Neurodevelopmental disorders (NDDs) encompass a heterogeneous group of chronic conditions characterized by impairments in development and function, significantly impacting quality of life. These disorders, including intellectual disability (ID), autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD), and others, manifest early in development and can persist throughout life. The etiology of NDDs is complex and heterogeneous, with genetic factors playing a significant role. Research suggests that NDDs may share overlapping genetic abnormalities, with neurexins (NRXNs), particularly NRXN1, emerging as a key susceptibility gene. NRXN1, crucial for synapse organization and transmission, has been implicated in various neurodevelopmental, psychiatric, and neuropsychological disorders. While the role of NRXN1 in synaptogenesis is well-studied, its role in neurogenesis remains debated, with some studies showing no effect on neurite outgrowth in NRXN1 mutant neurons and others reporting decreased neurite length and number. The prefrontal cortex (PFC), vital for executive functions, social cognition, and emotional regulation, is affected by NRXN1 deletion, potentially leading to dysfunction in these domains. This study aimed to investigate the behavioral and molecular consequences of PFC-specific NRXN1 knockdown in juvenile rats, focusing on anxiety-like behaviors, repetitive behaviors, and social interaction, and exploring the underlying proteomic changes.

Previous studies have linked NRXN1 mutations to ASD, ADHD, and schizophrenia. Mouse models with NRXN1 knockout have shown various behavioral alterations, including changes in repetitive grooming, motor learning, anxiety, and social approach, although results have varied depending on the mouse strain and the type of NRXN1 deletion (homozygous vs. heterozygous). Studies on the effects of NRXN1 on neurite outgrowth have yielded conflicting results. While some found no impact on neurite morphology in NRXN1 mutant human neurons, others demonstrated reduced neurite length and number in NRXN1+/- hiPSC-neurons. The PFC's importance in executive function, social cognition, and emotional regulation makes it a key region of interest in studying NRXN1's role in NDDs. Prior research has shown that NRXN1 deletion alters PFC metabolism and reduces the efficiency of functional brain networks.

This study used male Sprague Dawley rats (n=27) at 3 weeks of age. Rats were randomly assigned to three groups: wild type (WT), a negative control group (Sh-Nc), and an experimental group with NRXN1 knockdown in the PFC (Sh-NRXN1). Knockdown was achieved through stereotactic microinjection of adeno-associated virus (AAV) vectors targeting NRXN1 exon 1. Behavioral testing, including the open-field test (OFT) and three-chamber sociability test, was conducted 2 weeks post-injection to assess anxiety-like behavior, repetitive behavior (self-grooming), and social interaction. In vitro experiments used primary neuronal cultures from rat PFC, where NRXN1 knockdown was achieved using lentiviral vectors. Neurite outgrowth was analyzed using immunofluorescence staining of microtubule-associated protein 2 (MAP2), followed by quantitative analysis of total neurite length, length of the longest neurite, number of primary neurites, and branch points. Proteomic analysis using tandem mass tag (TMT) labeling and mass spectrometry (MS) was performed on PFC neuron samples from Sh-NRXN1 and control rats to identify differentially expressed proteins. Immunoblotting was used to validate the proteomic findings. Statistical analysis included one-way ANOVA, t-tests, and other appropriate methods.

NRXN1 knockdown was successfully achieved both in vivo and in vitro. Behavioral analysis revealed significant differences between the Sh-NRXN1 group and the control groups. Sh-NRXN1 rats showed increased rearing frequency in the OFT, suggesting increased exploration or possibly repetitive behavior, and spent less time in the center, indicating increased anxiety. Critically, self-grooming bouts and time were significantly increased in the Sh-NRXN1 group, demonstrating repetitive behavior. In the three-chamber sociability test, Sh-NRXN1 rats exhibited increased entries into the social chamber and increased contact with the social cylinder, despite not increasing their interaction time; suggesting frequent but brief interactions. This ‘hyperactive’ social interaction, combined with elevated anxiety and repetitive behavior, provides a distinct behavioral profile. In vitro, NRXN1 knockdown significantly reduced total neurite length and the number of branch points in primary PFC neurons, indicating impaired neurite outgrowth. Proteomic analysis identified 130 differentially expressed proteins (57 upregulated, 73 downregulated) in Sh-NRXN1 rats compared to controls. These proteins were enriched in pathways related to neuronal function, mental disorders, extracellular matrix, cell membrane, and morphological changes. Immunoblotting confirmed the downregulation of three selected proteins (ANXA1, ANXA4, GRB2) involved in cell morphology and membrane structure.

The findings demonstrate that PFC-specific NRXN1 knockdown in juvenile rats leads to a complex behavioral phenotype characterized by anxiety-like behavior, increased repetitive self-grooming, and altered social interaction. The observed ‘hyperactive’ social behavior in the Sh-NRXN1 group, though seemingly positive, could be interpreted as a manifestation of stereotyped behavior with reduced social content, potentially driven by the increased anxiety levels observed in these animals. The impairment in neurite outgrowth observed in vitro suggests that NRXN1 plays a direct role in neuronal development and that the behavioral changes observed may stem, at least in part, from structural alterations at the neuronal level. The proteomic findings highlight potential molecular mechanisms underlying these behavioral and morphological changes, implicating pathways related to neuronal function, and suggesting that impaired protein-protein interactions mediated by NRXN1 dysfunction might contribute to NDDs. Differences between this study and prior research might be due to differences in species (rats vs. mice), age (juvenile vs. adult), genetic background, and the type and extent of NRXN1 manipulation.

This study confirms the association between NRXN1 and abnormal behaviors in NDDs, providing detailed insights into the behavioral and molecular consequences of PFC-specific NRXN1 knockdown in juvenile rats. The findings highlight the complexity of NRXN1's role in neuronal development and behavior, suggesting its involvement in multiple pathways that might contribute to NDDs. Further research is needed to fully elucidate the molecular mechanisms linking NRXN1 dysfunction to the observed behavioral and morphological changes. Future studies could explore the specific roles of the differentially expressed proteins identified in this study and investigate the therapeutic potential of targeting these pathways.

The study used a rat model, which might not perfectly capture the complexities of human NDDs. The knockdown efficiency of NRXN1 was higher in vitro compared to in vivo, potentially limiting the generalizability of some findings. The study focused on a specific age range and a single strain of rats, limiting the generalizability of the findings across different developmental stages and genetic backgrounds. While proteomic analysis identified numerous differentially expressed proteins, further investigation is needed to fully understand their roles in the observed phenotypes.