Synapses are established with precision during brain development and are constantly remodeled as a consequence of synaptic activity in the adult networks. Synaptic dysfunction underlies the molecular basis of several neurodevelopmental disorders, such as autism spectrum disorders (ASD). Trans-synaptic adhesion systems can regulate synaptic function, as they organize pre- and postsynaptic protein complexes. One of these adhesion systems is formed by neurexins and neuroligins. These proteins promote the assembly and maturation of synapses through a bidirectional mechanism. In mammals, neurexins are encoded by three genes with two alternative promoters, which produce the long (alpha-neurexins) and the short (beta-neurexins) isoforms. In addition, alternative splicing in the extracellular domain contributes to generate hundreds of neurexins isoforms. Despite the high heterogeneity of the extracellular region, the cytoplasmic domain is common to all neurexin isoforms and it is thought to regulate intracellular signalling. The relevance of neurexins in neurodevelopmental disorders has been highlighted by the identification of mutations in neurexin genes in ASD. Recently, we have suggested a role for synaptic defects of beta-neurexin-1 as a risk factor for autism and mental retardation.
To characterize in cultured neurons the effect of a beta-neurexin-1 dominant negative mutant that lacks the cytoplasmic tail (HA-bNrxDC). To inhibit the function of beta-neurexin-1 in vivo by expressing the HA-bNrxDC mutant. To characterize the behavioral phenotype of a double transgenic mice expressing an inducible form of the HA-bNrx1DC mutant (TRE-HA-bNrx1DC/CamKII-tTA).
In vitro studies have been performed in hippocampal neurons at 10-14 DIV isolated from 18-19 embryonic day rat brains. For in vivo studies we have generated a transgenic mouse line that expresses a HA-tagged beta-neurexin-1 mutant lacking the cytoplasmic domain (HA-bNrx1DC) under the control of the inducible TRE promoter. The TRE-HA-bNrx1DC transgenic mice have been crossed with CAMKII –tTA animals to direct the expression of the mutant protein to glutamatergic terminals in vivo.
Our in vitro results suggest that HA-bNrx1DC mutant can function as a dominant negative mutant as it can be recruited to the membrane of glutamatergic synapses through interaction with neuroligin-1, but it inhibits intracellular signalling mediated by the cytoplasmic tail. In vivo we show expression of HA-bNrx1DC in the cortex and hippocampal formation by immunolocalization. Moreover, we have evaluated the behavioral consequences of the lack of beta-neurexin-1 function in TRE-HA-bNrx1DC/CamKII-tTA double transgenic mice.
Inducible expression of a beta-neurexin-1 dominant negative mutant might have implications in the study of autism, as it may help answering to what extent synaptic and behavioral defects due to beta-neurexin-1 dysfunction can be rescued by resuming normal beta-neurexin-1 function.