Human Motor Neurons With SOD1-G93A Mutation Generated From CRISPR/Cas9 Gene-Edited iPSCs Develop Pathological Features of Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by gradual degeneration and elimination of motor neurons (MNs) in the motor cortex, brainstem, and spinal cord. Some familial forms of ALS are caused by genetic mutations in superoxide dismutase 1 (SOD1) but the mechanisms driving MN disease are unclear. Identifying the naturally occurring pathology and understanding how this mutant SOD1 can affect MNs in translationally meaningful ways in a valid and reliable human cell model remains to be established. Here, using CRISPR/Cas9 genome editing system and human induced pluripotent stem cells (iPSCs), we generated highly pure, iPSCderived MNs with a SOD1-G93A missense mutation. With the wild-type cell line serving as an isogenic control and MNs from a patient-derived iPSC line with an SOD1-A4V mutation as a comparator, we identified pathological phenotypes relevant to ALS. The mutant MNs accumulated misfolded and aggregated forms of SOD1 in cell bodies and processes, including axons. They also developed distinctive axonal pathologies. Mutants had axonal swellings with shorter axon length and less numbers of branch points. Moreover, structural and molecular abnormalities in presynaptic and postsynaptic size and density were found in the mutants. Finally, functional studies with microelectrode array demonstrated that the individual mutant MNs exhibited decreased number of spikes and diminished network bursting, but increased burst duration. Taken together, we identified spontaneous disease phenotypes relevant to ALS in mutant SOD1 MNs from genome-edited and patient-derived iPSCs. Our findings demonstrate that SOD1 mutations in human MNs cause cell-autonomous proteinopathy, axonopathy, synaptic pathology, and aberrant neurotransmission.

Applications of CRISPR-Cas9 in Alzheimer’s Disease and Related Disorders

“…Standard CRISPR-Cas9 has been used to introduce various mutations into the SOD1 gene of iPSCs. The resulting cell lines developed some features associated with the disease [ 84 , 85 ]. Similar experiments have been conducted in vivo with the use of CRISPR-Cas9-mediated homologous recombination to generate multiple C. elegans models, each carrying an independent SOD1 mutation [ 82 ].…”

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

“…One such attempt has been recently reported wherein a group generated CRISPR-iPSC-based isogenic ALS disease models harboring a SOD1 missense mutation (G93A) by utilizing a CRISPR/Cas9-based targeted knock-in approach on healthy iPSC lines. The genome-edited iPSCs were subsequently differentiated into motor neurons, demonstrating the pathological signatures of intracellular misfolded SOD1 aggregate accumulation, such as reduced axonal length and branching, presynaptic and postsynaptic abnormalities, and anomalous electrical activity (Kim et al 2020a ). In another study, a group generated CRISPR/Cas9-mediated SQSTM1 knockout iPSC-derived cortical neurons and assessed its impact on neuronal physiology (Poon et al 2021 ).…”

Emerging hiPSC Models for Drug Discovery in Neurodegenerative Diseases

“…Interestingly, hiPSC-derived MNs with a SOD1 (G93A) missense mutation, generated by CRISPR/Cas9, developed axonal pathologies, such as axonal swellings with a shorter axon length and fewer branch points, in addition to abnormalities in presynaptic and postsynaptic size and density. The SOD1 mutation also led to a decrease in the frequency of action potentials and network bursting; however, burst duration increased [ 175 ]. Additionally, SOD1 mutant hiPSC-derived astrocytes have been shown to exhibit the upregulation of several proinflammatory genes in parallel to the downregulation of many genes associated with homeostatic functions [ 176 ].…”