Whole-organism screening in a zebrafish model of CLN2 disease identifies pregnenolone as a modulator of lysosomal functions with anti-epileptic properties

Lysosomal storage disorders (LSDs), a group of inherited genetic diseases, are often associated with early-onset neurodegeneration and refractory epileptic seizures. In CLN2 disease, an LSD caused by recessively inherited dysfunction of lysosomal serine protease Tripeptidyl Peptidase 1 (TPP1), lysosomes are functionally impaired through a characteristic accumulation of subcellular materials. Here, we develop and apply a whole-organism screening workflow in tpp1 ^-/- zebrafish to identify small molecules that suppress epileptic seizures — a hallmark of the human disease — in this model. Among 640 US Food and Drug Administration-approved drugs, pregnenolone, an endogenous precursor for steroid biosynthesis, efficiently suppresses seizures and cell death in tpp1 ^-/- zebrafish. Using a semi-automated high-content workflow, we further show that pregnenolone normalizes lysosomal architecture in tpp1 ^-/- zebrafish. Pregnenolone stimulates steroid hormone biosynthesis and related gene expression, which is dysregulated in tpp1 ^-/- zebrafish. Taken together, tpp1 ^-/- zebrafish are a suitable model to study CLN2 disease, in which we have identified pregnenolone as a candidate with therapeutic properties.