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Our work in this area has been funded by donations from the Storr Family Foundation through the Prader-Willi Syndrome Association. Based on our success we have submitted a National Institutes of Health (NIH) RO1 application with a goal of using genome editing in cell culture models to establish the role of Prader-Willi syndrome (PWS) imprinted genes in regulating hormone secretion and to determine the genetic, molecular, and cellular mechanisms involved in maturation and/or exocytosis of hormone and neuropeptide secretory granules. The NIH application includes Peter F. Drain, PhD, a collaborator at the University of Pittsburgh for his expertise in mechanisms of secretory granule (SG) maturation and exocytosis especially for insulin from β-cells.
In our NIH application, we describe a complex genetic locus critical for maturation or exocytosis of peptide hormone and peptide neurotransmitter SGs. PWS is a multisystem disorder caused by loss of function of a cluster of ~10 paternally-expressed, imprinted genes, with neonatal failure to thrive (FTT), childhood-onset hyperphagia, obesity, and deficits in growth hormone (GH), gonadotropin-releasing hormone (GnRH), and other hormones. In a PWS mouse model with FTT, we found profound pancreatic endocrine abnormalities characterized by an early developmental defect with increased islet-cell apoptosis and reduced β- and α-cell mass, deficient basal and glucose-stimulated insulin secretion suggestive of defective maturation or exocytosis of insulin SGs and leading to hypoinsulinemia, concurrent hypoglucagonemia with lack of a counter-regulatory response to hypoglycemia, and dysregulated expression of genes encoding hormones and proteins involved in hormone secretion. Based on the clinical and mouse data, we propose a hypothesis that PWS genes are required for endocrine cell secretory function. To overcome genetic and physiological complexities in animal models or human, we will use hormone-secreting cells in an in vitro system to determine the molecular and cellular mechanisms by which PWS genes control hormone secretion. Aims 1, 2, 3 will assess control of 1) insulin, 2) glucagon, and 3) GnRH secretion, each with four sub-Aims: A) CRISPR/Cas9 will be used to generate deletions of the PWS-orthologous domain in mouse and rat cell lines that specifically secrete insulin, glucagon, or GnRH and that co-express a fluorescent hormone biosensor within SGs; B) To demonstrate functional effects of PWS-deletions, we will assay secreted hormone levels in control and knockout cells; C) To determine the molecular pathways underlying hormone secretion deficits, we will perform whole transcriptome-microarrays on PWS-knockout and control cells to identify genes with abnormal expression levels or alternative mRNA splicing, mechanisms in which PWS-genes are implicated; D) To genetically dissect which PWS-gene is required for secretion of each hormone, we will use CRISPR/Cas9 to make smaller deletions of subsets or single genes within the PWS-imprinted domain, and assess as in B and C. Aim 4 will utilize isogenic control and PWS-knockout cells from Aims 1 through 3 that express a biosensor in insulin, glucagon, or GnRH SGs, to identify the stage(s) of SG maturation and/or exocytosis blocked in PWS-knockout cells. Specifically, we will assess trafficking (ER→ Golgi→SG) by live-cell confocal microscopy and immunohistochemistry, [Ca2+] in response to secretagogues by GCaMP and rhod-2 dyes, proteolytic maturation of pro-peptide to mature peptide by western blot analysis, and content of peptides per SG by ELISA. This study will identify how imprinted genes control SG maturation or exocytosis in endocrine cells, leading to insights into the pathogenesis and potential treatments for PWS and other disorders with abnormalities in these hormones, including type 2 diabetes and diseases of puberty or reproduction.
The Storr Family Foundation through the Prader-Willi Syndrome Association
Robert D. Nicholls, PhD
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