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Keywords:

  • Copper chaperone;
  • CCS;
  • Amyotrophic lateral sclerosis;
  • Superoxide dismutase;
  • Motor neuron

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

Abstract : Copper trafficking in mammalian cells is highly regulated. CCS is a copper chaperone that donates copper to the antioxidant enzyme copper/zinc superoxide dismutase 1 (SOD 1). Mutations of SOD1 are responsible for ~20% of familial amyotrophic lateral sclerosis (FALS). Monospecific antibodies were generated to evaluate the localization and cellular distribution of this copper chaperone in human and mouse brain as well as other organs. CCS is found to be ubiquitously expressed by multiple tissues and is present in particularly high concentrations in kidney and liver. In brain and spinal cord, CCS was found throughout the neuropil, with expression largely confined to neurons and some astrocytes. Like SOD1, CCS immunoreactivity was intense in Purkinje cells, deep cerebellar neurons, and pyramidal cortical neurons, whereas in spinal cord, CCS was highly expressed in motor neurons. In cortical neurons, CCS was present in the soma and proximal dendrites, as well as some axons. Although the distribution of CCS paralleled that of SOD1, there was a 12-30-fold molar excess of SOD1 over CCS. That both SOD1 and CCS are present, together, in cells that degenerate in ALS also emphasizes the potential role of CCS in mutant SOD1-mediated toxicity.

The cellular mechanisms that regulate the intracellular transport, trafficking, and storage of many transition metals have only recently been understood. Copper, a ubiquitous transition metal, is required for the biological activation of dioxygen, which is essential for the survival of all living organisms (Solomon and Lowery, 1993). Because the electron structure of copper allows a direct interaction with oxygen, copper functions as a facile cofactor in important redox reactions in many enzymes involved in key cellular processes (e.g., iron homeostasis, antioxidant defense, neurotransmitter biosynthesis, connective tissue formation, pigment production, and endocrine organ regulation) (Harris and Gitlin, 1996). However, copper can be very toxic. Thus, cells have developed mechanisms to ensure the proper intracellular transport and compartmentalization of this transition metal (Valentine and Gralla, 1997). The delivery of copper to specific proteins is mediated through distinct intracellular pathways of copper trafficking. Several copper carriers (or metal chaperones), soluble proteins that bind and deliver copper to specific intracellular proteins, have recently been identified. Glerum et al. (1996a,b) identified a small soluble protein (69 amino acids) in yeast, named COX17, that functions with mitochondrial proteins SCO1 and SCO2 to specifically deliver copper to cytochrome oxidase in the mitochondria. The laboratories of both Culotta and Gitlin (Lin and Culotta, 1995 ; Klomp et al., 1997) have cloned and identified a copper chaperone in yeast, termed ATX1 (72 amino acids), and its human homologue, HAH1 (68 amino acids), which specifically deliver copper to transport ATPases in the secretory pathway (Pufahl et al., 1997).

Recently, Culotta et al. (1997) identified a copper chaperone in yeast, termed LSY7 (249 amino acids), and its mammalian homologue, CCS (copper chaperone for superoxide dismutase ; 274 amino acids). Their studies demonstrated that CCS is able to specifically deliver copper to the antioxidant enzyme Cu2+/Zn+ superoxide dismutase (SOD1). Culotta et al. (1997) showed that CCS, encoded by a single-copy gene on chromosome 11, RNAs are found in all human tissues and cell types examined. Furthermore, the initial study showed that LYS7/CCS was essential for activity of yeast SOD1, and additional efforts have shown that both human SOD1 and familial amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants acquire copper through action of CCS (Corson et al., 1998). We now report a detailed tissue and cellular localization of CCS in human and rodent tissue, using multiple specific antibodies to CCS.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

