The Copper Chaperone CCS Is Abundant in Neurons and
Astrocytes in Human and Rodent Brain
*†Jeffrey D. Rothstein, *Margaret Dykes-Hoberg, ‡§Laura B. Corson, Mark Becker,
§¶Don W. Cleveland, †Donald L. Price, #Valeria Cizewski Culotta, and Phillip C. Wong
Departments of *Neurology, †Neuroscience, and #Environmental Sciences, and Neuropathology Laboratory and ‡Program in
Human Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland; and §Ludwig Institute for Cancer
Research and ¶Department of Neuroscience, University of California at San Diego, La Jolla, California, U.S.A.
Abstract: Copper trafﬁcking in mammalian cells is highly
regulated. CCS is a copper chaperone that donates cop-
per to the antioxidant enzyme copper/zinc superoxide
dismutase 1 (SOD 1). Mutations of SOD1 are responsible
for ϳ20% of familial amyotrophic lateral sclerosis (FALS).
Monospeciﬁc antibodies were generated to evaluate the
localization and cellular distribution of this copper chap-
erone 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. Key Words:
Copper chaperone —CCS—Amyotrophic lateral sclero-
sis—Superoxide dismutase—Motor neuron.
J. Neurochem. 72, 422– 429 (1999).
The cellular mechanisms that regulate the intracellular
transport, trafﬁcking, and storage of many transition met-
als have only recently been understood. Copper, a ubiq-
uitous 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 homeosta-
sis, 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 cop-
per 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 labora-
tories 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 Cu
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
Received July 2, 1998; revised manuscript received August 11,
1998; accepted August 26, 1998.
Address correspondence and reprint requests to Dr. J. D. Rothstein at
Department of Neurology, Meyer 6-113, Johns Hopkins University,
600 N. Wolfe St., Baltimore, MD 21287, U.S.A.
Abbreviations used: ALS, amyotrophic lateral sclerosis; CCS, cop-
per chaperone for superoxide dismutase; PAGE, polyacrylamide gel
electrophoresis; SDS, sodium dodecyl sulfate; SOD1, superoxide dis-
mutase; TBS, Tris-buffered saline.
Journal of Neurochemistry
Lippincott Williams & Wilkins, Philadelphia
© 1999 International Society for Neurochemistry