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Pre-Natal Manifestation of Systemic Developmental Abnormalities in Spinal Muscular Atrophy

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Pre-Natal Manifestation of Systemic Developmental Abnormalities in Spinal Muscular Atrophy Edinburgh Research Explorer Pre-natal manifestation of systemic developmental abnormalities in spinal muscular atrophy Citation for published version: Motyl, AAL, Faller, K, Groen, EJN, Kline, R, Eaton, S, Ledahawsky, L, Chaytow, H, Lamont, DJ, Wishart, T, Huang, Y & Gillingwater, T 2020, 'Pre-natal manifestation of systemic developmental abnormalities in spinal muscular atrophy', Human Molecular Genetics, vol. 29, no. 16, pp. 2674 - 2683. https://doi.org/10.1093/hmg/ddaa146 Digital Object Identifier (DOI): 10.1093/hmg/ddaa146 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Human Molecular Genetics General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 01. Oct. 2021 Human Molecular Genetics, 2020, Vol. 29, No. 16 2674–2683 doi: 10.1093/hmg/ddaa146 Advance Access Publication Date: 9 July 2020 General Article GENERAL ARTICLE Pre-natal manifestation of systemic developmental Downloaded from https://academic.oup.com/hmg/article/29/16/2674/5869328 by guest on 03 February 2021 abnormalities in spinal muscular atrophy Anna A.L. Motyl1,2, Kiterie M.E. Faller3, Ewout J.N. Groen4,†, Rachel A. Kline2,5, Samantha L. Eaton2,5, Leire M. Ledahawsky1,2, Helena Chaytow1,2, Douglas J. Lamont6, Thomas M. Wishart2,5, Yu-Ting Huang1,2,¶ and Thomas H. Gillingwater1,2,¶,*,‡ 1Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, UK, 2Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh EH16 4SB, UK, 3Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh EH25 9RG, UK, 4UMC Utrecht Brain Center, University Medical Center, Utrecht 3584 CG, The Netherlands, 5The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK and 6FingerPrints Proteomics Facility, University of Dundee, DD1 5EH, UK *To whom correspondence should be addressed at: University of Edinburgh, Old Medical School (Anatomy), Teviot Place, Edinburgh EH8 9AG, UK. Tel: +44 (0)131 6503724; Email: [email protected] Abstract Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). SMN-restoring therapies have recently emerged; however, preclinical and clinical studies revealed a limited therapeutic time window and systemic aspects of the disease. This raises a fundamental question of whether SMA has presymptomatic, developmental components to disease pathogenesis. We have addressed this by combining micro-computed tomography (μCT) and comparative proteomics to examine systemic pre-symptomatic changes in a prenatal mouse model of SMA. Quantitative μCT analyses revealed that SMA embryos were significantly smaller than littermate controls, indicative of general developmental delay. More specifically, cardiac ventricles were smaller in SMA hearts, whilst liver and brain remained unaffected. In order to explore the molecular consequences of SMN depletion during development, we generated comprehensive, high-resolution, proteomic profiles of neuronal and non-neuronal organs in SMA mouse embryos. Significant molecular perturbations were observed in all organs examined, highlighting tissue-specific prenatal molecular phenotypes in SMA. Together, our data demonstrate considerable systemic changes at an early, presymptomatic stage in SMA mice, revealing a significant developmental component to SMA pathogenesis. †Ewout J.N. Groen, http://orcid.org/0000-0002-2330-9444 ‡Thomas H. Gillingwater, http://orcid.org/0000-0002-0306-5577 ¶These authors contributed equally. Received: June 5, 2020. Revised: July 2, 2020. Accepted: July 6, 2020 © The Author(s) 2020. 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. 