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

Self-oligomerization Regulates Stability of Survival Motor Neuron Protein Isoforms by Sequestering an SCF\u3csup\u3eSlmb\u3c/sup\u3e Degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

A Direct Comparison of IV and ICV Delivery Methods for Gene Replacement Therapy in a Mouse Model of SMARD1

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal recessive disease caused by the loss of the ubiquitously expressed IGHMBP2 gene. SMARD1 causes degeneration of alpha-motor neurons, resulting in distal muscle weakness, diaphragm paralysis, and respiratory malfunction. We have reported that delivery of a low dose of AAV9-IGHMBP2 to the CNS results in a significant rescue of the SMARD1 mouse model (nmd). To examine how a delivery route can impact efficacy, a direct comparison of intravenous (IV) and intracerebroventricular (ICV) delivery of AAV9-IGHMBP2 was performed. Using a low-dose, both IV and ICV delivery routes led to a significant extension in survival and increased body weight. Conversely, only ICV-treated animals demonstrated improvements in the hindlimb muscle, neuromuscular junction, and motor function. The hindlimb phenotype of IV-treated mice resembled the untreated nmd mice. We investigated whether the increased survival of IV-treated nmd mice was the result of a positive impact on the cardiac function. Our results revealed that cardiac function and pathology were similarly improved in IV- and ICV-treated mice. We concluded that while IV delivery of a low dose does not improve the hindlimb phenotype and motor function, partial restoration of cardiac performance is sufficient to significantly extend survival. Keywords: gene therapy, IGHMBP2 delivery, cardiac defects, SMARD

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

Transcriptional changes which are common across all 4 screens on differentially vulnerable motor neurons.

<p>Transcriptional changes which are common across all 4 screens on differentially vulnerable motor neurons.</p

In vivo validation of transcripts in a <i>Drosophila</i> model of ALS8.

<p>A) Images show eyes from wild-type, DVAP-P58S (carrying ALS8 patient mutation), DVAP-P58S Dfd (AL8 patient mutation with decreased expression of Dfd, the Drosophila homologue of Hoxc4) and DVAP-P58S Dfd (AL8 patient mutation with decreased expression of Nrbp1) Drosophila. Note the decrease in eye size observed in DVAP-P58S flies. This phenotype was supressed by decreased expression of dfd, and enhanced by decreased expression of Nrbp1. B) Bar chart (Mean ± SEM) showing the area (mm²) of the eye in <i>Drosophila</i> lines which over or under express specified transcripts in DVAP-P58S flies. *** P<0.001, **P<0.01 by ANOVA with Holm-Sidak’s post hoc test. N = approx. 12 flies per group with each value reflecting average of 2 eyes per fly. C) Table details the transcripts which were identified as modifiers of the eye phenotype in DVAP-P58S flies, denoting the Drosphila official gene symbol, the official gene symbol of the mouse homologue and the directional change observed in vulnerable motor neurons in the independent transcriptional screens [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006680#pgen.1006680.ref016" target="_blank">16</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006680#pgen.1006680.ref019" target="_blank">19</a>]. For those transcripts which were decreased in vulnerable motor neurons, their expression was increased in DVAP-P58S flies, and for those transcripts which were increased in vulnerable motor neurons, their expression was decreased in DVAP-P58S flies.</p

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

Top 20 transcriptional changes identified between vulnerable (abdominal) and resistant (cranial) motor neurons from P10 wild-type mice.

<p>Top 20 transcriptional changes identified between vulnerable (abdominal) and resistant (cranial) motor neurons from P10 wild-type mice.</p