Polyclonal antipeptide antibodies

A synthetic peptide corresponding to the C-terminal region of human CCS protein (NH2-K262GRKESAQPPAHL274-COOH ; human CCS antibody) was synthesized on an Applied Biosystems 430A Peptide Synthesizer (Applied Biosystems, Foster City, CA, U.S.A.). After synthesis, the peptide was purified by using reversed-phase HPLC. The purified peptide was then coupled to thyroglobulin by using glutaraldehyde. Antisera were generated against the protein-conjugated synthetic peptide in New Zealand White rabbits (Covance, Denver, PA, U.S.A.), as previously described (Rothstein et al., 1994). The crude antisera were affinity purified (Blackstone et al., 1992 ; Rothstein et al., 1994) on a column prepared by coupling bovine serum albumin-conjugated CCS oligopeptide to Affi-Gel 15 (Bio-Rad, Grand Island, NY, U.S.A.), before their use in immunoblotting or immunocytochemistry. An additional CCS oligopeptide antibody was generated by immunizing rabbits with a keyhole limpet hemocyanin coupled CCS oligopeptide (WEERGRPIAGQGRKDS), corresponding to residues 252-267 of mouse CCS (mouse CCS antibody). The resulting serum was affinity purified over a peptide column as before (Rothstein et al., 1994).

To characterize the antibodies, CCS was expressed in yeast as described previously (Culotta et al., 1997). In brief, CCS cDNA was cloned into the pSM703 plasmid, placing CCS under control of the Saccharomyces cerevisiae PGK1 (phosphoglycerol kinase) promoter.

CCS fusion protein

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

A nearly full-length mouse CCS (encoding residues 31-274) from EST no. 553101 was subcloned into a hexahistidine bacterial expression plasmid (pRSET B ; Invitrogen). The hexahistidine-tagged CCS fusion protein was nickel purified to >95% (Qiagen) and used as a standard for quantifying accumulated CCS levels in mouse tissues.

Immunoblotting and immunohistochemistry

Preparation of tissue for gel electrophoresis and immunoblotting. Tissue was collected from freshly obtained rat/mouse brain and spinal cord, or human autopsy tissue. The tissue was dissected, and homogenized with a Brinkmann Polytron in ice-cold phosphate-buffered saline. In some experiments, tissue was homogenized in an ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 10% (wt/vol) sucrose, 20 U/ml aprotinin, 20 μg/ml antipain, 20 μg/ml leupeptin, 1 mM EDTA, and 5 mM EGTA. Homogenates were stored at 10 mg protein/ml at-70°C.

The specificity of the antibodies, and the regional distribution of the protein in brain and body organs, was evaluated by immunoblotting of the rodent or human tissue homogenates. Aliquots of tissues (5-20 μg of protein) were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) (8% polyacrylamide gels) and transferred by electroblotting to polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, MA, U.S.A.) by electroblotting (30 V, overnight). Blots were blocked (1 h) in 0.5% nonfat dry milk, 0.1% Tween 20, 50 mM Tris-buffered saline (TBS) at room temperature, then incubated (1 h) with affinity-purified CCS antibody at a 1 : 500 dilution (75 μg/ml) in blocking buffer. Blots were then washed, incubated (1 h) with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1 : 5,000) in blocking buffer), and finally washed with TBS. The immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL, U.S.A.).

Quantitation of CCS and SOD1 in mouse tissues

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

Protein extracts from tissues of a 9-month-old BL6 mouse were prepared as described (Bruijn et al., 1997). Equal amounts of protein were loaded on a 12% SDS-PAGE gel, transferred to nitrocellulose, probed with either a CCS peptide antibody or an SOD1 peptide antibody that recognizes mouse and human SOD1 with equal affinity (Borchelt et al., 1994), and quantitated by using 125I-protein A on a phosphoimager (Molecular Dynamics, CA, U.S.A.). A dilution series of the hexahistidine mouse CCS fusion protein or human SOD1 (Sigma) was used as a standard to quantify the level of accumulated CCS or SOD1 in the various tissues. Approximate molar ratios of SOD1 to CCS were determined by converting the percentage of total protein ratios to molar ratios by dividing by the molecular weights.

