CLN2 disease is caused by Tripeptidyl Peptidase 1 (TPP1) loss-of-function and treatments are limited so further potential therapeutics are needed. The zebrafish larval model of CLN2 disease (tpp1-/-) shows phenotypes that resemble the human disease, including neuronal and retinal degeneration, seizures and premature death (1). Using a combination of medium-throughput assays to measure the disease phenotypes, an FDA-approved drug library of 640 compounds was screened, identifying a single hit compound, pregnenolone, which demonstrates beneficial effects on seizure activity and cell death in tpp1-/- mutants.
Using live imaging of novel transgenic zebrafish expressing autophagosome and lysosome markers LC3 and Lamp1 respectively, tagged by ZsGreen fluorescent protein, we were able to reliably quantify lysosomal number, size and morphology, as well as autophagic flux in vivo in muscle. Results highlighted severe lysosomal abnormality and blocked autophagic flux in tpp1-/- mutants which was improved by pregnenolone. To explore the mechanism of action, we found that pregnenolone acts via a mTORC1/TFEB-independent mechanism to improve lysosome phenotypes and that the reduction in seizures is not due to pregnenolone inducing autophagy. Steroid hormone measurements indicated 7/11 steroids (including pregnenolone) were upregulated in tpp1-/- mutants, three of which were normalised by pregnenolone treatment. RNA Seq revealed significant enrichment in the cholesterol and steroid pathways in tpp1-/- mutants, which was stimulated by pregnenolone. LCAT, NPC2 and StARD3 mRNAs were all increased in tpp1-/- mutants and then reduced to normal levels by pregnenolone, suggesting that dysregulated cholesterol homeostasis may contribute to tpp1-/- pathology. Thus, we demonstrate the use of this model in identifying a potential novel therapeutic for CLN2 disease, pregnenolone, which leads to alleviation of lysosomal impairment and improved disease phenotypes, particularly seizures.