The Copper-transporting ATPases, Menkes and Wilson Disease Proteins, Have Distinct Roles in Adult and Developing Cerebellum

Abstract

Copper is essential for brain metabolism, serving as a cofactor to superoxide dismutase, dopamine-β-hydroxylase, amyloid precursor protein, ceruloplasmin, and other proteins required for normal brain function. The copper-transporting ATPases ATP7A and ATP7B play a central role in distribution of copper in the central nervous system; genetic mutations in ATP7A and ATP7B lead to severe neurodegenerative disorders, Menkes disease and Wilson disease, respectively. Although both ATP7A and ATP7B are required, their specific roles and regulation in the brain remain poorly understood. Using high-resolution imaging and functional assays, we demonstrate that ATP7A and ATP7B show cell-specific distribution in adult cerebellum, have distinct enzymatic characteristics, and are regulated differently during development. ATP7B is continuously expressed in Purkinje neurons (PN) where it delivers copper to the ferroxidase ceruloplasmin. ATP7A is a faster copper transporter than Wilson disease protein as evidenced by faster rates of catalytic reactions. The expression of ATP7A switches during development from PN to Bergmann glia, the cells supporting PN function in adult brain. Inactivation of ATP7B (Wilson disease protein) by gene knock-out induces a striking shift in the expression of the ATP7B target protein, ceruloplasmin, from PN to Bergmann glia, where ATP7A (Menkes disease protein) is present. The induced cell-specific change in expression restores copper delivery to ceruloplasmin via ATP7A. Overall, the results provide evidence for distinct functions of ATP7A and ATP7B in the cerebellum and illustrate a tight link between copper homeostasis in PN and Bergmann glia. Copper is essential for brain metabolism, serving as a cofactor to superoxide dismutase, dopamine-β-hydroxylase, amyloid precursor protein, ceruloplasmin, and other proteins required for normal brain function. The copper-transporting ATPases ATP7A and ATP7B play a central role in distribution of copper in the central nervous system; genetic mutations in ATP7A and ATP7B lead to severe neurodegenerative disorders, Menkes disease and Wilson disease, respectively. Although both ATP7A and ATP7B are required, their specific roles and regulation in the brain remain poorly understood. Using high-resolution imaging and functional assays, we demonstrate that ATP7A and ATP7B show cell-specific distribution in adult cerebellum, have distinct enzymatic characteristics, and are regulated differently during development. ATP7B is continuously expressed in Purkinje neurons (PN) where it delivers copper to the ferroxidase ceruloplasmin. ATP7A is a faster copper transporter than Wilson disease protein as evidenced by faster rates of catalytic reactions. The expression of ATP7A switches during development from PN to Bergmann glia, the cells supporting PN function in adult brain. Inactivation of ATP7B (Wilson disease protein) by gene knock-out induces a striking shift in the expression of the ATP7B target protein, ceruloplasmin, from PN to Bergmann glia, where ATP7A (Menkes disease protein) is present. The induced cell-specific change in expression restores copper delivery to ceruloplasmin via ATP7A. Overall, the results provide evidence for distinct functions of ATP7A and ATP7B in the cerebellum and illustrate a tight link between copper homeostasis in PN and Bergmann glia. Copper is an important nutrient for normal brain homeostasis serving as a cofactor of key metabolic enzymes involved in mitochondrial function, neurotransmitter biosynthesis, oxidative stress defense, and iron transport (1Frieden E. Clin. Physiol. Biochem. 1986; 4: 11-19PubMed Google Scholar). It plays additional and poorly understood roles in neuronal myelination (2Prodan C.I. Holland N.R. Wisdom P.J. Burstein S.A. Bottomley S.S. Neurology. 2002; 59: 1453-1456Crossref PubMed Scopus (113) Google Scholar) and in regulation or function of the prion and amyloid precursor proteins (3Multhaup G. Schlicksupp A. Hesse L. Beher D. Ruppert T. Masters C.L. Beyreuther K. Science. 1996; 271: 1406-1409Crossref PubMed Scopus (591) Google Scholar, 4Millhauser G.L. Acc. Chem. Res. 2004; 37: 79-85Crossref PubMed Scopus (359) Google Scholar). Abnormal copper homeostasis is the primary cause of Menkes disease (MNK) 1The abbreviations used are: MNK, Menkes disease; WND, Wilson disease; MNKP, MNK protein; WNDP, WND protein; CP, ceruloplasmin; PN, Purkinje neuron; BG, Bergmann glia; P, postnatal day. and Wilson disease (WND), severe metabolic disorders with marked neurological manifestations (5Scheinberg H. Gitlin J. Science. 