Progranulin–glucocerebrosidase complex regulates tau and alpha-synuclein inclusions to alter phenotypes in tauopathy

Many neurodegenerative disorders including Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD) are characterized by abnormal protein deposition and frequently show comorbid pathology. Progranulin (PGRN) is implicated not only in TDP-43 but in tau and alpha-synuclein proteinopathies. However, the underlying mechanisms are unknown. Here, we generated P301S tau transgenic mice with PGRN haploinsufficiency and loss and found that those mice exhibit exacerbated disinhibition phenotype while showing attenuated memory impairment, hippocampal atrophy, and transcriptomic changes. Remarkably, the phenotypic alteration was accompanied by an increase in tau inclusions, which are positive for alpha-synuclein, a PGRN binding partner beta-glucocerebrosidase (GCase), and its substrate glucosylceramide. GCase inhibition or PGRN deficiency enhanced tau aggregation induced by AD-derived tau seeds in neurons. In vitro, GCase and glucosylceramide promoted P301S tau aggregation. Similar co-pathology was observed in AD and FTLD-GRN patients. Overall design: 18 samples (hippocampus) from 6 genotypes with 3 biological replicates were individually processed and sequenced. Nuclei were isolated from hippocampi using Nuclei EZ Prep Kit (SIGMA #NUC-101). Libraries were prepared using Chromium Single Cell 3” Reagent Kits v3 (10X Genomics) . The libraries underwent HiSeq paired-end sequencing with a sequence depth of 200 million reads per sample using NovaSeq instrument (Ilumina).

诸多神经退行性疾病（包括阿尔茨海默病(Alzheimer's disease, AD)与额颞叶变性(frontotemporal lobar degeneration, FTLD)）均以异常蛋白质沉积为核心特征，且常伴随共病病理改变。前颗粒蛋白(progranulin, PGRN)不仅与TDP-43蛋白病相关，亦参与tau蛋白病及α-突触核蛋白(alpha-synuclein)蛋白病的发生发展，但具体分子机制至今尚未阐明。本研究构建了携带PGRN单倍体功能缺失与完全缺失的P301S tau转基因小鼠模型，结果显示该模型小鼠的去抑制表型显著加重，但记忆损伤、海马萎缩及转录组改变均得到缓解。值得注意的是，该表型改变伴随tau包涵体数量增多，且此类包涵体呈α-突触核蛋白、PGRN结合伴侣β-葡萄糖脑苷脂酶(beta-glucocerebrosidase, GCase)及其底物葡萄糖脑苷脂(glucosylceramide)阳性。在神经元中，抑制GCase活性或PGRN缺失均可增强阿尔茨海默病来源的tau种子诱导的tau蛋白聚集；体外实验进一步证实，GCase与葡萄糖脑苷脂可直接促进P301S tau蛋白聚集。在阿尔茨海默病及FTLD-GRN患者脑组织中，同样可观察到类似的共病理改变。实验整体设计：本研究纳入6种基因型的小鼠样本，共18份海马组织样本，每组设置3次生物学重复，所有样本均单独完成处理与测序。使用Nuclei EZ Prep试剂盒（Sigma #NUC-101）从海马组织中分离细胞核；采用Chromium单细胞3'试剂试剂盒v3（10X Genomics）构建测序文库；随后使用NovaSeq测序仪（Illumina）进行HiSeq双端测序，每份样本的测序深度为2亿条reads。