Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondrial DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol γ) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol γ exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol γ and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol γ reported here could have implications for forms of mtDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTP/dNTP ratios that are expected to prevail in such disease states, Pol γ enters a polymerase/exonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.

Background: Store-operated Ca 2ϩ entry is regulated by the sensor STIM1 and the channel ORAI1. Results: Deficiency alters mitochondrial shape and increases mitochondrial activity resulting in increased susceptibility to oxidative stress and cell death by nuclear translocation of apoptosis-inducing factor. Conclusion: Store-operated Ca 2ϩ entry regulates mitochondrial function and vulnerability. Significance: STIM1 plays a role in oxidative stress by regulating mitochondrial function.