Restoration of Progranulin Expression Rescues Cortical Neuron Generation in Induced Pluripotent Stem Cell Model of Frontotemporal Dementia. Homo sapiens

To understand how haploinsufficiency of progranulin (PGRN) protein causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSC) from patients carrying the GRNIVS1+5G>C mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD and known to express PGRN. Although generation of neuroprogenitors was unaffected, their further differentiation into neurons, especially CTIP2-, FOXP2- or TBR1-TUJ1 double positive cortical neurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of PGRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNAseq analysis confirmed reversal of altered gene expression profile following genetic correction. Wnt signaling pathway, one of the top defective pathways in FTD-iPSC-derived neurons coupled with its reversal following genetic correction, makes it an important candidate. Therefore, we demonstrate for the first time that PGRN haploinsufficiency hampers corticogenesis in vitro. Overall design: We profiled 6 samples: two biological replicates for 3 conditions. Condition 1 consists of neuronal progeny derived from human Embryonic Stem Cells. Condition 2 consists of neuronal progeny derived from induced pluripotent stem cells generated from patients carrying PGRN mutation. Condition 3 consists of neuronal progeny derived from induced pluripotent stem cells generated from patients carrying PGRN mutation, genetically modified to correct the PGRN defect.

为阐明前颗粒蛋白（progranulin, PGRN）单倍体不足引发额颞叶痴呆（frontotemporal dementia, FTD）的分子机制，我们从携带GRNIVS1+5G>C突变的患者样本中诱导生成了诱导多能干细胞（induced pluripotent stem cells, iPSC，下称FTD-iPSCs）。我们将FTD-iPSCs定向诱导分化为皮层神经元——这类细胞是FTD中受累最显著的细胞群，且已知表达PGRN蛋白。尽管神经祖细胞的生成过程未受影响，但FTD来源的神经子代细胞向神经元的进一步分化显著受阻，其中尤以CTIP2阴性、FOXP2阴性或TBR1-TUJ1双阳性的皮层神经元为甚。我们通过锌指核酸酶介导的方式，将PGRN互补DNA（complementary DNA, cDNA）整合至AAVS1位点，成功纠正了皮层神经发生缺陷，证实PGRN单倍体不足会导致皮层神经元生成效率低下。RNA测序（RNA sequencing, RNA-seq）分析显示，遗传修正后异常的基因表达谱得到逆转。Wnt信号通路是FTD-iPSC来源神经元中存在功能缺陷的核心通路之一，且该通路的异常可通过遗传修正得到逆转，因此其是潜在的关键调控靶点。综上，我们首次证实PGRN单倍体不足会在体外抑制皮层发生过程。

实验整体设计：我们对6份样本开展转录组分析，3种实验条件各设置2个生物学重复。条件1：源自人类胚胎干细胞（human Embryonic Stem Cells）的神经子代细胞；条件2：源自携带PGRN突变患者的诱导多能干细胞的神经子代细胞；条件3：源自携带PGRN突变患者的诱导多能干细胞、经遗传修饰纠正PGRN功能缺陷后的神经子代细胞。