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

Kiani, L. N.; Civiletto, G.; Lizzo, G.; Zdebik, A.; Brickell, G.; Mahmood, F.; Nalkos, D. D.; Michaelides, L.; Eldridge, P.; Young, E. M.; McPherson, H.; Campanella, M.; Gut, P.; Russell, C.

doi:10.1101/2025.08.26.670480

bioRxiv · 10.1101/2025.08.26.670480

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

Kiani, L. N.·Civiletto, G.·Lizzo, G.·Zdebik, A.·Brickell, G.·Mahmood, F.·Nalkos, D. D.·Michaelides, L.·Eldridge, P.·Young, E. M.·McPherson, H.·Campanella, M.·Gut, P.·Russell, C.

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Abstract

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.

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Kiani, L. N., Civiletto, G., Lizzo, G., Zdebik, A., Brickell, G., Mahmood, F., Nalkos, D. D., Michaelides, L., Eldridge, P., Young, E. M., McPherson, H., Campanella, M., Gut, P., Russell, C.. 2025-08-27. Whole-organism screening in a zebrafish model of CLN2 disease identifies pregnenolone as a modulator of lysosomal functions with anti-epileptic properties. https://doi.org/10.1101/2025.08.26.670480

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