Fig 1.
Intermuscular variability in the levels of neuromuscular junction pathology in the Smn2B/- mouse model of SMA.
A) Schematic diagram showing location of muscles which were innervated by either vulnerable or resistant motor neurons in a mouse model of SMA. Vulnerable muscles, as defined by increased neuromuscular junction (NMJ) pathology, include: external and internal oblique; transversus abdominis; and rectus abdominis. Resistant muscles, as defined by a low level of NMJ pathology, include: levator auris longus; auricularis superior; and adductor auris longus. B) Confocal micrographs showing NMJs with the pre-synaptic terminal labeled with antibodies against neurofilament (NF; Green) and synaptic vesicle protein 2 (green) and the muscle endplate labeled with alpha-bungarotoxin (red) from rectus abdominis and auricularis superior muscles. Note that in the wild-type abdominal and Smn2B/- cranial muscle, all endplates appear fully innervated where each endplate is covered by the pre-synaptic terminal labeled with SV2 and NF. In the rectus abdominis from the Smn2B/-, mouse there is evidence of significant NMJ pathology, as evidenced by endplates lacking a pre-synaptic terminal (white arrow heads). Scale bar = 40μm.
Table 1.
Top 20 transcriptional changes identified between vulnerable (abdominal) and resistant (cranial) motor neurons from P10 wild-type mice.
Table 2.
Top 5 functional clustering of transcriptional changes identified between vulnerable and resistant motor neurons.
Table 3.
Summary of independent screens on differentially vulnerable motor neurons.
Table 4.
Transcriptional changes which are common across all 4 screens on differentially vulnerable motor neurons.
Table 5.
Functional clustering of transcriptional changes which are common in 2 or more of the screens on differentially vulnerable motor neurons.
Fig 2.
Comparison of 4 independent screens on differentially vulnerable motor neurons reveals a large number of common transcriptional changes.
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 [16] and in the microarray study on differentially vulnerable motor neuron performed by Brockington et al., 2013 [17] (green), Kaplan et al., 2014 [19] (blue) and Hedlund et al., 2010 [18](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.
Table 6.
Mouse genes and their Drosophila homologue which were tested in the DVAPP-P58S Drosophila screen.
Fig 3.
In vivo validation of transcripts in a Drosophila model of ALS8.
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 Drosophila 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 [16–19]. 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.
Fig 4.
Overexpression of SNCA ameliorate phenotype and neuromuscular junction pathology in the Smn2B/- mouse model of SMA.
A, B) Kaplan Maier plot (A) and weight curve (B) showing profile of control (Smn2B/+; black) and untreated Smn2B/- (red) compared to mice treated with a low dose (1e11; blue) or high dose (3e11; green) of AAV9 expressing SNCA. Note that while the low dose only increases weight gain (Student’s t-test p < 0.0001, the high dose of AAV9-SNCA significantly increased weight (Student’s t-test p<0.0001) and lifespan in the Smn2B/- mouse model (Mantel-Cox Survival Curve Comparison Test p = 00027). C) Confocal micrographs showing NMJs with the pre-synaptic terminal labeled with antibodies against neurofilament (NF; red) and synaptic vesicle protein 2 (red) and the muscle endplate labeled with alpha-bungarotoxin (green) from the transversus abdominis muscle from P18 mice. Note the presence of fully (arrowhead) and partially (arrow) denervated endplates in the untreated Smn2B/- mouse which were less commonly observed in the Smn2B/- mouse treated with high dose AAV9-SNCA. Scale bar = 20μm. D) Bar chart ± SEM showing the increase in the percentage of fully occupied endplates in untreated Smn2B/- mice (black bars) compared to Smn2B/- treated with high dose AAV9-SNCA. *** P <0.001 by Mann-Whitney U test where n = 4/8 mice/muscles per group.