Immunocytochemistry

Mice (C57B6 ; n = 5) between the ages of 2 and 4 months (20-25 g) were anesthetized by intraperitoneal injection of chloral hydrate and perfused with phosphate-buffered saline (pH 7.4) followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains and spinal cords from rodents were postfixed for 1 h in the same fixative, then cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 24 h. Coronal or sagittal sections (40 μm) of brain and/or spinal cord were cut on a freezing Microtome (Zeiss ; 20-40 μm) and/or were transferred to cold TBS (pH 7.2). In addition, brain and spinal cord were obtained at autopsy from two humans who died of nonneurological disorders with postmortem intervals <7 h. The tissues were dissected and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 48 h, cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 24 h, and then frozen at -75°C. In some cases, paraformaldehyde-fixed human or rodent tissue was embedded in paraffin, then sectioned on a Zeiss Microtome (10 μm).

Frozen sections and delipidated, rehydrated paraffin sections were preincubated (1 h) with 4% normal goat serum diluted in 0.1% Triton X-100/TBS and were then incubated (48 h, 4°C) in the affinity-purified human CCS antibody (75 μg/ml) of IgG per milliliter in 0.1% Triton X-100, 2% normal goat serum, and TBS. Control sections were incubated as follows : (1) with the CCS antibody preadsorbed overnight with an excess (50 μM) of either synthetic CCS oligopeptide peptide ; (2) with the primary antisera omitted ; or (3) with the secondary antibody omitted. In some cases, immunoglobulin from rabbits obtained before antigen injections (preimmune antisera) was used. After primary antibody incubation, sections were rinsed (30 min) in TBS, incubated (1 h) with goat anti-rabbit (Cappel) diluted 1 : 200 in TBS with 2% normal goat serum and 0.1% Triton X-100. After rinsing in TBS, the sections were incubated (1 h) in rabbit peroxidase-antiperoxidase complex (Sternberger Monoclonals, Baltimore, MD, U.S.A.) diluted 1 : 300 in TBS with 2% normal goat serum. After the final incubation, sections were rinsed (30 min) in TBS, and the reaction product was developed by incubation in 0.03% diaminobenzidine/0.01% H2O2/TBS. Sections were rinsed in TBS, mounted onto gelatincoated slides, dehydrated in a series of graded ethanol solutions followed by xylene, and then coverslipped.

Characterization of the CCS antibody and protein in tissue

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

To produce an antibody that could specifically detect mammalian CCS, a 13-amino acid peptide from the carboxy-terminal region of the human protein was synthesized. This amino acid sequence does not share homology with other copper chaperones (e.g., COX17 and ATX1), SOD1, or any other peptide sequence in Gen-Bank. One rabbit was inoculated repeatedly and produced a high-titer response. After affinity purification, the antibody revealed strong immunoreactivity for an ~35-kDa protein in yeast strains expressing human CCS (Fig. 1A, lane 2) but lacking SOD1 and LYS7. This compares favorably with the predicated molecular mass for CCS of 29 kDa. Immunoreactivity was not seen in control yeast lacking both the LYS7 chaperone and SOD1 (Fig. 1A, lane 1), or yeast lacking LYS7 but containing SOD1 (Fig. 1A, lane 3). A nonspecific peptide was identified by the antibody only in yeast. The 35-kDa polypeptide was also seen in yeast expressing CCS and SOD1 but deleted in the LYS7 chaperone (Fig. 1A, lane 4), confirming its identification as the human CCS. In a similar manner, in tissue homogenates from human or mouse brain, the antibody bound specifically to a polypeptide of the size expected for CCS and identical to that seen in immunoblots from yeast expressing CCS (Fig. 1A, lanes 5 and 6). Coincubation with CCS oligopeptide eliminated immunoreactivity (Fig. 1A, lanes 7 and 8). Identification of the 35-kDa peptide was also observed with the keyhole limpet hemocyanin-conjugated mouse CCS oligopeptide antibody.

