Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Rescue strategies for reverting the phenotype caused by the autism-linked mutation R451C in neuroligin3: in vitro and in vivo approaches

Summary An increasing number of mutations associated with Autism Spectrum Disorders (ASDs) have been found in genes coding for synaptic proteins, including cell adhesion molecules located on the postsynaptic terminal, such as the Neuroligins. The substitution Arg451Cys (R451C) in Neuroligin3 is associated with ASDs and promotes the partial misfolding of the extracellular domain of the protein that is blocked in the endoplasmic reticulum rather than being trafficked to the cell membrane. The mutant protein in the endoplasmic reticulum induces a stress condition of the organelle. The reduced amount of R451C Neuroligin3 on the cell surface impairs synaptic function and normal social behaviors. We have selected compounds that belong to the glucocorticoid family, for their ability to promote the exit of the mutant protein from the endoplasmic reticulum and to improve the cell surface trafficking of R451C Neuroligin3 in HEK-293 cells. The treatment promotes the formation of artificial synapses, between HEK-293 expressing the R451C NLGN3 mutation and primary hippocampal neurons. The data obtained in HEK-293 were validated in a more physiological cellular system, consisting of neural progenitor cells that can be differentiated in astrocytes, and mixed neurons/astrocyte derived from the hippocampus of mice that endogenously express either the WT or the mutant R451C Neuroligin3. Our results show that the treatment promotes the recovery of endogenous levels of mutant Neuroligin3 and lowers the activation of the unfolded protein response. The effect of the treatment was subsequently tested in vivo on the mouse model, knock-in for R451C NLGN3. However, the chronic treatment with dexamethasone did not show the expected behavioral improvements and increase of NLGN3 levels. The effect of the treatment was subsequently tested in vivo on the mouse model, knock-in for R451C NLGN3. The chronic treatment with dexamethasone did not show the expected behavioral improvements and increase of NLGN3 levels. Finally, we characterized social behavior at different times during mice development. We found alterations in the ability to habituate to the same unfamiliar mouse in adolescent, young and adult NLGN3 mice. In adult mice, we observed the onset of aggressive behavior. The treatment with acute or chronic intranasal oxytocin recovered behavioral impairments.