Development and translation of therapies for spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterised by widespread loss of lower motor neurons from the spinal cord, leading to progressive weakness and muscle atrophy. SMA is largely caused by homozygous loss of the survival motor neuron (SMN) 1 gene, resulting in reduced levels of full-length SMN protein. Although no approved treatment is currently available for SMA, several clinical trials investigating different approaches to increase SMN levels are showing promising early results. Trials investigating the use of therapies targeting muscle strength and neuroprotective pathways are also in progress, generating the possibility of delivering combination therapies utilising both SMN-dependent and SMN-independent targets. Due to an increased understanding of the cellular and molecular consequences of SMN depletion, a second wave of therapies targeted at pathways downstream of SMN are currently undergoing preclinical development. As these therapies move forward towards the clinic, new treatment options are likely to become available, raising the potential to generate an effective ‘cure’ for SMA

Synaptic Protection in the Brain of WldS Mice Occurs Independently of Age but Is Sensitive to Gene-Dose

Disruption of synaptic connectivity is a significant early event in many neurodegenerative conditions affecting the aging CNS, including Alzheimer's disease and Parkinson's disease. Therapeutic approaches that protect synapses from degeneration in the aging brain offer the potential to slow or halt the progression of such conditions. A range of animal models expressing the slow Wallerian Degeneration (Wld(S)) gene show robust neuroprotection of synapses and axons from a wide variety of traumatic and genetic neurodegenerative stimuli in both the central and peripheral nervous systems, raising that possibility that Wld(S) may be useful as a neuroprotective agent in diseases with synaptic pathology. However, previous studies of neuromuscular junctions revealed significant negative effects of increasing age and positive effects of gene-dose on Wld(S)-mediated synaptic protection in the peripheral nervous system, raising doubts as to whether Wld(S) is capable of directly conferring synapse protection in the aging brain.We examined the influence of age and gene-dose on synaptic protection in the brain of mice expressing the Wld(S) gene using an established cortical lesion model to induce synaptic degeneration in the striatum. Synaptic protection was found to be sensitive to Wld(S) gene-dose, with heterozygous Wld(S) mice showing approximately half the level of protection observed in homozygous Wld(S) mice. Increasing age had no influence on levels of synaptic protection. In contrast to previous findings in the periphery, synapses in the brain of old Wld(S) mice were just as strongly protected as those in young mice.Our study demonstrates that Wld(S)-mediated synaptic protection in the CNS occurs independently of age, but is sensitive to gene dose. This suggests that the Wld(S) gene, and in particular its downstream endogenous effector pathways, may be potentially useful therapeutic agents for conferring synaptic protection in the aging brain

Restoration of SMN in Schwann cells reverses myelination defects and improves neuromuscular function in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, primarily affecting lower motor neurons. Recent evidence from SMA and related conditions suggests that glial cells can influence disease severity. Here, we investigated the role of glial cells in the peripheral nervous system by creating SMA mice selectively overexpressing SMN in myelinating Schwann cells (Smn(−/−);SMN2(tg/0);SMN1(SC)). Restoration of SMN protein levels restricted solely to Schwann cells reversed myelination defects, significantly improved neuromuscular function and ameliorated neuromuscular junction pathology in SMA mice. However, restoration of SMN in Schwann cells had no impact on motor neuron soma loss from the spinal cord or ongoing systemic and peripheral pathology. This study provides evidence for a defined, intrinsic contribution of glial cells to SMA disease pathogenesis and suggests that therapies designed to include Schwann cells in their target tissues are likely to be required in order to rescue myelination defects and associated disease symptoms

Morphologic and functional correlates of synaptic pathology in the cathepsin D knockout mouse model of congenital neuronal ceroid lipofuscinosis

