Regulation of copper transport crossing brain barrier systems by Cu-ATPases: effect of manganese exposure

Abstract

Regulation of cellular copper (Cu) homeostasis involves Cu-transporting ATPases (Cu-ATPases), i.e., ATP7A and ATP7B. The question as to how these Cu-ATPases in brain barrier systems transport Cu, i.e., toward brain parenchyma, cerebrospinal fluid (CSF), or blood, remained unanswered. This study was designed to characterize roles of Cu-ATPases in regulating Cu transport at the blood-brain barrier (BBB) and blood-CSF barrier (BCB) and to investigate how exposure to toxic manganese (Mn) altered the function of Cu-ATPases, thereby contributing to the etiology of Mn-induced parkinsonian disorder. Studies by quantitative real-time RT-PCR (qPCR), Western blot, and immunocytochemistry revealed that both Cu-ATPases expressed abundantly in BBB and BCB. Transport kinetic studies by in situ brain infusion and ventriculo-cisternal (VC) perfusion in Sprague Dawley rat suggested that the BBB was a major site for Cu entry into brain, whereas the BCB was a predominant route for Cu efflux from the CSF to blood. Confocal evidence showed that the presence of excess Cu or Mn in the choroid plexus cells led to ATP7A relocating toward the apical microvilli facing the CSF, but ATP7B toward the basolateral membrane facing blood. Mn exposure inhibited the production of both Cu-ATPases. Collectively, these data suggest that Cu is transported by the BBB from the blood to brain, which is mediated by ATP7A in brain capillary. By diffusion, Cu ions move from the interstitial fluid into the CSF, where they are taken up by the BCB. Within the choroidal epithelial cells, Cu ions are transported by ATP7B back to the blood. Mn exposure alters these processes, leading to Cu dyshomeostasis-associated neuronal injury.

Keywords: Cu-ATPases; blood-CSF barrier; blood-brain barrier; copper; copper transport; manganese.

FIG. 1.

Comparison of Atp7a and Atp7b mRNA expression levels in control brain capillaries and RBE4 cells. Relative mRNA levels of Atp7a and Atp7b in control brain capillaries and RBE4 cells were quantified by qPCR and expressed as the ratios of Atp7a/Gapdh or Atp7b/Gapdh. The data are representative of triplicate experiments. Data represent mean ± SD, n = 5–6; **p < 0.01.

FIG. 2.

Decrease in mRNA and protein expression levels of ATP7A and ATP7B in the in vivo brain capillary and in vitro RBE4 cells following Mn exposure. (I) In vivo subchronic exposure study: rats received either daily ip injection of 6 mg Mn/kg or saline (as control), 5 days per week, for 4 weeks. (A and B) Atp7a and Atp7b mRNA expression levels in the brain capillary were quantified by qPCR and expressed as the ratios of Atp7a/Gapdh or Atp7b/Gapdh. The data are representative of triplicate experiments. Data represent mean ± SD, n = 5; *p < 0.05, as compared with controls. (C and D). Representative Western blot autographs of Atp7a and Atp7b in the brain capillary. (E and F). Quantification of Western blot densitometry and statistical analysis. Ct: control group; Mn: Mn-exposed group. Data represent mean ± SD, n = 3; **p < 0.01, as compared with controls. (II) In vitro studies with RBE4 cells: (A and B) Atp7a and Atp7b mRNA expression levels in RBE4 cells were quantified by qPCR and expressed as the ratios of Atp7a/Gapdh or Atp7b/Gapdh. The data are representative of triplicate experiments. Data represent mean ± SD, n = 6; **p < 0.01, as compared with controls. (C and D) Representative Western blot autographs of Atp7a and Atp7b in RBE4 cells. (E and F) Quantification of Western blot densitometry and statistical analysis. Ct: control group; Mn: Mn-exposed group. Data represent mean ± SD, n = 3; **p < 0.01, as compared with controls.

FIG. 3.

Subcellular distribution of ATP7A and ATP7B in RBE4 cells following in vitro Mn exposure. (A) ATP7A localization in RBE4 cells. (a and d) ATP7A fluorescent signals; (b and e) DIC images; (c and f) merged images. (B) ATP7B localization in RBE4 cells. (a and d) ATP7B fluorescent signals; (b and e) DIC images; (c and f) merged images.

