Fig 2: Zfp106 suppresses GGGGCC-mediated neurotoxicity in Drosophila.(a) Schematics and Punnett squares from crosses of UAS-Zfp106; UAS-30R-GFP (left) and UAS-Zfp106; UAS-3R-GFP (right) transgenic flies to OK371-GAL4 flies. The Punnett squares show the number of flies that eclosed from the indicated crosses and show that expression of 30R-GFP resulted in failure of ~50% of flies to eclose (25 actual versus 54.5 predicted); this defect was completely suppressed by co-expression of Zfp106. The Chi-square test (χ2) was used to determine the significance of the differences between the observed and expected frequencies of the expected genotypes. (b) Locomotor activity of 28-day post-eclosion flies of the indicated genotypes assessed by rapid iterative negative geotaxis (RING) assay. Data are presented as the mean height climbed ± SEM. A two-way ANOVA analysis of the locomotor data revealed a significant effect of repeat expression (p<0.0001) and Zfp106 expression (p<0.01) on locomotor activity, with a significant interaction between the two factors (p<0.01); i.e. Zfp106 coexpression significantly suppresses neurotoxicity induced by expression of 30R (p<0.01) by Bonferroni's posthoc Multiple Comparison Test. n = 25 male flies for each genotype; ****p<0.0001, **p<0.01. (c, d, e) Representative NMJs of muscle 6/7 in abdominal segment A3 of third instar larvae of the indicated genotypes co-stained with anti-HRP (neuronal marker, red channel) and anti-Bruchpilot (Brp) (active zone marker, green channel). Staining of the active zone component Brp shows that expression of 30R causes a reduction in Brp-positive active zones [arrowheads in (d) compared to control in c]. Coexpression of Zfp106 suppressed the reduction in Brp-positive active zones caused by 30R [arrowheads in e compared to d]. (f) Quantification of active zones as total voxel occupancy by Brp signal at each terminal shows that Zfp106 significantly suppresses the reduction in active zone caused by expression of 30R. Data were analyzed by one-way ANOVA and Bonferroni's Multiple Comparison Test. ***p<0.001, ****p<0.0001.DOI: http://dx.doi.org/10.7554/eLife.19032.013

Fig 3: Zfp106 interacts with a network of other ALS-associated RNA-binding proteins.(a) Network representation of the Zfp106 interactome generated by Cytoscape. Zfp106 (yellow node) interacts with 39 proteins with top 5% MiST score (blue nodes). Curated protein-protein interactions from the CORUM database are indicated as nodes connected by green lines. (b) Gene Ontology (GO) Biological Process analysis of the Zfp106 interactome ranked by p value. The number of interacting proteins (top 5% by MiST score) in each category is indicated inside each bar. (c) Selected ALS-associated proteins within the top 5% MiST-scoring hits from the Zfp106 interactome are listed with putative functions associated with the pathobiology of ALS indicated.DOI: http://dx.doi.org/10.7554/eLife.19032.011

Fig 4: Zfp106 loss-of-function causes a severe neuromuscular phenotype in mice.(a–h) Evidence of profound neuromuscular pathology in Zfp106-null mice. H&E stained sections of quadriceps muscles (a–d) from 4-week old and 12-week old wild type (a, c) and Zfp106 knockout (b, d) mice are shown. Note the presence of grouped small angular fibers in knockout mice (dashed circle), already clearly evident by 4-weeks of age (b, arrowheads) compared to the rounder, more evenly-shaped fibers in controls (a). By 12-weeks (end stage disease), Zfp106 knockout mice have profound pathology and the presence of atrophic and degenerated muscle fibers with numerous inclusions (d, asterisks). Scale bar, 100 μm. ChAT immunohistochemical staining (e–h) of lumbar spinal cord sections from 8-week old wild type (e, g) and Zfp106 knockout (f, h) mice show a drastic reduction in the number of ChAT+ motor neurons. Arrowheads mark ChAT-expressing motor neurons in the ventral horn. Scale bar, 200 μm in all panels. (i) Quantification of ChAT-positive neurons in the lumbar spinal cord of wild type and Zfp106 knockout mice at 8 weeks of age shows a ~ 47% loss of motor neurons in Zfp106 knockout mice. n = 5 mice for each genotype. Data are expressed as percent of control ± SD and were analyzed by t-test. ***p<0.001. (j) Schematic of the HB9-3×FLAG-2×STREP-Zfp106-IRES-gfp (HB9-Zfp106) transgene. (k, l, m) Transgenic expression of Zfp106 in motor neurons suppresses the wasting phenotype (k), the reduction in grip strength (l) and the loss of lumbar spinal cord motor neurons (m) in Zfp106 knockout mice. Wasting data are expressed as the mean weight ± SEM. Grip strength data are expressed as the mean grip score ± SD; grip score: 1, 1–10 s; 2, 11–25 s; 3, 26–60 s; 4, 61–90 s; 5, > 90 s. ChAT-positive neuron counts are expressed as percent of control ± SD. n = 5 female mice at 8 weeks of age for each genotype. Data were analyzed by one-way ANOVA and Bonferroni's Multiple Comparison Test. *p<0.05; ***p<0.001, ****p<0.0001.DOI: http://dx.doi.org/10.7554/eLife.19032.005