Abstract
Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons, leading to muscle atrophy with spasticity and eventual death in 3–5 years after the disease onset. More than 50 mutations linked to ALS have been found in the gene TARDBP, encoding the protein TDP-43 that is the predominant component of neuronal inclusions in ALS. TDP-43 is an RNA binding protein with glycine-rich domains that binds to more than 6,000 RNAs in the human brain. However, ALS-related mutations do not appear to affect the function of these genes, indicating that a toxic gain-of-function may occur. We generated transgenic zebrafish lines expressing human TDP-43, either the wild-type form or the ALS-causative G348C mutation identified in a subset of ALS patients, with the transgene expression driven by an inducible heat shock promoter in order to bypass a potential early mortality. The expression of the mutant but not the wild-type human TDP-43 in zebrafish embryos induced a reduction of the locomotor activity in response to touch compared to controls and moderate axonopathy of the motor neurons of the spinal cord, with premature branching of the main axonal branch, recapitulating previous results obtained by mRNA injections. We used these lines to investigate transcriptomic changes due to the presence of mutant TDP-43 using RNA sequencing and have found 159 genes that are differentially expressed compared to control, with 67 genes up-regulated and 92 genes down-regulated. These transcriptomic changes are in line with recent transcriptomic data obtained in mouse models, indicating that these zebrafish transgenic lines are adequate to further study TDP-43-related ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disorder characterized by the loss of both upper motor neurons, located in the motor cortex and lower motor neurons, located in the anterior horn of the spinal cord and in the brainstem
In order to study the effects of mutant TDP-43, we have generated transgenic zebrafish lines expressing stably either human wildtype TDP-43 (TDP-43WT) or bearing the G348C mutations found in ALS patients from a French-Canadian population (TDP-43G348C) (Kabashi et al, 2008)
While heat shock of wild type embryos or expressing TDP-43WT had no influence on locomotion compared to wild type, the expression of TDP-43G348C led to a strong reduction of the swim distance, the swim duration and the maximum swim velocity compared to controls (Figure 2B), indicating that the motor neuron axonal defects had a strong impact on the physiology as seen by the severe negative influence on the locomotion of the TDP-43G348C embryos, as was previously reported (Kabashi et al, 2010, 2011; Laird et al, 2010; Vaccaro et al, 2012, 2013; Armstrong and Drapeau, 2013; Patten et al, 2017)
Summary
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disorder characterized by the loss of both upper motor neurons, located in the motor cortex and lower motor neurons, located in the anterior horn of the spinal cord and in the brainstem. TDP-43 is a predominantly nuclear protein, but was first linked to ALS and FTD as the main protein found in the ubiquitin-positive cytoplasmic inclusions in the neurons, including motor neurons, of ALS and of FTD patients, with a clearance of its usual nuclear localization (Arai et al, 2006; Neumann et al, 2006). Following this discovery, mutations in TARDBP were identified in around 5% of fALS cases, 1% of sALS, cases and 1% of FTD cases (Kabashi et al, 2008; Sreedharan et al, 2008; Caroppo et al, 2016). TDP-43 is known to bind to at least 6,000 mRNAs in the murine brain and to modify the expression level of at least 600 mRNAs (Polymenidou et al, 2011), and as such it was hypothesized that mutant TDP-43 may induce differentially expressed genes and isoforms, which may be part of the pathogenicity of ALS
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