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

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).

Rapid Diagnostic Test to Detect and Discriminate Infectious Hematopoietic Necrosis Virus (IHNV) Genogroups U and M to Aid Management of Pacific Northwest Salmonid Populations.

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonids in North America, Europe, and Asia that is phylogenetically classified into five major virus genogroups (U, M, L, E, and J). The geographic range of the U and M genogroup isolates overlap in the North American Columbia River Basin and Washington Coast region, where these genogroups pose different risks depending on the species of Pacific salmon (Oncorhynchus spp.). For certain management decisions, there is a need to both test for IHNV presence and rapidly determine the genogroup. Herein, we report the development and validation of a U/M multiplex reverse transcription, real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) protein gene. The new U/M RT-rPCR is a rapid, sensitive, and repeatable assay capable of specifically discriminating between North American U and M genogroup IHNV isolates. However, one M genogroup isolate obtained from commercially cultured Idaho rainbow trout (O. mykiss) showed reduced sensitivity with the RT-rPCR test, suggesting caution may be warranted before applying RT-rPCR as the sole surveillance test in areas associated with the Idaho trout industry. The new U/M assay had high diagnostic sensitivity (DSe > 94%) and specificity (DSp > 97%) in free-ranging adult Pacific salmon, when assessed relative to cell culture, the widely accepted reference standard, as well as the previously validated universal N RT-rPCR test. The high diagnostic performance of the new U/M assay indicates the test is suitable for surveillance, diagnosis, and confirmation of IHNV in Pacific salmon from the Pacific Northwest regions where the U and M genogroups overlap.

Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.

Wilson disease (WD) is a severe hepato-neurologic disorder that affects primarily children and young adults. WD is caused by mutations in ATP7B and subsequent copper overload. However, copper levels alone do not predict severity of the disease. We demonstrate that temporal and spatial distribution of copper in hepatocytes may play an important role in WD pathology. High resolution synchrotron-based x-ray fluorescence imaging in situ indicates that copper does not continuously accumulate in Atp7b(-/-) hepatocytes, but reaches a limit at 90-300 fmol. The lack of further accumulation is associated with the loss of copper transporter Ctr1 from the plasma membrane and the appearance of copper-loaded lymphocytes and extracellular copper deposits. The WD progression is characterized by changes in subcellular copper localization and transcriptome remodeling. The synchrotron-based x-ray fluorescence imaging and mRNA profiling both point to the key role of nucleus in the initial response to copper overload and suggest time-dependent sequestration of copper in deposits as a protective mechanism. The metabolic pathways, up-regulated in response to copper, show compartmentalization that parallels changes in subcellular copper concentration. In contrast, significant down-regulation of lipid metabolism is observed at all stages of WD irrespective of copper distribution. These observations suggest new stage-specific as well as general biomarkers for WD. The model for the dynamic role of copper in WD is proposed.