1952; 116: 484-485Crossref PubMed Scopus (208) Google Scholar, 6Bearne A.G. Ann. Hum. Genet. 1960; 24: 33-43Crossref PubMed Scopus (85) Google Scholar, 7Menkes J.H. Alter M. Stegleder G.K. Weakley D.R. Sung J.H. Pediatrics. 1962; 29: 764-779PubMed Google Scholar). Despite the essential role of copper, very little is currently known about copper transport in either normal or diseased human brain. As a result, the molecular mechanisms underlying the MNK- and WND-related neurodegeneration remain essentially uncharacterized. MNK and WND are associated with mutations in copper-transporting P-type ATPases ATP7A and ATP7B, respectively (We will refer to ATP7A and ATP7B as Menkes disease protein, MNKP, and Wilson disease protein, WNDP). MNKP and WNDP are central for the regulation of copper distribution in human cells, and their general functions are very similar. Both proteins utilize the energy of ATP hydrolysis to transport copper from the cytosol into the secretory pathway thus participating in the biosynthesis of copper-dependent enzymes. In peripheral tissues, MNKP was shown to transport copper to tyrosinase in melanocytes (8Petris M.J. Strausak D. Mercer J.F.B. Hum. Mol. Genet. 2000; 9: 2845-2851Crossref PubMed Scopus (188) Google Scholar), whereas WNDP delivers copper to a ferroxidase ceruloplasmin in the liver (9Terada K. Nakako T. Yang X.L. Iida M. Aiba N. Minamiya Y. Nakai M. Sakaki T. Miura N. Sugiyama T. J. Biol. Chem. 1998; 273: 1815-1820Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In addition, MNKP and WNDP regulate the intracellular copper concentration by trafficking to either plasma membrane (MNKP) or to vesicles (WNDP) and exporting excess copper from the cells (10Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (541) Google Scholar, 11Roelofsen H. Wolters H. Van-Luyn M.J. Miura N. Kuipers F. Vonk R.J. Gastroenterology. 2000; 119: 782-793Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In the central nervous system, the functional relationship between two copper-transporting ATPases is poorly understood. Current data suggest that MNKP controls the overall copper supply to the brain through its localization in choroid plexus (12Iwase T. Nishimura M. Sugimura H.H.I. Ozsawa F. Shinmura K. Suzuki M. Tanaka M. Kino I. Acta Neuropathol. 1996; 91: 482-488Crossref PubMed Scopus (40) Google Scholar) and also has a biosynthetic role in pituitary gland where it mediates copper delivery to peptidyl-glycine α-amidating monooxygenase (13Stevenson T.C. Ciccotosto G.D. Ma X.M. Mueller G.P. Mains R.E. Eipper B.A. J. Biol. Chem. 2003; 278: 12278-12284Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). What physiological functions the copper-transporting ATPases have in other brain regions and what the relationship is between MNKP and WNDP in either adult or developing brain remain unknown. Spatial and temporal differences in the expression of copper-transporting ATPases have been observed in tissues of mouse embryo (14Kuo Y.M. Gitschier J. Packman S. Hum. Mol. Genet. 1997; 7: 1043-1049Crossref Scopus (80) Google Scholar); however the developmental regulation of WNDP and MNKP has never been examined in detail and never within the CNS. To address these important issues, we have focused our attention on cerebellum, one of the critical regions of the brain expressing both MNKP and WNDP and several copper-requiring proteins. Using fluorescent imaging with a single cell resolution and functional analysis, we demonstrate that two copper-transporting ATPases, MNKP and WNDP, have distinct roles in cerebellum and that the copper distribution pathways in Purkinje neurons and Bergmann glia are tightly linked. These results have important implication for understanding of the Wilson disease and Menkes disease pathologies. Mouse Strains—The generation of Atp7b-/- mice has been described previously (15Buiakova O.I. Xu J. Lutsenko S. Zeitlin S. Das K. Das S. Ross B.M. Mekios C. Scheinberg I.H. Gilliam T.C. Hum. Mol. Genet. 