A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis

Senthilkumar Deivasigamani, Hemant Kumar Verma, Ryu Ueda, Anuradha Ratnaparkhi, Girish S Ratnaparkhi, Senthilkumar Deivasigamani, Hemant Kumar Verma, Ryu Ueda, Anuradha Ratnaparkhi, Girish S Ratnaparkhi

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression. The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.

Keywords: ALS; Drosophila RNAi screen; Neurodegeneration; TOR; VAP.

Conflict of interest statement

Competing interests: The authors have declared that no conflict of interest exists.

© 2014. Published by The Company of Biologists Ltd.

Figures

Fig. 1.. Scheme for the enhancer/suppressor screen.
Fig. 1.. Scheme for the enhancer/suppressor screen.
(A) A sensitized genetic background (sca-Gal4,UAS-dVAP) was used for a screen to identify interactors of dVAP. A recombinant stable line, expressing VAP in the sca domain was found to reduce the number of 10 macro chaetae (marked by arrowheads in w1118) to about 5–6 at 25°C. At an increased VAP dosage, at 28°C, the macro chaetae reduced to 0–1. Expressing dsRNA for VAP, in a dVAP over-expression background, led to a reversal of the phenotype with macro chaetae reverting to wild type levels both at 25°C and 28°C. Numbers at the top right hand corner of each picture are average macro chaetae, counted for ten females of the corresponding genotype. (B) Primary screening was done at both 25°C and 28°C. A sca-Gal4, UAS-VAP/Cyo recombinant, stable line was generated and females from that line were crossed to males with different transgenic RNAi inserts. Genes that lead to a further decrease of macro chaetae (from 5–6) at 25°C were deemed enhancers and genes that increased number of macro chaetae (from 0) at 28°C were considered to be suppressors. 2635 genes, encompassing 4600 individual lines were used for the primary screen. 930 genes showed change in the phenotype and were categorized as modifiers. 930 genes identified in the primary screen were used for a thorough, quantitative screening, with controls, at 25°C. Macrochaetae from ten F1 females were counted and compared to the base line 5.5 macro chaetae in the master control (sca-Gal4, UAS-VAP/+). Student's t-test was used to select lines that had significantly greater macro chaetae and these were considered bona-fide suppressors. Lines that did not meet our threshold for significance (p>0.01, Average macro chaetae <7.5) were discarded. F1 females with average macro chaetae <5.5 were compared to the related RNAi control (sca-Gal4/+; UAS-RNAi/+) at 25°C. Again, a Student's t-test was used to select lines above our threshold for significance (p<0.01, average macro chaetae <4). Starting with 2635 genes in the primary screen, the final numbers for enhancers and suppressors after comparison with controls and rigorous statistical analyses was 45 and 58 respectively. These genes were shortlisted for the validation process.
Fig. 2.. An integrated network of dVAP…
Fig. 2.. An integrated network of dVAP genetic interactors.
An extended network of dVAP network was built by integrating VAP interaction data from our screen (103 genes) with known physical interactors of dVAP. The extended network (not shown) includes 406 genes and 953 edges. (A) Analysis indicated that 36 modifiers (displayed as circles), which are a subset of the 103 modifiers discovered, interact physically among themselves. Blue connecting lines (edges) indicate physical interaction. (B) Drosophila homologs of known ALS causing loci, TBPH, Alsin and sod1 are suppressors of dVAP function and are part of the genetic network. The genotype for each cross is Sca-Gal4/+; UAS-VAP/UAS-Gene-RNAi. For each figure, average macro chaetae values from ten females are represented on the top right hand corner. (C) Thirty-five known physical interactors of VAP are a subset of the 2635 genes screened in this study. Of these, eight genes were found to be genetic interactors of VAP. SNAMA, tmod, lethal (1) G0222, epsilon-COP and Pex-19 are suppressors while Droj2, karyopherin beta3, Porin are enhancers.
Fig. 3.. The Drosophila NMJ is used…
Fig. 3.. The Drosophila NMJ is used to screen for interaction of modifiers with VAP(P58S).
Thirteen of the 103 modifiers discovered in our macro chaetae screen were tested in the larval muscle-4 NMJ for interaction with VAP(P58S). For this and subsequent figures, approximately 15 NMJs were dissected, stained (anti-HRP, red), imaged and measured for the average size of boutons (displayed in yellow at the top RHS of each figure). (A) A wild type (C155-Gal4/+) NMJ. The average bouton size is 3.987 (±0.03). Shown here and below is Z-series of a synapse rendered as maximum intensity projection. (B) Expression of UAS-VAP(P58S), using the C155-Gal4 driver increases the size of the boutons. (C–F) Knockdown of CG6048 (C), CG9172 (D), TBPH (E) and Nup75 (F) in a C155/UAS-VAP(P58S) background reverses the effect of the VAP(P58S) over-expression and rescues bouton size to wild-type levels. (G) Quantitation of bouton size (in micrometer) for the RNAi knockdown of each gene tested in a VAP(P58S) background (top panel) and in a wild type background (bottom panel). For this and subsequent NMJ figures, error bars represent standard errors of the mean (SEM). Scale bar: 5 µm. * indicates a p-value<0.01 (but >0.001), while ** indicates a p-value of <0.001.
