Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome
Anna Cariboni, … , Fanny Mann, Christiana Ruhrberg
Anna Cariboni, … , Fanny Mann, Christiana Ruhrberg
Published May 18, 2015
Citation Information: J Clin Invest. 2015;125(6):2413-2428. https://doi.org/10.1172/JCI78448.
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Research Article Development Endocrinology Genetics Neuroscience

Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome

Abstract

Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1–dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Authors

Anna Cariboni, Valentina André, Sophie Chauvet, Daniele Cassatella, Kathryn Davidson, Alessia Caramello, Alessandro Fantin, Pierre Bouloux, Fanny Mann, Christiana Ruhrberg

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Figure 4

The SEMA3ER619C mutation fails to rescue GT1-7 neuron survival.

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The SEMA3ER619C mutation fails to rescue GT1-7 neuron survival. (A and B...
(A and B) Generation of WT and mutant SEMA3E AP fusion proteins. COS-7 cells were transfected with a control expression vector or vectors encoding AP fused to human SEMA3E or mutant SEMA3ER619C. Cells were immunolabeled (A) or immunoblotted (B) with an antibody against SEMA3E. Cells and conditioned media contained a band of approximately 180 kDa, composed of the 95-kDa hSEMA3E and 85-kDa AP fragments. Tubulin was used as a loading control. (C) AP-SEMA3E bound WT, but not Plxnd1-null, tissue. Sagittal sections of E14.5 mouse cerebral cortex of the indicated genotypes were incubated with AP-SEMA3E. Note that SEMA3E bound blood vessels in WT, but not in Plxnd1-null, tissue. (D) AP-SEMA3E binding to GnRH neurons in vitro. The incubation of GT1-7 cells with COS cell–conditioned DMEM containing AP ligands demonstrated similar binding of WT AP-SEMA3E and mutant AP-SEMA3ER619C to GT1-7 cells, while AP alone did not bind the cells. (E) SEMA3ER619C was not neuroprotective for GT1-7 cells. PI and Hoechst staining shows that WT SEMA3E, but not SEMA3ER619C, mediated neuroprotection of serum-starved GT1-7 cells. The percentage of PI-positive cells relative to Hoechst-stained cells is shown as the mean ± SEM. n = 3; ***P < 0.001 by 1-way ANOVA. (F) The SEMA3ER619C mutation impaired PI3K-dependent AKT phosphorylation. Immunoblotting and graph quantitation show AKT (Ser473) phosphorylation relative to total AKT in serum-starved GT1-7 cells treated for 15 minutes with WT SEMA3E or SEMA3ER619C. n = 3; ***P < 0.001 by Student’s t test. Scale bars: 50 μm (A), 150 μm (C), 20 μm (D), 100 μm (E).