Comparison of independent screens on differentially vulnerable motor neurons reveals alpha-synuclein as a common modifier in motor neuron diseases

The term "motor neuron disease" encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an in vivo validation, we demonstrate that the manipulation of a significant number of these transcripts can modify the neurodegenerative phenotype observed in a Drosophila line carrying an ALS causing mutation. Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic modifiers, and demonstrated SNCA is a modifier of pathology in motor neuron disease

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA

Short-duration splice promoting compound enables a tunable mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. SMA results from insufficient survival motor neuron (SMN) protein due to alternative splicing. Antisense oligonucleotides, gene therapy and splicing modifiers recently received FDA approval. Although severe SMA transgenic mouse models have been beneficial for testing therapeutic efficacy, models mimicking milder cases that manifest post-infancy have proven challenging to develop. We established a titratable model of mild and moderate SMA using the splicing compound NVS-SM2. Administration for 30 d prevented development of the SMA phenotype in severe SMA mice, which typically show rapid weakness and succumb by postnatal day 11. Furthermore, administration at day eight resulted in phenotypic recovery. Remarkably, acute dosing limited to the first 3 d of life significantly enhanced survival in two severe SMA mice models, easing the burden on neonates and demonstrating the compound as suitable for evaluation of follow-on therapies without potential drug-drug interactions. This pharmacologically tunable SMA model represents a useful tool to investigate cellular and molecular pathogenesis at different stages of disease

Therapeutic targets for spinal muscular atrophy : Inhibiting the inhibitors

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Spinal muscular atrophy (SMA) is an autosomal recessive disorder that is a leading genetic cause of infantile death. SMA is the most common inherited motor neuron disease and occurs in approximately 1: 6,000 live births. The gene responsible for SMA is called survival motor neuron-1 (SMN1). A human-specific copy gene is present on the same region of chromosome 5q called SMN2. SMN2 is nearly identical to SMN1; however, mutations in SMN2 have no clinical consequence if SMN1 is retained. The reason why SMN2 cannot prevent disease development in the absence of SMN1 is that the majority of SMN2-derived transcripts are alternatively spliced, resulting in a truncated and unstable protein. The presence of SMN2 in all SMA patients is fundamental to the biology of the disease; however, from a translational perspective, targeting SMN2 may prove to be the most important therapeutic opportunity for all patients. The presence of SMN2 opens the door to a number of exciting therapeutic strategies, including anti-sense oligonucleotides (ASOs) that prevent the pathogenic SMN2 splicing event. Our efforts are focused on several repressor regions upstream and downstream of SMN2 exon 7. Importantly, when manipulating these repressor regions, hallmarks of the disease at the cellular level such as neuromuscular junction pathology in various SMA animal models are corrected. Currently, there are no approved SMA-specific compounds, and developing a broad array of therapeutic strategies to address this complex disease is essential. The development and design of highly-potent ASOs provide novel molecular targets for SMA therapeutics that can dramatically improve disease phenotype and extend patients' life span

Mouse genes and their Drosophila homologue which were tested in the DVAPP-P58S Drosophila screen.

<p>Mouse genes and their Drosophila homologue which were tested in the DVAPP-P58S Drosophila screen.</p

Comparison of 4 independent screens on differentially vulnerable motor neurons reveals a large number of common transcriptional changes.

<p>Scatter plot showing the fold change of transcripts which were differentially expressed in differentially vulnerable motor neurons in the RNAseq performed by Murray et al., 2015 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006680#pgen.1006680.ref016" target="_blank">16</a>] and in the microarray study on differentially vulnerable motor neuron performed by Brockington et al., 2013 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006680#pgen.1006680.ref017" target="_blank">17</a>] (green), Kaplan et al., 2014 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006680#pgen.1006680.ref019" target="_blank">19</a>] (blue) and Hedlund et al., 2010 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006680#pgen.1006680.ref018" target="_blank">18</a>](red). Numbers denote number of number of common transcriptional screens within each quadrant of the plot. Note that the majority of changes occur with a common directional change i.e. fall within the bottom left or top right quadrant of the graph.</p