Reactive oxygen species-mediated neurodegeneration is independent of the ryanodine receptor in Caernorhabditis elegans

Abstract

Despite the significant impacts on human health caused by neurodegeneration, our understanding of the degeneration process is incomplete. The nematode Caenorhabditis elegans is emerging as a genetic model organism well suited for identification of conserved cellular mechanisms and molecular pathways of neurodegeneration. Studies in the worm have identified factors that contribute to neurodegeneration, including excitotoxicity and stress due to reactive oxygen species (ROS). Disruption of the gene unc-68, which encodes the ryanodine receptor, abolishes excitotoxic cell death, indicating a role for calcium (Ca²⁺) signaling in neurodegeneration. We tested the requirement for unc-68 in ROS-mediated neurodegeneration using the genetically encoded photosensitizer KillerRed. Upon illumination of KillerRed expressing animals to produce ROS, we observed similar levels of degeneration in wild-type and unc-68 mutant strains. Our results indicate that ROS-mediated cell death is independent of unc-68 and suggest multiple molecular pathways of neurodegeneration.

Figure 1. KillerRed activation causes neurodegeneration

(A) KillerRed expressing animals were illuminated or kept in the dark (Control), then scored for GABA neuron function by determining the percentage of animals that displayed the shrinker phenotype. Data presented is the percentage of shrinkers ± 95% confidence intervals. Illumination resulted in a significant increase in the shrinker frequency. (B) Representative images of the nervous system from illuminated and control animals obtained imaged by fluorescent microscopy. Illumination resulted in beading of ventral and dorsal cords (arrows) as well as rounding of cell bodies (asterisk) that is characteristic of neurodegeneration.

Figure 2. unc-68 mutants are not GABA defective

The indicated types of worms were scored for the shrinker phenotype. Data presented is the frequency of shrinkers ± 95% confidence intervals. At least 30 worms were scored for each experiment. Wild-type and unc-68 mutant worms do not display the shrinker phenotype. By contrast GABA- worms, unc-47(e307) mutants, have a robust highly penetrant shrinker phenotype.

Figure 3. Molecular verification of unc-68(r1161) allele

Parallel single worm PCR reactions were performed to amplify the unc-68 gene using wild-type and unc-68(r1161) animals as template. The expected 8172 bp product was obtained from wild-type (arrowhead) animals. The r1161 allele is an ~7.2 kb deletion of unc-68 and the expected ~950 bp product was obtained upon amplification of unc-68(r1161) animals (arrow). Other bands are likely non-specific amplification products as no-template controls were empty.

Figure 4. unc-68 mutants are not defective in KillerRed-mediated neurodegeneration

Wild-type and unc-68 mutant animals were illuminated (+) or kept in the dark (−), then scored for the shrinker phenotype. Data presented in the frequency of shinker animals ± 95% confidence intervals. At least 100 animals were scored for each condition and unc-68 mutants represent a pool of e540 and r1161 alleles, both of which had similar levels of neurodegeneration.

Figure 5. Multiple pathways of neurodegeneration in C. elegans

ROS, excitotoxicity, and mitochondrial dysfunction can lead to neruodegeneration through molecular pathways that differ in their dependence on Ca²⁺.