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, 3 (6), e2334

A Drosophila Model of ALS: Human ALS-associated Mutation in VAP33A Suggests a Dominant Negative Mechanism

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A Drosophila Model of ALS: Human ALS-associated Mutation in VAP33A Suggests a Dominant Negative Mechanism

Anuradha Ratnaparkhi et al. PLoS One.

Abstract

ALS8 is caused by a dominant mutation in an evolutionarily conserved protein, VAPB (vesicle-associated membrane protein (VAMP)-associated membrane protein B)/ALS8). We have established a fly model of ALS8 using the corresponding mutation in Drosophila VAPB (dVAP33A) and examined the effects of this mutation on VAP function using genetic and morphological analyses. By simultaneously assessing the effects of VAP(wt) and VAP(P58S) on synaptic morphology and structure, we demonstrate that the phenotypes produced by neuronal expression of VAP(P58S) resemble VAP loss of function mutants and are opposite those of VAP overexpression, suggesting that VAP(P58S) may function as a dominant negative. This is brought about by aggregation of VAP(P58S) and recruitment of wild type VAP into these aggregates. Importantly, we also demonstrate that the ALS8 mutation in dVAP33A interferes with BMP signaling pathways at the neuromuscular junction, identifying a new mechanism underlying pathogenesis of ALS8. Furthermore, we show that mutant dVAP33A can serve as a powerful tool to identify genetic modifiers of VAPB. This new fly model of ALS, with its robust pathological phenotypes, should for the first time allow the power of unbiased screens in Drosophila to be applied to study of motor neuron diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. VAPwt and VAPP58S (VAPmut) have differential effects on synaptic morphology at the neuromuscular junction.
Shown are representative synapses on muscle 4 of 3rd instar larvae stained with anti-HRP, which labels the presynaptic membrane. The effect of neuronal overexpression of VAPwt and VAPP58S on bouton morphology was assessed using the pan-neural elav-GAL4 driver (A–C) and the more restricted OK6-GAL4 (D–F). (H–J) Effect of neuronal expression of wild type and mutant VAP on bouton size in VAPΔ166 animals. (A and D) Control synapses on muscle 4 (elav-GAL4/+ and OK6-GAL4/+ respectively). Neuronal overexpression of VAPwt results in smaller boutons (B and E), while expression of VAPP58S leads to an increase in bouton size (C and F). (G). Graphical analysis of bouton size effects. Here and throughout, values shown are mean±SEM. For both drivers, the average size of boutons in animals expressing VAPwt and VAPP58S was found to be significantly different not only with respect to one other but also controls (*, p<0.001, one way ANOVA with Sidak-Holm multiple comparison test). The bouton sizes measured was as follows (µm): elav-GAL4/+ = 4.03±0.14 (n = 16); elav-GAL4/VAPwt = 2.79±0.17 (n = 16); elav-GAL4/ VAPP58S = 4.45±0.08 (n = 24); OK6-GAL4/+ = 4.24±0.14 (n = 20); OK6-GAL4/VAPwt = 2.78±0.17 (n = 11); OK6-GAL4/ VAPP58S = 4.72±0.11 (n = 24). (H–J) A representative synapse on muscle 4 is shown in each panel. (H) In VAPΔ166 mutants, enlarged boutons are observed. (I) Neuronal expression of VAPwt using elav-GAL4 rescues this phenotype by reducing bouton size. (J) Expression of VAPP58S had no effect on bouton size. (K) Histogram comparing bouton size for the genotypes in panels H–K. Mean bouton diameter (µm) for VAPΔ166;UAS-VAPwt/elav-GAL4 (4.89±0.13 (n = 27)) was significantly different from VAPΔ166 (6.18±0.15 (n = 24); p<0.001, one way ANOVA with Sidak-Holm comparison). However, bouton size in VAPΔ166;UAS-VAPP58S/elav-GAL4 (5.9±0.24 (n = 30) did not differ significantly from VAPΔ166 . n.s., not significant. Scale bar, 10 µm.
Figure 2
Figure 2. VAPP58S appears to function as a dominant negative by inhibiting the activity of wild type VAP.
