Establishing a Mouse Model of NL3R617W-Associated Autism Spectrum Disorder for a Functional Study

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and limited behavior. Despite the association of numerous synaptic gene mutations with ASD, the presence of behavioral abnormalities in mice expressing autism-associated R617W mutation in synaptic adhesion protein neuroligin-3 (NL3) has not been established. This work focuses on establishing a mouse model of ASD caused by NL3 R617W missense mutation (NL3R617W) and characterizing and profiling the molecular as well as behavioral features of the animal model.

The expression and distribution of NL3R617W mutant protein in the 293T cell membrane and intracellular NL3 was detected by using immunofluorescence approach. Meanwhile, synaptic markers (Synapsin I, vesicular glutamate transporter (VGluT) I and vesicular γ-aminobutyric acid transporter (VGAT)) and synapse number were detected with a confocal fluorescence microscope. Thereafter, the effect on NL3R617W was verified. The expression of synaptic proteins, postsynaptic density protein-95 (PSD95) and Src homology domain and multiple ankyrin repeat domains protein 3 (SHANK3), was verified by Western blot. The interaction between NL3 and neurexin 1 (NRXN1) was studied by means of co-immunoprecipitation. The behavior of autistic mice induced by NL3R617W mutation was examined using the Morris water maze and the Y maze. NL3R617W mutant mice were assessed in the open field, and three-chamber test was conducted to assess and observe the presence of hyperactivity, repetitive behavior, friendliness, and social novelty.

The results indicated that the NL3 mutation could influence the interaction between NL3 and NRXN1, and inhibit the expression of VGluT I. Nevertheless, NL3 mutation would not influence the expression of NL3 on cell membrane, the intracellular distribution of NL3, or the endoplasmic reticulum retention. The outcomes of animal studies demonstrated that the ASD mice with NL3R617W exhibited a significant decrease in the capacity for spatial memory and exploration, as well as the expression levels of the postsynaptic scaffolding proteins, PSD95 and SHANK3 (p < 0.05). The number of excitatory synapses in hippocampal cornu ammonis (CA)1 and CA3 and the sensory cortex was also significantly reduced (p < 0.01). Compared to the control mice, the NL3R617W mutant mice were less active in the open field (p < 0.001), a finding consistent with the three-chamber test result showing reduced degree of activity. Furthermore, compared to the control mice, the NL3R617W mutant animals spent less time with stranger mice (p < 0.05).

NL3R617W mutation may inhibit the expression of postsynaptic scaffolding proteins by influencing the interaction with NRXN1, thus inhibiting synapse formation and reducing the number of excitatory synapses.