Copper efflux from murine microvascular cells requires expression of the menkes disease Cu-ATPase

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Copper transport is vital for brain function.
• Menkes disease is linked to mutations in the copper-transporting ATPase gene (Atp7a).
• Previous studies linked Atp7a to copper transport in PC12 and C6 glioma cells.

Purpose of the Study:

• To investigate the presence and function of the copper-transporting ATPase (Atp7a) in cerebrovascular endothelial (CVE) cells of the blood-brain barrier (BBB).
• To determine if Atp7a in CVE cells plays a role in regulating copper penetration into the brain.
• To explore the potential link between Atp7a dysfunction in CVE cells and low brain copper levels observed in Menkes disease.

Main Methods:

• Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify Atp7a cDNA fragments from murine CVE cells.
• Sequencing of amplified cDNA fragments to confirm homology with mouse Atp7a.
• Functional analysis using the sulfhydryl reagent p-chloromercuribenzoate (p-CMB) to assess copper efflux inhibition.

Main Results:

• Atp7a gene expression was confirmed in murine CVE cells.
• Amplified cDNA fragments showed high sequence homology (99.7-100%) with mouse Atp7a, covering critical domains (heavy metal binding and C-terminus).
• Copper efflux from CVE cells was inhibited by p-CMB, suggesting functional Atp7a activity.

Conclusions:

• A functional copper-transporting ATPase (Atp7a) is present in the blood-brain barrier (BBB) and controls copper entry into the brain.
• Defects in Atp7a within cerebrovascular endothelial cells are a likely primary cause of reduced brain copper levels in Menkes disease.