Hematopoietic cell-derived mitochondria transfer reduces morbidity and mortality in a mouse model of leigh syndrome

Abstract Introduction: Mitochondria transfer is a recently described phenomenon in which donor cells deliver mitochondria to acceptor cells. One possible consequence of mitochondria transfer is energetic support of neighboring cells; for example, exogenous healthy mitochondria can rescue cell-intrinsic defects in mitochondrial metabolism, as shown in cultured ρ0 cells or Ndufs4-/- peritoneal macrophages. Mitochondria transplantation has been explored in multiple settings, including ex vivo exposure of hematopoietic stem cells prior to autologous transplantation for patients with large-scale mitochondrial DNA mutations, as well as mitigation of ischemic injury to the heart, brain, and limbs. However, the therapeutic potential of mitochondria transfer-based therapies for inherited mitochondrial disorders remains unclear. Leigh syndrome (LS) is a fatal pediatric mitochondrial disease characterized by progressive neurodegeneration. No effective treatments exist for this devastating condition. Ndufs4-/- mice, which lack a subunit of complex I in the electron transport chain, serve as a robust LS model. We previously demonstrated that in vivo administration of wild-type mitochondria can restore aerobic respiration in Ndufs4-/- peritoneal macrophages. Immune cells are known to donate mitochondria to metabolically compromised cells in several tissues, raising the possibility that leveraging mitochondria transfer could ameliorate systemic mitochondrial disease. Methods: We evaluated mitochondria transfer-based strategies to improve morbidity and mortality in Ndufs4-/- mice. Two approaches were tested: (1) intraperitoneal injection of purified mitochondria from wild-type (WT) or Ndufs4-/- mice, either as a single administration or repeated weekly injections; and (2) bone marrow transplantation (BMT) from WT or Ndufs4-/- donors, including experiments using the mtDendra2Flox/Flox (PhAM Flox; referred to as mtD2F/F) mitochondrial reporter system. Metabolic function of peritoneal macrophages was assessed using Seahorse-based oxygen consumption rate (OCR) analysis. Whole-body metabolism was measured in metabolic cages, neurological performance was evaluated using the rotarod assay, and survival was monitored. Circulating and tissue-associated donor mitochondria were tracked to assess transfer to recipient immune and non-immune cells. Finally, cross-species experiments were performed using human mitochondria administered to Ndufs4-/- mice. Results: Single intraperitoneal injection of WT mitochondria into Ndufs4-/- mice increased both basal and maximal OCR in peritoneal macrophages, indicating improved mitochondrial respiration. Repeated weekly injections further enhanced whole-body energy expenditure and neuromotor performance, ultimately extending survival. In contrast, mitochondria derived from Ndufs4-/- mice failed to improve neurological function (median 84 vs 68 days, p=0.0052). BMT from WT donors led to the release of extracellular mitochondria into circulation and subsequent transfer to host immune (B cells, neutrophils) and non-immune (epithelial, endothelial, stromal) cells in blood, spleen, and liver. This transfer was associated with increased energy expenditure, improved rotarod performance, and significantly prolonged survival compared to Ndufs4-/- donor BMT (median 74 vs 40 days, p<0.0001). Importantly, cross-species administration of purified human mitochondria to Ndufs4-/- mice also resulted in enhanced neurological function and extended lifespan (median 80.5 vs 67.5 days, p=0.0079), supporting the translational potential of this approach. Conclusion: Our findings demonstrate that mitochondria transfer-based interventions, including systemic delivery of isolated WT mitochondria and BMT from WT donors, ameliorate morbidity and extend lifespan in the Ndufs4-/- model of Leigh syndrome. These benefits are linked to enhanced macrophage respiration, systemic metabolic improvements, and donor mitochondria transfer to recipient tissues. Moreover, successful cross-species rescue with human mitochondria highlights the potential of mitochondria transfer as a therapeutic strategy for primary mitochondrial diseases such as LS.