A novel multisystem approach uncovers conserved mitochondrial complex I disease phenotypes and suppressors

Technical Summary: A Novel Multisystem Approach Uncovers Conserved Mitochondrial Complex I Disease Phenotypes and Suppressors

Problem Statement
Mitochondriopathies, particularly those caused by mutations in Complex I (CI) of the electron transport chain, present a significant therapeutic challenge due to their heterogeneous clinical manifestations (e.g., Leigh syndrome), the vast number of pathogenic mutations, and the lack of animal models that accurately recapitulate the systemic nature of the disease. Current interventions are limited, and existing models often fail to correlate the severity of CI structural disruption with specific disease phenotypes. Furthermore, reliance on mammalian animal models is increasingly restricted by regulatory shifts favoring New Approach Methods (NAMs). There is an urgent need for reliable, multi-systemic pipelines that can identify conserved pathological endpoints and screen for disease modifiers without relying solely on traditional mammalian models.

Methodology
The authors developed a comprehensive multisystemic pipeline integrating non-mammalian in vivo models (Caenorhabditis elegans and zebrafish) with mammalian in vitro models (neuroblastoma SH-SY5Y cells, iPSC-derived neural progenitor cells [NPCs], midbrain dopaminergic neurons [mDANs], and brain organoids [MBOs]).

• Genetic Engineering: The study focused on the NDUFS1 gene, specifically introducing the pathogenic missense mutation c.691C>G (L231V) found in Leigh syndrome patients. This was achieved via CRISPR/Cas9 in SH-SY5Y cells and human iPSCs. In C. elegans, two orthologous point mutations were engineered into the nuo-5 gene (L234V and R412C) to mimic the human L231V and R408C variants. A zebrafish ndufs2 knockout model was also utilized.
• Phenotypic Characterization:
  • In Vitro: Researchers assessed cell proliferation, neuronal differentiation (neurite outgrowth), mitochondrial respiration (Seahorse XFe96), mitochondrial membrane potential (MMP), oxidative stress (ROS, lipid peroxidation, ferroptosis sensitivity), and electrophysiological activity (via High-Density Microelectrode Arrays).
  • In Vivo: C. elegans models were evaluated for lifespan, fertility, motility (thrashing and automated tracking), synaptic activity (aldicarb sensitivity), and mitophagy (using Rosella reporters). Zebrafish larvae were assessed for morphological defects (body length, liver size) and swimming activity.
• Intervention Screening: The pipeline was used to test the efficacy of dietary supplements, specifically lutein and vitamin B12 (cobalamin), in rescuing observed phenotypes across all model systems.

Key Results

1. Conserved Mitochondrial and Cellular Defects:

   • SH-SY5Y Cells: The NDUFS1 L231V mutation resulted in reduced protein expression (post-transcriptional) without altering transcript levels. Mutant cells exhibited slower proliferation, impaired neuronal differentiation, and defective CI assembly (reduced supercomplex formation) leading to isolated CI deficiency and reduced mitochondrial membrane potential. Notably, despite mitochondrial dysfunction, there was no overt redox imbalance or increased sensitivity to oxidative stressors (H2O2, DEM, glutamate) under basal conditions.
   • iPSC-Derived Models: Mutant NPCs showed premature differentiation and mitochondrial defects. Differentiated mDANs displayed reduced axonal branching and lower firing rates initially, which normalized over time, suggesting delayed maturation. Mutant brain organoids (MBOs) showed delayed growth and, crucially, exhibited increased epileptic-like firing rates upon potassium channel blockade (4-AP treatment).

2. In Vivo Phenotypes and Neurobehavioral Alterations:

   • C. elegans: The nuo-5 mutants displayed mutation-dependent severity (R412C > L234V). Phenotypes included delayed development, reduced fertility, and impaired motility. Interestingly, while some stress resistance parameters were unchanged, mutants showed increased sensitivity to ferroptosis. A key finding was the presence of "epileptic-like" behavior: mutants showed increased automated tracking activity but reduced effective thrashing and hypersensitivity to aldicarb, indicating neuromuscular dysfunction.
   • Zebrafish: ndufs2 mutants exhibited reduced body length, hepatomegaly (enlarged liver), and neuromuscular dysfunction.

3. Rescue by Dietary Interventions:

   • Lutein and Vitamin B12: Both compounds significantly rescued key pathological features across species. In C. elegans, they restored motility, reduced aldicarb sensitivity, and normalized the expression of synaptic genes (nlg-1, unc-17). In zebrafish, both compounds rescued body length and liver pathology.
   • Mechanism: The rescue effects were linked to non-cell-autonomous mechanisms, likely involving the gut-brain axis and the modulation of one-carbon metabolism and choline homeostasis, rather than direct antioxidant activity.

Significance and Claims
The paper claims three primary advances:

1. Establishment of a Robust NAM Pipeline: The study successfully integrates isogenic patient-specific models across varying biological complexities (from cells to whole organisms) to identify conserved disease endpoints. This approach aligns with regulatory shifts toward reducing mammalian animal use while maintaining translational relevance.
2. Identification of Epileptic-like Endophenotypes: The study identifies a cross-species "epileptic-like" state characterized by altered neuronal firing (in organoids) and paradoxical locomotor behavior (in worms). This provides a novel, quantifiable translational readout for CI deficiency that mirrors clinical neurological manifestations of Leigh syndrome.
3. Discovery of Conserved Metabolic Suppressors: The identification of lutein and vitamin B12 as effective suppressors suggests that CI deficiency pathogenesis involves conserved metabolic pathways (specifically one-carbon/choline metabolism) that are amenable to dietary modulation. The findings support the hypothesis that systemic, non-cell-autonomous mechanisms (potentially via the gut-brain axis) play a critical role in mitochondrial disease, offering a potential therapeutic strategy even in the presence of persistent mitochondrial defects.

The authors emphasize that their findings argue against oxidative stress being the primary driver of CI deficiency pathology in these models, highlighting instead the importance of metabolic rewiring and gene-environment interactions in disease heterogeneity and treatment response.