GLP-1R agonists demonstrate potential to treat Wolfram syndrome in human preclinical models

Abstract

Aims/hypothesis: Wolfram syndrome is a rare autosomal recessive disorder caused by pathogenic variants in the WFS1 gene. It is characterised by insulin-dependent diabetes mellitus, optic nerve atrophy, diabetes insipidus, hearing loss and neurodegeneration. Considering the unmet treatment need for this orphan disease, this study aimed to evaluate the therapeutic potential of glucagon-like peptide 1 receptor (GLP-1R) agonists under wolframin (WFS1) deficiency with a particular focus on human beta cells and neurons.

Methods: The effect of the GLP-1R agonists dulaglutide and exenatide was examined in Wfs1 knockout mice and in an array of human preclinical models of Wolfram syndrome, including WFS1-deficient human beta cells, human induced pluripotent stem cell (iPSC)-derived beta-like cells and neurons from control individuals and individuals affected by Wolfram syndrome, and humanised mice.

Results: Our study shows that the long-lasting GLP-1R agonist dulaglutide reverses impaired glucose tolerance in WFS1-deficient mice, and that exenatide and dulaglutide improve beta cell function and prevent apoptosis in different human WFS1-deficient models including iPSC-derived beta cells from people with Wolfram syndrome. Exenatide improved mitochondrial function, reduced oxidative stress and prevented apoptosis in Wolfram syndrome iPSC-derived neural precursors and cerebellar neurons.

Conclusions/interpretation: Our study provides novel evidence for the beneficial effect of GLP-1R agonists on WFS1-deficient human pancreatic beta cells and neurons, suggesting that these drugs may be considered as a treatment for individuals with Wolfram syndrome.

Keywords: GLP-1R agonists; Human pancreatic beta cells; Wolfram syndrome; iPSC-derived beta cells; iPSC-derived neurons.

Fig. 1

Dulaglutide treatment prevents glucose intolerance in WFS1-deficient mice. (a) Schematic representation of the experiment. Four-week-old Wfs1 KO and WT littermates were treated for 8 weeks with saline (Veh, n=6–7) or dulaglutide (1 mg/kg every 4 days, Dula, n=6–7). After 8 weeks, treatment was suspended for 10 days (washout, WO), and then resumed for 4 more weeks. Timing of metabolic tests (IPGTT and ITT) is indicated. (b, f, m, o) IPGTT glucose levels, (c, g, n, p) AUC and (d, h) insulin levels. (e, i) Insulinogenic index, (j) ITT glucose levels, (k) insulin sensitivity calculated as ITT area over the curve (AOC) and (l) beta cell function. Results are expressed as mean and SD. In the bars, circles and squares represent individual female mice, triangles and diamonds male mice. Extremities of floating bars are maximal and minimal values; horizontal line shows median. *p<0.05, **p<0.01, ***p<0.001 KO Veh vs WT Veh; ^††p<0.01, ^†††p<0.01 other time points vs T0 in KO Veh; ^‡p<0.05, ^‡‡p<0.01, ^‡‡‡p<0.001 other time points vs T0 in WT Veh; ^§p<0.05, ^§§p<0.01, ^§§§p<0.001 Dula vs Veh in KO, by two-way or one-way ANOVA (as suitable) followed by Sidak’s or Dunn’s correction for multiple comparisons. ΔI/ΔG, incremental insulin over incremental glucose; T0, time zero (treatment initiation)

Fig. 2

Dulaglutide treatment reverses diabetes in WFS1-deficient mice. (a) Schematic representation of the experiment. Seven-week-old (n=8) (b, c) or 20-week-old (n=6) (d, e) Wfs1 KO mice were treated for 4–12 weeks with dulaglutide (1 mg/kg every 4 days) and compared with age-matched WT littermates. (b, d) IPGTT glucose levels and (c, e) AUC of WT mice and dulaglutide-injected Wfs1 KO mice at baseline (T0) and after 4, 8 and 12 weeks of treatment. Results are expressed as mean and SD. In the bars circles and squares represent individual female mice, triangles and diamonds male mice. Extremities of floating bars are maximal and minimal values; horizontal line shows median. *p<0.05, **p<0.005, ***p<0.001 KO T0 vs WT ; ^§p<0.05, ^§§p<0.05, ^§§§p<0.001 Dula vs KO T0, by two-way or one-way ANOVA (as suitable) followed by Sidak’s or Dunn’s correction for multiple comparisons. Dula, dulaglutide; T0, time zero (treatment initiation)

