Seizures are regulated by ubiquitin-specific peptidase 9 X-linked (USP9X), a de-ubiquitinase

Abstract

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.

Fig 1. Prickle interactome.

We combined findings from our proteomics interaction experiment and public databases to generate a prickle interactome. We used the MetaCore (MetaCore, GeneGO Inc., St. Joseph, MI, USA) networking function and String database 9.1 to curate interaction maps of the proteins identified. Information for identified interactions is obtained from several sources including but not limited to genomic context, database imports (PPI and pathway databases), high-throughput experiments, co-expression, and text mining. We uploaded our lists of proteins from LC-MS/MS into the software programs and exported the networks into Cytoscape 2.7.0 for manipulation of the network appearance. (Nodes, circles; Edges, lines). Red lines correspond to interactions observed by our labs using yeast-2-hybrid and IP-MS approaches. The extended interactome was generated as we have previously described. [, –9, 18, 19] Prickle1 and Prickle2 interact with known synaptic proteins. The interaction with USP9X is novel.

Fig 2. PRICKLE interacts with USP9X via its carboxyl terminal.

A. PRICKLE1 and PRICKLE2 interact with endogenous Bcr, Tanc2, and Usp9x in NGF-differentiated PC12 cells. GFP immunoprecipitates from stable lines expressing GFP, GFP-PRICKLE1 or GFP-PRICKLE2 confirm that PRICKLE interacts with Bcr, Usp9x and Tanc2. B. Flag immunoprecipitates, from HEK293T cells overexpressing flag-tagged PRICKLE1 or PRICKLE2, show endogenous USP9X physically interacts with PRICKLE. C, D. Schematic of PRICKLE1(C) and PRICKLE2(D) constructs. Flag-immunoprecipitates from HEK293T cells overexpressing the indicated constructs were analyzed by anti-USP9X Western blotting. Both PRICKLE 1 and 2 interact with USP9X via their C-termini while BCR binding mapped to their N-termini.

Fig 3. USP9X stabilizes PRICKLE in HEK293T cells and the mouse brain.

A. Flag-immunoprecipitates from HEK293T cells overexpressing the indicated constructs show PRICKLE and USP9X interact via their carboxyl termini. B, C. USP9X deubiquitinates PRICKLE. Immunoprecipitates from HEK293T cells overexpressing the indicated constructs show PRICKLE ubiquitination in the presence of HA-Ubiquitin and deubiquitination/stabilization by Usp9x. IP (immunoprecipitates), IN (input). D. Loss of Usp9X affects Prickle2 (green) expression. Deletion of Usp9x results in decreased Prickle2 expression in the cortex (I), CA1 (II) and dentate gyrus (III) of 4-week old mice when compared to controls (IV, V, VI). n = 2. Scale bar:20μM. Nuclear stain = DAPI.

Fig 4. Genetic and pharmacological suppression of the seizure phenotype in prickle mutant flies.

(A–C) Reducing faf dosage with three separate loss-of-function alleles ((faf^B3, faf^BX3, faf^BX4; graphs A, B and C, respectively) suppresses the pk^sple-mediated seizure phenotype detected by the modified bang sensitivity assay. Six vials per genotype were assayed. D. Inhibition of faf activity with Degrasyn suppresses pk^sple-mediated seizure activity detected using the “fly flip” assay. Three vials per genotype were assayed. Note: +/+ = Oregon-R control flies; sple = pk^sple; sple/faf = pk^sple/+; +/faf. * = p < 0.05. Error bars = standard error. Each vial contains 5 males and 5 females.