Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to aging, as are Alzheimer's, Parkinson's and Huntington diseases (Gitler, Dhillon et al. 2017). ALS is a fatal disease characterized by a progressive and selective loss of upper and lower motor neurons. Patients with ALS usually die from respiratory failure 2 to 5 years after the onset of symptoms (Tao and Wu 2017, Oskarsson, Gendron et al. 2018). This disease appears late in age and usually affects individuals between 40 and 60 years old with an average of 55 years (Taylor, Brown et al. 2016).
About 90% of the cases are sporadic and 10% are familial (Zarei, Carr et al. 2015). More than 200,000 people are currently living worldwide with ALS with an incidence rate between 0.6 and 3.8 per 100,000 person-year according to recent studies (Arthur, Calvo et al. 2016, Longinetti and Fang 2019). Unfortunately, ALS is incurable and there are no effective treatments available for people living with the disease. Indeed, no treatment has made it possible to significantly slow or stop the progression of the disease. Despite many clinical trials, the only drugs approved by the US FDA are Riluzole and Radicava (Edaravone) (Cruz 2018). Both of these drugs are meant to slow the progression of the disease and extend the life expectancy of patients (Abe, Itoyama et al. 2014). The glutamatergic neurotransmission inhibitor, Riluzole, has a modest effect on survival (average extended lifespan of 2–3 months) whereas the antioxidant drug, Edaravone, appears to be effective in slowing the progression of ALS in the early stages of the disease (Jaiswal 2019). However, recent population studies have shown that Riluzole may help prolong survival from 6 to 19 months compared to the previous clinical trials (Andrews, Jackson et al. 2020).
In the past few decades, research has made it possible to associate several genetic abnormalities to ALS but the most common are the following. Mutations in the C9orf72 gene represent the largest proportion (30–40%) of patients with familial ALS (Balendra and Isaacs 2018, Shi, Lin et al. 2018). Mutations in the SOD1 (superoxide dismutase 1) gene represent around 15–20% of the cases and mutations in the FUS (Fused in Sarcoma) oncogene account for approximately 5% of familial ALS cases (Blair, Williams et al. 2010, Moller, Bauer et al. 2017, Pansarasa, Bordoni et al. 2018). Finally, about 5% of the cases have mutations in the TAR DNA Binding Protein-43 (TARDBP) gene, which codes for the TDP-43 protein (Valdmanis, Daoud et al. 2009, Mejzini, Flynn et al. 2019).
TDP-43 (TAR DNA binding protein-43) is a RNA/DNA binding protein with a molecular weight of 43 kDa. This protein is highly conserved, expressed ubiquitously and is part of the large hnRNP family (heterogeneous nuclear ribonucleoprotein) (Van Deerlin, Leverenz et al. 2008). This family includes proteins which can bind to RNA in a sequence-specific manner with the presence of RRMs (RNA recognition motifs). TDP-43 contains 414 amino acids (aa) and its coding gene, TARDBP, is located on chromosome 1. This protein includes an N-terminal region (1-102 aa) with a nuclear localization signal (NLS, 82–98 aa), two RRMs: RRM1 (104–176 aa) and RRM2 (192–262 aa), a nuclear export signal (NES, 239–250 aa), and a C-terminal region (274–414 aa) which contains a domain rich in glutamine and asparagine (345–366 aa), and a glycine-rich region (366–414 aa) (Fig. 1A). The C-terminal region seems to be particularly involved in the pathology of TDP-43 in ALS. As a prion-like domain, this region is disorganized and predisposed to pathological aggregation (Dhakal, Wyant et al. 2020). Also, the majority of mutations and phosphorylation sites associated with ALS are located in this C-terminal region (Prasad, Bharathi et al. 2019).
TDP-43 is mainly located in the nucleus where it performs its main functions, but it can also shuttle to the cytoplasm to play other roles. This protein has many functions including being involved in transcription, splicing, translation as well as stabilization, maturation and transport of mRNA (Prasad, Bharathi et al. 2019). TDP-43 binds to approximately 6,000 mRNA transcripts, representing almost 30% of the entire transcriptome (Polymenidou, Lagier-Tourenne et al. 2012).
During stress, TDP-43 assembles into stress granules (SG) in the cytoplasm, and is involved in the assembly and maintenance of the integrity of these structures (Hergesheimer, Chami et al. 2019). In pathological conditions like ALS, the cytoplasmic concentration of TDP-43 increases, and the protein is hyperphosphorylated, ubiquitinated and cleaved which leads to the formation of inclusion bodies (Scotter, Chen et al. 2015). These inclusions can be particularly damaging for cells such as motor neurons (Prasad, Bharathi et al. 2019).
Years of research have made it possible to learn more about ALS and different factors implicated. However, despite the advancement of knowledge on this proteinopathy, ALS remains complex and multifactorial, making the causes of this disease still largely unknown. To be able to study the molecular mechanisms, the signaling pathways involved in ALS and possibly develop effective treatments, the development of research models is essential.
To do so, we turned to the model organism Caernorhabditis elegans. This nematode has several advantages including a small size (1 mm at adulthood), a compact genome, hermaphroditic-self reproduction, rapid development cycle (3 to 5 days at 20°C), simple genetic manipulation, a well-studied simple nervous system as well as constant transparency during its short lifespan (3–4 weeks) (Sulston and Horvitz 1977, Johnson and Wood 1982, Altun and Hall 2009). Depending on the bioinformatics approach used, between 60–80% of human genes have an ortholog in C. elegans (Kaletta and Hengartner 2006, Shaye and Greenwald 2011). Thus, the conservation of genes, molecules and genetic interactions in this model is a clear advantage for studying genetics. Additionally, the C. elegans genome contains an orthologue of TARDBP, named tdp-1. The resulting TDP-1 protein has 414 amino acids (isoform c), 38% of homology and conserved domains with the human protein except it lacks the glycine-rich domain (Fig. 1C). TDP-1 is primarily nuclear but can shuttle to the cytoplasm, is expressed in most tissues and has conserved functions. Like TDP-43, TDP-1 exhibits RNA binding activity and plays a role in the regulation of protein homeostasis (Zhang, Hwang et al. 2012). TDP-1 also plays a role in adult lifespan, hyperosmotic response, response to oxidative stress and several other processes (Vaccaro, Tauffenberger et al. 2012).
We wanted to develop a C. elegans model reflecting an identical mutation found in patients and subsequent mouse models to aid translational studies (Arnold, Ling et al. 2013, White, Kim et al. 2018). We generated transgenic Caenorhabditis elegans models expressing human wild-type TDP-43(WT) and mutant TDP-43(Q331K) in GABAergic motor neurons (Fig. 1AB). The human Q331K mutation is located in the glycine-rich domain, where the majority of mutations are found in ALS patients (Prasad, Bharathi et al. 2019).
We also obtained by mutagenesis and CRISPR-Cas9 a physiologically accurate model based on the mutation R393C in tdp-1 (Fig. 1D). This endogenous mutation is located in the equivalent C. elegans domain, the C-terminal domain. Our objective was to characterize these models and determine if they can recapitulate key aspects of ALS disease such as motor deficits and age-dependent neurodegeneration causing paralysis. We believe that the TDP-1 model may reflect more precisely the physiological expression of the gene in the human disease because of its mutation in an endogenous gene and in the absence of potential artefacts resulting from overexpression. To do so, we explored the impact of these TDP-43/TDP-1 mutations on the physiological aspects of the models and assessed the repercussions of these genetic defects on the integrity of the motor nervous system.