Fig. 3

WFS1 silencing sensitises human beta cells to ER stress. WFS1 was silenced or not (siCT) in EndoC-βH1 cells using two siRNAs (siWFS1#1 and #2). (a) Representative western blot and (b) densitometric quantification of WFS1 knockdown 72 h after transfection. GAPDH was used as a control for protein loading. (c) Apoptosis assessed by Hoechst 33342/propidium iodide staining in control and WFS1-silenced EndoC-βH1 cells exposed or not for 24 h to thapsigargin, tunicamycin or brefeldin. Data points represent independent experiments. Extremities of floating bars are maximal and minimal values; horizontal line shows median. (d–l) Time course of tunicamycin exposure in EndoC-βH1 cells silenced for WFS1 for 48 h. WFS1, ATF3, CHOP, BIP and XBP1s mRNA expression was measured by real-time PCR (d–h) and normalised to reference gene ACTB. WFS1, ATF3 and BiP expression was examined by western blot and normalised to the reference protein GAPDH (i–l). Results are means ± SEM of n=3 (real-time PCR) or n=4 (western blots) independent experiments, and are expressed as fold of the highest value in each experiment. *p<0.05, **p<0.01, ***p<0.001 siWFS1#1 vs siCT, §p<0.05, §§p<0.01, §§§p<0.001 siWFS1#2 vs siCT; ^†p<0.05, ^††p<0.01, ^†††p<0.001 treated vs CT or vs time 0 h in WFS1-deficient cells; ^‡p<0.05, ^‡‡p<0.01, ^‡‡‡p<0.001 treated vs CT or vs time 0 h in control cells. Data were analysed by one-way or two-way ANOVA (as suitable), followed by Sidak’s or Dunnett’s test for multiple comparisons. Bref, brefeldin; CT, control untreated; Thap, thapsigargin; Tuni, tunicamycin

Fig. 4

Exenatide protects human beta cells from ER stress-induced apoptosis. WFS1 was silenced in EndoC-βH1 cells (a–g) or dispersed human islets (h–i) using siRNAs (siWFS1#1 and #2). At 24 h after transfection, the cells were pretreated or not for 2 h or 24 h, respectively, with exenatide (50 nmol/l), dulaglutide (50 mmol/l) or forskolin (20 μmol/l), and then exposed or not (CT) for 24 (a, b), 16 (c–g) or 48 h (h, i), respectively, to tunicamycin alone or in combination with exenatide, forskolin or dulaglutide. (a, h) WFS1 mRNA expression by real-time PCR. (b, i) Apoptosis evaluated by Hoechst 33342/propidium iodide staining. (c–g) Western blot data. WFS1, JunB, ATF3 and BiP protein expression was normalised to the geometric mean of the reference proteins tubulin and GAPDH, and expressed as fold of the highest value in each experiment. Data points represent independent experiments. Extremities of the floating bars are maximal and minimal values; horizontal line shows median. *p<0.05, **p<0.01, ***p<0.001 siWFS1#1 or siWFS#2 vs the same treatment in siCT; ^††p<0.01, ^†††p<0.001 treated vs CT in WFS1-silenced cells; ^‡p<0.05, ^‡‡p<0.01, ^‡‡‡p<0.001 treated vs CT in control cells; ^§p<0.05, ^§§p<0.05, ^§§§p<0.001 Tuni+Ex or Tuni+Fk vs Tuni, by one-way ANOVA followed by Sidak’s or Tukey’s test for multiple comparisons. CT, control (vehicle); Du, dulaglutide; Ex, exenatide; Fk, forskolin; Tuni, tunicamycin

Fig. 5

Exenatide protects iPSC-derived Wolfram syndrome beta-like cells from ER stress-induced apoptosis. Wolfram syndrome iPSCs (Wolf-2010-11.1, Wolf-2011-13.2 and Wolf-2010-9.4) and the isogenic control line (Wolf-9.4-Corr-2G6.1) were differentiated into beta-like cells. (a) Representative images and (b) quantification of dispersed stage 7 aggregate cells stained for insulin (green), glucagon (red) and somatostatin (pink). Polyhormonal cells (Poly) are coloured yellow. Nuclei were stained with DAPI. Scale bar, 20 μm. Whole (c–e) or dispersed (f) aggregates were pretreated or not for 24 h with exenatide (50 nmol/l) or forskolin (20 μmol/l) and then exposed or not (CT) for 48 h to tunicamycin (Tuni), alone or combined with exenatide (Ex) or forskolin (Fk). (c, d) Time course of early and late apoptosis in whole control (c) or WFS1 (d) aggregates assessed by RealTime-Glo Annexin V. Results are expressed as fold of basal apoptosis in each condition. (e) AUC of (c) and (d). (f) Apoptosis determined by Hoechst 33342/propidium iodide staining in dispersed aggregates. Circles represent differentiations of Wolf-9.4-Corr-2G6.1, triangles Wolf-2010-11.1, diamonds Wolf-2011-13.2 and squares Wolf-2010-9.4. Extremities of floating bars are maximal and minimal values; horizontal line shows median. *p<0.05, **p<0.01 WFS1 vs control; ^†p<0.05, ^†††p<0.001 Tuni vs CT in WFS1 cells; ^‡‡p<0.01 Tuni vs CT in control cells; ^§p<0.05, ^§§p<0.01, ^§§§p<0.001 Fk vs CT, Tuni+Ex or Tuni+Fk vs Tuni, by one-way or two-way ANOVA or mixed effects analysis followed by the two-stage step-up method of Benjamini, Krieger and Yekutieli or Tukey’s test for multiple comparisons. CT, control (vehicle); GCG, glucagon; INS, insulin; SST, somatostatin

