Figure 1.
ALFA-1 is the orthologue of C9ORF72 in C. elegans.
(A) Protein sequence alignment using Clustal W and BoxShade of C9ORF72 isoform 1 and ALFA-1 isoform 1. Overall, these sequences share 26% identify and 59 % similarity.
(B) ALFA-1 has two predicted transcripts and the ok3062 deletion mutation spans exons 3 and 4 for both transcripts.
(B) RT-PCR confirming the complete loss of expression of the alfa-1 transcripts. act-3 was used as a control.
Figure 2.
Age-dependent motility defects and neurodegeneration in alfa-1(ok3062) mutants.
(A) alfa-1(ok3062) mutants showed motility defects leading to paralysis of 60% of the population by day 12 of adulthood compared to 20% for N2 worms (P<0.0001).
(B) alfa-1(ok3062) worms are more sensitive to aldicarb-induced paralysis than N2 worms (P<0.0001).
(C) Percentage of wild type N2 or alfa-1(ok3062) worms displaying a swimming-induced paralysis phenotype after 8 hours in liquid culture (** P<0.001).
(D) Example of gap (indicated by arrow) along a neuronal process in animals expressing the unc-47p::GFP reporter.
(E) Quantification of neurodegeneration at day 9 of adulthood associated with alfa-1(ok3062) in different neuronal populations including cholinergic neurons marked by unc-17p::GFP, dopaminergic neurons visualized with dat-1p::GFP, or GABAergic neurons revealed by unc-47p::GFP. Significant neurodegeneration was observed in the GABAergic neurons of alfa-1(ok3062) mutants (**P<0.001).
Figure 3.
alfa-1(ok3062) mutants are sensitive to osmotic stress.
(A) In thermal stress resistance assays, alfa-1(ok3062) mutants were indistinguishable from wild type N2 worms, and daf-2(e1370) were not statistically different alfa-1(ok3062);daf-2(e1370) mutants.
(B) In oxidative stress resistance assays, alfa-1(ok3062) mutants were indistinguishable from wild type N2 worms, and daf-2(e1370) were not statistically different alfa-1(ok3062);daf-2(e1370) mutants.
(C) alfa-1(ok3062) mutants were more sensitive to osmotic stress than N2 worms (P<0.005), while alfa-1(ok3062);daf-2(e1370) worms are slightly more sensitive when compared to daf-2(e1370) worms alone.
(D) The difference in sensitivity between alfa-1(ok3062);daf-2(e1370) and daf-2(e1370) increases at 500 mM NaCl (P<0.005). At 600 mM NaCl, the effect of NaCl is too drastic to see a difference.
(E) During thermal stress, alfa-1(ok3062) worms are not more sensitive to neurodegeneration than the unc-47p::GFP worms.
(F) When exposed to 400 mM NaCl, alfa-1(ok3062) worms had a higher rate of neurodegeneration than unc-47p::GFP worms (*P<0.05).
Figure 4.
Genetic interactions between alfa-1(ok3062), TDP-43, and FUS.
(A) TDP-43A315T; alfa-1(ok3062) worms had a higher rate of paralysis that either TDP-43A315T or alfa-1(ok3062) worms alone (P<0.005).
(B) FUSS57∆ worms, alfa-1(ok3062) worms, and FUSS57∆; alfa-1(ok3062) worms showed similar rates of paralysis.