Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+-channels with the secretory apparatus, but little is known about how these components are organized. We have shown over the past years that α-neurexins are synaptic cell-surface molecules that are required for Ca2+-triggered exocytosis. Mice lacking all three α-neurexins die early postnatally. The most striking feature of the triple knockout phenotype is the dramatic reduction in spontaneous and evoked neurotransmission at both glutamatergic and GABAergic synapses, and is presumably due to an impaired function of high voltage-activated Ca2+ channels.
Transgenic rescue experiments on the null-mutant background confirmed that α-neurexins perform a non-redundant role in Ca2+-dependent neurotransmitter release, which requires their long extracellular domains and can not be undertaken by the shorter β-neurexins. In spite of the dramatic functional defects, only subtle morphological changes were found in the brains of KOs, limited to a moderate decrease in neuropil and area density of type II (presumably inhibitory) synapses. In addition to their role at central synapses, α-neurexins also affect transmission at the neuromuscular junction, and contribute substantially to Ca2+-triggered release of secretory granules from endocrine cells.
In this project, we are currently the important question how high voltage-dependent Ca2+ channels are involved in the process and which aspect of their function is affected by α-neurexins To this end, we investigate, for example, the subcellular targeting of neurexins and Ca2+ channels to the synapse, and try to determine the respective molecular determinants and interaction partners involved. We show that neurexins are targeted to synapses via a new type of transport vesicle in primary hippocampal neurons and in transgenic animals that were previously shown to rescue the null-mutant phenotype. The absence of bassoon labeling and the ubiquitous distribution of these punctae demonstrate that they are not PTVs but colocalization and cosedimentation assays with RIM1α suggest that they may carry additional components of the exocytotic machinery.
Using mutagenesis of N- and C-terminal sequence motifs and identification of loss-of-function and gain-of-function mutations, we found that efficient plasma membrane insertion of neurexins occurs predominately in the axonal/synaptic compartment and requires their PDZ-binding motif. Our data suggest that neurexins, in contrast to their postsynaptic binding partner neuroligins, are delivered to synapses via transport vesicles in a polarized targeting process that is regulated by their C-terminal interactions with PDZ-domain containing binding partners.