Coordinate control of endoplasmic reticulum chaperone biosynthesis and peptide hormone secretion revealed by a β-cell model of Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a multisystem disorder caused by loss of expression of a cluster of paternally-expressed, imprinted genes. Neonatal failure to thrive is followed by childhood-onset hyperphagia, obesity, neurobehavioral abnormalities, and hormonal deficits. Prior evidence from a mouse model with a deletion of the orthologous PWS-domain identified abnormal pancreatic islet development with deficient insulin secretion, hypoglucagonemia, and postnatal onset of progressive, lethal hypoglycemia. To investigate PWS-genes in β-cell secretory function, we used CRISPR/Cas9 genome-editing to generate isogenic, clonal INS-1 insulinoma lines with 3.16 Mb deletions of the silent, maternal (control) or active, paternal (PWS) alleles. A significant reduction in basal and glucose-stimulated insulin secretion signifies a cell autonomous insulin secretion deficit in PWS β-cells. Parallel proteome and transcriptome studies revealed reduced levels of secreted peptides and for eleven endoplasmic reticulum (ER) chaperones, including HSPA5 and HSP90B1. In contrast to dosage compensation previously seen for ER chaperones in Hspa5 or Hsp90b1 gene knockouts, compensation is precluded by the widespread deficiency of ER chaperones in PWS cells. Remarkably, but consistent with reduced ER chaperone levels, PWS β-cells are more sensitive to ER stress activation of all three regulatory pathways (XBP1, eIF2α-P, ATF6-N). Therefore, a coordinated, chronic deficit of ER chaperones in PWS β-cells is hypothesized to lead to a delay in ER transit and/or folding of insulin and other cargo along the secretory pathway. These findings provide insight into the pathophysiological basis of hormone deficits in PWS and indicate key roles for PWS-imprinted genes in β-cell secretory function.

普拉德-威利综合征（Prader-Willi syndrome, PWS）是一类由父本表达的印记基因簇表达缺失引发的多系统疾病。该病患者新生儿期表现为生长发育不良，随后儿童期出现食欲亢进、肥胖、神经行为异常以及激素缺乏。此前有研究通过携带同源PWS区域缺失的小鼠模型发现，该模型小鼠存在胰岛发育异常，伴胰岛素分泌不足、低胰高血糖素血症，且出生后出现进行性致死性低血糖。为探究PWS相关基因在β细胞分泌功能中的作用，本研究采用CRISPR/Cas9基因组编辑技术，构建了携带3.16 Mb片段缺失的同基因克隆化INS-1胰岛素瘤细胞系，分别针对沉默的母本等位基因（对照组）与活跃的父本等位基因（PWS模型组）进行编辑。结果显示，PWS模型组β细胞的基础及葡萄糖刺激后的胰岛素分泌量显著降低，提示其存在细胞自主性的胰岛素分泌缺陷。平行开展的蛋白质组与转录组研究表明，PWS模型组的分泌肽水平下调，且包括HSPA5、HSP90B1在内的11种内质网（endoplasmic reticulum, ER）分子伴侣的表达量均显著降低。与此前在Hspa5或Hsp90b1基因敲除模型中观察到的内质网分子伴侣剂量补偿效应不同，PWS细胞中内质网分子伴侣的广泛缺乏阻断了该补偿机制。值得注意的是，与内质网分子伴侣水平降低的结果一致，PWS β细胞对三条调控通路（XBP1、eIF2α-P、ATF6-N）的内质网应激激活更为敏感。据此，研究团队推测，PWS β细胞中内质网分子伴侣的协同性慢性缺乏，会导致胰岛素及其他分泌cargo在内质网的转运和/或折叠过程出现延迟。本研究揭示了PWS激素缺乏的病理生理基础，并阐明了PWS印记基因在β细胞分泌功能中的关键作用。