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
The Journal of Neuroscience, June 15, 2002, 22(12):4842–4849
challenge (Wellington et al., 2000). However, these initial studies
did not evaluate whether altered Ca2?homeostasis would influ-
ence the generation of toxic fragments. In many forms of cell death,
both caspases and calpains are activated (Nakagawa and Yuan,
2000; Wang, 2000; Blomgren et al., 2001). Furthermore, despite the
difference in cleavage-site specificity, an increasing number of
cellular proteins are found to be dually susceptible to caspases and
calpains, including ?- and ?-spectrin, calmodulin-dependent pro-
tein kinases, and tau (Wang, 2000). Here, we report that Htt is a
substrate of caspases and calpains. We show that cleavage of Htt by
calpain is polyglutamine repeat-dependent, with increasing length
of the tract correlating with increased susceptibility of Htt to
cleavage. The Htt fragments generated from calpain cleavage are
smaller than those generated from caspase cleavage and therefore
are more toxic to cells (Hackam et al., 1998). Calpain activation is
detected in human HD tissue but not in age-matched controls. The
total levels of both active and inactive calpains are increased in HD
patients when compared with age-matched controls. Some of the
cleavage products in HD tissue are similar in size to those gener-
ated by recombinant calpains.
MATERIALS AND METHODS
Cell culture. Superfect reagent (Qiagen, Valencia, CA) was used for
transient transfections in human embryonic kidney 293T cells with plas-
mids described previously (Goldberg et al., 1996; Martindale et al., 1998;
Wellington et al., 2000) and included wild-type and expanded full-length
huntingtin (Htt15 and Htt44, respectively), caspase-resistant huntingtin
(Htt15 D513A, D552A, D586A), and the wild-type and expanded
N-terminal fragment of Htt (Htt 3949-15 and Htt 3949-138). Thapsigar-
gin (2.5 ?M; Sigma, St. Louis, MO) was added 24 hr after transfection,
and cells were lysed and collected at 48 hr.
Western blot analysis. Cell pellets or human brain tissue (Harvard
Brain Tissue Resource Center, Belmont, MA) were homogenized in
NP-40 lysis buffer (0.1% NP-40, 50 mM HEPES, pH 7.4, 250 mM NaCl,
and 5 mM EDTA) or RIPA (10 mM Tris, pH 8.0, 150 mM NaCl, 1%
Triton X-100, 1% deoxycholate, 0.1% SDS, and 5 mM EDTA) with
protease inhibitors (complete mini, Roche, Mannheim, Germany;
Z-VAD, Sigma) (Ellerby et al., 1999a,b). Controls were prepared by
treating Htt-transfected lysates in lysis buffer with 5 ?M DTT, 10 mM
CaCl2, and 3 ?M m-calpain (Sigma) for 5 min at 30°C. Lysate proteins
were resolved on a 12% polyacrylamide gel, transferred to a polyvinyli-
dene difluoride membrane, and probed with monoclonal Htt 2166 (3.5
?g/ml; Chemicon, Temecula, CA), calpain small subunit 3083 (9 ?g/ml;
Chemicon), and ?-calpain 3104 (3.5 ?g/ml; Chemicon) antibodies. Im-
munoblots were developed with a peroxidase-conjugated secondary an-
tibody and enhanced chemiluminescence.
In vitro protein synthesis and cleavage. The Htt 1955-15, Htt 3949-15,
and Htt 3949-138 constructs were translated with a TnT-coupled kit
(Promega, Madison, WI) and the products were incubated with
?-calpain (Calbiochem, La Jolla, CA) or m-calpain (Sigma) in NP-40
lysis buffer with 5 ?M DTT and no added 3 ?M or 10 mM CaCl2at 30°C
for 5 min or for the time indicated in Figure 2C. Calpain inhibitor I
(Bachem, Bubendorf, Switzerland) was added where indicated. Reac-
tions were terminated by addition of EDTA, SDS sample buffer, and
boiling. Control caspase-2, caspase-3, and caspase-6 cleavage products
were produced as described previously (Ellerby et al., 1999a,b).
