The synapse is essential for normal neuronal communication and synaptic abnormalities could underlie many neuronal pathologies leading to such diseases as epilepsy and autism. Recent reports suggest that the Wnt signaling pathway is essential for normal synaptic development and function. However the role of specific Wnt ligands and their downstream signaling molecules play in synapse formation and function remain unclear. PRICKLE1 (PK1) and PRICKLE2 (PK2) are downstream Wnt signaling molecules which are suggested to play essential roles in neuronal function. PK1 was recently shown to be mutated in three large families with epilepsy, and Pk2 interacts with post-synaptic density 95 and subunits of the NMDA receptor. Although it seems clear that PK1 and PK2 are critical for normal neuronal function, their role in synaptic function and animal behavior remain to be investigated. In Aim1, we show that mutations in prickle (pk) genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PK1 or PK2. In overexpression assays in zebrafish, pk mutations resulted in aberrant pk function. A seizure phenotype was present in the Pk1-null mutant mouse, two Pk1 point mutant (missense and nonsense) mice, and a Pk2-null mutant mouse. Drosophila with pk mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. In aim 2, we describe two families with ASD-specific mutations in the non-canonical Wnt gene PK2. These mutations reduced the co-localization of the human PK2 protein with PSD-95, another protein implicated in ASDs. Studying Pk2 function in mice, we found that disrupting Pk2 in mouse hippocampal neurons reduced dendrite branching, synapse number, and post-synaptic density size. Consistent with these findings, disrupting Pk2 decreased the frequency and size of spontaneous miniature synaptic currents. Interestingly, these phenotypes were rescued by wild-type human PK2, but not the ASD-associated PK2 mutants suggesting that these mutations cause a critical loss of PK2 function. Behavioral studies in Pk2-/- mice suggest that loss of Pk2 function lead to ASD-like behaviors. These studies provide new insight into the biological roles of PK2, its behavioral importance, and firmly link non-canonical Wnt signaling abnormalities and ASDs. Together, Aim1 and 2 show that the Pk proteins are critical regulators of normal neuronal function and suggest that Pk2 could be a link between epilepsy and autism.

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.