Abstract
ABSTRACTPatient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by loss of motoneurons (MNs) in the spinal cord and brain, leading to progressive muscle atrophy
By using these new in vitro models, the authors show that the incorporation of mutated fused in sarcoma/translocated in liposarcoma (FUS) into cytoplasmic stress granules occurs in both undifferentiated induced pluripotent stem cells (iPSCs) and iPSC-derived motoneurons when these are subject to various ALS-related cellular stressors
Implications and future directions The iPSC-based in vitro model described here allows the evaluation of the effects of patient-specific ALS-associated FUS mutations on ALSrelevant cell types, e.g. motoneurons
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by loss of motoneurons (MNs) in the spinal cord and brain, leading to progressive muscle atrophy. FUS and TDP-43 are both RNA-binding proteins with a role in multiple steps of RNA processing (Lagier-Tourenne et al, 2010) This suggests a common mechanism underlying their involvement in ALS. The majority of cases are sporadic; several genes have been recently linked to ALS, including the fused in sarcoma/translocated in liposarcoma (FUS) gene. Whereas wild-type FUS protein is predominantly nuclear, ALS-associated mutations cause its cytoplasmic delocalization. In vitro studies have been mainly carried out in cancer cell lines that ectopically express mutant or wild-type proteins These cell systems do not recapitulate the complexity of the motoneuron and its microenvironment and/or imply non-physiological levels of protein, which has hampered an accurate molecular analysis of ALS pathogenesis
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