Hynes-Allen, A. | Search bioRxiv

bioRxiv2026

Tissue-wide metabolic buffering confers resilience to mosaic mitochondrial dysfunction

Mitochondria and oxidative phosphorylation (OxPhos) are essential for cellular homeostasis. However, the phenotypes caused by mitochondrial dysfunction often display remarkable tissue-specificity. What determines the susceptibility of individual cells to metabolic or mitochondrial defects in a complex in vivo tissue context, remains largely unknown. We find that neural stem cells (NSCs) in the developing Drosophila brain can maintain normal proliferation despite severe cell-autonomous OxPhos-dysfunction, provided that sufficient neighbouring cells remain metabolically intact. This tissue-wide buffering progressively fails as the proportion of NSCs with OxPhos dysfunction increases, indicating that the phenotypic threshold for mitochondrial dysfunction is an emergent property of a tissue rather than only of individual cells or cell-types. Mechanistically, we find that OxPhos-deficient NSCs activate a stress-response associated with ATF4/crc-transcriptional activation. NSCs upregulate lactate dehydrogenase (LDH) expression to maintain glycolysis, but their proliferation remains limited by NAD+ regeneration rather than by ATP production. Non-cell-autonomous rescue of NSC-proliferation depends on LDH-dependent NAD+-production in a brain-wide glial network connected by gap junctions and the glutamate/aspartate-transporter Eaat1. These findings demonstrate that the phenotypic threshold for mitochondrial dysfunction is determined by tissue-wide spare metabolic capacity rather than only of individual cells or cell-types. Tissue heterogeneity thus provides resilience to metabolic dysfunction, evidencing key benefits of diversity, and suggesting new therapeutic strategies to enhance endogenous metabolic buffering.

developmental biology↗

bioRxiv2025

MitoPerturb-Seq identifies common and gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy

Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.

genomics↗