Mutations in the cathepsin D (CTSD) gene cause an aggressive neurodegenerative disease (congenital neuronal ceroid lipofuscinosis) that leads to early death. Recent evidence suggests that presynaptic abnormalities play a major role in the pathogenesis of CTSD deficiencies. To identify the early events that lead to synaptic alterations, we investigated synaptic ultrastructure and function in pre-symptomatic CTSD knock-out (Ctsd(−/−)) mice. Electron microscopy revealed that there were significantly greater numbers of readily releasable synaptic vesicles present in Ctsd(−/−) mice than in wild-type control mice as early as postnatal day 16. The size of this synaptic vesicle pool continued to increase with disease progression in the hippocampus and thalamus of the Ctsd(−/−) mice. Electrophysiology revealed a markedly decreased frequency of miniature excitatory postsynaptic currents (EPSCs) with no effect on pair-pulse modulation of the evoked EPSPs in the hippocampus of Ctsd(−/−) mice. The reduced miniature EPSC frequency was observed before the appearance of epilepsy or any morphological sign of synaptic degeneration. Taken together, the data indicate that CTSD is required for normal synaptic function, and that a failure in synaptic trafficking or recycling may be an early and important pathological mechanism in Ctsd(−/−) mice; these presynaptic abnormalities may initiate synaptic degeneration in advance of subsequent neuronal loss

Survival Motor Neuron (SMN) protein is required for normal mouse liver development

We would like to thank Lucas Fraga who helped with primer design and Alison Thomson for tissue collection. We would also like to acknowledge the Microscopy and Histology Core Facility at the University of Aberdeen for the use of their facilities. SHP is funded by Anatomical Society, Euan MacDonald Centre for Motor Neurone Disease Research and an Elphinstone Scholarship for ES from the University of Aberdeen. THG is funded by SMA Trust (UK SMA Research Consortium award), Muscular Dystrophy UK, and Anatomical Society (PhD Studentship). FM is funded by Medical Research Council, SMA-Europe and the National Institute for Health Research Biomedical Research Centre and Great Ormond Street Hospital Children’s Charity. HZ is funded by SMA-Europe and the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. Corrigendum: Survival Motor Neuron (SMN) protein is required for normal mouse liver development Published online: 10 November 2016 DOI: 10.1038/srep35898Peer reviewedPublisher PDFOthe

Survival of motor neurone protein is required for normal postnatal development of the spleen

Funding S.H.P. received an Anatomical Society PhD Studentship award for A.K.T. T.H.G. received an Anatomical Society PhD Studentship award for R.A.P. and funding from the SMA Trust (UK SMA Research Consortium), Euan MacDonald Centre for Motor Neurone Disease Research, and Muscular Dystrophy UK. K.J.S received funding for pathologic studies in human subjects from NICHD grant R01-HD054599.Peer reviewedPostprin

Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo

Lamin A/C dysregulation contributes to cardiac pathology in a mouse model of severe spinal muscular atrophy

Cardiac pathology is emerging as a prominent systemic feature of spinal muscular atrophy (SMA), but little is known about the underlying molecular pathways. Using quantitative proteomics analysis, we demonstrate widespread molecular defects in heart tissue from the Taiwanese mouse model of severe SMA. We identify increased levels of lamin A/C as a robust molecular phenotype in the heart of SMA mice and show that lamin A/C dysregulation is also apparent in SMA patient fibroblast cells and other tissues from SMA mice. Lamin A/C expression was regulated in vitro by knockdown of the E1 ubiquitination factor ubiquitin-like modifier activating enzyme 1, a key downstream mediator of SMN-dependent disease pathways, converging on β-catenin signaling. Increased levels of lamin A are known to increase the rigidity of nuclei, inevitably disrupting contractile activity in cardiomyocytes. The increased lamin A/C levels in the hearts of SMA mice therefore provide a likely mechanism explaining morphological and functional cardiac defects, leading to blood pooling. Therapeutic strategies directed at lamin A/C may therefore offer a new approach to target cardiac pathology in SMA

Morphological characteristics of motor neurons do not determine their relative susceptibility to degeneration in a mouse model of severe spinal muscular atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice - including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA