Activation of the unfolded protein response is associated with impaired granulopoiesis in transgenic mice expressing mutant Elane

Abstract

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNAlike ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane.

Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

“…Unfortunately, mutant-ELANE knockin mice fail to reproduce the abnormal granulopoiesis, as observed in SCN patients (18,19). Moreover, BM cells are not an ideal experimental tool, since it is difficult to obtain sufficient nucleated cells due to the invasiveness of the aspiration procedure in pediatric patients with rare BM-failure syndromes.…”

“…Two different models of mutant ELANE knockin mice showed no neutropenia basally or after chemotherapyinduced stress (18,19). One of these mice only developed neutropenia after administration of a potent proteasome inhibitor but not after silencing the most relevant UPR sensor, Perk (19).…”

Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice

“…In addition, it is noteworthy that knock-in mice with single ELA2 mutations found in patients do not result in a baseline neutropenia in these animals. 42,43 This implies that effects on multiple granule proteins may be required for this effect in neutrophils. However, to date we have not been able to show that the engineered MBP-1 and EPX loci 16,17 actually generate truncated or aberrant polypeptides (our unpublished observations) and the suggestion that specific events need to coordinately happen in two genes limits the probability of this explanation.…”

A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation

“…We and others have suggested activation of the unfolded protein response (UPR) by misfolded mutated NE protein in myeloid progenitors as one possible mechanism. [15][16][17] However, it is unknown whether activation of the UPR is the only mechanism responsible for the differentiation block in CN myeloid progenitors.…”