Altered ATP7A expression and other compensatory responses in a murine model of Menkes disease

Abstract

Mutations in the copper-transporter ATP7A lead to severe neurodegeneration in the mottled brindled hemizygous male (MoBr/y) mouse and human patients with Menkes disease. Our earlier studies demonstrated cell-type- and -stage-specific changes in ATP7A protein expression during postnatal neurodevelopment. Here we examined copper and cuproenzyme levels in MoBr/y mice to search for compensatory responses. While all MoBr/y neocortical subcellular fractions had decreased copper levels, the greatest decrease (8-fold) was observed in cytosol. Immunostaining for ATP7A revealed decreased levels in MoBr/y hippocampal pyramidal and cerebellar Purkinje neurons. In contrast, an upregulation of ATP7A protein occurred in MoBr/y endothelial cells, perhaps to compensate for a lack of copper in the neuropil. MoBr/y astrocytes and microglia increased their physical association with the blood-brain barrier. No alterations in ATP7A levels were observed in ependymal cells, arguing for specificity in the alteration observed at the blood-brain barrier. The decreased expression of ATP7A protein in MoBr/y Purkinje cells was associated with impaired synaptogenesis and dramatic cytoskeletal dysfunction. Immunoblotting failed to reveal any compensatory increase in levels of ATP7B. While total levels of several cuproenzymes (peptide-amidating monooxygenase, SOD1, and SOD3) were unaltered in the MoBr/y brain, levels of amidated cholecystokinin (CCK8) and amidated pituitary adenylate cyclase-activating polypeptide (PACAP38) were reduced in a tissue-specific fashion. The compensatory changes observed in the neurovascular unit provide insight into the success of copper injections within a defined neurodevelopmental period.

Figure 1

ATP7A and ATP7B levels in Mo^Br/y brain. A. SDS-solubilized protein (20 μg) prepared from the indicated tissues from P8 wild-type male and Mo^Br/y mice was fractionated by SDS-PAGE: neocortex (Ctx), hippocampus (Hip), olfactory bulb (OB), cerebellum (Cbm) and hypothalamus (Hyp). The membrane was probed with antisera directed against ATP7A and β_III-tubulin. Quantification of ATP7A signal intensity is shown. The error bars indicate the range from duplicate experiments, each was normalized to P8 wild-type neocortex. B. Validation of ATP7B antiserum via immunoblotting. Extracts of baculovirus-infected Sf9 cells expressing purified ATP7B (+Con) and homogenates (40 μg total protein) from P8 and adult cerebellum were fractionated and the membrane was incubated with a 1:500 dilution of ATP7B antiserum, blocked antiserum or preimmune serum. C. ATP7B immunoblot of SDS-solubilized homogenates from P8 wild-type and Mo^Br/y brain. Quantification of ATP7B signal intensity is shown. The error bars indicate the range from duplicate experiments normalized to wild-type neocortex.

Figure 2

Copper content of Mo^Br/y cytosol is reduced more than particulate fractions. Pools of P4 wild-type and Mo^Br/y neocortex were fractionated by differential centrifugation as described in Materials and Methods. Copper concentrations were measured by atomic absorption spectroscopy. Two independent sets of samples were analyzed. A. Copper content of subcellular fractions normalized to total protein. P1, the initial low speed pellet, contains cell debris and a complex mixture of organelles. B. ATP7A immunoblot (20 μg protein) of homogenate and subcellular fractions. C. PHM activity of subcellular fractions normalized to total protein. D. SOD1 immunoblot (20 μg protein) of homogenate and subcellular fractions. Blots shown are representative of at least two experiments.

Figure 3

Peptide amidation is differentially impaired in Mo^Br/y brain. A. CCK8-NH₂ radioimmunoassay from P8 and P12 wild-type and Mo^Br/y neocortex (Ctx), hippocampus (Hip), olfactory bulb (OB), cerebellum (Cbm) and hypothalamus (Hyp). B. PACAP38-NH₂ radioimmunoassay from P8 and P12 wild-type and Mo^Br/y brain and liver (Lvr, negative control). Peptide levels were normalized to total protein. Each radioimmunoassay was performed in triplicate with pooled samples from three individual animals. *, p < 0.05 as determined by two-tailed student’s t test.

