Investigating cellular dysfunction related to lysosomal impairment in CLN2 disease

溶酶体是细胞废物降解和再循环的中心，也是重要的代谢信号中枢。这两种功能都对细胞内环境的稳定有重要影响，研究溶酶体贮积症（LSDs）为了解溶酶体的功能和溶酶体损伤对细胞的广泛影响提供了有价值的见解。本研究关注CLN 2疾病中的溶酶体损伤，CLN 2疾病是一种由溶酶体丝氨酸蛋白酶三肽基肽酶1（TPP 1）的Reconstructedinherited功能障碍引起的LSD。本课题主要利用CLN 2病（tpp 1-/-）斑马鱼模型，研究Tpp 1缺陷导致的溶酶体损伤相关的细胞功能障碍，并提出了一种新的转基因斑马鱼品系，其自噬体和溶酶体标记物分别为LC 3和Lamp 1，用ZsGreen荧光蛋白标记。利用这些细胞系，我能够可靠地定量体内溶酶体的数量、大小和形态，以及自噬通量。这个数据集突出了tpp 1-/-突变斑马鱼中严重的溶酶体异常，以及自噬通量的阻断和自噬体位置的改变。tpp 1-/-突变斑马鱼中溶酶体损伤对细胞内稳态的影响通过一种关键代谢调节剂的过度活跃、雷帕霉素复合物的哺乳动物靶蛋白1（mTORC 1）和通过tpp 1-/-突变斑马鱼的全基因组RNA测序检测到的基因表达的大量变化以及健康同胞来证明。此外，我观察到用双烯醇酮治疗后溶酶体表型的改善，一种先前被鉴定为减少TPP 1-/-小鼠斑马鱼癫痫发作的化合物。我继续探索了双烯醇酮的潜在机制及其对疾病发病机制的见解，特别关注溶酶体生物学，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.