Leigh syndrome Cell Models for Research

Leigh syndrome (LS) is a rare, progressive neurodegenerative disorder with an estimated incidence of 1 in 40,000 live births, though higher in certain populations (e.g., Saguenay-Lac-Saint-Jean, Quebec, Canada). The disease typically manifests in infancy or early childhood, with a median survival of 2-3 years after onset, although some patients survive into adolescence or adulthood. The clinical presentation includes developmental regression, hypotonia, ataxia, ophthalmoplegia, and characteristic bilateral symmetrical lesions in the basal ganglia and brainstem. There is no cure, and current management is supportive, making the development of effective therapies a critical unmet need. (WHO, 2023; NCI, 2023)

Leigh syndrome is an ideal model for studying mitochondrial dysfunction and neurodegeneration. It is caused by mutations in over 75 genes, primarily affecting mitochondrial respiratory chain complexes, providing a diverse genetic landscape for mechanistic studies. Public datasets, such as the Leigh Syndrome International Registry and the Mitochondrial Disease Sequence Data Resource (MSeqDR), offer valuable clinical and genomic data. Open questions include the precise molecular mechanisms linking mitochondrial dysfunction to neuronal death, the role of glial cells, and the identification of potential therapeutic targets. Gene-edited cell models are essential for dissecting these pathways and testing novel interventions.

• • Leigh syndrome results from defective mitochondrial energy production, primarily affecting the oxidative phosphorylation (OXPHOS) system. Key pathways include:
• • Mitochondrial respiratory chain dysfunction: Mutations in nuclear or mitochondrial genes encoding subunits of complexes I, II, III, IV, and V impair ATP synthesis.
• • Reactive oxygen species (ROS) overproduction: Defective electron transport leads to increased ROS, causing oxidative damage to mitochondrial DNA, proteins, and lipids.
• • Apoptosis and necrosis: Mitochondrial dysfunction triggers cell death pathways, particularly in high-energy-demand neurons.
• • Mitochondrial dynamics and quality control: Impaired fusion/fission and mitophagy contribute to mitochondrial accumulation and cellular damage.

Data from TCGA, COSMIC, ClinVar, and MSeqDR.

• • Mitochondrial dysfunction in Leigh syndrome affects multiple signaling networks:
• • AMPK/mTOR pathway: Energy stress activates AMPK, which inhibits mTORC1, leading to reduced protein synthesis and autophagy.
• • PI3K/AKT pathway: Impaired ATP production reduces AKT phosphorylation, affecting cell survival and growth.
• • NF-κB pathway: ROS activates NF-κB, promoting inflammation.
• • HIF-1α pathway: Mitochondrial ROS stabilizes HIF-1α, inducing glycolytic genes.
• • Calcium signaling: Mitochondrial dysfunction alters calcium buffering, affecting synaptic transmission and neuronal excitability.

Organoids recapitulate cell-cell interactions and allow long-term studies of neuronal degeneration.

• • Animal models for Leigh syndrome include:
• • Ndufs4 knockout mouse: Global or neuron-specific knockout, recapitulates encephalopathy and early lethality.
• • Surf1 knockout mouse: Shows complex IV deficiency and mild phenotype.
• • MT-ATP6 mutant mouse: Generated by mitochondrial DNA editing, exhibits late-onset symptoms.
• • Zebrafish models: Used for high-throughput drug screening.
• • Drosophila models: Useful for genetic modifier screens.

CRISPR-based gene editing enables the creation of isogenic cell lines with precise mutations in Leigh syndrome-associated genes. For example, a NDUFS4 knockout cell line can be generated in a wild-type background to study complex I deficiency, while a MT-ATP6 knock-in cell line can introduce the m.8993T>C mutation to model ATP synthase dysfunction. These models are commercially available and sequence-verified, ensuring reproducibility and accelerating research. They are essential for studying disease mechanisms, screening drug candidates, and developing gene therapies.

Gene-edited cell lines are used to validate the functional impact of disease-associated variants. For example, CRISPR knockout of NDUFS4 in HEK293 cells leads to reduced complex I activity and increased ROS production, confirming its role in mitochondrial dysfunction. Knock-in of pathogenic mutations allows the study of specific molecular consequences. These models are also used in CRISPR screens to identify genetic modifiers that rescue mitochondrial defects.

Isogenic pairs (wild-type vs. mutant) are used in high-throughput screens to identify compounds that selectively target mutant cells. For example, a screen using NDUFS4 knockout cells may identify drugs that improve mitochondrial function or reduce oxidative stress. These models also help study resistance mechanisms to potential therapies, such as gene therapy vectors.

CRISPR-engineered cells are used in synthetic lethality screens to identify genes that become essential when mitochondrial function is impaired. This can reveal novel biomarkers and therapeutic targets. For instance, a screen in SURF1 knockout cells may identify genes involved in compensatory pathways that could be targeted to selectively kill mutant cells.