Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome
Lisa C. Burnett, … , Dieter Egli, Rudolph L. Leibel
Lisa C. Burnett, … , Dieter Egli, Rudolph L. Leibel
Published December 12, 2016
Citation Information: J Clin Invest. 2017;127(1):293-305. https://doi.org/10.1172/JCI88648.
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Research Article Endocrinology Genetics

Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome

Abstract

Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell–derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p–/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p–/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p–/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH–releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.

Authors

Lisa C. Burnett, Charles A. LeDuc, Carlos R. Sulsona, Daniel Paull, Richard Rausch, Sanaa Eddiry, Jayne F. Martin Carli, Michael V. Morabito, Alicja A. Skowronski, Gabriela Hubner, Matthew Zimmer, Liheng Wang, Robert Day, Brynn Levy, Ilene Fennoy, Beatrice Dubern, Christine Poitou, Karine Clement, Merlin G. Butler, Michael Rosenbaum, Jean Pierre Salles, Maithe Tauber, Daniel J. Driscoll, Dieter Egli, Rudolph L. Leibel

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Figure 1

NHLH2 and PCSK1 are reduced in PWS iPSC-derived neurons and Snord116p–/m+ (DEL) hypothalami.

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NHLH2 and PCSK1 are reduced in PWS iPSC-derived neurons and Snord116p–/...
(A) Diagram of the PWS locus. Maternally expressed genes are shown in pink, paternally expressed genes in blue, non-imprinted genes in green. Protein-coding genes are shown as ovals, snoRNAs as rectangles, long noncoding RNAs as triangles, imprinting center as a diamond. Not drawn to scale. cen, centromere; tel, telomere. (B–F) Gene expression in the PWS locus following neuron differentiation (n = 7 control [CON], n = 1 PWS MD [2 clones used], n = 3 PWS LD). (G) RNA sequencing identified a downregulation in PCSK1 in PWS neurons (n = 7 CON, n = 1 PWS MD [2 clones used], n = 2 PWS LD). This heatmap is also shown in Supplemental Figure 4A and includes the full list of all genes differentially expressed. (H and J) PCSK1 and NHLH2 gene expression levels from an independent differentiation experiment, as measured by qRT-PCR (n = 7 CON, n = 1 PWS MD [2 clones used], n = 3 PWS LD). (I and K) Quantification of PC1 and NHLH2 protein levels in iPSC-derived neurons (n = 5 CON [3 lines], n = 2 PWS LD, n = 1 PWS MD). (L and M) Food intake after 5 hours of refeeding (n = 6 WT, n = 5 DEL). (N–R) Transcript levels in hypothalami at fasting and refeeding (n = 11 WT, n = 13 DEL, overnight fasted; n = 15 WT, n = 14 DEL, 5-hour refed). All data are expressed as mean ± SEM. B–F were analyzed with Kruskal-Wallis with post hoc Dunn’s multiple comparison test; comparisons are against unaffected controls. L and M were analyzed with a 2-tailed, type 3 (assumes unequal variance) Student’s t test. N, P, and Q were analyzed with 1-way ANOVA with Tukey’s post hoc test. (O) WT fast and DEL fast were compared with a 2-tailed, type 3 Student’s t test. (R) WT refed and DEL refed were compared with a 2-tailed, type 3 Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.