Multi-omic Analysis of an Mucolipidosis II Neuronal Cell Model Uncovers Involvement of Pathways Related to Neurodegeneration and Drug Metabolism

Defining the molecular consequences of lysosomal dysfunction in neuronal cell types remains an area of investigation that is needed to understand many underappreciated phenotypes associated with lysosomal disorders. Here we characterize GNPTAB-knockout DAOY medulloblastoma cells using different genetic and proteomic approaches, with a focus on how altered gene expression and cell surface abundance of glycoproteins may explain emerging neurological issues in individual with mucolipidosis (ML). The two knockout clones that were characterized demonstrated all the biochemical hallmarks of this disease, including loss of intracellular glycosidase activity due to impaired mannose 6-phosphate-dependent lysosomal sorting, lysosomal cholesterol accumulation, and increased markers of autophagic dysfunction. RNA sequencing identified altered transcript abundance of several neuronal markers and genes involved in drug metabolism and transport, and neurodegeneration-related pathways. Using selective exo-enzymatic labeling (SEEL) coupled with proteomics to profile cell surface glycoproteins, we demonstrated altered abundance of several glycoproteins in the knockout cells. Most striking was increased abundance of the amyloid precursor protein and apolipoprotein B, indicating that loss of GlcNAc-1-phosphotransferase function in these cells corresponds with elevation in proteins associated with neurodegeneration. The implication of these findings on lysosomal disease pathogenesis and the emerging neurological manifestations of GNPTAB-related disorders is discussed To investigate the effects of GNPTAB knockout on the phenotype of neuronal cells, we established two knockout clones derived from Daoy medulloblastoma cells. We then performed gene expression profiling of the parental cells and both knockout clones from RNA-seq collected in quadruplicate from each cell line. Differential gene expression analysis and pathway enrichment from RNA-seq data were performed comparing the clones to parental control.

明确神经元细胞类型中溶酶体功能障碍（lysosomal dysfunction）的分子效应，仍是理解与溶酶体疾病相关的诸多未被充分认知的表型的关键研究方向。
本研究采用多种遗传与蛋白质组学（proteomics）方法，对GNPTAB敲除（GNPTAB-knockout）的DAOY髓母细胞瘤细胞进行表征，重点探究基因表达改变与糖蛋白（glycoprotein）细胞表面丰度变化如何解释粘脂贮积症（mucolipidosis, ML）患者出现的新发神经症状。
经表征的两株敲除克隆株均展现出该疾病的全部生化特征：包括因6-磷酸甘露糖（mannose 6-phosphate）依赖型溶酶体分选受损导致的胞内糖苷酶活性丧失、溶酶体胆固醇蓄积，以及自噬功能障碍（autophagic dysfunction）标志物水平升高。
RNA测序（RNA sequencing）结果显示，多个神经元标志物基因、药物代谢与转运相关基因，以及神经退行性相关通路（neurodegeneration-related pathways）的转录本（transcript）丰度发生改变。
本研究采用选择性外酶标记（selective exo-enzymatic labeling, SEEL）结合蛋白质组学技术对细胞表面糖蛋白进行谱分析，结果显示敲除细胞中多种糖蛋白的丰度发生改变。
最为显著的是淀粉样前体蛋白（amyloid precursor protein）与载脂蛋白B（apolipoprotein B）的丰度升高，这表明该细胞中GlcNAc-1-磷酸转移酶（GlcNAc-1-phosphotransferase）功能丧失，与神经退行性相关蛋白水平升高存在关联。
本研究讨论了上述发现对溶酶体疾病发病机制以及GNPTAB相关疾病新发神经症状的启示。
为探究GNPTAB敲除对神经元细胞表型的影响，我们构建了两株源自Daoy髓母细胞瘤细胞的敲除克隆株。
随后，我们对亲本细胞与两株敲除克隆株进行了基因表达谱分析，所有细胞系的RNA测序数据均设置四重生物学重复（quadruplicate）。
我们将敲除克隆株与亲本对照细胞的RNA测序数据进行了差异基因表达分析与通路富集分析。