2674 Human Molecular Genetics, 2020, Vol. 29, No. 16 2675 Introduction SMA mouse embryos are smaller than littermate controls Spinal muscular atrophy (SMA) is the second most common autosomal recessive disorder in humans, after cystic fibrosis: We first asked whether anatomical defects could be detected with 2% of people carrying an SMA-associated mutation, this in SMA embryos compared to littermate controls. To do so, we disease affects 1 in 6000–10 000 live births and is the leading performed micro-computed tomography (μCT) imaging of whole genetic cause of infant death (1,2). SMA is primarily a neurode- embryos from a single litter of mice at E14.5 (Fig. 1A, i-iv). Initial generative disease characterized by loss of lower motor neurons anatomical, qualitative assessment of the embryos confirmed and muscle atrophy (3–5). In 98% of cases, SMA arises from a the presence and standard location of all major body organs and homozygous mutation in the survival motor neuron 1 gene (SMN1) systems in both SMA and littermate animals. Using the scans, (6), leading to insufficient production of full-length SMN protein. we then segmented the images to facilitate volumetric mea- SMN is ubiquitously expressed in all cell types, including during surement and 3D reconstruction of regions of interest (Supple- development and is crucial for survival (7,8). Mouse embryos mentary Material, Movie 1). We measured the volumes of whole in which Smn has been genetically knocked out die around the embryos as well as brain, liver and the cardiac ventricles (Fig. 1B, Downloaded from https://academic.oup.com/hmg/article/29/16/2674/5869328 by guest on 03 February 2021 peri-implantation stage (9). Supplementary Material, Movies 2–5). Volumetric quantification In late 2016, Spinraza® (nusinersen) became the first FDA- revealed that SMA embryos were significantly smaller than their approved drug to treat SMA (5,10–14). The FDA subsequently littermate controls (Fig. 1C; unpaired t-test, n =4,P = 0.0396). Vol- approved Zolgensma® (onasemnogene abeparvovec-xioi) in ume measurements of liver and brain revealed no difference May 2019 (15,16). Both of these treatments work by increasing between SMA and control embryos, whereas the cardiac ven- the levels of full-length SMN protein, the former through an tricles were significantly smaller in the SMA animals (Fig. 1C; antisense oligonucleotide and the latter using AAV9-delivered unpaired t-test, n =4, P = 0.0059). Taken together, these experi- gene replacement therapy. However, despite the success of both ments showed that SMA embryos differ from controls in total Spinraza® and Zolgensma® in improving outcomes in SMA body size, suggesting a developmental delay in SMA embryos patients, their efficacy is highly variable from one patient to compared to littermate controls, and that internal organs are another, and they modify disease course rather than providing differentially affected. acure(10,15,16). ® One likely explanation for the modest efficacy of Spinraza Cardiac ventricles do not show overt pathology and Zolgensma® in some patients is the timing and targeting of therapy delivery. Thus, results from clinical trials suggest that As cardiac ventricles were found to be significantly smaller in treatment should be performed as early as possible to obtain SMA mice (Fig. 1C), we next examined whether they exhibited maximal therapeutic benefit (10,17). This finding is supported by any structural changes known to be representative of cardiac pre-clinical animal studies, which have revealed a narrow post- pathology, and which are known to occur in the heart of SMA natal time window for the successful restoration of SMN in SMA mice at later developmental stages. For example, Shababi et al. mice (18–20). Moreover, several lines of evidence suggest that (28) described thinning of the interventricular septum (IVS) in systemic delivery of therapies, rather than targeting of motor the E17.5 heart of SMA embryos of the same model. Using neurons in the spinal cord, is required for maximal therapeutic orientation-adjusted transverse μCT images, we performed mor- benefit (21–23). phometric measurements of the ventricles. We examined the Despite our growing awareness of the need for early thera- width of the IVS and thickness of the left ventricular wall. peutic intervention in SMA, we still do not know where and when Despite the smaller volume of SMA hearts at E14.5, no overt the disease first manifests. The majority of pre-clinical studies structural defects were identified (Fig. 2A, A’, C and D). To verify have been performed in post-natal animals. However, several this finding, we performed the same measurements on H&E- studies support the hypothesis that SMA, particularly in its stained transverse cryosections. Again, structural defects were most severe forms, may present with pre-symptomatic, prenatal not observed in the heart (Fig. 2B, B’, C and D), suggesting
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