Calpain Activation in Huntington’s Disease
Juliette Gafni and Lisa M. Ellerby
Buck Institute for Age Research, Novato, California 94945
Huntington’s disease (HD) is a neurodegenerative disorder
caused by a CAG expansion that results in elongation of the
polyglutamine tract at the N terminus of huntingtin (Htt). Abnor-
mal proteolytic processing of mutant Htt has been implicated
as a critical step in the initiation of HD. The protease(s) involved
in this process has not been fully characterized. Here we report
that activated calpain was detected in the caudate of human
HD tissue but not in age-matched controls. In addition, one of
the major N-terminal Htt proteolytic fragments found in human
HD tissue appears to be derived from calpain cleavage. Htt
fragments in HD lysates were similar in size to those produced
by exposure of in vitro-translated Htt to exogenous calpain.
Incubation of in vitro-translated Htt with calpain generated a
cascade of cleavage events with an initial intermediate cleav-
age product at 72 kDa and a final cleavage product at 47 kDa.
The rate of cleavage of Htt by calpain was polyglutamine-
length-dependent. These results suggest that cleavage of Htt in
human HD tissue is mediated in part by the Ca2?-activated
neutral protease, calpain.
Key words: huntingtin; Huntington’s disease; calpain; pro-
teases; triplet repeat disease; neurodegeneration
Huntington’s disease (HD) is an autosomal-dominant neurode-
generative disease caused by a CAG expansion in the huntingtin
gene (htt) (Huntington’s Disease Collaborative Research Group,
1993). The essential neuropathological characteristic of HD is the
loss of medium spiny neurons in the caudate nucleus and the
cortical projection neurons in layers V and VI (Cudkowicz and
Kowall, 1990; Hedreen et al., 1991; Albin, 1995). Expression of
truncated forms of mutant huntingtin protein (Htt) and not the
full-length protein induces cell death by apoptosis (Martindale et
al., 1998). This led to the hypothesis that toxic protein fragments
derived from full-length mutant Htt are required for disease
initiation (DiFiglia et al., 1997; Li and Li, 1998; Hackam et al.,
1999; Miyashita et al., 1999; Ona et al., 1999; Peters et al., 1999;
Sanchez et al., 1999; Wellington et al., 2000). One family of
proteases that promote the cleavage of Htt and other polyglu-
tamine expansion disease proteins is the cell death proteases,
caspases (Goldberg et al., 1996; Martindale et al., 1998; Welling-
ton et al., 1998, 2000; Ellerby et al., 1999a,b). However, one of the
principal pathways of neurotoxicity in the mammalian brain is
glutamate excitotoxicity, which depends on excessive Ca2?influx
into the cell. In neurons, this pathway is often accompanied by the
activation of cysteine proteases from both the caspase and calpain
family (Wang, 2000). Multiple lines of evidence suggest that
alterations in intracellular Ca2?levels play a role in HD
In an HD mouse model expressing full-length expanded hu-
man Htt, resting Ca2?levels are increased by almost twofold in
CA1 pyramidal neurons (Hodgson et al., 1999). Transgenic mice
expressing full-length mutant Htt show significantly reduced syn-
aptic vesicular uptake of glutamate (Li et al., 2000). NMDA
receptor currents are also enhanced in in vitro and in vivo HD
models (Chen et al., 1999). Because the NMDA receptor is
glutamate-sensitive, Ca2?-permeable, and expressed in the me-
dium spiny neostriatal neurons targeted in HD, it follows that
intracellular Ca2?levels may increase through increased NMDA
receptor-mediated signaling. A third line of evidence linking
Ca2?dysregulation to HD is that the levels of proteins involved
in Ca2?regulation are altered in HD patients and mouse models
(Hodgson et al., 1999; Luthi-Carter et al., 2000). Given these
multiple lines of evidence linking HD with alterations in Ca2?
homeostasis, we investigated whether the Ca2?responsive pro-
tease, calpain, plays a role in the cleavage of Htt in HD.
Calpains are a family of Ca2?-dependent intracellular cysteine
proteases, including the ubiquitously expressed ?- and m-calpains.
?-Calpain requires micromolar levels of Ca2?, whereas m-calpain
requires millimolar levels of Ca2?for activation. Both ?- and
m-calpains are heterodimeric and consist of a distinct large 80
kDa catalytic subunit and a common small 28 kDa regulatory
subunit. The addition of Ca2?results in the autolytic processing
of the catalytic subunit of ?-calpain from an 80 kDa protein to a
76 kDa protein, whereas activation of the m-calpain catalytic
subunit results in 20 amino acids being removed from the 80 kDa
protein N terminus. The small calpain regulatory subunit is
converted from a 28 kDa protein to a 21 kDa polypeptide with
increased Ca2?levels. The physiological roles and possible func-
tional distinctions of ?- and m-calpains remain unclear, but
suggested functions include participation in cell division and
migration (Huttenlocher et al., 1997), integrin-mediated signal
transduction, and apoptosis (Kulkarni et al., 1999).
In vitro, we have shown that Htt is cleaved by caspases at three
sites, yielding N-terminal fragments of 70, 75, and 80 kDa (Well-
ington et al., 2000). These fragments are also generated when Htt
is incubated with apoptotic extracts or cells (Hackam et al., 1998;
Martindale et al., 1998). Mutation of the caspase sites in Htt
prevented the accumulation of these fragments during apoptotic
Received Jan. 8, 2002; revised March 22, 2002; accepted March 29, 2002.
This work was supported by National Institutes of Health Grant NS40251
(L.M.E.), the Huntington’s Disease Society of America, and the Multiple Dystrophy
Association. The human brain tissue was provided by the Harvard Brain Tissue
Resource Center (United States Public Health Service Grant MN/NS31862). We
thank Drs. Greenberg, Hermel, LaFevre-Bernt, and Sarah Lamson for critical
comments and Dr. Michael Hayden for the htt vectors.
Correspondence should be addressed to Dr. Lisa M. Ellerby, Buck Institute for
Age Research, 8001 Redwood Boulevard, Novato, CA 94945. E-mail: lellerby@
Copyright © 2002 Society for Neuroscience 0270-6474/02/224842-08$15.00/0
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