Investigating the role of aberrant protein-protein interactions in familial ALS pathogenesis
using iPSC-derived motor neurons
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that selectively affects motor neurons. Approximately 5 to 10 % of cases are familial (fALS) and result from inherited genetic mutations, such as those affecting the gene Fused in Sarcoma (FUS). The FUS protein is a crucial regulator of multiple cellular functions, including stress granule (SG) formation as well as RNA binding and processing. Aggregates of FUS are a hallmark of FUS-ALS, but the role of these aggregates in disease pathogenesis is not clear. This question remains unresolved because the majority of studies have used cellular models incapable of recapitulating the complex biology of motor neurons (MNs). As a result, no therapeutics are available to prevent or slow ALS pathogenesis. Induced pluripotent stem cells (iPSCs) provide a revolutionary approach to model ALS because they can be differentiated into a theoretically limitless number of patient-specific MNs.
In order to investigate ALS in vitro, our lab previously used CRISPR/Cas9-mediated gene editing to generate isogenic WT and P525L FUS-eGFP iPSCs. In line with existing reports, P525L FUS showed preferential abnormal cytoplasmic localization, and high content imaging performed on arsenite-stressed cells linked the mutation to altered SG dynamics. Using the GFP sequence as an affinity tag, we performed a preliminary pull down experiment on iPSC-derived MNs to explore FUS interactors, and identified protein partners interacting differentially with WT and P525L FUS. Because some of the detected proteins have been associated with ALS when mutated, we consider this as a strong clue that something relevant for the disease is happening at this level, and speculate that such interactions are integral to the induction of FUS-ALS. We propose to further investigate protein-protein interactions in this model to identify which players may be crucial for disease pathogenesis.
To determine if ALS pathology is induced via gain- or loss-of-function, we will either knock down or overexpress these proteins in iPSC-derived MNs, and evaluate the impact on ALS pathological phenotypes. Our hope is to shed some light on the mechanisms involved in disease pathogenesis, thereby facilitating the discovery of novel therapeutic approaches. Since pathological aggregation of proteins is a key feature of ALS, - and FUS inclusions have been reported also in sporadic cases, indicating a common pathological denominator - it may be possible, in the future, to develop these findings into therapeutics for most ALS patients.