Investigating cellular dysfunction related to lysosomal impairment in CLN2 disease

溶酶体是细胞废物降解和回收的中心，也是重要的代谢信号中枢。溶酶体储存障碍(LSD)的研究为了解溶酶体的功能以及溶酶体损伤时广泛存在的细胞影响提供了有价值的见解。本研究的重点是CLN2病的溶酶体损伤，这是一种由溶酶体丝氨酸蛋白酶三肽基多肽酶1(TPP1)隐性遗传功能障碍引起的LSD。本研究主要利用斑马鱼的CLN2病模型(TPP1-/-)，旨在研究由于TPP1基因缺失导致的与溶酶体损伤相关的细胞功能障碍。利用这些品系，我能够可靠地定量溶酶体的数量、大小和形态，以及活体中的自噬通量。这个数据集突出了TPP1-/-突变斑马鱼中溶酶体的严重异常，以及自噬通量的受阻和自噬体位的改变。溶酶体损伤对TPP1-/-突变斑马鱼细胞动态平衡的影响被证明是从一个关键的代谢调节因子，哺乳动物雷帕霉素复合体靶标1(MTORC1)的过度活动以及通过对TPP1-/-突变斑马鱼的全基因组RNA测序检测到的一些基因表达的变化来证明的。此外，我观察到溶酶体表型的改善与孕烯醇酮的治疗，一种先前被发现可以减少TPP1-/-突变斑马鱼的癫痫发作的化合物。我继续探索孕烯醇酮的潜在机制，以及它为疾病发病机制提供的见解，特别是溶酶体生物学、自噬和胆固醇代谢。

The lysosome is the centre of cellular waste degradation and recycling, as well as an importantmetabolic signaling hub. Both functions have a major influence over cellular homeostasis.Studying lysosomal storage disorders (LSDs) provides valuable insights into functions of thelysosome and the widespread cellular impact during lysosomal impairment. This study focuseson lysosomal impairment in CLN2 disease, an LSD caused by recessively inheriteddysfunction of lysosomal serine protease Tripeptidyl Peptidase 1 (TPP1). Predominantly usinga zebrafish model of CLN2 disease (tpp1-/-), the aim of this project was to investigate thecellular dysfunction related to lysosomal impairment as a consequence of Tpp1 deficiency.I present novel transgenic zebrafish lines with autophagosome and lysosome markers, LC3 andLamp1 respectively, tagged by ZsGreen fluorescent protein. Using these lines, I was able toreliably quantify lysosomal number, size and morphology, as well as autophagic flux in vivo.This dataset highlights severe lysosomal abnormality in tpp1-/- mutant zebrafish, alongsideblocked autophagic flux and alterations in autophagosome position. The impact of lysosomalimpairment on cellular homeostasis in tpp1-/- mutant zebrafish is evidenced by hyperactivity ofa key metabolic regulator, mammalian target of rapamycin complex 1 (mTORC1) and anumber of changes in gene expression detected by whole genome RNA Sequencing of tpp1-/-mutant zebrafish, alongside healthy siblings.Furthermore, I observed improvement in the lysosomal phenotype with treatment ofpregnenolone, a compound previously identified to reduce seizures in the tpp1-/- mutantzebrafish. I went on to explore the potential mechanisms of pregnenolone and the insights itprovides into disease pathogenesis, with a particular focus on lysosomal biology, autophagyand cholesterol metabolism.