Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans

Abstract

An expansion of the hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the Caenorhabditis elegans C9ORF72 orthologue, F18A1.6, also called alfa-1. alfa-1 mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. alfa-1 mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of alfa-1 were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of alfa-1/C9ORF72 expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.

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-43 ^A315T; alfa-1(ok3062) worms had a higher rate of paralysis that either TDP-43 ^A315T or alfa-1(ok3062) worms alone (P<0.005). (B) FUS ^S57∆ worms, alfa-1(ok3062) worms, and FUS ^S57∆; alfa-1(ok3062) worms showed similar rates of paralysis.