(A) Histogram comparing the rescue from lethality of VAPΔ20 (null mutant) and VAPΔ166 (partial deletion), using neuronal expression of VAPwt, VAPP58S, or a combination of both. The ordinate represents the percentage of mutant hemizygous males obtained (i.e., VAP mutant hemizygous males/VAP mutant hemizygous males+balancer chromosome males). Each bar represents the mean obtained from at least 2–3 independent experiments. Both VAPP58S and VAPwt are able to rescue the larval lethality observed in VAPΔ20 and VAPΔ166 mutants. However, the percentage of mutant males obtained with VAPwt is higher (72.9±7.3 for VAPΔ20 and 61.5±1.2 for VAPΔ166) as compared with VAPP58S (45.0±3.1 for VAPΔ20 and 46.6±0.06 for VAPΔ166). For each mutant, the value for wild type rescue differed significantly from mutant (p<0.05 for. Δ20 and 0.01 for Δ166; ANOVA with Sidak-Holm test). Rescue of lethality is also obtained by co-expressing VAPP58S and VAPwt. In this case, the percentage of mutant males obtained was similar to that observed with VAPP58S alone (45.7±3.3 for VAPΔ20 and 40.3±2.7 for VAPΔ166). However, for each mutant, rescue using the recombined VAPP58S, VAPwt chromosome differed significantly from that obtained using VAPwt alone (p<0.05 for Δ20 and 0.01 for Δ166, ANOVA with Sidak-Holm test). n.s., not significant. (B–E) VAPP58S suppresses the thoracic bristle phenotype produced by expression of VAPwt using Sca-GAL4. (B) Shown in this panel is the thorax of a control (sca-GAL4/+) adult fly with a stereotyped pattern and number of dorsal macrochaetae (arrows). (C) In Sca-GAL4/UAS-VAPwt animals, the dorsal macrochaetae either fail to develop or are dramatically reduced in number. (D) Expression of VAPP58S has no effect on the development or number of bristles. (E) However, co-expression of VAPP58S with VAPwt results in a suppression of the bristle loss phenotype caused due to overexpression of VAPwt.
Figure 3
Figure 3. VAPP58S forms ubiquitinated aggregates and induces aggregation of VAPwt in vivo.
(A–C) Confocal images of 3rd instar larval muscles stained with anti-VAP. (A) A control animal (G14-GAL4/+). VAP expression is observed at the neuromuscular junction and in underlying muscles 6 and 7. (B) G14-GAL4/UAS-VAPwt. Increased VAP immunoreactivity is observed with expression of VAPwt. (C) G14-GAL4/UAS-VAPP58S. Punctate staining of ALS-associated mutant VAP is observed. (D) Staining with anti-VAP (red) and anti-ubiquitin (Ubi; green) of animals expressing VAPP58S in the muscle. Mutant VAP forms aggregates that appeared as puncta through out the muscle (red). Ubiquitin immunoreactive puncta also are observed (green) that largely colocalized with the VAP aggregates (merged image). (E–G) Staining with anti-myc (green) and anti-HA (red) of animals expressing myc-VAPwt and HA-VAPP58S in the muscle. (E) Similar to untagged VAPwt and VAPP58S transgenes, expression of myc-VAPwt appears diffuse and cytoplasmic. (F) HA-VAPP58S forms aggregates that appear punctate throughout the muscle. (G) Muscle field of a 3rd instar larva expressing both myc-VAPwt and HA-VAPP58S. Expression of myc-VAPwt (green) appears punctate and colocalizes with HA-VAPP58S aggregates (merged image). Scale bar, 20 µm.
Figure 4
Figure 4. VAPP58S disrupts microtubule organization.
Representative synapses on muscle 6–7 of elav-GAL4/+ (A), VAPΔ166 (B), elav-GAL4/ UAS-VAPwt (C) and elav-GAL4/UAS-VAPP58S animals stained with anti-HRP to recognize the presynaptic membrane (red) and anti-Futsch (green). Anti-Futsch labels stable presynaptic microtubules. (A) In control animals, anti-Futsch staining appears thread-like (arrow). At distal boutons, the staining appears more punctate or diffuse (arrowhead). (B) In VAPΔ166 animals, Futsch expression fills up the cytoplasmic region inside boutons (asterisks). (C) In elav-GAL4/UAS-VAPwt animals, the anti-Futsch staining appears similar to control animals, although some boutons show disorganized microtubule morphology (asterisk). (D) In elav-GAL4/UAS-VAPP58S, more boutons have a disorganized microtubule morphology (asterisk). (E) Quantitation of microtubule morphology assessed using anti-Futsch. The percentage of boutons of the muscle 6-7 synapse exhibiting disorganized microtubule phenotypes was calculated. Values: Controls: 34.0±1.5% (n = 26 synapses). elav-GAL4/ UAS-VAPwt: 37.0±2.6 % (n = 25 synapses). VAPΔ166 (63.4±5.4; n = 15 synapses). elav-GAL4/UAS-VAPP58S (53.3±2.1 %; n = 40 synapses). The black brackets above the histogram indicate all significantly different relationships (*, p<0.001, ANOVA with Kruskal-Wallis test). The red brackets indicate non significant relationships. There was no significant difference in the percentage of boutons with abnormal microtubule morphology between VAPΔ166 and elav-GAL4/UAS-VAPP58S, nor was there a significant difference between elav-GAL4/UAS-VAPwt and the control. Scale bar, 20 µm.
