USP9X in Cortical Development and Behaviour

Abstract

Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) are two of the most common neurodevelopmental disorders classified by the Diagnostic and Statistical Manual of Mental disorders 5th Edition. Both are often comorbid, meaning that they often exist in the same patient. Due to this, it has been hypothesised that there may be a common cause for ID and ASD, a common disruption in the same neurodevelopmental pathway. A gene that has been implicated in both the pathogenesis of ID and ASD is the Ubiquitin-Specific Protease 9 XLinked (USP9X). Targets of USP9X and USP9X itself have been found to be part of mTOR, TGFb, and WNT signalling pathways, all equally important pathways in the developing brain. In addition, deletion of Usp9x in mice has been found to have immense effects on the postnatal development of the hippocampus. Recently, a study reported that Usp9x-null mice had learning and memory deficits, similar to that seen in ID patients with USP9X mutations. However, to date, there has been no extensive study into how the deletion of Usp9x in mice affects cortical development nor investigated if any ASD-related behaviour manifested from the deletion. Therefore, this study set out to address those questions by deleting Usp9x from the dorsal telencephalon of mice and used them as a model system to investigate the role of Usp9x in cortical development and behaviour. Adult male mice where Usp9x has been deleted (Usp9x-/y) and their control littermates (Usp9x+/y) were used in this project. Firstly, the adult neocortex was characterised using haematoxylin staining, where measurements were made in several areas of the neocortex. Results indicate that the neocortex were minimally affected with the deletion of Usp9x; the thickness of most areas remained not significantly different to controls, barring the retrosplenial cortex being thicker in Usp9x-/y mice compared to Usp9x+/y mice. However, the deletion of Usp9x lead to a significantly smaller corpus callosum compared to controls. Following that, the axonal initiation ability of neurons lacking Usp9x was investigated by culturing neurons from embryonic mouse cortices. Usp9x-/y cortical neurons were found to grow axons normally, comparable to the controls. To characterise any changes in behaviour as a result of deleting Usp9x from the dorsal telencephalon, Usp9x-/y mice were subjected to a battery of behavioural assays. The first set of assays measured anxiety-related behaviour, including open-field test, elevated plus maze (EPM), light/dark box, and long-term readings using the Phenomaster. The results showed that Usp9x-/y mice displayed hyperactivity and low anxiety behaviour in short-term open field, EPM, and light/dark tests. However, Usp9x-/y mice also showed high anxiety-like traits in the home-cage Phenomaster readings, indicating that Usp9x-/y mice may have chronic, long-term anxiety. Next, Usp9x-/y mice were subjected to several tests that measured the three diagnostic traits of ASD, impaired communication, deficits in social interaction, and repetitive behaviour. To test for communication, Usp9x-/y pups were recorded for their ultrasonic vocalisations (USVs). Usp9x-/y pups were found to have changes in communication after being separated from the dams. Next, Usp9x-/y mice were tested for any impairments in their socialisation skills. The mice were subjected to the standard three-chambered sociability test and a five-day social habituation/dishabituation test. Results from both tests indicated that Usp9x-/y mice have low social interest, confirming one of the most indicative traits of ASD is present in Usp9x-/y mice. However, Usp9x-/y mice did not display any repetitive behaviour when tested using the Y-maze. Despite that, based on these results, the morphological and behavioural phenotypes seen in the Usp9x-/y mouse model correspond to the symptoms seen in the human patients. This study shows that Usp9x-/y mouse model has the potential to be used as a model organism to explore therapeutic avenues, such as drug or compound screening.