Analysis of mutations in synaptic adhesion molecules involved in neurodevelopmental disorders: cell mechanisms of endoplasmic reticulum retention and unfolded protein response activation

Several forms of monogenic autism spectrum disorders are associated to mutations in the genes encoding for the postsynaptic cell adhesion molecules, Neuroligins. The autism-linked substitution of the arginine at position 451 by a cysteine (R451C) in Neuroligin3 induces local misfolding of the extracellular domain, causing partial retention in the endoplasmic reticulum. The accumulation of misfolded proteins in the endoplasmic reticulum can eventually result in stress conditions and ultimately in the activation of the unfolded protein response. We have generated a PC12 Tet-On cell system with inducible expression of either wild type or R451C Neuroligin3. In this system, we show that the over-expression of R451C NLGN3 leads to the activation of the unfolded protein response, both in proliferative and neuronal-differentiating conditions. The knockin mouse strain expressing R451C Neuroligin3 is currently considered a model for studying a monogenic form of autism spectrum disorders. We have characterized the effect of the mutation on Neuroligin3 protein levels during development from embryonic stage E12 to postnatal day P60, observing in the knockin mice lower Neuroligin3 protein levels and a delay in the expression of the protein. We showed that the endogenous mutant Neuroligin3 is partially retained in the endoplasmic reticulum as observed in vitro. In the R451C Neuroligin3 migrates in two different bands, representing the incompletely glycosylated ER-retained protein and the mature glycosylated form that traffics to the cell membrane. We have investigated the activation of the unfolded protein response in vivo, in the R451C Neuroligin3 mouse model and showed the Unfolded protein response and autism spectrum disorders upregulation of the two main targets, BiP and CHOP, in total brain and cerebellum extracts from both adult and embryonic knockin mice. BiP protein levels and the phosphorylation of eIF2α were significantly increased only in the cerebellum of adult knockin mice in comparison to wild type, in agreement with the mRNA data. Unfolded protein response signaling has been reported to regulate synaptic function and plasticity. The AMPA-mediated glutamatergic currents were studied in the cerebellum, where we observed a significant increase in miniature excitatory synaptic currents in Purkinje cells of the knockin in comparison to the wild type mice. The final aim of this thesis was focused on selecting molecules from a library of chemical compounds, acting in correcting the defective trafficking of mutant Neuroligin3. The cellbased screening used HEK293 stably transfected with a truncated and fluorescent form of R451C. We have identified one compound active on improving selectively the trafficking of the R451C along the secretory pathway. The effects caused by this compound are promising for evaluating in vivo the rescue of the behavioral and functional phenotype described for the R451C Knockin mouse. This molecule, or molecules structurally correlated, could be used for designing therapeutic strategies for monogenic forms of autism characterized by the retention of misfolded Neuroligins within the endoplasmic reticulum. In conclusion, we provide a link, both in vitro and in vivo, between UPR activation and a form of monogenic ASD caused the R451C misfolding mutation in Neuroligin3. We have identified in the cerebellum of the knockin mouse model expressing R451C Neuroligin3, the specific brain region where UPR targets and modulators are regulated. Lately this region has been implicated in cognitive and emotional traits typical of the autistic Unfolded protein response and autism spectrum disorders phenotype. Since UPR mediators are involved in neuronal plasticity, the activation of UPR in cerebellum can lead to neuronal circuits alterations and consequently have a role in the autistic phenotype.