SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy

Thomas M. Wishart 1 2 3 Jack P.-W. Huang 1 2 3 Lyndsay M. Murray 1 2 3 Douglas J. Lamont 0 2 Chantal A. Mutsaers 1 2 3 Jenny Ross 1 2 3 Pascal Geldsetzer 1 2 3 Olaf Ansorge 2 4 Kevin Talbot 2 Simon H. Parson 1 2 3 Thomas H. Gillingwater 1 2 3 0 'FingerPrints' Proteomics Facility, College of Life Sciences, University of Dundee , Dundee, UK 1 Centre for Integrative Physiology, University of Edinburgh Medical School , Edinburgh, UK 2 Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK 3 Euan MacDonald Centre for Motor Neurone Disease Research 4 Department of Neuropathology, John Radcliffe Hospital , Oxford, UK Reduced expression of the survival motor neuron (SMN) gene causes the childhood motor neuron disease spinal muscular atrophy (SMA). Low levels of ubiquitously expressed SMN protein result in the degeneration of lower motor neurons, but it remains unclear whether other regions of the nervous system are also affected. Here we show that reduced levels of SMN lead to impaired perinatal brain development in a mouse model of severe SMA. Regionally selective changes in brain morphology were apparent in areas normally associated with higher SMN levels in the healthy postnatal brain, including the hippocampus, and were associated with decreased cell density, reduced cell proliferation and impaired hippocampal neurogenesis. A comparative proteomics analysis of the hippocampus from SMA and wild-type littermate mice revealed widespread modifications in expression levels of proteins regulating cellular proliferation, migration and development when SMN levels were reduced. This study reveals novel roles for SMN protein in brain development and maintenance and provides the first insights into cellular and molecular pathways disrupted in the brain in a severe form of SMA. - INTRODUCTION Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant mortality, is an autosomal-recessive neuromuscular disorder with a carrier frequency of 1:50 and an annual incidence of around 1:10 000 live births (1). SMA is classically characterized by degeneration and loss of large alpha motor neurons in the ventral horn of the spinal cord and paralysis of proximal muscles in the limbs and trunk, resulting from denervation at the neuromuscular junction (2 5). The most severe and common form of SMA, accounting for 50% of SMA diagnoses (SMA type I; Werdnig Hoffmann syndrome), leads to disease onset in the first 6 months of life, no achievement of sitting without support and death within the first 2 years of life (5,6). The vast majority of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene, leading to reduced expression levels of full-length SMN protein (7). SMN protein is known to play a critical role in several core cellular pathways, including snRNP biogenesis and pre-mRNA splicing (reviewed in 8,9). Previous studies have suggested that motor neuron-specific changes in splicing pathways may be responsible for tissue-specific targeting in SMA (10), although recent evidence has shown that these changes are likely to occur as a consequence, rather than a cause, of the disease (11). Although SMN protein is ubiquitously expressed in tissues and organs across a range of different species, including rats, monkeys and humans (12), the extent to which SMN deficiency leads specifically to targeted pathological changes in the neuromuscular system remains unclear. For example, the fact that significant levels of SMN protein have previously been identified in the brain (12,13) raises the possibility that reduced levels of expression may have effects on non-motor regions of the nervous system. Here, we show that reduced levels of SMN protein result in regionally selective, impaired brain development in a mouse model of severe SMA [Smn2/2;SMN2 mice (14)]. In highly affected regions, such as the hippocampus, SMN deficiency resulted in lower cell density, decreased cell proliferation and decreased postnatal neurogenesis. Proteomic analysis of the hippocampus from SMA mice revealed changes in molecular pathways involved in cellular proliferation, migration and development. Regionally specific modifications in brain morphology reveal abnormal postnatal brain development in a mouse model of severe SMA To establish whether reduced levels of SMN protein influence brain morphology, we examined postnatal brain development in an established mouse model of severe SMA [Smn2/2;SMN2 mice (14)]. A comparison of whole, freshly dissected brains from Smn2/2;SMN2 mice with brains from non-affected, wild-type littermates (Smn+/+;SMN2 mice) at a late-symptomatic disease time point (postnatal Day 5; P5) revealed a significant reduction in brain size and weight (Fig. 1C and D). Equivalent analysis at a pre-symptomatic time point (P1) revealed that brains from Smn2/2;SMN2 mice were indistinguishable from those of control littermates and were of an almost identical weight (Fig. 1A and B). Thus, gross morphological changes were apparent in the brains of mice with a severe form of SMA at late-symptomatic ages. Higher-resolution morphological assessment of Nisslstained coronal brain sections from Smn2/2;SMN2 mice at pre- and late-symptomatic time points revealed region-specific effects of reduced SMN levels. Qualitatively, the most striking morphological changes were observed in the hippocampus and particularly the hippocampal dentate gyrus. This region was noticeably smaller in Smn2/2;SMN2 mice at latesymptomatic time points (Fig. 2). Quantitative assessment confirmed our qualitative observations, as by P6 the area of the hippocampal dentate gyrus was 50% smaller in Smn2/2;SMN2 mice compared with wild-type littermates (Fig. 2E). Statistical comparisons of hippocampal dentate gyrus area in Smn2/2;SMN2 and littermate control mice confirmed a significant effect of the reduction in SMN levels [two-way ANOVA; P , 0.001; F(1,35) 20.45]. Interestingly, previous reports have cited the hippocampus as a brain region likely to be particularly susceptible to SMA pathology in human patients with severe forms of SMA (15). More modest morphological reductions were observed in the primary motor cortex (M1) of Smn2/2;SMN2 mice. These subtle morphological differences were present at the time of birth (pre-symptomatically) and persisted through latesymptomatic stages where they became statistically significant (Fig. 2A and B). Despite this decrease in motor cortex thickness, the primary somatosensory cortex (S1BF) remained unaffected in Smn2/2;SMN2 mice at all stages of disease progression [Fig. 3; two-way ANOVA; P . 0.05; F(1,36) 3.93], suggesting that distinct cortical areas were differentially affected. Importantly, the finding that not all brain regions were equally affected in Smn2/2;SMN2 mice confirmed that the developmental changes identified were likely to represent specific vulnerability of regions such as the hippocampus, rather than simply oc (...truncated)