1999; 8: 1665-1671Crossref PubMed Scopus (168) Google Scholar). The Atp7b-/- and wild-type C57BLx129SV/SvEv mice were housed at the OHSU Department of Comparative Medicine according to the National Institutes of Health guidelines on the use of laboratory and experimental animals. Food and water were provided ad libitum. Animals were either perfused with 4% paraformaldehyde for the in situ hybridization studies or euthanized by CO2 inhalation for Western blotting experiments, and the livers and cerebellum were quickly removed. Mice of either sex were used for the experiments. Unless specified, adult wild-type and Atp7b-/- mice were 10 weeks of age. In Situ Hybridization—The brains were removed following perfusion, soaked for 24 h in 4% paraformaldehyde, 20% sucrose, then 30% sucrose and then embedded in Optimal Cutting Temperature compound (Tissue-Tek, Torrance, CA) at -20 °C. Sagittal sections of 20 μm were sliced and placed onto gelatin/poly-l-lysine-coated slides. The slides were treated with 1% hydrogen peroxide (H2O2) in phosphate-buffered saline, blocked with an avidin/biotin blocking kit following manufacturer's instructions (Biomeda, Foster City, CA), and then washed with phosphate-buffered saline/diethyl pyrocarbonate, 100 mm glycine/diethyl pyrocarbonate and 0.3% Triton X-100. Following treatment with 1 μg/ml proteinase K for 15 min, slides were incubated with 4% paraformaldehyde followed by a 10-min incubation with 0.25% anhydride in 0.1 m triethanolamine. For prehybridization, slides were incubated in hybridization solution (2× sodium chloride and sodium citrate solution (SSC), 50% deionized formamide, 1% Sarkosyl, 10% dextran sulfate, 1× Denhardt's solution, 50 mm phosphate buffer, 250 μg/ml yeast tRNA, 500 μg/ml salmon sperm DNA, and 50 μm dithiothreitol at 37 °C for 2 h. After prehybridization, biotin-labeled oligonucleotide probes at 200 ng/ml were added and incubated overnight at 37 °C in a humidity chamber. Excess probes were removed by stringent washes in SSC, blocked in blocking buffer (TBT, PerkinElmer Life Sciences), and incubated with streptavidin-horseradish peroxidase. For signal detection, fluorescein isothiocyanate and cynanine 3 (Cy3) Tyramide Signal Amplification system kits were used (Life Sciences, Rockville, MA). Samples were then mounted using Vectashield with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and analyzed using a Zeiss LSM510 confocal microscope (Carl Zeiss, Gottingen, Germany). Immunohistochemistry—The 20-μm sagittal tissue sections were rehydrated in phosphate-buffered saline, blocked in blocking buffer (5% serum of secondary antibody, 0.2% Triton X-100, 1% bovine serum albumin in phosphate-buffered saline). Slides were then incubated overnight at 4 °C with primary antibody diluted 1:5000 in dilution buffer (0.25% bovine serum albumin, 0.2% Triton X-100 in phosphate-buffered saline). Following washes, slides were incubated for 1 h in fluorescently labeled secondary antibody (Alexa Fluor 488 donkey antirat IgG for anti-WNDP or Alexa Fluor 555 goat anti-rabbit IgG for anti-MNKP or Alexa Fluor 555 donkey anti-goat for anti-ceruloplasmin (CP), Molecular Probes, Eugene, OR). Sections were washed, mounted using Vectashield with 4′,6-diamidino-2-phenylindole, and analyzed using a Zeiss confocal scanning microscope. Expression of WNDP and MNKP in Sf9 Cells—Expression of WNDP in Sf9 cells has been described previously (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). To the expressing MNKP, the MNKP was into the using The was using the kit as described previously (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). Expression of MNKP in Sf9 cells was by using anti-MNKP that were of of MNKP and of WNDP and MNKP using was as described previously (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar) using of the MNKP or WNDP in the 20 mm 200 mm mm buffer on the and ATP of were as (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). For the experiments, the proteins were for 2 with 2 μm and the of was as the ATP was then added to and the was to on at the on The proteins were then and their of was analyzed following and as described previously (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). To copper of catalytic 50 of or membrane were in 200 of buffer 250 μm and incubated on for 15 was for 10 at and the was in 200 of buffer 100 μm 100 μm and as in The was incubated on for 15 and then was as described using with 2 μm for 4 on of and Using Western mm and cerebellum from the wild-type or Atp7b-/- mice were in 500 of the buffer, mm m sucrose, and 1 of Samples were for 10 at 500 The was and the was for at For liver 50 of was and for cerebellum, of membrane was for on were to and using antibody The Expression of MNKP and WNDP in the localization of MNKP and WNDP in cerebellum, we analyzed the distribution of their using in situ hybridization with oligonucleotide probes for MNKP and two probes for WNDP were to the of the were used as a probes for MNKP used or of Bergmann glia, whereas the probes signal In the