USP9X encodes a substrate-specific deubiquitylating enzyme known to regulate neural progenitor cells of the developing brain. A recent large-scale X-exome re-sequencing effort of X-linked intellectual disability (ID) families identified 3 unique USP9X variants which segregated with the disorder. To investigate USP9X as novel intellectual disability gene and potential pathogenic mechanism(s) we isolated both primary neural stem cells and neurons from the embryonic brain of the Usp9x knock-out mouse model.
Using the well established neurosphere assay to investigate neural stem/precursor cells in-vitro, we isolated neural stem cells from both control and Usp9x knock-out mice and used these cells to model neuronal migration. In the absence of Usp9x there was a 42% reduction of in-vitro neuronal migration. To establish if the USP9X ID variants also result in aberrant neuronal migration, we nucleofected control and null Usp9x neural stem cells with USP9X ID variant constructs. The neuronal migration defect was unable to be rescued by 2/3 of the variants, suggesting a possible pathogenic mechanism in USP9X ID patients. In the absence of Usp9x, we also identified a 43% reduction in primary axonal growth and arborisation of hippocampal neurons. Re-expression of the three USP9X ID variant constructs in Usp9x null neurons was unable to rescue the axonal defects.
2D-DIGE followed by mass spectrometry on cortical neurons isolated from control and knock-out embryonic brains identified 28 deregulated proteins. Identified deregulated proteins included genes involved in human neuronal migration disorders, whilst others have roles in neuronal polarity and axon growth. These results suggest cellular processes affected in USP9X ID patients and further support the results from the in-vitro neuronal migration assay.
Together our data identifies USP9X as a novel ID gene, revealing new roles for USP9X in neuronal development. Our results highlight the usefulness of neural stem cell models to identify pathogenic mechanisms of ID.