Chemical chaperone treatment reduces intracellular accumulation of mutant collagen IV and ameliorates the cellular phenotype of a COL4A2 mutation that causes haemorrhagic stroke

Human Molecular Genetics, 2014, Vol. 23, No. 2 doi:10.1093/hmg/ddt418 Advance Access published on September 2, 2013 283–292 Chemical chaperone treatment reduces intracellular accumulation of mutant collagen IV and ameliorates the cellular phenotype of a COL4A2 mutation that causes haemorrhagic stroke Lydia S. Murray1, Yinhui Lu2, Aislynn Taggart1, Nicole Van Regemorter3, Catheline Vilain3, Marc Abramowicz3, Karl E. Kadler2 and Tom Van Agtmael1,∗ 1 Received May 3, 2013; Revised August 13, 2013; Accepted August 23, 2013 Haemorrhagic stroke accounts for ∼20% of stroke cases and porencephaly is a clinical consequence of perinatal cerebral haemorrhaging. Here, we report the identification of a novel dominant G702D mutation in the collagen domain of COL4A2 (collagen IV alpha chain 2) in a family displaying porencephaly with reduced penetrance. COL4A2 is the obligatory protein partner of COL4A1 but in contrast to most COL4A1 mutations, the COL4A2 mutation does not lead to eye or kidney disease. Analysis of dermal biopsies from a patient and his unaffected father, who also carries the mutation, revealed that both display basement membrane (BM) defects. Intriguingly, defective collagen IV incorporation into the dermal BM was observed in the patient only and was associated with endoplasmic reticulum (ER) retention of COL4A2 in primary dermal fibroblasts. This intracellular accumulation led to ER stress, unfolded protein response activation, reduced cell proliferation and increased apoptosis. Interestingly, the absence of ER retention of COL4A2 and ER stress in cells from the unaffected father indicate that accumulation and/or clearance of mutant COL4A2 from the ER may be a critical modifier for disease development. Our analysis also revealed that mutant collagen IV is degraded via the proteasome. Importantly, treatment of patient cells with a chemical chaperone decreased intracellular COL4A2 levels, ER stress and apoptosis, demonstrating that reducing intracellular collagen accumulation can ameliorate the cellular phenotype of COL4A2 mutations. Importantly, these data highlight that manipulation of chaperone levels, intracellular collagen accumulation and ER stress are potential therapeutic options for collagen IV diseases including haemorrhagic stroke. INTRODUCTION Collagen IV is a major component of the basement membrane (BM), an extracellular matrix structure that provides support and compartmentalization to tissues as well as influences cell behaviour. In the vasculature, a BM surrounds vascular smooth muscle cells and separate them from endothelial cells. Vertebrates express six collagen IV polypeptide chains (a1[IV] –a6[IV]) encoded by the genes COL4A1– COL4A6. These polypeptide chains contain a central collagen domain, characterized by Gly-X-Y repeats in which every third amino acid is a glycine residue. In the endoplasmic reticulum (ER), three chains interact to form triple helical protomers and the glycine residues of the collagen domain are critical for helix formation (1). Three distinct protomers occur in vertebrates: a1.a1.a2(IV), a3.a4.a5(IV) and a5.a5.a6(IV)(1), with a1.a1.a2(IV) being present in vascular BMs. ∗ To whom correspondence should be addressed at: Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK. Tel: +44 1413306200; Fax: +44 1413305481; Email: tom.vanagtmael@ glasgow.ac.uk # The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 2Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK 3Department of Medical Genetics, Hopital Erasme – Université Libre de Bruxelles, Brussels, Belgium 284 Human Molecular Genetics, 2014, Vol. 23, No. 2 accumulation, ER stress and apoptosis. Taken together, these results indicate that the ability of cells to cope with mutant collagen folding and ER-retention may be a critical modifier of collagen IV diseases and potentially represent future therapeutic targets. RESULTS A COL4A2 mutation leads to haemorrhagic stroke and porencephaly Previous identification of COL4A1 mutations in patients with porencephaly (12) led us to perform candidate gene mutation analysis on a large pedigree presenting with autosomal dominant porencephaly with reduced penetrance (see Fig. 1 in 13 and Fig. 1A). While sequence analysis of patient IV:21, who has a large porencephalic cyst, excluded COL4A1 mutations, it identified a base pair change in exon 28 of COL4A2 (Fig. 1B) that is predicted to substitute a glycine residue of a Gly-X-Y repeat for aspartic acid (G702D) (Fig. 1C). Sequence analysis of additional family members confirmed segregation of the mutation with the phenotype and that the mutation was absent in 100 unrelated ethnically matched healthy controls, providing strong evidence that COL4A2 G702D represents the causative mutation. The glycine residue is highly conserved (Fig. 1C) and located in the collagen domain of COL4A2, which forms the triple helical domain of the a1.a1.a2(IV) protomer. Renal and ocular examinations did not reveal any kidney or eye defects characteristic of COL4A1 disease within this pedigree. Moreover, obligate carriers (Fig. 1A) did not exhibit any signs of subclinical cerebrovascular disease (see Fig. 3 in 13), in contrast to the vascular abnormalities observed using MRI in almost 100% of asymptomatic carriers of COL4A1 mutations (2). Thus, the reduced penetrance of the cerebral haemorrhaging and absence of extra-vascular defects suggest that COL4A2 mutations may lead to a milder disease than COL4A1 mutations. COL4A2G702D leads to BM defects To investigate if defects in the BM contribute to the disease mechanism, dermal biopsies were collected from a patient (IV:21 in Fig. 1A), his unaffected father, who carries the mutation (III:8 in Fig. 1A), and an ethnically matched control. Despite the absence of gross skin defects, electron microscopy (EM) analysis revealed focal BM defects including thinning, Figure 1. (A) A pedigree shows familial dominant porencephaly with reduced penetrance (adapted from Fig. 1 in 13). Individuals analysed are indicated by black arrows and affected individuals are indicated by black symbols. (B) Electropherogram of the COL4A2G702D mutation in the cDNA of patient, unaffected father and WT: control (asterisk indicates mutation). (C) Protein alignment of affected highly conserved glycine residue (grey box and arrow) (Mut: patient; HS: human, MM: mouse, RN: rat, GG: chicken). Alignmen (...truncated)