Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons
Liheng Wang, Yang Liu, George Stratigopoulos, Sunil Panigrahi, Lina Sui, Yiying Zhang, Charles A. Leduc, Hannah J. Glover, Maria Caterina De Rosa, Lisa C. Burnett, Damian J. Williams, Linshan Shang, Robin Goland, Stephen H. Tsang, Sharon Wardlaw, Dieter Egli, Deyou Zheng, Claudia A. Doege, Rudolph L. Leibel
Liheng Wang, Yang Liu, George Stratigopoulos, Sunil Panigrahi, Lina Sui, Yiying Zhang, Charles A. Leduc, Hannah J. Glover, Maria Caterina De Rosa, Lisa C. Burnett, Damian J. Williams, Linshan Shang, Robin Goland, Stephen H. Tsang, Sharon Wardlaw, Dieter Egli, Deyou Zheng, Claudia A. Doege, Rudolph L. Leibel
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Research Article

Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons

Abstract

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell–derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC–derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.

Authors

Liheng Wang, Yang Liu, George Stratigopoulos, Sunil Panigrahi, Lina Sui, Yiying Zhang, Charles A. Leduc, Hannah J. Glover, Maria Caterina De Rosa, Lisa C. Burnett, Damian J. Williams, Linshan Shang, Robin Goland, Stephen H. Tsang, Sharon Wardlaw, Dieter Egli, Deyou Zheng, Claudia A. Doege, Rudolph L. Leibel

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Figure 5

BBS mutations impair leptin signaling in RFP-LEPR transgenic human fibroblasts and iPSC-derived hypothalamic neurons.

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BBS mutations impair leptin signaling in RFP-LEPR transgenic human fibro...
(A–D) Leptin signaling is impaired in RFP-LEPR transgenic BBS fibroblasts. Control 1, control 2, BBS1A, BBS1B, BBS10A, and BBS1B + FLAG-BBS1 RFP-LEPR transgenic fibroblasts were treated with 0 or 0.5 μg/mL leptin (30 minutes) after overnight serum starvation. Leptin signaling was assessed by Western blot (WB) of p-STAT3 levels. STAT3, RFP, and GSK3β were also probed (A and C). In C, the 2 p-STAT3 blot images are from short (top) and long time exposures. (B and D) Quantification of p-STAT3/STAT3 from A and C. p-STAT3/STAT3 was further normalized to RFP-LEPR/GSK3β (n = 1). (E) Leptin signaling can be rescued in BBS1B RFP-LEPR transgenic fibroblasts by overexpressing FLAG-BBS1. BBS1B and BBS1B + FLAG-BBS1 RFP-LEPR transgenic fibroblasts were treated as in C and stained with anti–p-STAT3 and Hoechst. Arrows indicate p-STAT3–positive cells. Scale bar: 200 μm. (F and G) Leptin signaling is disturbed in BBS iPSC–derived hypothalamic neurons. Day 34 iPSC-derived hypothalamic neurons were serum starved overnight and treated with 0 or 1 μg/mL leptin for 30 minutes. (F) p-STAT3, STAT3, and actin were examined by WB. (G) Quantification of WB in F (n = 2–3). (H and I) Leptin signaling, as measured by immunostaining of p-STAT3, is disrupted in BBS iPSC–derived hypothalamic neurons. (H) Day 34 neurons were treated with leptin and stained with anti-αMSH, anti–p-STAT3, and Hoechst. Scale bar: 20 μm. (I) Quantification of p-STAT3+ POMC neurons in H (n = 3). **P < 0.01 by 2-way ANOVA followed by Bonferroni’s multiple-comparison test. (J) Overexpression of BBS10 increases total LEPR proteins. 293FT cells were cotransfected with mouse LEPRB-Myc (mLeprb-Myc) and GFP or FLAG-BBS1 or FLAG-BBS10 for 24 hours. Myc, FLAG, and actin were probed. Arrows indicate FLAG-BBS1 and FLAG-BBS10 bands. (K) Quantification of mLeprb-Myc in J. ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple-comparison test. NS, no significant difference.