image

Figure 1. Characterization of CCS affinity-purified polyclonal antibody in yeast, human, and rat tissues. A : Specificity of CCS antibody on immunoblots of yeast alone (lane 1), expressing CCS (lane 2), yeast without LYS7 protein (lane 3), yeast without LYS7 protein but expressing CCS (lane 4). The yeast alone strain is a ▵SOD1, ▵LYS7 strain. Immunoblotting demonstrates the specificity of antibodies to CCS in human and rat brain tissue (lanes 5 and 6). Detergent-soluble extracts (20 μg) of human and rat cortex were subjected to SDS-PAGE and immunoblotted with CCS antibody. Immunoreactivity in both tissues was completely abolished when antibodies were preabsorbed with 50 μM synthetic CCS oligopeptide peptide before immunoblotting (lanes 7 and 8). B : Quantitative analysis of CCS and SOD1 in brain tissue. Varying amounts (in nanograms) of pure CCS or SOD1 were immunoblotted (first four lanes ; nanograms of pure protein) and compared with various tissue extracts. The molar ratios of SOD1 to CCS are shown below the lanes for each tissue examined. C : Comparison of CCS proteins in brain tissue from various animal species compared with pure CCS expressed by yeast (lane 2). Except for chicken brain (lane 7), antisera to pure CCS identified a single polypeptide with similar electrophoretic migration properties in human (lane 3), rat (lane 4), mouse (lane 5), and bovine brain (lane 6).

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To evaluate the specificity of the antibody in animal organisms, brain homogenates from multiple species were examined (Fig. 1C). Antibodies to human CCS identified the same polypeptide in rat (lane 4), mouse (lane 5), and bovine brain (lane 6) tissue. A single polypeptide was also identified in chicken brain (lane 7), although the apparent molecular weight was smaller relative to the other brain specimens. This may reflect an alternate form of CCS.

Because CCS serves as a copper chaperone to SOD1, we evaluated the abundance of CCS relative to SOD1 in mouse tissue (Fig. 1B), using an antibody to mouse CCS. It is not surprising that CCS (like SOD1) is found in most tissues analyzed and is particularly abundant in liver and kidney (see below). Levels of accumulated CCS range from 0.004 to 0.012% of total cellular protein, which is about seven- to 14-fold less than SOD1 levels, resulting in a 12-30-fold molar excess of SOD1 over CCS.

Localization of CCS in human tissue nervous system

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

Immunoblots from brain regions demonstrated a similar level of protein expression in all human brain and spinal cord regions (human CCS antibody ; Fig. 2A). In a similar manner, when tissues from various human organs were examined, there appeared to be a comparable and ubiquitous expression of CCS (Fig. 2B), except for much lower levels of expression in pancreas and spleen. The low pancreatic expression may be due to proteolytic digestion, and it was mirrored by low SOD1 expression (not shown). This is similar to the extensive expression of SOD1 in many tissues reported by others (Fridovich, 1986). It is interesting that in several tissues including cerebellum, kidney, and liver, an additional polypeptide, ~5 kDa larger than CCS, was identified with both antibodies. Because immunoreactivity for this protein was also eliminated by preincubation with CCS oligopeptide, this higher immunoreactive polypeptide could be an alternate form of CCS or a CCS-related protein uniquely expressed by these tissues.

image

Figure 2. Distribution of CCS in human CNS and body organs. Aliquots (20 μg of protein/lane) of tissue homogenates from isolated brain regions (A) or various body (B) organs were subjected to SDS-PAGE and immunoblotted with antibodies to CCS (1:400 dilution).

