Forward Genetic Screen in Caenorhabditis elegans Suggests F57A10.2 and acp-4 As Suppressors of C9ORF72 Related Phenotypes

Abstract

An abnormally expanded GGGGCC repeat in C9ORF72 is the most frequent causal mutation associated with amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration (FTLD). Both gain-of-function (gf) and loss-of-function (lf) mechanisms have been involved in C9ORF72 related ALS/FTLD. The gf mechanism of C9ORF72 has been studied in various animal models but not in C. elegans. In the present study, we described mutant C9ORF72 modeling in C. elegans and report the finding of two suppressor genes. We made transgenes containing 9 or 29 repeats of GGGGCC in C9ORF72, driven by either the hsp-16 promoters or the unc-119 promoter. Transgenic worms were made to carry such transgenes. Phenotypic analysis of those animals revealed that P_hsp-16::(G4C2)₂₉::GFP transgenic animals (EAB 135) displayed severe paralysis by the second day of adulthood, followed by lethality, which phenotypes were less severe in P_hsp-16::(G4C2)₉::GFP transgenic animals (EAB242), and absent in control strains expressing empty vectors. Suppressor genes of this locomotor phenotype were pursued by introducing mutations with ethyl methanesulfonate in EAB135, screening mutant strains that moved faster than EAB135 by a food-ring assay, identifying mutations by whole-genome sequencing and testing the underlying mechanism of the suppressor genes either by employing RNA interference studies or C. elegans genetics. Three mutant strains, EAB164, EAB165 and EAB167, were identified. Eight suppressor genes carrying nonsense/canonical splicing site mutations were confirmed, among which a nonsense mutation of F57A10.2/VAMP was found in all three mutant strains, and a nonsense mutation of acp-4/ACP2 was only found in EAB164. Knock down/out of those two genes in EAB135 animals by feeding RNAi/introducing a known acp-4 null allele phenocopied the suppression of the C9ORF72 variant related movement defect in the mutant strains. Translational conformation in a mammalian system is required, but our worm data suggest that altering acp-4/ACP2 encoding lysosomal acid phosphatase may provide a potential therapeutic method of reducing acp-4/ACP2 levels, as opposed or complementary to directly reducing C9ORF72, to relieve C9ORF72-ALS phenotypes. It also suggests that the C9ORF72-ALS/FTLD may share a pathophysiologic mechanism with vesicle-associated membrane protein-associated protein B, a homolog of F57A10.2/VAMP, which is a proven ALS8 gene.

Keywords: ACP2; ALS-FTLD; C9ORF72; Caenorhabditis elegans; VAPB; amyotrophic lateral sclerosis.

Figure 1

A forward genetic screen identified F57A10.2/VAMP and acp-4/ACP2 as suppressors of C9ORF72 pathology in C. elegans. Generally (G4C2)₂₉ or P_hsp animals produced more severe deficit phenotypes than (G4C2)₉ or P_unc119, except for the brood size assay. EAB164 animals carrying both mutations of F57A10.2/VAMP and acp-4/ACP2 had the most suppression of all tested phenotypes. Student t-tests were performed to generate P-values. (A) Transgenes and transgenic C. elegans lines. (B) Animals expressing C9ORF72 disease-related fragments had delayed developmental phenotypes and suppressors from the screen partially relieved the defect. N2 (wide type), EAB141, EAB144, EAB137, EAB136, EAB242, and EAB135, and suppressors, EAB164, EAB165, and EAB167 were staged for 4 days from about 50 eggs/strain at room temperature. At least three experiments were performed in each case, and a typical experimental result is shown. For the P_hsp animals, EAB135 had the highest lethality on Day 4, compared to strain EAB 242, and both had higher lethality than EAB141 and N2 animals. The lethality was partially relieved in EAB164, EAB165, and EAB167. A similar trend was seen in P_unc119animals. (C) Transgenic animals displayed reduced brood sizes. EAB135, EAB136, and EAB137 animals had similar degrees of reduced brood sizes compared to N2, EAB141, and EAB144 animals. For example, EAB135 and EAB136 strains had 139 and 123 brood sizes, compared to 272 and 279 in EAB141 and EAB144 strains, respectively. At least 15 animals per strain were scored in a single experiment, and three independent experiments were performed. (D) Transgenic animals showed age-dependent locomotion defects in swimming assays. Thrashes/20 s were recorded. Statistically significant differences were found between and among strains, as below, Day 1: EAB135 vs. EAB242 (46 vs. 65, P < 0.05); Day 10: both EAB135 and EAB242 vs. EAB141 (5 and 31 vs. 62, P < 0.05 and < 0.001, respectively). Three independent experiments were performed in each case. In each experiment, about 30 animals per strain were scored. (E) Transgenic strain EAB135 had the most severely impaired life span, followed by EAB136 and EAB242 animals. EAB141, EAB144, and EAB137 had a life span similar to N2 animals. Three experiments were independently performed in each case. Results of a typical experiment are shown, using 35 animals per strain, in which EAB135 animals had a reduced life span of 17 days, compared to about 26 days for the EAB141 strain.

