2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA

Boris Pantic,Mikel Muñoz-Oreja,Diego Perez-Rodriguez,Antonella Spinazzola,Uxoa Fernandez-Pelayo,Lodovica Vergani,Michael G Hanna,Robert D S Pitceathly,Thanh-Mai Julie Dang,Robert Mcfarland,Amaia Lopez De Arbina,Ian J Holt,Robert W Taylor,Marina Villar-Fernandez,Mara Mennuni,Daniel Ives,Iain G Johnston

doi:10.1038/s41467-021-26829-0

Abstract

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication.

Highlights

Pathological variants of human mitochondrial DNA typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood
We show that 2DG and another glucose analogue, 5-thioglucose (5TG), select wild-type mitochondrial DNA (mtDNA) molecules in multiple cell types and restore the mitochondrial respiratory capacity. 2DG selectively inhibits the replication of the mutant mtDNA, as does glutamine restriction, but not glucose deprivation or inhibition of glycolysis
Segregation to wild-type mtDNA was independent of cellular proliferation, as 2DG slowed cell growth in mutant and control cells (Supplementary Fig. 2c), and 2DG and 5TG decreased the mutant load in fully confluent P1 and P2 fibroblasts, again accompanied by restoration of oxidative phosphorylation (OXPHOS) protein levels (Fig. 1j and Supplementary Fig. 2d)

Summary

Introduction

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. In the case of the most common pathological variant, m.3243A>G, which produces dysfunctional mt-tRNALeu(UUR), the mutant molecules are selected in most cell lines[4,11,12], whereas A549 adenocarcinoma can occasionally select the wild-type mtDNAs5,6. We inferred that restricting glycolysis should favour mitochondrial fitness, and promote the selection of wild-type mtDNA. The glucose analogue 2Deoxy-D-glucose (2DG) inhibits glycolysis[17] and restricts glutamine utilization[18] making it a strong candidate to select wild-type over deleterious mtDNA variants. We show that 2DG and another glucose analogue, 5-thioglucose (5TG), select wild-type mtDNA molecules in multiple cell types and restore the mitochondrial respiratory capacity. Metabolite usage for mtDNA replication depends on mitochondrial fitness and it can be leveraged to favour the propagation of functional mtDNAs

Methods

Results

Conclusion