were in Purkinje neurons from Bergmann glia and PN was using the The marked between the MNKP and WNDP to a specific distribution of MNKP and WNDP in adult used for the in situ hybridization of probes used in fluorescent in situ in a used for the in situ hybridization of probes used for in fluorescent in situ in a was by the experiments. The anti-MNKP antibody cell the molecular as for was also to the of the is to in cerebellum The of WNDP protein in Purkinje neurons was The anti-WNDP antibody PN to the of WNDP with and to the expression of a PN the transporter 2002; PubMed Scopus Google Scholar, J. 1996; PubMed Google Scholar) in the adult cerebellum, MNKP is expressed in Bergmann glia, whereas WNDP is in Purkinje with to a of of the functions of MNKP and WNDP in peripheral tissues is to copper to the secretory pathway for into copper-requiring enzymes. is a copper-dependent is essential for iron homeostasis in the central nervous system Y. H. M. Gitlin S. A. PubMed Scopus Google Scholar, R.J. J. S. J. 2002; PubMed Google Scholar) and is in the cerebellum Gitlin J. Clin. 1996; PubMed Scopus Google Scholar). To of the copper-transporting ATPases is involved in copper delivery for biosynthesis, we the distribution of MNKP and WNDP with that of that in the cerebellum is expressed in Purkinje neurons with a in the cells and the in Bergmann glia of WNDP and that WNDP in observed of MNKP and CP, that the role of MNKP is distinct from that of The Expression of MNKP studies using mice to MNKP expression in Purkinje neurons Y. H. T. N. Nishimura M. Gitschier J. Packman S. Res. 1997; PubMed Scopus Google Scholar) in to our of MNKP to Bergmann glia in the cerebellum of adult of developmental in MNKP To we examined the localization of MNKP in the cerebellum at the and of postnatal development. also the MNKP of distribution with the expression of WNDP at the These of MNKP in developing and adult cerebellum and also differences in the regulation of expression of MNKP and WNDP during development. MNKP is in Purkinje where WNDP is also expressed that the and development of these cells the of both copper-transporting The localization of MNKP and WNDP in Purkinje neurons is also at and at a marked change in MNKP In to Purkinje the MNKP in Bergmann glia and is to Bergmann glia in the adult In WNDP is in PN development and the change in WNDP during development is an in the of an of WNDP of WNDP and MNKP The of both MNKP and WNDP in PN during development to functions of the copper-transporting ATPases in developing associated with their in MNKP and WNDP have distinct for the transport to a regulation of intracellular of the MNKP and WNDP in PN localization of these are to we to the functional of MNKP and studies have that MNKP have than WNDP (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, I. J. Strausak D. Camakaris J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these were using and MNKP I. J. Strausak D. Camakaris J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) WNDP (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google and expression in cell of To these we expressed both MNKP and WNDP in Sf9 cells using and then enzymatic of two copper ATPases in their The key in the MNKP and WNDP enzymatic are and then are with the and of copper from the (16Tsivkovskii R. Eisses J.F. Kaplan J. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, I. J. Strausak D. Camakaris J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Both in and the of these used to the rates of MNKP and that of MNKP by ATP is faster than WNDP and min, The is also faster for MNKP with for WNDP, These results suggest a catalytic for ATP hydrolysis copper by To that the distinct of are to differences in the protein of WNDP and MNKP, we an ATP of catalytic for these two proteins. These that the for ATP is essentially for WNDP and MNKP and that both MNKP and WNDP the catalytic with the for copper, required for of is for WNDP than for MNKP μm with that WNDP copper than MNKP The of WNDP in the of Expression from Purkinje to Bergmann and WNDP are very 50% in the our data demonstrate that these proteins have cell-specific in their developmental expression and have distinct functional the as to or is functional of these copper in the To address we Atp7b-/- knock-out mice that functional WNDP (15Buiakova O.I. Xu J. Lutsenko S. Zeitlin S. Das K. Das S. Ross B.M. Mekios C. Scheinberg I.H. Gilliam T.C. Hum. Mol. Genet. 