Figure 5
Figure 5. Neuronal overexpression of VAPwt but not VAPP58S leads a reduction in active zones.
(A–C) Confocal images of synapses at muscle 4 stained with anti-HRP (red) and nc82, which labels the active zone protein bruchpilot (brp, green). Control (A) and elav-GAL4/UAS-VAPP58S (C) animals showed similar numbers of nc82 immunoreactive puncta per synapse. In contrast, such puncta were fewer in elav-GAL4/UAS-VAPwt animals (B). (D) A histogram showing the difference in number of nc82 positive puncta in all 3 genotypes. In elav-GAL4/UAS-VAPwt animals, an average of 142±14 puncta per synapse were observed (n = 18) as compared to 199±8.4 (n = 17) and 199±12 (n = 16) in control and elav-GAL4/UAS-VAPP58S animals, respectively (*, different from other two genotypes, p<0.001, ANOVA with Sidak-Holm test). n.s., not significant. Scale bar, 10 µm
Figure 6
Figure 6. Ultrastructural analysis of the neuromuscular junction of animals overexpressing VAPwt and VAPP58S.
(A–D) Electron micrographs depicting cross sections of synaptic boutons in control (A), elav-GAL4/UAS-VAPwt (B), and elav-GAL4/UAS-VAPP58S larvae (C and D). Specialized membrane folds present on the postsynaptic surface called the subsynaptic reticulum (SSR), and active zones (asterisks) that include presynaptic T-bars are indicated. As compared to control boutons (A), boutons from animals overexpressing wild type VAP are smaller and contain fewer neurotransmitter vesicles (B). Occasionally, fusion of adjacent boutons was observed (arrow). (C and D) Boutons from elav-GAL4/VAPP58S animals are shown. In addition to the smaller neurotransmitter containing vesicles, a few large vesicles were observed (C, dark arrow). Electron dense T-body-like structures associated with clusters of vesicles are often observed within the cytoplasm of the bouton (C, white arrow and inset). Abnormal electron dense structures are also found in the SSR close to the post-synaptic membrane (C and D, white arrowhead; inset in D).
Figure 7
Figure 7. VAPP58S impairs BMP signalling at the synapse.
A–C, representative confocal images of the synapse at muscle 6–7 stained with phosphorylated SMAD (pMAD; red) and bruchpilot (nc82, green), a component of the active zone. (A) In control animals (G14-GAL4/+) postsynaptic expression of pMAD coincides with the expression of presynaptic Bruchpilot as shown in the merged image. (B) In G14-GAL4 /UAS-VAPwt synapses, more intense pMAD staining is observed, indicative of enhanced BMP signalling. (C). In G14-GAL4/UAS-VAPP58S animals, pMAD immunoreactive puncta are less intense (arrow) as compared to the controls indicating reduced BMP signaling. Scale bar, 10 µm.
Figure 8
Figure 8. Genetic interaction between VAP and BMP signalling: Expression of dominant negative thickvein suppresses the VAPwt bristle phenotype.
(A–C) Thoraces of control (A), sca-GAL4/VAPwt (B) and sca-GAL4/VAPwt, DNtkv animals (C). (A) In control animals, the bristles are invariant in number and position. Circled region indicates the 10 bristles used for analysis. (B and D) Expression of VAPwt using sca-GAL4 leads to loss of bristles. On average, 2–3 bristles are observed in these animals. (C and D) Expression of dominant negative thickvein (DN-tkv) results in a significant suppression of the bristle loss phenotype caused by overexpression of VAPwt (p<0.001, ANOVA with Sidak-Holm test). This was observed with two independent lines of DN-Tkv referred to as L1 and L2; an average of 6 and 5 bristles respectively, were observed in these animals (D). n.s., not significant.

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