FIG. 4.

Comparison of Atp7a and Atp7b mRNA expression levels in freshly isolated choroid plexus tissues and choroidal Z310 cells. Relative mRNA levels of Atp7a and Atp7b in the control choroid plexus and Z310 cells were quantified by qPCR and expressed as the ratios of Atp7a/Gapdh or Atp7b/Gapdh. The data are representative of triplicate experiments. Data represent mean ± SD, n = 5–6; **p < 0.01.

FIG. 5.

Decreased mRNA and protein levels of ATP7A and ATP7B in the in vivo choroid plexus tissues and in vitro choroidal Z310 cells following Mn exposure. (I) In vivo subchronic exposure study: (A and B) Atp7a and Atp7b mRNA expression levels in the choroid plexus were quantified by qPCR and expressed as the ratios of Atp7a/Gapdh or Atp7b/Gapdh. The data are representative of triplicate experiments. Data represent mean ± SD, n = 6; *p < 0.05, **p < 0.01 as compared with controls. (C and D) Representative Western blot autographs of Atp7a and Atp7b in plexus tissues. (E and F) Quantification of Western blot densitometry and statistical analysis. Ct: control group; Mn: Mn-exposed group. Data represent mean ± SD, n = 3; *p < 0.05, as compared with controls. (II) In vitro studies with Z310 cells: (A and B) Relative mRNA levels of Atp7a and Atp7b were quantified by qPCR and expressed as the ratios of Atp7a/Gapdh or Atp7b/Gapdh. The data are representative of triplicate experiments. Data represent mean ± SD, n = 6; *p < 0.05, **p < 0.01, as compared with controls. (C and D) Representative Western blot autographs of ATP7A and ATP7B in Z310 cells. (E and F) Quantification of Western blot densitometry and statistical analysis. Ct: control group; Mn: Mn-exposed group. Data represent mean ± SD, n = 3; **p < 0.01, as compared with controls.

FIG. 6.

Subcellular trafficking of ATP7A and ATP7B in the choroid plexus. (A) Subcellular trafficking of ATP7A in freshly isolated choroid plexus tissues following in vitro Cu incubation. (a, d, and g) ATP7A fluorescent signals; (b, e, and h) DIC images; (c, f, and i) merged images. (B) Subcellular trafficking of ATP7B in freshly isolated choroid plexus tissues following in vitro Cu incubation. (a, d, and g) ATP7B fluorescent signals; (b, e, and h) DIC images; (c, f, and i) merged images. (C) Subcellular trafficking of ATP7A in choroid plexus tissues following in vivo subchronic Mn exposure. (a and d) ATP7A fluorescent signals; (b and e) DIC images; (c and f) merged images. (D) Subcellular trafficking of ATP7B in choroid plexus tissues following in vivo subchronic Mn exposure. (a and d) ATP7B fluorescent signals; (b and e) DIC images; (c and f) merged images. Arrow head: pointing the apical membrane of choroidal epithelia facing the CSF; arrow: pointing the basolateral membrane of choroidal epithelia facing the blood

FIG. 7.

Subcellular distributions of ATP7A and ATP7B in choroidal epithelial Z310 cells following in vitro Mn exposure. (A) ATP7A studies. (a and d) ATP7A fluorescent signals; (b and e) DIC images; and (c and f) merged images. (B) ATP7B studies. (a and d) ATP7B fluorescent signals; (b and e) DIC images; (c and f) merged images. Ct: control group, Mn: Mn-exposed group.

FIG. 8.

Decreased Cu clearance by the BCB following in vivo subchronic Mn exposure. (A) % ¹⁴C radioactivity in the CSF outflow. (B) % ⁶⁴Cu radioactivity in the CSF outflow. (C) The data of last five time points of the outflow radioactivity during steady-state were used to quantify the steady state of ¹⁴C, ⁶⁴Cu, and ⁶⁴Cu/¹⁴C in the outflow of CSF. Data represent means ± SD, n = 3; *p < 0.05, when compared with the control.