Disruption of copper homeostasis due to a mutation of Atp7a delays the onset of prion disease

Abstract

Copper influences the pathogenesis of prion disease, but whether it is beneficial or detrimental remains controversial. Copper homeostasis is also essential for normal physiology, as highlighted by the spectrum of diseases caused by disruption of the copper transporting enzymes ATP7A and ATP7B. Here, by using a forward genetics approach in mice, we describe the isolation of three alleles of Atp7a, each with different phenotypic consequences. The mildest of the three, Atp7a^brown, was insufficient to cause lethality in hemizygotes or mottling of the coat in heterozygotes, but did lead to coat hypopigmentation and reduced copper content in the brains of hemizygous males. When challenged with Rocky Mountain Laboratory scrapie, the onset of prion disease was delayed in Atp7a^brown mice, and significantly less proteinase-resistant prion protein was found in the brains of moribund Atp7a^brown mice compared with WT littermates. Our results establish that ATP7A-mediated copper homeostasis is important for the formation of pathogenic proteinase-resistant prion protein.

Prion diseases are fatal infectious neurodegenerative diseases caused by the misfolding and aggregation of the cellular prion protein, PrP, into the β-sheet–rich, proteinase-resistant, disease-associated form, PrP^res. Diseases caused by prions include Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep, and chronic wasting disease in cervids, yet the normal function of cellular prion protein remains a mystery. PrP is known to bind copper, and copper can induce expression of the PrP-encoding gene Prnp (1–4). Histidine residues within the highly conserved octarepeat of PrP bind copper (5–8), and additional copies of the octarepeat within PrP are associated with early-onset prion disease. Octarepeat expansion can increase the copper-binding affinity of PrP by as much as 10-fold, suggesting that copper plays a significant role in prion pathogenesis (8). Copper also binds to histidines within region 95–115 of PrP.

The role of copper in prion disease pathogenesis is complex, and interpretations to date have been contradictory. Binding of copper to PrP destabilizes the native α-helical PrP and leads to an increase in β-sheet structure and conversion to the proteinase-resistant form (9–13), yet it has been reported that copper inhibits PrP^res amplification (14). Copper is known to promote prion infectivity (15), yet it is also important in limiting neuropathology caused by reactive oxygen species (16). Although copper chelation delays disease onset in mice (17), similar delays are observed in mice and hamsters maintained on a high-copper diet (18, 19). Mice fed a copper-depleted diet are also reported to be more sensitive to prion pathogenesis following i.p. inoculation (18).

The physiological consequences of copper deficiency were first recognized among a group of sheep in western Australia, which were noticed for their “stringy” coats, anemia, and the fact that they consistently gave birth to ataxic lambs (20). The affected ewes were subsequently found to be copper-deficient, and drenching them with copper sulfate solution would reverse anemia, improve their coat condition, and prevent ataxia in their lambs (21). A similar link was established many years later in man, when patients with Menkes disease, an X-linked neurodegenerative condition, were discovered to be copper-deficient (22).

Menkes disease is caused by mutation of ATP7A, which encodes the P1B-type ATPase ATP7A (23). Along with the closely related ATPase ATP7B, ATP7A is a critical transporter of copper. Mutation of ATP7A is known to cause at least three distinct disorders (24), including Menkes disease (25–27), occipital horn syndrome (considered to be a milder form of Menkes disease) (28), and isolated distal motor neuropathy (29). Conversely, mutation of ATP7B causes Wilson disease (30, 31), which is characterized by neuronal and hepatic pathologic processes associated with copper accumulation.

Copper homeostasis is essential for two reasons. First, copper is an essential cofactor for enzymes such as superoxide dismutase, dopamine hydroxylase, tyrosinase, and lysyl oxidase, accounting for the characteristic defects in neurological function, pigmentation, and connective tissue formation seen in Menkes disease (32). Conversely, copper can promote the production of reactive oxygen species, and is therefore highly toxic in excess.

