The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Abstract

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

Copper trafficking to the secretory pathway

“…This observation illustrates the primary role of ATP7A in maintaining the cellular Cu balance and also suggests that the transport of Cu to the secretory pathway restricts Cu entry to other compartments. 105 One may conclude that CCS in the cytosol and the Cu chaperones for mitochondria have to compete with Atox1 for their quota of Cu. This conclusion is somewhat problematic because the mitochondria respiratory function is paramount for cell survival, whereas cells with a disrupted Cu flow to secretory pathway can survive and proliferate.…”

The mitochondrion: a central architect of copper homeostasis

“…Importantly, the matrix copper pool is dynamic and can expand in size, 30, 40 arguing that mitochondria are candidates for the sequestration of copper from the cytosol under conditions of cellular copper overload. However, mitochondrial ultrastructure and function are severely perturbed in the livers of Wilson disease (WD) patients due to increased mitochondrial copper levels, 41–43 emphasizing that there is an upper limit to the amount of copper that can be safely stored within the organelle.…”

“…36, 37 If copper accumulates in the matrix in a non-ligated form it could easily cause damage by non-specific interactions or by perturbing redox balance. 8, 40 In fact, the mitochondrial damage observed in WD livers can be prevented in a rat model of the disease by exogenous treatment with methanobactin, a copper chelator which dramatically reduces the mitochondrial copper burden. 44 …”

“…Matrix redox balance is nonetheless perturbed by mutations in ATP7A or ATP7B , which lead to mitochondrial hyper accumulation of copper and organelle dysfunction. 40, 43 However, whether GSH participates in copper delivery to mitochondria, whether the GSH:GSSG ratio affects copper trafficking within the organelle and whether any of these activities are impaired in copper handling disorders remain important, open questions.…”

“…109 Fusion of individual organelles into a reticular network is a common stress response in cells to protect the overall health of their mitochondrial population, 107 and a GSH deficiency has been reported in skeletal muscle of mitochondrial disease patients with electron transport chain defects. 110 While gross mitochondrial morphology does not appear to differ between control, SCO1 , SCO2 and ATP7A patient fibroblasts, 39, 40 a potential relationship between redox-dependent changes in mitochondrial dynamics and copper homeostasis has yet to be explored in mechanistic detail in more applicable disease models and contexts. It is imperative that future studies like these also consider the possible interplay between redox and the release of mitochondria-derived vesicles, which were originally identified by the McBride group and are now known to deliver specific, selective and physiologically relevant cargo to peroxisomes and endolysosomes.…”