Figure 2

The (G4C2)₂₉ transgene is toxic. Percent lethality on day 10 of adulthood at three different temperatures are shown. The wild-type N2 (WT) strain displayed little or no lethality at any temperature tested. Strain EAB135, carrying the (G4C2)₂₉ transgene, displayed much greater lethality at 20°C or 25°C than strain EAB242, carrying the (G4C2)₉ transgene (both P < 0.01).

Figure 3

The nuclearpore transportation (NPT) pathway might be impaired in P_hsp−16::(G4C2)_9/29::GFP transgenic animals. (A) Heat shock-driven expression of P_hsp−16::GFP transgenes. Animals were cultured at 20°C and then exposed to 31°C heat shock for 1 h (right panels) or to no heat shock (left panels). GFP was significantly expressed after heat shock in EAB141 animals, but was not much enhanced in EAB242 and EAB135 animals. Transgenic constructs were co-injected with a Pmyo-3::DsRed2 marker. Scale bars are 100 μm. (B) At the L4 stage, N2 and EAB242 animals were treated with NPP-10/NUP98 RNAi and RAN-2 RNAi for 72 h. NPP-10 RNAi reduced thrashes/20 s from 36.3 to 10.4 in N2s and from 35.1 to 19.8 in EAB242s (%Changes: 71.3% vs. 43.6%). RAN-2 RNAi reduced the thrashes to 9.7 in N2s and 17.1 in EAB242s (%Changes: 73.3% vs. 51.3%). L4440 control RNAi did not affect thrashes (36.3 and 35.1 of N2 and EAB242, respectively).

Figure 4

Suppressor genes F57A10.2 and acp-4 may exert their effects on C9ORF72 associated pathology via a loss-of-function mechanism. (A) EAB164, EAB165, and EAB167 were identified as suppressors of the slow movement phenotype of EAB135. All three suppressors were associated with improved swimming activity in thrashing assays using Day 1, Day 3, and Day 5 adults, compared to EAB135 animals. Strain EAB164 was the most significantly improved suppressor strain, showing almost similar movement ability to N2 animals. (B) Downregulation of suppressor gene F57A10.2 in EAB135 animals, by feeding RNAi, produced improved movement in swimming assays, with swimming resembling activity in strains carrying the potential relative null alleles. Total numbers of 50–98 animals were used in each condition. Three experiments were performed in each case. ^*P < 0.05, ^**P < 0.0002, ^***P < 0.00001. (C) We constructed EAB158 carrying acp-4 (lf,gk833833) on EAB135 background. EAB158 animals moved significantly better than both EAB135 and EAB157 (gk833833). Total numbers of 75–98 animals were used in each condition. Three experiments were performed at room temperature in each case. ^**P < 0.0002.