4-Sodium phenyl butyric acid has both efficacy and counter-indicative effects in the treatment of Col4a1 disease

Human Molecular Genetics, 2019, Vol. 28, No. 4 628–638 doi: 10.1093/hmg/ddy369 Advance Access Publication Date: 22 October 2018 General Article GENERAL ARTICLE counter-indicative effects in the treatment of Col4a1 disease Frances E. Jones1,‡ , Lydia S. Murray1,‡ , Sarah McNeilly1 , Afshan Dean1 , Alisha Aman1 , Yinhui Lu2 , Nija Nikolova1 , Ruben Malomgré1 , Karen Horsburgh3 , William M. Holmes4 , Karl E. Kadler2 and Tom Van Agtmael1,*,† 1 Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK, 2 Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine & Health, University of Manchester, Manchester, M13 9PT, UK, 3 Centre for Discovery Brain Sciences, Medical School, University of Edinburgh, Edinburgh, EH16 4SB, UK and 4 Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK *To whom correspondence should be addressed at: Institute of Cardiovascular and Medical Sciences, Davidson Building, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK. Tel: +44 (0)1413306200; Email: Abstract Mutations in the collagen genes COL4A1 and COL4A2 cause Mendelian eye, kidney and cerebrovascular disease including intracerebral haemorrhage (ICH), and common collagen IV variants are a risk factor for sporadic ICH. COL4A1 and COL4A2 mutations cause endoplasmic reticulum (ER) stress and basement membrane (BM) defects, and recent data suggest an association of ER stress with ICH due to a COL4A2 mutation. However, the potential of ER stress as a therapeutic target for the multi-systemic COL4A1 pathologies remains unclear. We performed a preventative oral treatment of Col4a1 mutant mice with the chemical chaperone phenyl butyric acid (PBA), which reduced adult ICH. Importantly, treatment of adult mice with the established disease also reduced ICH. However, PBA treatment did not alter eye and kidney defects, establishing tissue-specific outcomes of targeting Col4a1-derived ER stress, and therefore this treatment may not be applicable for patients with eye and renal disease. While PBA treatment reduced ER stress and increased collagen IV incorporation into BMs, the persistence of defects in BM structure and reduced ability of the BM to withstand mechanical stress indicate that PBA may be counter-indicative for pathologies caused by matrix defects. These data establish that treatment for COL4A1 disease requires a multipronged treatment approach that restores both ER homeostasis and matrix defects. Alleviating ER stress is a valid therapeutic target for preventing and treating established adult ICH, but collagen IV patients will require stratification based on their clinical presentation and mechanism of their mutations. † Tom Van Agtmael, http://orcid.org/0000-0003-4282-449X ‡ These authors contributed equally. Received: August 7, 2018. Revised: October 10, 2018. Accepted: October 11, 2018 © The Author(s) 2018. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 628 4-Sodium phenyl butyric acid has both efficacy and Human Molecular Genetics, 2019, Vol. 28, No. 4 Introduction mechanical stress. This highlights the need for patient stratification for such treatment approaches based on both their clinical presentation and knowledge of the underlying mechanism of their specific mutations. Results Targeting ER stress as a preventative treatment for ICH As defects due to COL4A1 mutations can present in childhood, a therapeutic approach will likely be long-term and would preferentially be orally administered. To model this preventative treatment, we treated Col4a1+/SVC mice (18,34) orally with PBA from conception, by treatment of pregnant dams, and ICH was determined in 5-month-old adult mice, treated daily (Fig. 1A). Magnetic resonance imaging (MRI) of the midbrain revealed that most ICH is centred towards the basal ganglia and, importantly, a ∼50% reduction in bleed volume of PBAtreated Col4a1+/SVC mice (Fig. 1B and C). As PBA did not affect the number of haemorrhages, this reduced severity is obtained by modulating bleed volume (Fig. 1C). Histopathological analysis using Prussian blue stain for hemosiderin (Perl’s staining) confirmed the MRI data (Fig. 1D and E). Col4a1+/SVC display increased levels of Iba1, a marker for neuroglial activation that is the initial step in the central nervous system inflammatory response following stroke (35), which was reduced in treated mice (Fig. 1F and G), supporting reduced cerebrovascular disease severity and neuroinflammation. Tail cuff plethysmography revealed no significant change in systolic blood pressure in mutant mice (Supplementary Material, Fig. S1), indicating the reduction in ICH is not dependent on altered vascular haemostasis. Modification of eye and renal defects Col4a1 renal disease encompasses glomerular defects including hypertrophy of Bowman’s capsule and glomerulocystic kidney disease (18,21,22), as well as tubular defects that are associated with polyuria (increased urine production) (22). Preventative PBA treatment reduced polyuria (Fig. 2A). However, both untreated and treated mice displayed defects of the parietal epithelium in Bowman’s capsule with a cuboidal appearance of epithelial cells, suggestive of epithelial cell activation (21) (Fig. 2B), and/or formation of multiple cell layers (18,21,22): ∼74% (75/102) and ∼73% (70/96) of Bowman’s capsules of untreated and treated mice, respectively (Fig. 2B, C and E). Col4a1 glomerulocystic kidney disease includes retraction of the capillary tuft (21), which was detected in ∼18% (19/103) of glomeruli in untreated mice and ∼15% (14/94) of treated mice (Fig. 2B, D and F). The occurrence of capillary tuft retraction in Col4a1+/SVC appears to occur in glomeruli that do not appear to exhibit the parietal epithelial cell defect (Fig. 2B). Evidence of atrophy of the medulla also remained (Supplementary Material, Fig. S2B). These data indicate a differential response whereby Bowman’s capsule and glomerular defects are recalcitrant to PBA treatment while polyuria appears reduced. In the eye, Col4a1 mutations can lead to ASD encompassing corneal clouding, cataracts, iris hypoplasia and buphthalmos (enlargement of the anterior chamber) on slit lamp examination (Fig. 2I) (5,18,20). PBA-treated and untreated mice showed iris– corneal adhesions, dysgenic ciliary body and reduced or absence of trabecular meshwork (Fig. 2G and H). Severity scoring revealed PBA treatment did not alter ASD severity (Fig. 2H), while slit lamp analysis revealed iris hypoplasia, corneal clouding and Diseases caused by mutations in extracellular matrix components a (...truncated)