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Sections from human cortex, hippocampus, and cerebellum were immunostained with antibody against CCS. All tissues were examined from patient material with a short postmortem delay (4-8 h) to minimize complications arising from postmortem protein degradation. CCS immunoreactivity was generally found through the neuropil, with considerably less staining seen in white matter (Fig. 3A). CCS immunoreactivity was primarily identified in neurons. In cortex, CCS immunoreactivity was readily discernible in large and small pyramidal neurons along with small neurons in all layers, as well as small astrocytes in gray and white matter. The staining was specific, as preincubation of the antibody with the immunizing peptide (50 μM) completely eliminated reactivity (Fig. 3B). Higher magnification revealed a predominantly punctate (particulate) pattern, readily visualized throughout the perikarya and proximal dendrites (Fig. 3C). This pattern of staining is quite comparable with that seen for SOD1 (Pardo et al., 1995). Nonpyramidal neurons typically had less staining in cortex. In a similar manner, in hippocampus, immunoreactive protein was found throughout the neuropil (Fig. 3D). It is interesting that granule cell neurons in the dentate gyrus were almost devoid of immunostaining, but intense immunoreactivity could be seen in occasional pyramidal cells in the dentate hilus (Fig. 3E). In the caudate nucleus, medium aspiny neurons were conspicuously immunoreactive along with large neurons in the globus pallidus (not shown), similar to those seen previously for SOD1 (Pardo et al., 1995). Immunolocalization in the cerebellum was also enriched for certain neuronal populations (Fig. 3F). Cerebellar granule cells were without immunoreactivity, but cerebellar Purkinje cells were intensely immunoreactive for CCS (Fig. 3G). A punctate, peridendritic staining pattern on cerebellar dendrites was highly suggestive of presynaptic or bouton localization.

image

Figure 3. Regional and cellular localization of CCS in human nervous tissue. A : CCS distribution in human frontal cortex. Immunoreactivity was found diffusely in neuropil and large and small pyramidal neurons (bar = 1 mm). B : Adjacent section immunostained with CCS antibody preadsorbed to the immunizing peptide (50 μM). C : Cortical neurons demonstrate soma and proximal dendrite immunoreactivity ; arrow indicates immunoreactive dendrite. The cellular localization was typically particulate (bar = 0.2 mm). D : Hippocampal dentate gyrus demonstrated modest CCS immunoreactivity in granule cells (bar = 2 mm). E : It is noteworthy that some small pyramidal and stellate neurons in the hilus were intensely immunoreactive for CCS (arrow), but others were almost devoid of immunostaining (asterisk) (bar = 0.2 mm). F : In cerebellar cortex CCS immunoreactivity was largely restricted to Purkinje cell layer (arrowheads), with an obvious lack of immunostaining in the cell-dense granule cell layer (gr) (bar = 2 mm). G : Purkinje cells were intensely immunopositive for CCS ; astrocytes (arrowhead) were also occasionally immunoreactive (bar = 0.2 mm).

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Localization of CCS in mouse nervous system compared with SOD1 immunolocalization

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

CCS distribution in mouse brain regions including cortex, striatum, cerebellum, and spinal cord, like human tissue, was fairly uniform (Fig. 4A). Most mouse organ tissue also had comparable levels of CCS, although lower levels were found in heart, muscle, and spleen (Fig. 4B).

image

Figure 4. Distribution of CCS in mouse CNS and body organs. Aliquots (20 μg of protein/lane) of tissue homogenates from isolated brain regions (A) or various body organs (B) were subjected to SDS-PAGE and immunoblotted with antibodies to CCS (1 : 400 dilution).

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To explore the distribution in greater detail, parallel localization studies were performed on mouse brain sections stained alternately for CCS and SOD1. Sagittal, thick (40 μm) sections of whole brains illustrate the distinctive general distribution of CCS (Fig. 5A) and SOD1 (Fig. 6A). CCS immunoreactivity was generally high throughout all brain regions and spinal cord but was largely absent from white matter tracts (Fig. 5A and E). In a similar manner, SOD1 had an almost identical general distribution (Fig. 6A and E). Controls, including CCS and SOD1 antibodies preabsorbed against their respective synthetic peptide (CCS) or protein (SOD1) (Figs. 5G and 6G), and omission of primary antibody, showed no immunostaining of any cellular elements (not shown). CCS and SOD1 immunoreactivity was generally neuronal (Fig. 5B-D and F), although lighter immunoreactivity in astrocytes could be observed. CCS immunoreactivity was observed in some axons including spinal cord white matter tracts and descending corticostriatal pathways (not shown), as has previously been reported for SOD1 (Pardo et al., 1995). The regional expression of CCS, however, was not uniform. Hippocampal neurons, including granule cells, had a modest expression (Fig. 5C) similar to the immunoreactivity for SOD1 in hippocampus (Fig. 6C). Certain neuronal populations were intensely immunoreactive for both SOD1 and CCS, i.e., midline thalamic neurons (Figs. 5D and 6D), cerebellar Purkinje cells, and deep cerebellar neurons (Figs. 5E and 6E). Large motor neurons were also strongly CCS immunoreactive (Fig. 5F) as well as SOD1 immunoreactive (Fig. 6F). Finally, ependymal cells of the spinal cord central canal were also intensely immunoreactive for CCS (not shown).

