WHY IS COPPER HOMEOSTASIS ESSENTIAL FOR BRAIN DEVELOPMENT?

Maintaining copper homeostasis is vital for brain development. The ability of copper to cycle between two oxidation states is utilized by enzymes for brain development. In vivo and cell culture models help in investigating the regulation of intercellular copper distribution.

For in vivo observation, a developing chick embryonic spinal cord was chosen and transverse sections of chick embryos were analyzed. A precision sensor was used in this embryo to find dramatic differences in use of copper between developing and fully developed mature neurons. It was studied that differentiated motor neurons had higher levels of peptidyl-glycine--monooxygenase which indicated the increased demand for copper in the secretory pathway.

Copper delivery to the secretory pathway is mediated by Atox1. ATP7A is another copper transporter that mediated systematic copper absorption and provides cuproenzymes in the trans Golgi network(TGN) with network. Cellular copper content in differentiated SHSY5Y cells(BDNF) is higher than in non-differentiating. Protein levels for ATP7A and Atox1 increase upon further differentiation. The ATP7A remained unaffected when HEK293 cells were with RA. Hence, suggesting that they play a vital role in neuronal differentiation. ATP7A is localized within TGN and vesicular structures. To regulate copper homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane. ATP7A trafficking to the plasma membrane is elevated to increase copper load and is reversed when copper concentrations are lowered.

Gsh GSSG(oxidized glutathione) is used to quantitatively measure redox status of cytosolic glutathione. After analyzing eight embryos(HH20 stage) in the independent experiments,it was concluded that differentiated postmitotic cells have larger fractions of glutathione than cycling cells. Further analysis of embryos at HH23 stage demonstrated the time modulation of cytosolic glutathione correlating with neuronal differentiation. The redox status of cytosolic glutathione during differentiating is evaluated using SHSY5Y cells. Sequential treatment with H2O2 and DTT verified Grx1-roGFP2 ability to respond to redox change. It was observed neuronal differentiation increased GSH GSSG ratio consistently with a shift towards more reduced state of glutathione pair observed in the roGFP experiments.

To test whether reduction of Atox1 metal-binding site increases copper delivery to secretory vesicles,copper efflux from differentiated and non-differentiated cells were compared. It was found that copper export from cells increases upon differentiation. Secretion of DBH(dopamine beta-hydroxylase) measured before and after treatment of the cells with BCNU. It was studied that BCNU treatment decreased the amount of secreted DBH suggesting that BCNU treatment decreased copper delivery to the secretory pathaway.

CONCLUSION: It has been technically challenging to study individual redox nodes. However this intensive research on individual redox nodes help us in understanding the correlation between glutathione levels and neuronal differention. This research paves a way for further analysis of cell cycle progression.