Abstracts

Rationale: Angelman syndrome (AS) is a devastating genetic disorder characterized by severe mental retardation and a characteristic behavioral phenotype. UBE3A, an E6-AP ubiquitin protein ligase, was identified as the locus mutated in AS. Seizures, abnormal electroencephalography (EEG), and microcephaly are observed in nearly 80% of AS patients. The most suitable drug therapy for treating seizures in AS patients remains a subject of debate and would benefit greatly from a better understanding of the underlying pathology. Toward this goal, we initiated studies using a simple vertebrate amenable to genetic manipulation and high-throughput experimental analysis e.g., zebrafish (Danio rerio). Methods: Zebrafish (mindbomb, line #hi909) were obtained from the Zebrafish International Resource Center (Eugene, OR). Mindbomb (mib) mutants carry a ubiquitin E3 ligase (UBE3A) mutation and were initially identified in a large-scale mutagenesis screen. Fish were mated and offspring analyzed at 3 days post-fertilization (dpf). Electrographic activity was monitored by using a field-recording electrode placed in the forebrain or optic tectum of agar-immobilized fish. For gene array analysis, zebrafish larvae were sorted by morphology, total RNA was extracted using TRIzol® Reagent,and quantified using GeneQuant® spectrophotometer. We pooled total RNA from 10 WT fish (control) and 10 mib mutants (experimental). We hybridized a total of 6 microarrays, including a dye swap for each tissue. Results: Zebrafish mindbomb (mib) mutants carry mutations for a gene that encodes a ubiquitin E3 ligase required for Notch activation. First, we examined mib mutants for potential seizure activity. Because the mindbomb mutant is characterized be a severe neurogenic phenotype manifesting as a “bumpy” microcephalic head, small eyes and body curled dorsally at 3 dpf, we sorted fish into mib mutant and wild-type (WT) sibling groups. WT sibling controls did not show any signs of burst discharge activity at this age (n = 9). In contrast, large-amplitude poly-spike burst discharge activity was recorded in all mib mutants at 3 dpf (n = 21). Second, we used a microarray to compare gene expression patterns between mib mutants (n = 10) and age-matched WT sibling controls (n = 10). Hybridization, scanning and analysis were completed by the NIH Neuroscience Micro-array Consortium (http://arrayconsortium.tgen.org/np2/home.do) using an Affymetrix zebrafish genome array with ~14,900 Danio rerio transcripts. In mib mutants, we identified a significant down-regulation of four genes in the inhibitory circuit pathway e.g., glutamate decarboxylase, GABA transporter-1, calbindin and parvalbumin. Conclusions: A zebrafish UBE3A mutant (mindbomb) was shown, for the first time, to exhibit seizure activity. Gene profiling using this zebrafish model of Angelman syndrome uncovered deficits in genes critical to GABA-mediated synaptic transmission and suggest that this deficit could underlie seizures associated with this disorder.