Here we describe the isolation of an Atp7a mutant mouse strain with an inherited copper deficiency. In hemizygous males, a 60% reduction in cerebral copper content was associated with delayed PrP^res conversion and prion pathogenesis.

Results

Positional Cloning of brown Phenotype.

During the course of a C57BL/6J mouse mutagenesis program, we identified a male G3 mouse with a brown coat. This phenotype, designated simply as brown, was not seen in either parent, suggesting that it was inherited as an autosomal or X-linked recessive trait (ref. 33; http://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=469). To determine the chromosomal location of the causative mutation, the index brown male was outcrossed to C3H/HeN females, and then backcrossed to his F1 daughters. Among a total of 17 offspring, six were phenotypically normal (one female, five male) and 11 shared the coat color phenotype of their sire (seven female, four male). Each mouse was genotyped at 128 polymorphic markers across the genome, and individual genotypes were tabulated to calculate logarithm (base 10) of odds scores at each marker. This revealed a single peak score of 3.86 at DXMit172, flanked by the distal marker DXMit114 and proximal marker DXMit121 (Fig. 1 A and B). Within this 62.5-Mb interval lay 358 protein-coding and 22 miRNA genes, three of which had previously been described to affect coat pigmentation when mutated in the mouse (Atp7a, Eda, and Htr2c). As a result of an absence of ectodermal dysplasia phenotypes [commonly associated with Eda mutants (34)] or postnatal growth retardation [as seen in an Htr2c mutant (35)], all coding exons and splice junctions of Atp7a were sequenced. A single T-to-C missense transition was detected at X:103283830, corresponding to position 1570 of the Atp7a transcript, in exon 5 of 23 total exons (Fig. 1C). The mutation is predicted to result in an isoleucine to threonine substitution at amino acid 483 of the ATP7A protein.

Fig. 1.

A hemizygous viable allele of Atp7a. (A) Linkage mapping of the brown mutation to chromosome X. (B) Haplotype groups of affected and unaffected F2 progeny, isolating the brown mutation to distal chromosome X. (C) DNA sequence chromatogram indicates the mutated nucleotide in Atp7a. (D) Sequence chromatograms of two independent alleles of Atp7a: Tigrou and Tigrou-like. (E) Comparison of the coat color phenotypes of three Atp7a mutants. (F) Predicted relationship between ATP7A activity and tyrosinase-mediated pigmentation. (G) Predicted ATP7A topology and domain structure, indicating the relative positions of each mutation. MBD1 to MBD6 represent the N-terminal metal binding domains, with the copper-binding cysteines indicated. Critical residues thought to bind copper in transmembrane domains 6, 7, and 8 are shown. The canonical actuator (marked as “A”) domain and phosphorylation (“P”) domain residues are also indicated. The dileucine (LL) and PDZ (PSD-95/Discs-large/ZO-1) binding motif (PBM) in the C-terminal tail are necessary for subcellular localization of ATP7A. Conversion of ATP to ADP by the nucleotide-binding (“N”) domain leads to phosphorylation of the P-domain and copper transport. The A-domain dephosphorylates the P-domain. Numbers 1, 2, and 3 indicate the order of copper binding and transport.

Allelic Series of Atp7a Mutations.

Although not the case for Atp7a^brown, heterozygous Atp7a mutants typically present with variegated coat color, a consequence of random X-inactivation in melanocyte precursors (36). Examples of this phenotype were previously observed in our mutagenesis program, and given their similarity to the classic mottled mutants (37, 38), were sequenced at the Atp7a locus. The mutants Tigrou and Tigrou-like were both found to harbor Atp7a mutations (Fig. 1D), respectively creating a premature termination codon at position 469, and an alanine-to-valine substitution at codon 998. Hemizygous Tigrou males were occasionally observed, lacked pigmentation (except at their extremities), and died within 3 wk of birth (Fig. 1E). Tigrou-like appeared to be entirely lethal in utero, as hemizygous males were not observed postpartum. Similar to Tigrou-like and Tigrou, the vast majority of Atp7a alleles described in mouse are hemizygous lethal in utero or shortly after birth (39). Atp7a^Blotchy and the Pewter series of alleles are all hemizygous viable, yet, unlike brown, cause variegated pigmentation in heterozygous form, making brown a uniquely mild allele in the Atp7a allelic series.

