MitoCellPhe reveals mitochondrial morphologies in single fibroblasts and clustered stem cells.

Mitochondria are dynamic organelles that differ significantly in their morphologies across cell types, reflecting specific cellular needs and stages in development. Despite the wide biological significance in disease and in health, delineating mitochondrial morphologies in complex systems remains challenging. Here, we present the Mitochondrial Cellular Phenotype (MitoCellPhe) tool developed for quantifying mitochondrial morphologies and demonstrate its utility in delineating differences in mitochondrial morphologies in a human fibroblast and human induced pluripotent stem cell (hiPSC) line. MitoCellPhe generates 24 parameters, allowing for a comprehensive analysis of mitochondrial structures and importantly allows for quantification to be performed on mitochondria in images containing single cells or clusters of cells. With this tool, we were able to validate previous findings that show networks of mitochondria in healthy fibroblast cell lines and a more fragmented morphology in hiPSCs. Using images generated from control and diseased fibroblasts and hiPSCs, we also demonstrate the efficacy of the toolset in delineating differences in morphologies between healthy and the diseased state in both stem cell (hiPSC) and differentiated fibroblast cells. Our results demonstrate that MitoCellPhe enables high-throughput, sensitive, detailed, and quantitative mitochondrial morphological assessment and thus enables better biological insights into mitochondrial dynamics in health and disease.

Quantifying Mitochondrial Dynamics in Patient Fibroblasts with Multiple Developmental Defects and Mitochondrial Disorders.

Mitochondria are dynamic organelles that undergo rounds of fission and fusion and exhibit a wide range of morphologies that contribute to the regulation of different signaling pathways and various cellular functions. It is important to understand the differences between mitochondrial structure in health and disease so that therapies can be developed to maintain the homeostatic balance of mitochondrial dynamics. Mitochondrial disorders are multisystemic and characterized by complex and variable clinical pathologies. The dynamics of mitochondria in mitochondrial disorders is thus worthy of investigation. Therefore, in this study, we performed a comprehensive analysis of mitochondrial dynamics in ten patient-derived fibroblasts containing different mutations and deletions associated with various mitochondrial disorders. Our results suggest that the most predominant morphological signature for mitochondria in the diseased state is fragmentation, with eight out of the ten cell lines exhibiting characteristics consistent with fragmented mitochondria. To our knowledge, this is the first comprehensive study that quantifies mitochondrial dynamics in cell lines with a wide array of developmental and mitochondrial disorders. A more thorough analysis of the correlations between mitochondrial dynamics, mitochondrial genome perturbations, and bioenergetic dysfunction will aid in identifying unique morphological signatures of various mitochondrial disorders in the future.

Evaluating the Bioenergetics Health Index Ratio in Leigh Syndrome Fibroblasts to Understand Disease Severity.

Several pediatric mitochondrial disorders, including Leigh syndrome (LS), impact mitochondrial (mt) genetics, development, and metabolism, leading to complex pathologies and energy failure. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed a glycolytic bioenergetics health index (BHI) for measuring mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in complex V or complex I (T10158C, T12706C). A high percentage (>90%) of pathogenic mtDNA in cells affecting complex V and a low percentage (<39%) of pathogenic mtDNA in cells affecting complex I was quantified. Levels of defective enzyme activities of the electron transport chain correlated with the percentage of pathogenic mtDNA. Subsequent bioenergetics assays showed cell lines relied on both OXPHOS and glycolysis for meeting energy requirements. Results suggest that whereas the precise mechanism of LS has not been elucidated, a multi-pronged approach taking into consideration the specific pathogenic mtDNA variant, glycolytic BHI, and the composite BHI (average ratio of oxphos to glycolysis) can aid in better understanding the factors influencing disease severity in LS.

Leigh Syndrome: A Tale of Two Genomes.

Leigh syndrome is a rare, complex, and incurable early onset (typically infant or early childhood) mitochondrial disorder with both phenotypic and genetic heterogeneity. The heterogeneous nature of this disorder, based in part on the complexity of mitochondrial genetics, and the significant interactions between the nuclear and mitochondrial genomes has made it particularly challenging to research and develop therapies. This review article discusses some of the advances that have been made in the field to date. While the prognosis is poor with no current substantial treatment options, multiple studies are underway to understand the etiology, pathogenesis, and pathophysiology of Leigh syndrome. With advances in available research tools leading to a better understanding of the mitochondria in health and disease, there is hope for novel treatment options in the future.

Cell-Permeable Succinate Increases Mitochondrial Membrane Potential and Glycolysis in Leigh Syndrome Patient Fibroblasts.

Mitochondrial disorders represent a large group of severe genetic disorders mainly impacting organ systems with high energy requirements. Leigh syndrome (LS) is a classic example of a mitochondrial disorder resulting from pathogenic mutations that disrupt oxidative phosphorylation capacities. Currently, evidence-based therapy directed towards treating LS is sparse. Recently, the cell-permeant substrates responsible for regulating the electron transport chain have gained attention as therapeutic agents for mitochondrial diseases. We explored the therapeutic effects of introducing tricarboxylic acid cycle (TCA) intermediate substrate, succinate, as a cell-permeable prodrug NV118, to alleviate some of the mitochondrial dysfunction in LS. The results suggest that a 24-hour treatment with prodrug NV118 elicited an upregulation of glycolysis and mitochondrial membrane potential while inhibiting intracellular reactive oxygen species in LS cells. The results from this study suggest an important role for TCA intermediates for treating mitochondrial dysfunction in LS. We show, here, that NV118 could serve as a therapeutic agent for LS resulting from mutations in mtDNA in complex I and complex V dysfunctions.