Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons
Liheng Wang, … , Claudia A. Doege, Rudolph L. Leibel
Liheng Wang, … , Claudia A. Doege, Rudolph L. Leibel
Published February 25, 2021
Citation Information: J Clin Invest. 2021;131(8):e146287. https://doi.org/10.1172/JCI146287.
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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 4

BBS1 and BBS10 bind to the insulin receptor and influence insulin signaling.

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BBS1 and BBS10 bind to the insulin receptor and influence insulin signal...
(A) BBS mutations disrupt insulin signaling in human fibroblasts. Western blot (WB) analysis of insulin signaling as indicated by phosphorylation of AKT in control and BBS fibroblasts. Fibroblasts were serum starved overnight and treated with 0 or 1 μg/mL insulin for 30 minutes. AKT, p-AKT Thr308, and p-AKT Ser473 were probed. (B) Quantification of p-AKT (Thr308)/AKT from A (n = 1). (C) BBS mutations abrogate insulin signaling in iPSC-derived TUJ1+ neurons. Control, BBS1A, and BBS10A iPSC–derived neurons (day 30) were serum starved overnight and treated with 0 or 1 μg/mL insulin for 30 minutes. p-AKT Ser473, AKT, and α-tubulin (A-TUB) were probed. (D) Quantification of p-AKT/AKT from WB in C (n = 1). (E) Diminished insulin signaling in neurons derived from 2 BBS10 shRNA-knocked-down iPSC lines as indicated. iPSC-derived TUJ1+ neurons (day 30) were serum starved overnight and treated with 0 or 1 μg/mL insulin for 30 minutes. p-AKT Ser473, AKT, and actin were examined by WB. (F) Quantification of p-AKT/AKT from WB in E (n = 2–3). (G) BBS10 disrupts insulin signaling by disturbing phosphorylation of the insulin receptor (IR) in day 12 neuronal progenitors (NPs). WB analysis of insulin signaling molecules as indicated in control, BBS10A, and BBS10A-FLAG-BBS10 transgenic NPs after 30-minute treatment with 0.01 and 0.1 μg/mL insulin. FLAG was used to confirm the overexpression of WT BBS10. (H and I) Quantification of p-AKT/AKT (H) (n = 1) and p-IR/IR (I) (n = 3) in D. **P < 0.01 by 1-way ANOVA followed by Tukey’s multiple-comparison test. (J) Coimmunoprecipitation (IP) confirms the interaction between BBS proteins and the IR. 293FT cells were transfected with CD615-GFP or CD615-3×FLAG-BBS1-GFP or CD615-3×FLAG-BBS10-GFP for 48 hours before insulin (1 μg/mL) treatment (30 minutes). IRβ, p-AKT Ser473, and FLAG were probed in FLAG IP samples and total input.