A single copy transgenic mutant FUS strain reproduces age-dependent ALS phenotypes in C. elegans

Abstract

Mutations in the human DNA/RNA binding protein FUS are associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration, including some aggressive and juvenile onset forms. Cytoplasmic inclusions of human FUS proteins are observed in various neurodegenerative disorders, such as Huntington's disease or spinocerebellar ataxia, suggesting that FUS proteinopathy may be a key player in neurodegeneration. To better understand the pathogenic mechanisms of FUS, we created single copy transgenic Caenorhabditis elegans strains expressing full-length, untagged human FUS in the worm's GABAergic neurons. These transgenic worms expressing human mutant FUS (mFUS) display the same ALS-associated phenotypes than our previous multiple copy transgenic model, including adult-onset age-dependent loss of motility, progressive paralysis and GABAergic neurodegeneration. These phenotypes are distinct from the transgenic worms expressing human wild-type FUS (wtFUS). We introduce here our C. elegans single copy transgenic for human mutant FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.

Figure 1. Expression of single copy transgenic human mutant FUS reproduces age-dependent ALS phenotypes in C. elegans

(A) Validation ofhuman FUStransgene expression by qPCR (taqman). B) Adult worms were scored daily on solid media for paralysis phenotype. Transgenic mFUS worms show increased progressive paralysis over 12 days compared to transgenic wtFUS worms (p< 0.0001). (C) Expression of mFUS does not affect lifespan compared to wtFUS expression. (D) Synaptic cholinergic transmission has been evaluated by exposing day 1 adult worms to the cholinesterase inhibitor aldicarb. Worms were scored over 4 hours for aldicarb induced paralysis. Day 1 adult mFUS worms were hypersensitive to aldicarb treatment compared to either wtFUS or N2 (p< 0.0001). (E) Motility was further assessed in liquid M9 media by using the PhylumTech WMicrotrackerOne instrument. Adult day 1 mFUS worms have decreased motility capacity in liquid media over 10 hours compared to wtFUS and N2 (p< 0.0001). (F) Representative photo of an adult day 9 old unc-47p::mCherry;FUS worm with a degeneration event in the ventral neuronal processes of the GABAergic motor neurons (Highlighted in the magnified frame). (G) Quantification of neurodegeneration in transgenic worms at days 1, 5 and 9 of adulthood. At day 5 and 9 of adulthood mFUS worms show significantly higher neurodegeneration than wtFUS worms (p< 0.0001). (H) Representative photo of an adult day 9 old unc-47p::mCherry;mtFUS worm with a dorsal degeneration event in the axon of the GABAergic motor ­neurons (Highlighted in the magnified frame). (I) Quantification of dorsal axonal degeneration events in adult day 9 worms. Dorsal axonal degeneration can be observed more frequently in mFUS worms compared to wtFUS or unc-47p::mCherry control worms (p< 0.0001). (J) Representation of GABAergic neuronal health in the transgenic FUS populations. mFUS worms have a higher percentage of different degenerative events in the day 9 of adulthood (Ventral, dorsal or both ventro-dorsal degeneration).