image

Figure 5. Distribution of CCS in mouse nervous system. A : Sagittal section of mouse brain illustrates the regional distribution of CCS (bar = 5 mm). B : In cerebral cortex, large and small neurons are CCS immunopositive (bar = 1 mm). C : Hippocampal CCS is localized generally to neuropil, with a modest presence in granule cell neurons of the dentate gyrus (A and B) (bar = 0.5 mm). D : Neurons in midline thalamic nuclei were strongly immunoreactive for CCS (bar = 0.2 mm). E : In cerebellum, CCS was localized to the Purkinje cell and molecular layers (arrowheads), along with deep cerebellar nuclei (arrow) (bar = 0.5 mm). F : Spinal cord motor neurons (arrows) are strongly immunoreactive for CCS compared with smaller interneurons (arrowheads) (bar = 0.2 mm). G : Immunoreactivity in tissue (cortex) was completely abolished when antibodies (75 μg/ml) were preabsorbed with 50 μM CCS oligopeptide before immunostaining. ob, olfactory bulb ; s, striatum ; c, cortex ; h, hippocampus ; cc, cerebellar cortex ; m, molecular layer ; P, Purkinje cell layer ; g, granule cell layer ; DCN, deep cerebellar nuclei.

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image

Figure 6. Distribution of SOD1 in mouse nervous system. A : Sagittal section of mouse brain illustrates the regional distribution of SOD1 compared with CCS (bar = 5 mm). B : In cerebral cortex, large and small neurons are SOD1 immunopositive (bar = 1 mm). C : Hippocampal SOD1 is strongly localized to neurons throughout the hippocampus (bar = 0.5 mm). D : Neurons in midline thalamic nuclei were strongly immunoreactive for SOD1 (bar = 0.2 mm). E : In cerebellum, SOD1 was intensely localized to Purkinje cells (arrowheads), to rare granule cell neurons, and to deep cerebellar nuclei neurons (arrows) (bar = 0.5 mm). F : Spinal cord motor neurons (arrows) are strongly immunoreactive for SOD1 compared with smaller, less reactive interneurons (arrowheads) (bar = 0.2 mm). G : Immunoreactivity in tissue (cortex) was completely abolished when antibodies (1 : 1,000) (Wong et al., 1995) were preabsorbed with 50 μM SOD1 peptide before immunostaining. ob, olfactory bulb ; s, striatum ; c, cortex ; h, hippocampus ; cc, cerebellar cortex ; m, molecular layer ; P, Purkinje cell layer ; g, granule cell layer ; DCN, deep cerebellar nuclei.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

SOD1 plays a major role as a cellular antioxidant by detoxifying superoxide radicals, and it requires copper in its active site for this enzymatic activity. In vitro, pure SOD1 protein can acquire copper, by direct addition of copper ions. However, in vivo, both yeast (Culotta et al., 1997) and human (Corson et al., 1998) apoproteins appear to acquire copper through the action of a copper chaperone. In yeast, this recently identified copper chaperone, designated as LYS7, is required for SOD1 to be enzymatically active. The human homologue of LYS7, known as CCS, has a 28% amino acid identity to LYS7 (Culotta et al., 1997). Studies in yeast also demonstrate that CCS is required to provide copper to activate SOD1. Our efforts here are consistent with such a requirement, with CCS levels and distribution paralleling those for SOD1.

