Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome
Anna Cariboni, Valentina André, Sophie Chauvet, Daniele Cassatella, Kathryn Davidson, Alessia Caramello, Alessandro Fantin, Pierre Bouloux, Fanny Mann, Christiana Ruhrberg
Anna Cariboni, Valentina André, Sophie Chauvet, Daniele Cassatella, Kathryn Davidson, Alessia Caramello, Alessandro Fantin, Pierre Bouloux, Fanny Mann, Christiana Ruhrberg
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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 2

SEMA3E promotes GnRH neuron survival via PLXND1 and PI3 kinase activation.

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SEMA3E promotes GnRH neuron survival via PLXND1 and PI3 kinase activatio...
(A and B) SEMA3E protects GT1-7 but not GN11 cells from death induced by serum starvation. Serum-starved GN11 (top row) and GT1-7 (bottom row) cells were treated with serum or SEMA3E. Dying cells were visualized by PI staining in the presence of Hoechst nuclear counterstain (A) to determine the proportion of PI-positive cells relative to all Hoechst-stained cells, shown as the mean ± SEM; n = 3; **P < 0.01 and ***P < 0.001 by 1-way ANOVA (B). (C and D) Blocking PLXND1 abolished SEMA3E-mediated neuroprotection of GT1-7 cells. Immunofluorescence staining with PLXND1 antibody (αPLXND1) showed that GT1-7 cells expressed PLXND1 (left panel, C). PI and Hoechst staining showed that αPLXND1, but not control IgG, inhibited SEMA3E-mediated neuroprotection in GT1-7 cells (right 2 panels, C). Proportion of PI-positive cells in all Hoechst-stained cells (D), shown as the mean ± SEM; n = 3; **P < 0.01 by 1-way ANOVA. (E and F) Blocking PI3K abolished SEMA3E-mediated neuroprotection of GT1-7 cells. PI and Hoechst staining showed that LY294 did not affect cell death under control conditions (left 2 panels, E), but inhibited SEMA3E-mediated neuroprotection in GT1-7 cells (right 2 panels, E). Proportion of PI-positive cells in all Hoechst-stained cells (F), shown as the mean ± SEM. n = 3; **P < 0.001 by 1-way ANOVA. (G and H) SEMA3E promoted PI3K-dependent AKT phosphorylation in GT1-7 neurons. Immunoblotting shows increased levels of phosphorylated AKT (p-AKT) (Ser473) relative to total AKT in serum-starved GT1-7 cells treated with SEMA3E, and LY294 abolished SEMA3E-induced AKT phosphorylation. n = 3; **P < 0.01 by Student’s t test. Scale bars: 10 μm (left panel, C); 25 μm (all other panels). The red line in H indicates the p-AKT/AKT ratio in untreated serum-starved GT1-7 cells, which was set to 1.