Fig. 6

Dulaglutide improves Wolfram syndrome iPSC beta-like cell function. (a) Schematic representation of the experiment. iPSC-derived stage 7 control (Wolf-9.4-Corr-2G6.1, n=3) or Wolfram syndrome aggregates (Wolf-2010-9.4, n=3 and Wolf-2010-11.1, n=1 differentiation) were transplanted under the kidney capsule of Rag2 KO mice (2 mice per differentiation). (b–e) IPGTT glucose levels and human C-peptide secretion evaluated 7 and 14 weeks after transplantation (n=6 mice transplanted with control aggregates, and n=8 mice transplanted with WFS1 aggregates). (f–h) Fourteen weeks after transplantation one mouse from each pair was randomised to either the dulaglutide (1 mg/kg every 4 days) or saline (Veh) group for 12 weeks. Results are expressed as mean and SD. (f) Non-fasting human C-peptide levels in WFS1-transplanted mice after 12 weeks of dulaglutide or vehicle administration. Paired symbols represent iPSC beta cells from one differentiation. Wolf-2010-9.4 is represented with squares and Wolf-2010-11.1 with triangles. Insulin secretion by control (g) and WFS1-deficient (h) grafts during in situ kidney perifusion with medium containing 0 (G0) or 20 mmol/l glucose (G20) alone or combined with forskolin, diazoxide, gliclazide or KCl. WFS1, n=4 mice per group; control, n=2 per group. ^†p<0.05, ^†††p<0.001 other time points vs T0 in WFS1; ^‡p<0.05, ^‡‡p<0.01, ^‡‡‡p<0.001 other time points vs T0 in control; *p<0.05 control vs WFS1; ^§§p<0.01, ^§§§p<0.001 WFS1 Veh vs WFS1 Dula by two-way ANOVA with Sidak’s or Dunnet’s correction for multiple comparisons. Dula, dulaglutide; DZ, diazoxide; GCZ, glicazide; K30, potassium chloride 30 mmol/l; T0, time zero. Panel (a) was generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)

Fig. 7

Exenatide protects Wolfram syndrome iPSC-derived neural precursors and cerebellar neurons. iPSCs Wolf 2010-9.4, Wolf 2010-11.1 and Wolf-2010-07.1 were differentiated into cerebellar neuron-like cells. (a) Differentiation timeline with representative phase contrast images of iPSCs (scale bar, 120 μm), neural rosettes, NPCs and cerebellar neuron-like cells (scale bars, 50 μm). (b) Representative immunofluorescence images for key markers OCT4, nestin, vimentin, PAX6, β-tubulin III, synaptophysin, GFAP, KIRRE2, ZIC1 and calbindin at day 12 of the first differentiation, in early NPCs (passage 1, P1) and at days 10 and 21 of the second differentiation. Scale bars, 30 μm. (c) Mitochondrial function by Seahorse in NPCs (n=3) and cerebellar neuron-like cells (n=3) exposed or not for 72 h to exenatide (500 nmol/l) or forskolin (20 μmol/l). Mitochondrial respiration was measured basally and after sequential injection of 20 mmol/l glucose, ATP synthase inhibitor oligomycin (5 μmol/l), oxidative phosphorylation uncoupler FCCP (4 μmol/l), and electron transport chain inhibitors rotenone and antimycin (1 μmol/l). Results are shown as means ± SEM of the two cell types together. (d, e) WFS1 iPSC-derived NPCs (squares) and cerebellar neuron-like cells (triangles) were exposed or not (CT) for 48 h to tunicamycin, alone or together with exenatide or forskolin. (d) Oxidative stress was measured by HPF. Menadione-treated cells were used as a positive control. (e) Apoptosis was determined by Hoechst 33342/propidium iodide. Extremities of floating bars are maximal and minimal values; horizontal line shows median. Symbols with the same colour come from one differentiation. *p<0.05, **p<0.01, ***p<0.001 Ex vs CT, ^†p<0.05, ^†††p<0.001 treated vs CT; ^§§p<0.01, ^§§§p<0.001 Tuni vs Tuni+Ex or Tuni+Fk; ^¶p<0.05 Ex vs CT, by two-way or one-way ANOVA followed by Dunnet or Holm–Sidak correction for multiple comparisons. CT, control (vehicle); Ex, exenatide; Fk, forskolin; GFAP, glial fibrillary acidic protein; P, passage; PAX6, paired box protein 6; Pos, positive control; Tuni, tunicamycin