Mechanisms ofprickle1afunction in zebrafish epilepsy and retinal neurogenesis

Abstract

SUMMARYEpilepsy is a complex neurological disorder characterized by unprovoked seizures. The etiology is heterogeneous with both genetic and environmental causes. Genes that regulate neurotransmitters and ion channels in the central nervous system have been associated with epilepsy. However, a recent screening in human epilepsy patients identified mutations in the PRICKLE1 (PK1) locus, highlighting a potentially novel mechanism underlying seizures. PK1 is a core component of the planar cell polarity network that regulates tissue polarity. Zebrafish studies have shown that Pk1 coordinates cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet how dysfunction of Pk1 relates to epilepsy is unknown. To address the mechanism underlying epileptogenesis, we used zebrafish to characterize Pk1a function and epilepsy-related mutant forms. We show that knockdown of pk1a activity sensitizes zebrafish larva to a convulsant drug. To model defects in the central nervous system, we used the retina and found that pk1a knockdown induces neurite outgrowth defects; yet visual function is maintained. Furthermore, we characterized the functional and biochemical properties of the PK1 mutant forms identified in human patients. Functional analyses demonstrate that the wild-type Pk1a partially suppresses the gene knockdown retinal defects but not the mutant forms. Biochemical analysis reveals increased ubiquitylation of one mutant form and decreased translational efficiency of another mutant form compared with the wild-type Pk1a. Taken together, our results indicate that mutation of human PK1 could lead to defects in neurodevelopment and signal processing, providing insight into seizure predisposition in these patients.

High-throughput behavioral assay to investigate seizure sensitivity in zebrafish implicatesZFHX3in epilepsy

“…We performed the behavior assay on pk1a morphant larvae along with age-matched, uninjected controls. We analyzed the data with SEIZR and demonstrated that SEIZR recognized the pk1a morphants as being seizure-sensitized (Figure 3B, p=0.027) in agreement with previously published findings (Mei et al, 2013). Thus, SEIZR efficiently and accurately analyzes differences in larval activity between groups.…”

“…With WT larvae, we observe increased activity with PTZ dose (Figure 3A, Spearman rank correlation of r=0.551, p<0.0001). Consistent with our previously published work (Mei et al, 2013), the 5 mM PTZ dose showed the most consistent response (smallest variation between larvae) and, therefore, this dose was used for further analyses.…”

“…To further validate the behavior assay and gene manipulation techniques used in conjunction with SEIZR, we tested a previously validated epilepsy gene, pk1a (Bassuk et al, 2008; Mei et al, 2013; H. Tao et al, 2011).…”

“…Using behavioral assays, work from our group has confirmed the role of the prickle 1 ( pk1 ) and 2 ( pk2 ), genes that are associated with seizures in humans (Bassuk et al, 2008; Hirotaka Tao et al, 2009). Specifically in pk1 and pk2 morphant larvae (larvae treated with antisense morpholino oligoes, MOs), we observed hyperactive motility in response to the seizure-inducing drug, pentylenetetrazol (PTZ) (Mei et al, 2014; Mei, Wu, Bassuk, & Slusarski, 2013); such a sensitized response to PTZ implies that normal levels of Pk1 and Pk2 activity directly or indirectly mitigate the risk of epileptic seizures and loss of function enhances the risk.…”

prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies

“…Coupling genetic analysis of human epilepsy cases with functional validation in animal models, we previously showed that mutations in human PRICKLE1 and PRICKLE2, as well as mutations in mouse, zebrafish and fly prickle (pk) orthologs, increase susceptibility to seizures (1)(2)(3). We recently showed that both mouse and fly Prickle can associate with Synapsin (4).…”