Based on the pigmentation phenotype of heterozygotes and lethality in hemizygotes, the three alleles could be arranged in a decreasing order of functional severity, with Tigrou-like predicted to cause the most severe copper deficiency and brown the least (Fig. 1F; protein schematic shown in Fig. 1G).

Although one might expect the Tigrou nonsense allele to be more detrimental than the A998V missense mutation of Tigrou-like, skipping of a nonsense mutation-harboring exon has been observed in a patient with Menkes disease (40). In the case of Tigrou, such a splice product would remove exon 5 yet remain in frame, but would do so at the expense of the fifth metal binding domain. Nevertheless, the effects of each missense mutation were consistent with PolyPhen-2 predictions, with respective scores of 1 and 0.476 for Tigrou-like and brown.

Reduced Quantities of Copper and Delayed Onset of Prion Disease.

The brains of Atp7a^brown/Y and Atp7a^+/Y littermates were analyzed for copper content. The amount of copper in the brains of Atp7a^brown/Y mice was reduced by 60% compared with Atp7a^+/Y littermates [1.6 ± 0.2 μg Cu²⁺/g of brain (mean ± SEM) vs. 3.9 ± 0.4 μg Cu²⁺/g; P < 0.0001; Fig. 2A].

Fig. 2.

Reduced copper content in Atp7a^brown mutant brains is associated with prolonged survival after intracranial RML scrapie infection. (A) The amount of copper in the brains of uninfected Atp7a^+/Y and Atp7a^brown/Y littermates was determined by ICP-MS (n = 5; error bars represent SEM). (B) Atp7a^+/Y and Atp7a^brown/Y mice were inoculated intracranially with RML scrapie and killed when clinical signs of prion disease were observed (n = 10). (C) The presence of PrP^res in the brains of clinically ill Atp7a^+/Y and Atp7a^brown/Y mice was confirmed by Western blot (n = 3). Similar results to those shown were obtained for all other samples analyzed in each experiment.

Atp7a^brown/Y and Atp7a^+/Y littermates were inoculated intracranially with the Rocky Mountain Laboratory (RML) strain of mouse scrapie. All 10 Atp7a^+/Y mice became ill with clinical signs of scrapie, including altered gait, kyphosis, ataxia, disorientation, and lethargy within 161 d after infection. In contrast, only one of 10 Atp7a^brown/Y mice developed disease within the same period. The remaining Atp7a^brown/Y mice became terminally ill by 180 d after infection (P < 0.0001; Fig. 2B). Moribund mice from both groups were killed, and their brains were harvested for detection of abnormally folded PrP^res protein (Fig. 2C). Proteinase K-resistant PrP was detected by Western blot in the brains of clinically ill Atp7a^+/Y (Fig. 2C, lanes 6, 8, and 10) and Atp7a^brown/Y mice (Fig. 2C, lanes 12, 14, and 16), confirming prion disease. No proteinase K-resistant PrP was detected in the brains of uninfected Atp7a^+/Y or Atp7a^brown/Y mice (Fig. 2C, lanes 2 and 4).

Histological analysis showed that the brains of clinically ill Atp7a^brown/Y and Atp7a^+/Y mice displayed spongiosis typical of transmissible spongiform encephalopathies, particularly in the cerebellum (Fig. 3, Top). Brain sections from both RML scrapie-infected Atp7a^+/Y and Atp7a^brown/Y mice displayed considerable loss of MAP2-immunoreactive dendrites in the neocortex and hippocampus (Fig. 3, Middle), most apparent in the CA1–CA3 regions, suggestive of selective damage to pyramidal neurons. Analysis of reactive astrocytes by GFAP staining showed that both groups of RML scrapie-infected mice also showed extensive astrogliosis (Fig. 3, Bottom). Consistent with the widespread neurodegenerative alterations observed by MAP2 and GFAP staining, analysis of neuronal cells with NeuN showed rarefaction and loss of neurons in the CA1–CA3 regions in both RML scrapie-infected groups, and was accompanied by microgliosis as determined by Iba1 staining (not shown).

