Tracing the molecular pathways underlying ALS disease
The terminal neurodegenerative disease amyotrophic lateral sclerosis (ALS) results in the loss of the motor neurons in the brain and spinal cord that control voluntary muscles. It is the commonest motor neuron disease. “Sadly, most patients die two to five years after diagnosis and there are only a few approved drugs known to help some patients, with many failed clinical trials,” says Edor Kabashi, project coordinator of the ALS-Networks project, which was funded by the European Research Council. Over the last two decades researchers have identified over a dozen genes with pathogenic mutations in some ALS patients. Expressed in most cell types, these were thought to be unrelated to ALS but are now central to the quest for new therapeutics. ALS-Networks wanted to identify common pathways where these mutated genes might interact, revealing how altered genetic expressions might degenerate motor neurons. The project focused on four genes commonly mutated in ALS. “The pathways that we identified appear to be shared not only in our models, but also in other animal models and in induced motor neurons from ALS patients. I’m certain that we are on the right path to major therapeutic advances,” adds Kabashi from France’s National Institute of Health and Medical Research (Inserm), the project host.
Transgenic zebra fish models
As zebra fish also have the same genes mutated and deregulated in ALS patients, they make good model organisms. Using Crispr/Cas9 techniques, the project targeted specific regions of the zebra fish genome to delete the ALS genes and/or to introduce mutations found in ALS patients. This created a series of zebra fish deletion mutants with which to study the four ALS genes under investigation: C9orf72, SQSTM1, TDP-43 and FUS. Vertebrate zebra fish models for C9orf72 helped identify a pathological marker for ALS. “We found that overexpressing proteins called dipeptide repeats, exacerbates motor damage under certain genetic conditions, leading to neurodegeneration,” notes Kabashi. Combining techniques, such as purification and omics analysis of motor neurons from these models, identified two shared pathways that contribute to motor neuron decline. Firstly, compromised mitochondria metabolism – key to maintaining cellular energy. Secondly, disrupted autophagy, cellular removal of old or damaged components. The team also developed a zebra fish model with a deactivated FUS gene which was shown to have reduced swimming abilities and increased mortality compared to controls. Studying how genetic interactions might influence disease-causing genetic variants – and so present risk factors – in collaboration with international consortia, the team developed zebra fish models for two additional ALS genes, GPX3 and NUP50. “We are really excited to study these variants further, as they appear to be involved in two key ALS pathogenic mechanisms, oxidative stress and nucleocytoplasmic transport,” explains Kabashi. A model was also developed for TBK1, another gene recently identified by the team as also present in ALS patients (a paper is currently in preparation). Inactivation of this gene leads to motor neuron degeneration and activation of cell death
Common pathways
The researchers continue to seek commonly deregulated motor neuron pathways as targets for therapies to reduce the suffering and improve quality of life for ALS patients. “For both commonly mutated genes, C9orf72 and FUS, we’ve already identified compounds that can reduce motor neuron and muscle degeneration,” adds Kabashi. Kabashi looks forward to defining specific markers that are altered in zebra fish models. Similarly, the fluorescent sorting of cells known to be affected in ALS, will enable cell-specific alterations to be further defined. “Identifying these markers could ultimately advance ALS gene therapy, which works well in spinal muscular atrophy, a neurodevelopmental condition that also targets motor neurons and muscle,” concludes Kabashi
Keywords
ALS-Networks, ALS, motor neuron, brain, spinal cord, muscles, gene, zebra fish, cell, mutated, therapeutics, mitochondria metabolism, autophagy
