A soluble endoplasmic reticulum factor as regenerative therapy for Wolfram syndrome

Abstract

Endoplasmic reticulum (ER) stress-mediated cell death is an emerging target for human chronic disorders, including neurodegeneration and diabetes. However, there is currently no treatment for preventing ER stress-mediated cell death. Here, we show that mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor secreted from ER stressed cells, prevents ER stress-mediated β cell death and enhances β cell proliferation in cell and mouse models of Wolfram syndrome, a prototype of ER disorders. Our results indicate that molecular pathways regulated by MANF are promising therapeutic targets for regenerative therapy of ER stress-related disorders, including diabetes, retinal degeneration, neurodegeneration, and Wolfram syndrome.

Fig. 1. MANF expression and secretion are induced by ER calcium depletion leading to suppression of ER stress-mediated cell death.

a qPCR analysis monitoring Manf mRNA expression levels in INS-1 832/13 cells treated with thapsigargin (TG) 10 nM for 24 h, 0.1 µM or 0.5 µM for 6 h. UT untreated (n = 3, ***P = 0.0001, ****P < 0.0001). b Western blot analysis monitoring extracellular and intracellular MANF levels. INS-1 832/13 cells were treated with 0.5 µM of TG for indicated times. c Left panel: western blot for evaluating the expression level of MANF protein in wild type (WT) and Manf knockout (Manf-KO) INS-1 832/13 cells. Right panel: caspase-3/7 activity normalized to cell viability in INS-1 832/13 cells treated with or without TG (0.1 µM for 4 h) (n = 8, ****P < 0.0001). d Western blot of cleaved caspase-3 in INS-1 832/13 cells pretreated with or without recombinant MANF peptide (5 µg/ml) for 24 h, and then challenged with TG (0.5 µM) for 6 h. Quantification of immunoblot analysis is shown in the right panel (n = 4, ****P < 0.0001). e The caspase-3/7 activity assay in INS-1 832/13 cells pretreated with recombinant MANF peptide (5 µg/ml) for 24 h, and then challenged with TG 0.5 µM for 6 h. f Left panel: western blot for evaluating the expression level of MANF protein in control (Ctrl) and MANF overexpressed INS-1 832/13 (MANF-OE) whole cell lysate and supernatant. Right panel: qPCR analysis monitoring the expression levels of Chop, sXbp1, Trb3, and Bip mRNA in Ctrl and MANF-OE INS-1 832/13 cells challenged with TG 0.5 µM for 6 h (n = 3, *P < 0.05, **P < 0.01, ****P < 0.0001).

Fig. 2. Effect of MANF on glucose-stimulated insulin secretion.

a Doxycycline-inducible shRNA directed against Wfs1 (INS-1 DOX-shWfs1) cells were treated with or without MANF peptide (5 µg/ml) for 24 h, and then treated with doxycycline (DOX). Insulin release was measured at basal (5.5 mM) glucose and stimulatory (16.7 mM) glucose conditions (n = 3, not significant). b Cellular insulin contents were measured after the 24 h pretreatment with MANF peptide (5 µg/ml) followed by DOX treatment (n = 3, not significant). c Glucose-stimulated insulin secretion on control (Ctrl) and MANF overexpressed INS-1 832/13 cells (MANF-OE). Insulin release was measured at 5.5 and 16.7 mM glucose conditions (n = 3, not significant). d Primary islets isolated from wild type (WT) and β cell-specific Wfs1 knockout mice (βWfs1^(−/−)) were pretreated with MANF peptide (5 µg/ml) for 24 h. Insulin release was measured at 5.5 mM and 16.7 mM glucose (n = 3, not significant).

Fig. 3. MANF activates proliferation of β cells.

The BrdU assay monitoring the proliferation of human primary islets treated with or without MANF peptide (5 µg/ml) for 5 days (n = 4, *P < 0.05).

Fig. 4. MANF attenuates cell death and activates cell proliferation in β cell models of Wolfram syndrome.

a Immunoblot analysis of cleaved caspase-3 and actin in doxycycline-inducible shRNA directed against Wfs1 (INS-1 DOX-shWfs1) cells. INS-1 DOX-shWfs1 cells were untreated or pretreated with MANF peptide (5 μg/ml) for 24 h, and then treated with doxycycline (DOX) for Wfs1 suppression. The quantified ratio of cleaved caspase-3 is shown in the right panel (n = 3, *P < 0.05). b Caspase-3/7 activity assay in INS-1 DOX-shWfs1 cells. INS-1 DOX-shWfs1 cells were untreated or pretreated with MANF peptide (5 μg/ml) for 24 h, and then treated with or without DOX for another 48 h (n = 3, **P < 0.01). c BrdU assay of β cell-specific Wfs1 knockout mice (βWfs1^(−/−)) primary islets. The isolated islets were treated with or without MANF peptide (5 µg/ml) for 5 days (n = 4, *P < 0.05). d qPCR analysis monitoring the expression levels of Chop, sXbp1, Trb3, and Bip mRNA in INS-1 DOX-shWfs1 cells. The cells were treated with or without MANF peptide (5 μg/ml) for 24 h, and then treated with DOX (n = 3, *P < 0.05).

Fig. 5. Overexpression of MANF by AAV9 enhances β cell proliferation in a mouse model of Wolfram syndrome.

a Top panels: double immunofluorescence staining of insulin (green fluorescence) and MANF (red fluorescence) on pancreatic tissue sections from β cell-specific Wfs1 knockout mice (βWfs1^(−/−)) taken 5 weeks after intraperitoneal injection of AAV-control (AAV9-CBA-IRES-GFP) or AAV9-MANF (AAV9-CBA-MANF-IRES-GFP) vector (n = 6 each group). Middle panels: double immunofluorescence staining of insulin (green fluorescence) and Ki67 (red fluorescence) of pancreatic sections from βWfs1^(−/−) mice taken 5 weeks after intraperitoneal injection of AAV-control or AAV9-MANF vector (n = 6 in each group). Bottom panels: double immunofluorescence staining of insulin (green fluorescence) and TUNEL staining (red fluorescence) of pancreatic sections from βWfs1^(−/−) mice taken 5 weeks after intraperitoneal injection of AAV-control or AAV9-MANF vector (n = 6 each group). The magnification is ×10 for each image, and ×40 for the right upper images. b Quantification of Ki67-positive β cells in AAV-control or AAV9-MANF injected βWfs1^(−/−) mice (AAV-control, n = 5; AAV9-MANF, n = 6; *P < 0.05). c Quantification of β cell mass in AAV-control or AAV9-MANF injected βWfs1^(−/−) mice (n = 6 in each group, not significant).