Fig. 3.

Normal spongiosis, dendrite loss, and astrogliosis in the brains of RML scrapie-infected Atp7a^brown mutants. Brain sections from uninfected Atp7a^+/Y, RML scrapie-infected Atp7a^+/Y, and RML scrapie-infected Atp7a^brown/Y mice killed when clinically ill were stained with H&E, antibodies against the dendrite marker MAP2, and antibodies against the astroglial marker GFAP. Arrows indicate areas of MAP2 staining and highlight dendrite loss in the RML scrapie-infected sections. Similar results to those shown were obtained for all other samples in each group (n = 3).

However, substantially less PrP^res was detected by immunohistochemistry in the brains of clinically ill Atp7a^brown/Y mice compared with those of Atp7a^+/Y (Fig. 4A). Upon further analysis, when equal amounts of protein from the brains of clinically ill mice were analyzed by Western blot (Fig. 4B), 41% less total PrP (P < 0.01; Fig. 4C) and 50% less PrP^res (P < 0.01; Fig. 4D) was detected in the brains of infected Atp7a^brown/Y mice compared with those of Atp7a^+/Y. No significant difference was seen between the amount of PrP in the brains of uninfected Atp7a^brown/Y and Atp7a^+/Y mice (Fig. 4 B and C).

Fig. 4.

Reduced PrP and PrP^res in the brains of RML scrapie-infected Atp7a^brown mutants. (A) Brain sections from clinically ill RML scrapie-infected Atp7a^+/Y and Atp7a^brown/Y mice were stained for proteinase K-resistant PrP (i.e., PrP^res; n = 3). Low-magnification (Upper) and high-magnification (Lower) images are shown. (B) Representative Western blots of PrP and PrP^res levels in the brains of uninfected and RML scrapie-infected Atp7a^+/Y (lanes 1–4) and Atp7a^brown/Y (lanes 5–8) mice. (C and D) Quantification of total PrP (C) and PrP^res (D) in the brains of uninfected and RML scrapie-infected Atp7a^+/Y and Atp7a^brown/Y mice as determined by Western blot by using GE ImageQuant software (n = 5; error bars represent SEM). Similar results to those shown were obtained for all other samples analyzed in each experiment.

In summary, we observed widespread degeneration in the cortex, hippocampus, basal ganglia, and cerebellum of RML scrapie-infected Atp7a^+/Y and Atp7a^brown/Y mice. The degree of neuronal loss, astrocytosis, and microgliosis in infected Atp7a^brown/Y mice was equivalent to that observed in infected Atp7a^+/Y mice, yet the amount of PrP^res in the brains of Atp7a^+/Y mice was significantly higher than in their Atp7a^brown/Y littermates, as measured by Western blotting and immunohistochemistry.

Discussion

This report describes the characterization of a hypomorphic allele of the Menkes disease gene orthologue, Atp7a, characterized by coat hypopigmentation and an insufficiency of copper in the brain. The Atp7a^brown mutation significantly increased the incubation period of intracranial RML scrapie infection, supporting earlier reports that copper chelation can delay the onset of scrapie disease (17). Atp7a^brown mutant mice had twice the reduction in brain copper content compared with mice treated with copper chelation, with the incubation period extended in a commensurate manner (10% longer vs. 7% after chelation) (17).

