A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

J Kim ,Amaia Paredes‐Redondo,Silvia Torelli,Francesco Catapano,Christin Luft,Sandra Louzada,Pentao Liu,Elizabeth Stevens,Fengtang Yang,Pierpaolo Ala,Yung‐Yao Lin,Evangelos Konstantinidis,Beiyuan Fu,David Ryan,Aw Edith Chan,Francesco Muntoni,Beatrice Lana,Robin Ketteler,David L Selwood ,Derek L Stemple

doi:10.15252/embr.201947967

Abstract

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α‐dystroglycan‐laminin interaction due to defective glycosylation of α‐dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human‐ and neural‐specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin‐related protein gene) mutation. We showed that CRISPR/Cas9‐mediated gene correction of FKRP restored glycosylation of α‐dystroglycan in iPSC‐derived cortical neurons, whereas targeted gene mutation of FKRP in wild‐type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α‐dystroglycan. Using human FKRP‐iPSC‐derived neural cells for hit validation, we demonstrated that compound 4‐(4‐bromophenyl)‐6‐ethylsulfanyl‐2‐oxo‐3,4‐dihydro‐1H‐pyridine‐5‐carbonitrile (4BPPNit) significantly augmented glycosylation of α‐dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC‐derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

Highlights

Post-translational processing of dystroglycan is crucial for its function as an extracellular matrix (ECM) receptor in a variety of fetal and adult tissues
Generation and characterization of FKRP-induced pluripotent stem cells (iPSCs) derived from a dystroglycanopathy patient with central nervous system (CNS) abnormalities
The initial characterization of five independent FKRP-iPSC clonal lines by qPCR revealed the gene expression of pluripotency markers, such as NANOG, OCT4 and REX1 (Appendix Fig S1). Among these clonal iPSC lines, we focused on the FKRP-iPSC line

Summary

Introduction

Post-translational processing of dystroglycan is crucial for its function as an extracellular matrix (ECM) receptor in a variety of fetal and adult tissues. The mature a-dystroglycan is a heavily glycosylated peripheral membrane protein with molecular weight range from 100 to 156 kDa, dependent on tissue-specific glycosylation. The b-dystroglycan is a 43-kDa transmembrane protein that links to the actin cytoskeleton via interaction with dystrophin [1]. Defective O-linked glycosylation of a-dystroglycan is a common pathological hallmark associated with number of genetic syndromes, encompassing symptoms from muscular dystrophies to ocular defects, cognitive deficits, and structural cortical malformations (cobblestone lissencephaly) in the central nervous system (CNS). This group of autosomal recessive disorders are commonly referred to as secondary dystroglycanopathies [2,3]. The effort of identifying causative gene mutations in dystroglycanopathies has shed light on a novel mammalian glycosylation pathway [4,5]

Methods

Results

Conclusion