1999; 8: 1665-1671Crossref PubMed Scopus (168) Google Scholar). that the of WNDP the delivery of copper to in Purkinje neurons for MNKP to the copper delivery to it has to expressed in PN of in Bergmann glia. the that in adult Atp7b-/- the MNKP to Bergmann glia In the distribution of is its expression in Purkinje neurons of wild-type is in Bergmann glia in the Atp7b-/- that expression on the of a functional in the The in the expression of to Bergmann glia was by using and MNKP, or and the Bergmann glia of both MNKP and CP, and of and was observed in the cerebellum of the Atp7b-/- mice PN was that the of copper-transporting by WNDP induces a in the expression of from Purkinje neurons to Bergmann glia, where the other copper MNKP, is It that the change in expression from the neurons to the Bergmann glia copper delivery to via To we the known of to two protein to the and analyzed on a M. Gitlin J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the wild-type liver and cerebellum, the and are both As in the Atp7b-/- liver is copper transport by WNDP is and MNKP is expressed in the In in the cerebellum of the Atp7b-/- is little change in the of with the the of WNDP, copper is to ceruloplasmin, via in copper homeostasis have marked for brain the copper distribution pathways in the normal and diseased brain remain essentially uncharacterized. results on a functional relationship between two copper MNKP and WNDP, in the that MNKP and WNDP are both in adult cerebellum, and their functions in of the brain are is on cell-specific localization of MNKP and WNDP Bergmann glia and Purkinje of the copper-requiring with WNDP, with MNKP, and distinct functional and developmental regulation of the that the function of WNDP in cerebellum is to copper to the copper-dependent ferroxidase CP, whereas the function of MNKP is to regulate copper concentration in Bergmann glia and also to copper for delivery to Purkinje The localization of MNKP in Bergmann glia is a and important It has been by two situ hybridization and with a single cell in to localization in cell we also MNKP in peripheral of The peripheral localization of protein in to Res. Mol. Res. 2000; PubMed Scopus Google Scholar), as of the MNKP protein is in the cell The of MNKP in is to by the previously localization of MNKP in PN Y. H. Y. M. T. J. 1998; PubMed Scopus Google Scholar) to developmental in the expression of protein, we in the The in expression of MNKP from PN to of the of between and PN is A. A. M. D. J. Google Scholar). In the adult cerebellum, PN the of PN and to these cells A. A. M. D. J. Google Scholar, A. T. J. K. M. A. T. M. M. Y. S. A. 2000; PubMed Scopus Google Scholar). copper is to PN is currently it is to that MNKP in Bergmann glia copper for by other cells, by the role of MNKP in where the role of MNKP is to copper for by other the 2 weeks MNKP is in PN with both ATPases are in developing neurons is a of the functional of MNKP and WNDP The that MNKP is a faster copper it both catalytic and faster than we that MNKP plays a primary role of excess important in functional of MNKP and WNDP is their for The of WNDP for copper is than that for MNKP, in where is essentially copper J. J. 2004; Scopus Google Scholar). The and of WNDP for copper are with its biosynthetic function. The delivery to the secretory pathway on the of WNDP for copper It is important to the relationship between of MNKP and WNDP and their intracellular functions is an our results to specific and roles of MNKP and WNDP in the wild-type it that in the Atp7b-/- we observed functional of WNDP by The of copper delivery to in the cerebellum of these mice as a of a striking shift in expression from Purkinje neurons to Bergmann glia, that functional of copper in these Inactivation of WNDP, is associated with copper in the cytosol and copper delivery to in the is to Bergmann glia in of the from Purkinje neurons either of of expression or in the the of and is an important of our is an essential metabolic plays an important role in iron transport in the central nervous system Y. H. M. Gitlin S. A. PubMed Scopus Google Scholar, R.J. J. S. J. 2002; PubMed Google Scholar). in in marked of iron in Purkinje neurons and neurodegeneration H. 2003; PubMed Scopus Google Scholar). The of and MNKP in Bergmann glia in Atp7b-/- mice restores copper delivery to ceruloplasmin, for the of WNDP and the function. The of marked neurological in the cerebellum of the Atp7b-/- mice and manifestations in the disease to the of functional for the generation of the anti-WNDP antibody, Eipper for the of the anti-MNKP antibody used for experiments, and for the antibody and for the MNKP and and for and with the imaging of Lutsenko and and for the and