[
International Worm Meeting,
2021]
Amyotrophic Lateral Sclerosis (ALS) is characterized by the progressive degeneration of neurons, resulting in chronic muscular atrophy. In 25-40% of familial ALS cases, an inappropriately expanded GGGGCC repeat is found in the first intron of the C9ORF72 gene, resulting in neuron degeneration and eventually death. We generated a new C. elegans model expressing 30 copies of GGGGCC nucleotide repeats at high levels specifically in 8 sensory glutamatergic neurons. Back-filling of the PHA and PHB phasmid sensory neurons with a fluorescent dye revealed that 80% to 90% of 30x(GGGGCC) animals lose neurons or have defective neuron process, but 3x(GGGGCC) animals were relatively unaffected. Given the highly penetrant defect we were able to use this simple assay and perform an unbiased screen to identify genetic suppressors of neurodegeneration. Using EMS, we identified 28 potential suppressor lines in which degeneration of phasmid neurons was partially rescued (between 30 to 70% intact neurons). Of these 28 lines, 23 of them are clearly independent isolate, suggesting they contain unique suppressors mutations. As a secondary screen of the suppressor and to facilitate gene identification, we established a genetic scheme allowing for each line 1/ to test if the EMS hit affected a gene rather than the 30x(GGGGCC) insertion and 2/ Establish independent sub lines carrying the suppressors, named Suppressor lines, and independent lines that derivate from the Suppressor line, named Sibling lines. These Sibling lines have a similar genetic backgrounds i.e. other genes affected by the EMS treatment but will fail to rescue the 30x(GGGGCC) related neuron degeneration, meaning they don't carry the suppressor anymore. These different lines will be sequenced and by using the subtraction method (https://doi.org/10.1534/g3.117.300135) we will establish a list of candidate genes that suppress the neuron degeneration caused by GGGGCC nucleotide expansion. Further tests, using mutants of these candidate genes, will then allow to have crucial insights on genetic modifiers and pathways allowing to alleviate neuron degeneration in ALS.
Mahapatra, Animesh, Walsh, Melissa, Tsai, Jason, Hart, Anne, Lins, Jeremy, Yanagi, Katherine, Stinson, Loraina, Kruskop, Jane, Osborne, Jennifer
[
International Worm Meeting,
2021]
Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset neurodegenerative disorders that results from the selective and progressive degeneration of cortical and spinal motor neurons. Mutations over 20 genes including RNA binding proteins, FUS and TDP43, and a free radical scavenger, superoxide dismutase 1 (SOD1) cause ALS. Although there is a well-established genetic component of ALS, it is still unclear why mutations in genes encoding functionally diverse proteins cause motor neuron degeneration. Identifying modifiers of neurodegeneration in C. elegans ALS models may provide insight into the mechanisms behind the selective and progressive neurodegeneration in ALS. Here, we have undertaken a classical forward genetic screen to identify suppressors of glutamatergic neuron degeneration. After exposure to oxidative stress, SOD-1G85R knock-in mutants have both glutamatergic and cholinergic neuron degeneration (PMC6200258). We mutagenized SOD-1G85R animals and screened for suppressors of glutamatergic neuron degeneration. After whole genome sequencing, we found that an RNA binding protein, hit in three independently isolated lines, suppresses cholinergic and glutamatergic neuron degeneration. Understanding the relationship between SOD1 and RNA binding proteins will provide insight if there are common mechanisms underlying neurodegeneration in ALS and has the potential to facilitate the development of treatments.