A comprehensive small interfering RNA screen identifies signaling pathways required for gephyrin clustering

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• Gephyrin is a postsynaptic scaffold protein crucial for stabilizing GABA(A) receptors at inhibitory synapses.
• Understanding the regulation of gephyrin clustering is vital for comprehending inhibitory neurotransmission.

Purpose of the Study:

• To identify protein kinases involved in gephyrin cluster stabilization using a kinome-wide screen.
• To elucidate the signaling pathways regulating gephyrin clustering and its functional consequences.

Main Methods:

• A kinome-wide siRNA screen in HeLa cells to identify kinases regulating gephyrin clustering.
• Validation of candidate kinases in primary rat hippocampal neurons.
• Analysis of signaling pathways including MAPK, PI3K-Akt, and mTOR.
• Assessment of GABAergic signaling and neuronal network activity.

Main Results:

• Identified 12 candidate kinases, including FGFR1, TrkB, TrkC, and components of MAPK and mTOR pathways.
• Demonstrated that brain-derived neurotrophic factor (BDNF) modulates gephyrin clustering via MAPK signaling.
• Showed BDNF activates mTOR and represses GSK3β, influencing gephyrin and GABA(A) receptor clustering.
• Rapamycin treatment (mTOR antagonist) resulted in neuronal network disinhibition.

Conclusions:

• BDNF regulates gephyrin clustering through both MAPK and PI3K-Akt-mTOR signaling pathways.
• Disruption of BDNF-mTOR signaling impairs GABA(A) receptor clustering and GABAergic function.
• Targeting mTOR signaling affects neuronal network activity, highlighting its role in synaptic function.