Within the CNS, CCS is present in many neuronal populations, and it is noteworthy that it appears to be selectively enriched in a subset of neurons, in particular, motor neurons in the spinal cord, pyramidal neurons in the cortex, and cerebellar Purkinje cells. The immunoreactivity was also occasionally seen within astrocytes. Much of the CCS immunoreactivity is restricted to the cytoplasm of the perikarya, dendrites, and some axons. Because immunocytochemistry cannot be accurately quantified, we are not able to determine the proportion of CCS in these compartments, although the soma appears to have the highest concentration. The immunoreactivity appears to be localized to discrete structures in the cytoplasm, but their identification awaits more detailed immunoelectron microscopy.

Because CCS is a chaperone for SOD1, a comparative analysis of the two proteins is instrumental in understanding their physiological relationship. Although SOD1 protein is known to be an extremely abundant cellular protein (Pardo et al., 1995), quantitatively, CCS is ~15-30-fold less abundant than SOD1, as might be expected if the action of CCS is catalytic. The immunolocalization studies do suggest a biochemical relationship between both proteins ; the cellular distribution and relative cellular expression of CCS appear to parallel those of SOD1. In brain, both CCS and SOD1 are largely restricted to neurons and appear to be enriched in similar neuronal populations. For example, Purkinje cells have a much greater immunoreactivity for both CCS and SOD1, compared with the adjacent, immunonegative, cerebellar granule cells. Future studies are necessary to further define the biochemical and regulatory relationship between CCS and SOD1.

Understanding the biology of CCS and SOD1 is important to unraveling the pathogenesis of disorders related to SOD1. Mutations of SOD1 have been found in almost 20% of familial ALS (Siddique and Deng, 1996). ALS is an adult-onset, progressive, and fatal neurological disorder classically characterized by death of cortical, brainstem, and spinal cord motor neurons (Kuncl et al., 1992 ; Rowland, 1994). Although most ALS has a sporadic onset, about 5-10% of ALS is inherited. Transgenic mice expressing mutant human SOD1 have been shown to reliably develop a progressive motor neuron disease that has many (although not all) of the clinical and pathological features of ALS (Gurney et al., 1994 ; Ripps et al., 1995 ; Wong et al., 1995 ; Bruijn et al., 1997). Furthermore, the ability of several drugs to retard the disease progression in these mice has been mirrored in human clinical trials (Bensimon et al., 1994 ; Gurney et al., 1994 ; Lacomblez et al., 1996 ; Miller et al., 1996). Taken together, these studies make this animal model an extremely useful and accurate model of the human disease.

These efforts have shown that the ALS-linked mutants cause disease through some, as yet, unidentified toxic property. Multiple hypotheses have been generated regarding the possible toxic property imbued by these mutations, including generation of peroxynitrite and increased peroxidase activity. Neither a loss of activity nor a loss of copper binding is a property of these mutant enzymes (Corson et al., 1998). In fact, the only common property of the mutant enzymes yet identified, at least as studied in yeast systems, is that they retain some copper binding and interact with CCS to acquire this copper (Culotta et al., 1997 ; Corson et al., 1998). This suggests that copper chaperone—SOD1 interactions could be a component of mutant SOD1-mediated toxicity. That both SOD1 and CCS are present together in cells that degenerate in ALS emphasizes the potential role of CCS in mutant SOD1-mediated toxicity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. CCS fusion protein
  5. Quantitation of CCS and SOD1 in mouse tissues
  6. RESULTS
  7. Characterization of the CCS antibody and protein in tissue
  8. Localization of CCS in human tissue nervous system
  9. Localization of CCS in mouse nervous system compared with SOD1 immunolocalization
  10. DISCUSSION
  11. Acknowledgements

This study was supported by National Institutes of Health grants NS33958 (J.D.R.), AG12992 (J.D.R.), and GM50016 (V.C.C.), along with support from the Muscular Dystrophy Association (J.D.R.) and the ALS Association (J.D.R. and V.C.C.).

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