Figure 4

ATP7A expression in Mo^Br/y hippocampus is decreased in pyramidal neurons and increased in adjacent microvessels. Sagittal sections from the CA1/2 region of P8 wild-type and Mo^Br/y hippocampus were stained simultaneously for ATP7A (red), an endothelial cell marker [Ricinus communis agglutinin-1 (RCA-1); green] and DNA (TO-PRO-3; blue) and examined via laser scanning confocal microscopy. A. Low power images. In wild-type hippocampus, staining for ATP7A is apparent in CA1 and CA2 neuronal cell bodies with no detectable immunoreactivity in the RCA-1 positive microvasculature (arrows). In Mo^Br/y hippocampus, ATP7A immunoreactivity is decreased in the pyramidal cell layer but increased in the surrounding capillary endothelium (vessels ≤ 10 μm in diameter) (arrows). The borders of stratum pyramidale (sp) are outlined with white dotted lines, and the demarcation between CA1 and CA2 is indicated with diagonal intersections. B. High power images. Little ATP7A immunoreactivity is apparent in brain microvessels of capillary diameter from wild-type hippocampus. ATP7A is detected in a perinuclear location (arrows) in Mo^Br/y microvessels; DG, dentate gyrus; gc, granule cell layer of the dentate gyrus; sl-m, stratum lucidum-moleculare; so, stratum oriens; sr, stratum radiatum; Scale bars: A, 50 μm; B, 20 μm.

Figure 5

ATP7A expression is unaltered in larger diameter blood vessels and ependymal cells/choroid plexus. A. Sagittal sections from the dentate gyrus of P8 wild-type and Mo^Br/y hippocampus were stained simultaneously for ATP7A (red), the vascular smooth muscle/pericyte marker α-smooth muscle actin (green) and DNA (blue). ATP7A is present at equal levels in wild-type and Mo^Br/y endothelial cells lining larger diameter vessels. B. Sagittal sections of P8 wild-type and Mo^Br/y lateral ventricle were stained for ATP7A (red), the cis-to-medial Golgi marker GM130 (green) and DNA (blue). ATP7A is expressed at highest levels in wild-type and Mo^Br/y ependyma and segregates to a perinuclear compartment (arrowheads in insets) distinct from GM130 (arrows in insets); Scale bars: A, 20 μm; B, 10 μm.

Figure 6

Increased neurovascular unit activity in Mo^Br/y brain. A. P8 wild-type and Mo^Br/y hippocampi were stained simultaneously for ATP7A (red), the astrocyte marker GFAP (green) and DNA (blue). In wild-type hippocampus, astrocytic end-feet contact the basal surface of endothelial cells (arrows). In Mo^Br/y hippocampus, astrocytic end-feet are recruited to the cerebrovasculature as displayed by enhanced levels of GFAP immunoreactivity at these basal contacts (arrows). B. P8 wild-type and Mo^Br/y cerebella were stained simultaneously for ATP7A (red), the microglial marker CD11b (green) and DNA (blue). In wild-type mice, there is minimal contact of quiescent microglia with the cerebrovasculature. In contrast, in Mo^Br/y mice, contact of perivascular microglia with the cerebrovasculature is enhanced (arrowheads). These observations are typical of the three P8 wild-type and three Mo^Br/y animals examined; similar observations were noted in P11 wild-type and Mo^Br/y brain (data not shown); gc, granule cell layer of the dentate gyrus; Scale bars: 20 μm.

Figure 7

Decreased expression of mutant ATP7A and postsynaptic dysfunction in Mo^Br/y cerebellar Purkinje neurons. A. Wild-type and Mo^Br/y cerebella were stained simultaneously for ATP7A (red), MAP2 (green) and DNA (blue). In wild-type tissue, ATP7A immunoreactivity is intense in Purkinje neurons in a perinuclear location and extends into proximal dendrites (arrow). Mutant ATP7A expression is reduced in Mo^Br/y Purkinje cells; Mo^Br/y Purkinje cell somata are outlined in white dotted lines. B. Wild-type and Mo^Br/y cerebella were stained simultaneously for ATP7A (red), PSD-95 (green) and DNA (blue). The punctate PSD-95 distribution apparent in wild-type tissue (arrowheads) is absent from Mo^Br/y Purkinje cell dendrites. C. Wild-type and Mo^Br/y cerebella were stained simultaneously for ATP7A (red), bassoon (green) and DNA (blue). Bassoon staining is most evident in the molecular layer, outlining the dendritic arbor of wild-type Purkinje cells (white outline; arrowheads in inset). In Mo^Br/y cerebellum, bassoon immunoreactivity surrounding Purkinje cell bodies is evident (arrowheads in inset). Three wild-type and three Mo^Br/y animals were examined at P11; similar observations were noted in three wild-type and Mo^Br/y animals at P8 (data not shown); EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; PL, Purkinje layer; Scale bars: A,C, 50 μm; B, 10 μm.

Figure 8

Decreased expression of mutant ATP7A and axonal pathology in Mo^Br/y cerebellar Purkinje neurons. A. Sections of P11 wild-type and Mo^Br/y cerebellum were stained simultaneously with antisera to ATP7A (red), NF200 (green; to identify Purkinje cell axons) and DNA (blue). B. Maximum projection images of a 0.3 μm Z-step series representing the total section thickness of 15 μm. Purkinje cell axons are indicated by arrowheads. C. P11 wild-type and Mo^Br/y cerebellar sections were stained simultaneously for βIV-spectrin (red), NF200 (green) and nuclei (blue). Axon initial segments are identified by arrows; Scale bars: A, 50 μm; B,C, 20 μm.