Induced Pluripotent Stem Cell-Derived Motor Neurons from Amyotrophic Lateral Sclerosis (ALS) Patients Carrying Different Superoxide Dismutase 1 Mutations Recapitulate Pathological Features of ALS
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal neurodegenerative disease with no effective treatment currently available. The exact pathogenesis of ALS remains unclear, but recent molecular and genetic discoveries have provided important insights into the common pathological processes underlying the disease. Among the genetic mutations associated with ALS, mutations in the superoxide dismutase 1 (SOD1) gene have been extensively studied due to their early discovery and widespread use in murine models. Evidence suggests that SOD1 mutations contribute to ALS through toxic properties such as protein aggregation, mitochondrial dysfunction, and calcium dysregulation. Despite significant advances in understanding the disease, translating experimental observations into effective therapies has been challenging, partly due to the lack of appropriate human cell-based models that can accurately recapitulate disease mechanisms.
Induced pluripotent stem cells (iPSCs) offer a promising alternative to traditional models. iPSCs derived from patients with genetic mutations naturally occurring in ALS can potentially manifest disease phenotypes, providing a valuable tool for studying disease mechanisms and identifying therapeutic targets. In this study, iPSCs were generated from two familial ALS (FALS) patients carrying different SOD1 mutations (SOD1-V14M and SOD1-C111Y) and differentiated into motor neurons (MNs). The study aimed to explore the expression of mutant SOD1 protein in these MNs, investigate intracellular calcium levels, and assess lactate dehydrogenase (LDH) activity during the differentiation process. The findings demonstrate that MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis, offering a cell-based disease model to further elucidate disease mechanisms and explore potential therapies.
Introduction
ALS is characterized by the progressive degeneration of motor neurons, leading to muscle weakness, paralysis, and ultimately death. While the majority of ALS cases are sporadic (SALS), approximately 10% are familial (FALS), with mutations in the SOD1 gene being one of the most common causes of FALS. SOD1 is an enzyme that plays a crucial role in protecting cells from oxidative stress by converting superoxide radicals into oxygen and hydrogen peroxide. Mutations in SOD1 lead to the formation of toxic protein aggregates, mitochondrial dysfunction, and disturbances in calcium homeostasis, all of which contribute to motor neuron degeneration.
Despite extensive research, the precise mechanisms by which SOD1 mutations cause ALS remain incompletely understood. Animal models, particularly transgenic mice expressing mutant SOD1, have been instrumental in studying ALS. However, these models do not fully replicate the complexity of human ALS, highlighting the need for human cell-based models. iPSCs derived from ALS patients offer a unique opportunity to study the disease in a human context, as they can be differentiated into the cell types affected in ALS, such as motor neurons.
Methods
The study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee of Peking University Third Hospital. Written informed consent was obtained from all participants. Skin fibroblasts were collected from two FALS patients carrying SOD1-V14M and SOD1-C111Y mutations, as well as from four healthy controls. The fibroblasts were reprogrammed into iPSCs using retroviral vectors encoding the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). The iPSCs were then differentiated into motor neurons using a modified protocol involving embryoid body formation and exposure to retinoic acid and sonic hedgehog (SHH).
Pluripotency of the iPSCs was confirmed through expression of pluripotency markers (SSEA-4, TRA1-60, TRA1-81, Nanog, OCT3/4, and SOX2), bisulfite genomic sequencing, and teratoma formation in immunodeficient mice. The differentiation of iPSCs into motor neurons was confirmed by immunofluorescence staining for motor neuron-specific markers (TUJ1, HB9, and ISL1). SOD1 protein levels were measured using Western blotting, intracellular calcium levels were assessed using the calcium-specific fluorescent dye Fluo 3-AM, and LDH activity was measured using an LDH assay kit.
Results
The iPSCs generated from the two FALS patients were successfully differentiated into motor neurons expressing motor neuron-specific markers. There were no significant differences in the number of HB9- or ISL1-positive motor neurons between the patient-derived and control-derived iPSCs, indicating that the SOD1 mutations did not impair the ability of the iPSCs to differentiate into motor neurons.
Analysis of SOD1 protein levels revealed that both the iPSCs and motor neurons derived from the FALS patients exhibited higher levels of SOD1 compared to the controls. This finding is consistent with the hypothesis that mutant SOD1 accumulates in motor neurons, contributing to the pathogenesis of ALS. Interestingly, there was no significant difference in LDH activity between the patient-derived and control-derived motor neurons, suggesting that the SOD1 mutations did not increase cell death during the differentiation process.
Measurement of intracellular calcium levels showed that motor neurons derived from the FALS patients had significantly higher calcium levels compared to those derived from controls. This finding supports the notion that calcium dysregulation is a key feature of ALS pathogenesis, potentially contributing to motor neuron degeneration. The increased calcium levels in the patient-derived motor neurons may result from mitochondrial dysfunction, as mitochondria play a crucial role in calcium homeostasis.
Discussion
The study demonstrates that iPSCs derived from FALS patients carrying SOD1 mutations can be differentiated into motor neurons that recapitulate key pathological features of ALS, including increased SOD1 protein levels and calcium dysregulation. These findings provide a valuable human cell-based model for studying the mechanisms underlying ALS and for testing potential therapeutic interventions.
The increased SOD1 protein levels observed in the patient-derived motor neurons are consistent with previous studies showing that mutant SOD1 forms toxic aggregates in motor neurons. These aggregates may disrupt cellular processes, leading to motor neuron degeneration. The study also highlights the role of calcium dysregulation in ALS pathogenesis. Elevated intracellular calcium levels in motor neurons can lead to mitochondrial dysfunction, oxidative stress, and ultimately cell death. The findings suggest that targeting calcium dysregulation may be a promising therapeutic strategy for ALS.
The study has several limitations. First, the sample size was small, with only two FALS patients and four healthy controls included in the analysis. Future studies should include a larger number of patients to confirm the findings. Second, the study focused on two specific SOD1 mutations (SOD1-V14M and SOD1-C111Y). It is possible that other SOD1 mutations or mutations in other genes associated with ALS may have different effects on motor neuron function. Finally, while the study provides valuable insights into the mechanisms underlying ALS, further research is needed to determine how these findings can be translated into effective therapies.
Conclusion
In conclusion, the study demonstrates that motor neurons derived from iPSCs generated from FALS patients carrying SOD1 mutations recapitulate key pathological features of ALS, including increased SOD1 protein levels and calcium dysregulation. These findings provide a valuable human cell-based model for studying the mechanisms underlying ALS and for testing potential therapeutic interventions. The study highlights the importance of calcium dysregulation in ALS pathogenesis and suggests that targeting calcium homeostasis may be a promising therapeutic strategy. Future studies should focus on expanding the sample size and exploring the effects of other genetic mutations associated with ALS.
doi.org/10.1097/CM9.0000000000001693
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