Abstract
Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.
Highlights
Brain development requires a fine-tuned copper homoeostasis
We have previously found that the copper chaperone Atox[1] can be reversibly oxidized within a physiologically relevant range of redox potentials[10,11], and hypothesized that such reversible oxidation might modulate intracellular copper fluxes
Our results suggest that changes in the redox status of cytosolic glutathione may have significant effect on the metabolic activity of secretory pathway by altering maturation and function of the resident copper-dependent enzymes
Summary
Differentiated motor neurons have higher levels of PAM. To test whether copper utilization changes upon neuronal differentiation we used two different experimental models. Larger fraction of reduced glutathione implies an increased availability of nicotinamide adenine dinucleotide phosphate (NADPH), the main cellular reductant maintaining the redox status of GSH To test this prediction we measured levels of NADPH in non-differentiated and differentiated SHSY-5Y cells and calculated the NADPH/NADP þ ratio. We treated differentiated cells with buthyl-cnitrosourea (BCNU) to artificially induce glutathione oxidation This treatment increased fraction of oxidized Atox[1] from 19 to 79% (Fig. 5c), establishing that the redox status of Atox[1] is regulated by cytosolic glutathione. Oxidation of Atox[1] metal-binding site is expected to decrease copper delivery to secretory vesicles, whereas reduction would facilitate this process To test this prediction, we compared copper efflux from differentiated and non-differentiated cells as a measure of copper sorting to the secretory pathway. BCNU treatment and associated glutathione oxidation decreases copper delivery to the secretory pathway
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