Neuron-specific changes in gene expression caused by NPC1 deficiency

Background: Niemann-Pick type C is a rare autosomal recessive lysosomal storage disorder presenting aggravating neurologic symptoms due degeneration of specific types of CNS neurons. At present, it is not well understood how neurons react to NPC1 deficiency and why some neuronal cell types are more vulnerable than others. Purpose: We took aimed to uncover how a specific type of CNS neuron that can be highly purified reacts to NPC1 deficiency based on changes in gene expression. Methods: Retinal ganglion cells were purified from individual one-week old Balb/c mice homozygous for a mutant NPC1 allele (NPC1m1N) and wildtype littermates (n = 4 mice each genotype) using immunopanning. Total RNA was isolated from acutely isolated neurons and subjected to RNAseq using 4 biological replicates for each genotype. Results: Our analysis revealed a strong downregulation of transcripts known to be decreased in mutant mice including Npc1 and Calb1 thus validating our approach. We observed a strong upregulation of genes for cellular cholesterol accretion and the downregulation of those for cholesterol release. Other changes including downregulation genes involved in the immune response and synaptic components. Conclusions: The observed changes suggest that neurons already at one week of age sense a cholesterol deficit because lipids accumulate in the endosomal-lysosomal system and cannot be redistributed intracellularly. Overall design: Gene expression analysis by RNAseq in retinal ganglion cells acutely purified from eight-days-old NPC1-deficient mice and wildtype littermates

背景：尼曼-匹克C型病（Niemann-Pick type C）是一种罕见的常染色体隐性溶酶体贮积症（lysosomal storage disorder），因特定类型的中枢神经系统（Central Nervous System, CNS）神经元变性，引发进行性加重的神经系统症状。目前学界尚未明确神经元如何应对NPC1基因缺陷，以及为何部分神经元亚型较其他亚型更易受损。
目的：本研究旨在通过基因表达变化分析，阐明一种可高度纯化的中枢神经系统神经元亚型对NPC1缺陷的响应机制。
方法：采用免疫淘选法（immunopanning），分别从4只纯合NPC1等位基因突变型（NPC1m1N）1周龄BALB/c小鼠，以及4只同窝野生型小鼠（每组n=4）中分离纯化视网膜神经节细胞。从急性分离的神经元中提取总RNA，对每个基因型设置4个生物学重复样本进行RNA测序（RNAseq）。
结果：分析结果显示，已知在突变小鼠中表达下调的转录本（包括Npc1与Calb1）出现显著下调，验证了本实验方法的有效性。研究团队观察到参与细胞胆固醇蓄积的基因显著上调，而参与胆固醇释放的基因则出现下调。此外还发现免疫应答相关基因与突触组分相关基因存在下调现象。
结论：本研究观察到的变化提示，仅1周龄的神经元即可感知胆固醇缺乏——脂质在内体-溶酶体系统中蓄积，无法在细胞内进行重新分布。
整体实验设计：通过RNA测序对8日龄NPC1缺陷型小鼠及其同窝野生型小鼠的急性纯化视网膜神经节细胞进行基因表达分析。