Epilepsy and Autism Spectrum disorders (ASD) are both complex neurodevelopmental disorders which share approximately 30% comorbidity. Epilepsy is characterized by unprovoked recurrent seizures and affects ~1% of the population while ASDs are characterized by deficits in language, social, and behavior and found 1 in 68 people. Variants in synaptic genes suggest disruptions in synaptic regulation underlie both conditions. PRICKLE1 and PRICKLE2 are known core WNT/ PCP genes implicated in Progressive myoclonic epilepsy in families and in the general population. Humans, mice, zebrafish, and Drosophila with disrupted Prickle exhibit epileptic behavior and other neurological deficits. Prickle is implicated in several aspects of neuronal development and mutated proteins display aberrant activity in vivo and in vitro. Recently, variations in PRICKLE1 were associated with ASDs in humans. The mechanisms by which PRICKLE could contribute to ASDs are unknown. Results presented here show Prickle1+/- mice exhibit ASD-like behavior. Prickle1 associates with Synapsin I; a phosphoprotein important for synaptogenesis, axonogenesis, and neurotransmitter release. Mutant R104QPRICKLE1 protein causes a reduction in sizes of dense-core vesicles in neuronal-like PC12 cells. Results indicate PRICKLE1 may be associated with ASDs and possibly involved in synaptic homeostasis. Prickle deregulation has also been associated with neural tube defects and cancers. The mechanism(s) by which Prickle is regulated is incompletely understood. To further elucidate the role of PRICKLE in disease, immunoprecipitates from PRICKLE-expressing stable tetracycline-regulated neuronal-like PC12 cells were identified by mass spectrometry. The deubiquitinating enzyme USP9X was identified as a novel interacting PRICKLE protein. USP9X is a substrate-specific deubiquitinating enzyme implicated in several aspects of neuronal development, associated with X-linked intellectual disability and a candidate gene for epilepsy. Results show that USP9X robustly deubiquitinates and protects PRICKLE from proteasomal degradation. USP9X variants found in the ARRA ASD cohort directly associates USP9X with ASDs. The identified USP9X mutations delete the PRICKLE-interacting domain and provide a possible mechanism for PRICKLE deregulation. Already a target for treating cancer, USP9X can serve as a therapeutic target to regulate PRICKLE levels.

The Hippo signalling pathway is an evolutionarily conserved kinase cascade responsible for the cell proliferation, tissue growth and apoptosis during development and its dysregulation contributes to tumourigenesis. This signalling pathway was initially discovered in Drosophila and soon after that, it was shown to be highly conserved in mammals. The core Lats kinases of this tumour suppressive pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Latest reports revealed various E3 ubiquitin ligases to negatively regulate the Hippo pathway through ubiquitination, yet few deubiquitinating enzymes have been described. In the present study, we report USP9X deubiquitinating enzyme as an essential regulator of the central components of this pathway. USP9X interacted strongly with Lats2 kinase and to a lesser extent with WW45, Kibra and Angiomotin family proteins. The knockdown of USP9X resulted in notable downregulation and destabilisation of Lats kinase and to lesser extents WW45, Kibra and Amot. This resulted in enhanced nuclear localisation of YAP and TAZ accompanied with activation of their target genes, CTGF and CYR61. USP9X was shown to stabilise Hippo components through its deubiquitinating activity. USP9X enzyme defective mutant lost the activity to stabilise Lats2, WW45, Kibra and Angiomotins through deubiquitination, leading to their ubiquitination. In the absence of USP9X, cells exhibited epithelial to mesenchymal transition phenotype and additionally gained anchorage-independent growth in soft agar. Moreover, USP9X knockdown disrupted acinar organisation of breast cells in three-dimensional acini cultures. In addition, YAP/TAZ target gene activation in USP9X knockdown cells could be rescued by knockdown of YAP, TAZ and TEAD2. Lastly, USP9X protein expression showed a positive correlation with Lats kinases, but negative correlation with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. The results strongly indicate that USP9X cooperates with Lats2 and other important Hippo components to suppress tumour growth.