in conditions like stroke and brain trauma.