In addition to the delay in disease onset, the amount of PrP^res in the brains of clinically ill Atp7a^brown/Y mice was significantly lower than that of clinically ill Atp7a^+/Y mice by Western blot and immunohistochemistry, consistent with previous reports implicating copper in the conversion of PrP to PrP^res (17). Nevertheless, the amount of neuronal loss and astrocytosis was similar in both groups in the cortex, hippocampus, cerebellum, and brainstem, indicating that residual PrP^res remains capable of causing neurodegeneration and death. The existence of proteinase-sensitive infectious prion protein (PrP^Sc) has been reported by others, and copper is known to influence the proteinase resistance of PrP (41–43). It is possible that, although accumulated PrP^Sc in a copper-deficient environment may be more sensitive to digestion by proteinases, it may remain toxic enough to cause disease. Equivalent pathology might also be a result of the reduced activity of neuroprotective copper-dependent enzymes such as SOD1. Others have previously reported that copper induces the expression of PrP in vivo (3, 4), yet we observed no difference in the amount of PrP in the brains of uninfected Atp7a^brown/Y and Atp7a^+/Y mice.

Our results are consistent with earlier reports of copper-induced PrP proteinase resistance (6, 7, 44) and the delay of disease onset by copper chelation (17). Although copper chelation or an Atp7a mutation can delay the onset of prion disease, it cannot prevent it, even given the 60% reduction in brain copper observed in Atp7a^brown mice. A reduction as great as this also has the potential to cause clinically undesirable side effects, and, as such, copper chelation therapy in humans should be approached cautiously.

Materials and Methods

Mice and Positional Cloning.

Atp7a^brown, Atp7a^Tigrou, and Atp7a^Tigrou-like alleles were all generated on a pure C57BL/6J background by N-ethyl-N-nitrosourea mutagenesis as previously described (45). The index brown mutant (C57BL/6J, male) was outcrossed to C3H/HeN females (Taconic), and F1 daughters were backcrossed to their father. Mice were grouped into mutant and WT cohorts (11 and six mice, respectively) based on coat color. Individual mice were typed at 128 microsatellite markers spaced across the genome (46), with expected and observed genotype frequencies used to calculate logarithm of odds scores at each position. Atp7a gene amplicons from WT, brown, Tigrou, and Tigrou-like genomic DNA were sequenced by using an ABI 3730xl capillary sequencer. The deleterious effect of Atp7a mutations were predicted by using the HumDiv-trained PolyPhen-2 server (version 2.1.0) (47). C57BL/6J males used for mutagenesis were obtained from The Jackson Laboratory. All other C57BL/6J mice were obtained from The Scripps Research Institute breeding colony. All animal procedures were in accordance with guidelines of the institutional animal care and use committee of The Scripps Research Institute.

Scrapie Inoculation.

Mice were inoculated intracranially with 30 μL of a 2% (wt/vol) RML C57BL/6J mouse scrapie brain homogenate in PBS solution. Inoculated mice were monitored daily for clinical signs of scrapie, including weight loss, lethargy, ataxia, and paralysis. Mice showing severe clinical signs of scrapie were killed, and brains were taken for biochemical and histological analysis.

Western Blotting.

Brains from scrapie-infected and noninfected control mice were homogenized in PBS solution at 100 mg of tissue/mL. Homogenate was then diluted with an equal volume of 1% Triton X-100 (Sigma) in PBS solution, sonicated, and cleared by centrifugation at 500 × g for 15 min. For diagnostic purposes, an equal volume of 5% brain homogenate from each mouse was untreated or treated with 50 μg/mL of proteinase K in 1% Triton X-100 solution for 1 h at 37 °C. For quantitative purposes, total protein concentration of each sample was determined by bicinchoninic acid assay (Pierce), and 20 μg of total protein from each sample was untreated or treated with 50 μg/mL of proteinase K. Reactions were stopped by adding an equal volume of 2× SDS loading buffer (Invitrogen) and heating to 95 °C for 5 min. The samples were then run on a 16% Tris-glycine gel (Invitrogen) and transferred to Invitrolon PVDF membrane (Invitrogen). The membrane was blocked and all antibodies were diluted in 5% nonfat milk. Human anti-PrP antibody D13 was used at 3 μg/mL to detect PrP, followed by peroxidase-conjugated goat anti-human antibody (Jackson ImmunoResearch) used at a 1:10,000 dilution. For quantitative Western blots, a mouse anti–α-tubulin antibody was used at 1:10,000, followed by peroxidase-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) used at 1:5,000. Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and exposed to X-ray film or imaged by using a GE ImageQuant LAS 4000. Signal intensities were analyzed with GE ImageQuantTL software.

