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

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABA(A) receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)-Akt also activates mTOR and represses GSK3ß, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF-mTOR signaling is associated with reduced GABA(A) receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K-Akt-mTOR signaling.

Receptor tyrosine kinase EphA7 is required for interneuron connectivity at specific subcellular compartments of granule cells.

Neuronal transmission is regulated by the local circuitry which is composed of principal neurons targeted at different subcellular compartments by a variety of interneurons. However, mechanisms that contribute to the subcellular localisation and maintenance of GABAergic interneuron terminals are poorly understood. Stabilization of GABAergic synapses depends on clustering of the postsynaptic scaffolding protein gephyrin and its interaction with the guanine nucleotide exchange factor collybistin. Lentiviral knockdown experiments in adult rats indicated that the receptor tyrosine kinase EphA7 is required for the stabilisation of basket cell terminals on proximal dendritic and somatic compartments of granular cells of the dentate gyrus. EphA7 deficiency and concomitant destabilisation of GABAergic synapses correlated with impaired long-term potentiation and reduced hippocampal learning. Reduced GABAergic innervation may be explained by an impact of EphA7 on gephyrin clustering. Overexpression or ephrin stimulation of EphA7 induced gephyrin clustering dependent on the mechanistic target of rapamycin (mTOR) which is an interaction partner of gephyrin. Gephyrin interactions with mTOR become released after mTOR activation while enhanced interaction with the guanine nucleotide exchange factor collybistin was observed in parallel. In conclusion, EphA7 regulates gephyrin clustering and the maintenance of inhibitory synaptic connectivity via mTOR signalling.

Organisation and control of neuronal connectivity and myelination by cell adhesion molecule neurofascin.

The neuronal cell adhesion molecule neurofascin is expressed in highly complex temporally and spatially regulated patterns. Accordingly, many different functions have been described including control of neurite outgrowth, clustering of protein complexes at the axon initial segments as well as at the nodes of Ranvier and axoglial contact formation at paranodal segments. At the molecular level, neurofascin provides a link between extracellular interactions of many different interaction partners and cytoskeletal components or signal transduction. Such interactions are subject to intimate regulation by alternative splicing and posttranslational modification. The versatile functional aspects of neurofascin interactions pose it at a central position for the shaping and maintenance of neural circuitry and synaptic contacts which are implicated in nervous system disorders.