Fig. 4.. dTOR, the fly homolog of…
Fig. 4.. dTOR, the fly homolog of mTOR modifies the VAP(P58S) over-expression phenotype at the NMJ.
(A) VAP(P58S) over-expression using C155-Gal4 leads to larger boutons at the NMJ. (B) Knockdown of Tor by RNAi in VAP(P58S) background restores the bouton size to wild type levels. (C) Knocking down Tor using RNAi did not have any effect on bouton size. (D) Over-expression of TOR-TED alone using C155-Gal4 resulted in increased bouton size. (E) Reduction of TOR activity by expressing a dominant negative form of TOR, TOR-TED reversed the increase in bouton size caused by UAS-VAP(P58S). Scale bar: 5 µm. (F) Quantitation of rescue in bouton size in VAP(P58S) background in response to reduced TOR.
Fig. 5.. Increased TOR signaling activates its…
Fig. 5.. Increased TOR signaling activates its downstream component S6K.
(A) Over-expression of constitutively active S6K using C155-Gal4 in a wild type background did not affect the bouton size. (B) Over-expression of constitutively active S6K using C155-Gal4 in a VAP(P58S) over-expression background did not reduce the bouton size. (C) Reducing S6K activity using dominant negative form did not have significant effect on bouton size. (D) Reducing S6K activity using dominant negative form rescued the increased bouton size in a VAP(P58S) background. Average size of boutons from about 15 NMJs is displayed in yellow at the top right of each figure. Scale bar: 5 µm. (E) Quantitation of effect of S6K on the bouton size in presence and absence of VAP(P58S).
Fig. 6.. Increased TSC activity reverses the…
Fig. 6.. Increased TSC activity reverses the VAP(P58S) bouton phenotype.
(A–D) Over-expression and knocking down of Rheb had no effect on bouton size in the VAP(P58S) background. (E,F) Increasing Tsc activity by co-expressing Tsc1,2 rescued the bouton size in VAP(P58S) background (F) while Tsc1,2 over-expression by itself did not have any effect on bouton size (E). (G,H) Knocking down Tsc1 in VAP(P58S) background did not rescue the bouton size. Scale bar: 5 µm. Average size of boutons from about 15 NMJs is displayed in yellow at the top right of each figure. (I) Quantitation of effect of perturbations in Tsc and Rheb levels on bouton size.
Fig. 7.. Decreased TSC activity and increased…
Fig. 7.. Decreased TSC activity and increased S6K activity reverses the VAP(wt) small bouton phenotype.
(A) VAP wild type over-expression using C155-Gal4 leads to reduced bouton size. (B–D) Reducing TOR pathway activity by over-expressing Tsc1,2 (B) or TOR dominant negative (D) does not affect the bouton size in the VAP wild type over-expression background. However, increasing TOR pathway activity using Tsc1 knock down increased the bouton size to wild types levels (C). (E–H) TOR downstream components can alter the bouton size in VAP wild type over-expression. Over-expression of a constitutively active form of S6K (E) and constitutively active Thor (G) rescues the bouton size, while the dominant negative form does not (F). Over-expression of Atg1 along VAP wild type reduced the bouton size further (H). Scale bar: 5 µm. Average size of boutons from about 15 NMJs is displayed in yellow at the top right of each figure. (I) Quantitation of effect of TOR pathway and its downstream components in VAP wild type over-expression mediated bouton size. Error bars represent SEM. * indicates a p-value<0.01 (but >0.001), while ** indicates a p-value of <0.001.
Fig. 8.. Rapamycin feeding mitigates VAP(P58S) bouton…
Fig. 8.. Rapamycin feeding mitigates VAP(P58S) bouton phenotype.
(A,B) C155-Gal4 larvae fed with DMSO (A) and 200 nM Rapamycin (B). (C,D) C155>VAP(P58S) larvae fed with DMSO (C) and 200 nM Rapamycin (D). Rapamycin, a chemical inhibitor of TOR effectively restores bouton size to control levels in VAP(P58S) animals. (E) Quantitation of effects of Rapamycin on VAP(P58S) expressing animals. (F) A model for the effect of VAP(P58S) expression on TOR. Genetic interactors (grey filled circles or rounded squares) of VAP(P58S) suggests an up-regulation of Tor (red arrows) in neurons while VAP(wt) expression appears to have opposite effects (blue arrow). Rapamycin feeding of larvae reverses VAP(P58S) phenotypes, pointing to an increase in TOR signaling in VAP(P58S) expressing neurons.

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