Analysis of Brain Copper Content.

Brains were analyzed individually for copper content using inductively coupled plasma mass spectroscopy (ICP-MS). Whole brain was homogenized in ddH₂O at 100 mg of tissue/mL. The tissue homogenate was cleared by ultrasonication and centrifugation at 500 × g for 15 min. One milliliter of cleared brain homogenate was digested with 3 mL of 70% nitric acid by using a microwave acid digestion vessel (Parr). Each sample was microwaved for 30 s at full power in a 1,200-W household microwave oven and allowed to cool for 30 min before opening. Separate microwave digestion vessels were used for mutant and WT samples. The digested sample was then diluted 1:2 with ddH₂O before analysis. Standard concentrations of Cu used were 0, 10, 25, 50, 75, 100, and 200 parts per billion. Each standard and sample contained 100 parts per billion yttrium as an internal standard. Each sample was analyzed three times, with 2% (vol/vol) nitric acid run for 2 min between each sample. The ICP-MS was recalibrated, and standards were rerun after every 15 samples.

Histology and PrP^res Immunohistochemistry.

Brain hemispheres were fixed in 4% paraformaldehyde containing zinc for a minimum of 24 h before being dehydrated, embedded in paraffin wax, cut into 6-μm sections, and placed on glass slides. Sections were deparaffinized with xylene and rehydrated with ethanol and water. For H&E staining, sections were stained with Shandon hematoxylin (Thermo Scientific) and differentiated in 1% HCl (in 70% isopropanol). Hematoxylin-stained sections were blued with Scott tap water substitute before being stained with Shandon eosin (Thermo Scientific), dehydrated with ethanol, cleared with xylene, and mounted with Permount. For PrP^res immunohistochemistry, antigen retrieval was performed by autoclaving in PBS solution for 12 min at 250 °F. Proteinase-sensitive PrP was removed by treatment with 50 μg/mL of proteinase K for 20 min at room temperature. The sections were then blocked with 10% normal goat serum overnight, avidin–biotin blocked (Vector Laboratories), and incubated in 3% hydrogen peroxide for 20 min. PrP was detected with 3 μg/mL antibody D13, biotinylated anti-human IgG (Jackson ImmunoResearch), and avidin-conjugated peroxidase (Jackson ImmunoResearch). After PrP staining, sections were counterstained with Shandon hematoxylin and Scott tap water substitute. The sections were dehydrated with ethanol and xylene and mounted using Permount (Fisher Scientific).

MAP2, GFAP, NeuN, and Iba1 Immunohistochemistry.

Vibratome sections (40 μm) were immunolabeled overnight with antibodies against the neuronal marker NeuN (1:1,000; Millipore), the astroglial marker GFAP (1:500; Millipore), the dendritic marker MAP2 (1:200; Millipore), and the microglial cell marker Iba1 (1:500; Wako), followed by incubation with species-appropriate secondary antibodies conjugated to HRP or FITC (1:2,000; Vector Laboratories). Sections labeled with HRP (NeuN, Iba1, and GFAP) were incubated with DAB and analyzed with a bright-field digital Olympus microscope. Signal intensities were assessed by optical density measurements by using the Image Quant 1.43 program (National Institutes of Health) and corrected against background. FITC-labeled sections (i.e., MAP2) were analyzed with an MRC1024 laser scanning confocal microscope (Bio-Rad).