Neuroligins are postsynaptic proteins that can induce presynaptic differentiation. These single-transmembrane-domain proteins feature a long extracellular sequence and a short intracellular sequence. The rodent genome contains 4 genes that encode Neuroligins, Neuroligin1–Neuroligin4, and, intriguingly, these proteins localize at distinct types of synapses: Whereas Neuroligin1 localizes at excitatory synapses, Neuroligin2 and Neuroligin4 localize at inhibitory synapses, and Neuroligin3 is found at both excitatory and inhibitory synapses. Moreover, mutations in Neuroligin3 and Neuroligin4 are associated with autism in humans. However, the molecular mechanism underlying these phenomena is poorly understood.

To investigate the molecular mechanisms of synaptogenesis and autism, we identifi...[ Read more ]

Neuroligins are postsynaptic proteins that can induce presynaptic differentiation. These single-transmembrane-domain proteins feature a long extracellular sequence and a short intracellular sequence. The rodent genome contains 4 genes that encode Neuroligins, Neuroligin1–Neuroligin4, and, intriguingly, these proteins localize at distinct types of synapses: Whereas Neuroligin1 localizes at excitatory synapses, Neuroligin2 and Neuroligin4 localize at inhibitory synapses, and Neuroligin3 is found at both excitatory and inhibitory synapses. Moreover, mutations in Neuroligin3 and Neuroligin4 are associated with autism in humans. However, the molecular mechanism underlying these phenomena is poorly understood.

To investigate the molecular mechanisms of synaptogenesis and autism, we identified novel Neuroligin-binding proteins by means of mass spectrometry and yeast two-hybrid screening. In the yeast two-hybrid screening, 2 NEDD4-family proteins were identified: ITCH and WWP2. The human and rodent genomes contain 9 genes that encode NEDD4-family proteins, which are HECT-type E3 ubiquitin ligases that contain WW domains. Our results showed that some of these proteins interact with Neuroligin2 through their WW domains and ubiquitinate Neuroligin2. Interestingly, among the 9 NEDD4-family proteins, only ITCH induces marked ubiquitin-dependent endocytosis of Neuroligin2, which results in a substantial reduction of cell-surface levels of Neuroligin2. Moreover, Neuroligins are differentially regulated by ubiquitin-dependent endocytosis. Whereas Neuroligin1 and Neuroligin2 are regulated by ubiquitination, Neuorligin3 is not, because it lacks a ubiquitination site. Ubiquitination of Neuroligins by ITCH causes a reduction of synapses in cultured neurons. Conversely, knockdown of ITCH or inhibition of its ubiquitin-ligase activity causes morphological changes in synapses. In summary, our results identify ubiquitin-dependent endocytosis of Neuroligins as a novel molecular mechanism of synapse elimination.