Abstracts

Rationale: Mutations in STXBP1, the gene encoding syntaxin-binding protein 1, are prevalent factors in causing neurodevelopmental disorders and epilepsy that manifest in at least 1 in 30, 000 patients. With mortality rates surpassing 50%, diminished quality of life, reports of developmental regression in adulthood and inadequate treatment options, there is an urgent need for additional research into STXBP1 Disorders. To better understand how STXBP1 mutations contribute to these diverse phenotypes, we used CRISPR-generated stxbp1a^-/- and stxbp1b^-/- zebrafish (Grone et al. 2016) to systematically study brain activity, behavior, and metabolism.

Methods: Adult male and female heterozygotes for stxbp1a or stxbp1b were crossed to generate wild type, heterozygote and homozygote larvae. Experiments were performed at 5 days post-fertilization (dpf) (n > 500 fish). Local field potential (LFP) recording in combination with fiber photometry were used to monitor brain activity in larvae expressing a genetically encoded calcium indicator (HuC:2B:GCaMP6s). Flash-frozen WT and stxbp1a^-/- larvae were used for untargeted metabolomics analysis (Creative Proteomics). Advanced locomotor video acquisition was achieved using a high-resolution Multi-Camera Array Microscope (MCAMä) from Ramona Optics. All larvae were genotyped post hoc.

Results: stxbp1a^-/- zebrafish larvae replicate neurodevelopmental features of STXBP1 Disorders, such as atypical muscle tone (unresponsiveness to startle stimuli and body rigidity) and severe movement disorder (significant decreases in total distance traveled). Advanced activity metrics including tail and head angle measurements in combination with machine-learning algorithms further confirmed these movement disorder phenotypes. Untargeted metabolomics identified an array of small-molecule metabolites that were down-regulated in stxbp1a^-/- larvae compared to age-matched WT controls. stxbp1b^-/- zebrafish larvae exhibit epileptic phenotypes evidenced by spontaneous electrographic seizure activity in LFP recordings and synchronized increased fluctuations in calcium signaling.

Conclusions: Our findings demonstrate that zebrafish STXBP1 models uniquely present motor impairments (stxbp1a^-/-) or epileptic phenotypes (stxbp1b^-/-) representing the spectrum of clinical phenotypes seen in these patients. An integrated neuro-omics approach for analysis of these zebrafish offers an unprecedented opportunity for exploration of STXBP1 pathophysiology and therapy development.

Funding: NIH/NINDS grants R01-NS096976, R01-HD102071, R21-NS138525 and U54-NS117170 (to S.C.B.) and Savoy Foundation of Canada (to P.W-F)