Immunocytochemistry. Formalin-fixed human caudate tissue (Harvard
Brain Tissue Resource Center) was embedded in paraffin, sectioned, and
deparaffinized with xylene. Antigen retrieval was performed by micro-
waving sections in 10 mM citrate buffer, pH 6.0, for 5 min. Primary anti-
bodies were as follows: monoclonal calpain regulatory subunit [14 ?g/ml;
monoclonal antibody (mAb) 3083; Chemicon]; polyclonal m-calpain cata-
lytic subunit (2 ?g/ml; sc-7533; Santa Cruz Biotechnology, Santa Cruz,
CA); monoclonal ?-calpain catalytic subunit (7 ?g/ml; mAb 3104;
Chemicon); and monoclonal Htt (7 ?g/ml; mAb 2166; Chemicon). Rab-
bit IgG (2 ?g/ml) or goat IgG (2 ?g/ml) was used as a negative control.
Biotinylated secondary antibody (6 ?g/ml; Vector Laboratories, Burlin-
game, CA) was incubated for 1 hr at 37°C followed by signal amplifica-
tion with biotin/avidin and diaminobenzidine visualization. Mayers
hematoxylin (American MasterTech, Lodi, CA) was used as a counter-
Htt is cleaved by calpains into three major N-terminal
We have shown previously that caspases cleave Htt to produce
N-terminal fragments that are toxic to cells (Hackam et al., 1998;
Martindale et al., 1998; Wellington et al., 2000). Caspases cleave
Htt in a 76 aa region to produce 70, 75, and 80 kDa N-terminal
fragments (Wellington et al., 2000). Here we show that Htt is also
cleaved in vitro by ?-calpain and m-calpain (Fig. 1) and that some
of the N-terminal fragments produced are smaller in length than
those derived from caspase cleavage and therefore more toxic
(Hackam et al., 1999). We initially evaluated calpain cleavage of
Htt by treating the in vitro-translated, [35S]-labeled, N-terminal
Htt fragment (Htt 1955-15) with either ?-calpain or m-calpain.
Lower concentrations of calpains produce cleavage fragments at
67 and 62 kDa, whereas higher concentrations of calpains pro-
duce a cleavage product at 47 kDa (Fig. 1A,B). ?-Calpain and
m-calpain cleaved Htt at the same sites (Fig. 1A,B). ?-Calpain
cleaved Htt in the presence of low (3 ?M) and high (10 mM) Ca2?,
m-calpain cleaved Htt only in the presence of high Ca2?(10 mM),
and preincubation with calpain inhibitor 1 (30 ?M) completely
blocked cleavage of Htt by both calpains (Fig. 1C).
To further evaluate the calpain cleavage sites of Htt with
respect to caspase cleavage, we used a larger Htt construct en-
coding a 1211 aa fragment because it contains all three previously
mapped caspase sites (Wellington et al., 2000). Treatment of in
vitro-translated, [35S]-labeled, N-terminal Htt 3949-15 with cal-
pain produced three fragments of 72, 67, and 62 kDa (Fig. 1D).
Previous work with recombinant caspases shows that caspase-3
cleaves Htt at D513 and D552, caspase-2 cleaves at D552, and
caspase-6 cleaves at D586 (Fig. 1E). As shown in Figure 1D,
calpain generated a 72 kDa fragment that migrates between the
caspase-3 Htt fragment at 70 kDa and the caspase-2 Htt fragment
at 75 kDa. Therefore, one of the calpain sites lies between amino
acids 513 and 552 of Htt. Additional experiments with the ex-
panded forms of in vitro-translated Htt showed that three of the
calpain cleavage products (72, 67, and 62 kDa) contain the poly-
glutamine tract (Fig. 2), whereas the smallest fragment (47 kDa)
does not (data not shown). In summary, calpain treatment of Htt
produces three N-terminal cleavage products (72, 67, and 62 kDa)
and one C-terminal cleavage fragment (47 kDa) derived from the
N-terminal portion of Htt.
Predicted calpain cleavage sites
Previous studies have mapped the caspase sites in Htt (Welling-
ton et al., 2000), which are shown in Figure 1E. Using this
information and known substrate cleavage sites for calpain, we
can predict likely calpain cleavage sites in Htt for the N-terminal
cleavage products. Based on amino acid sequence, the caspase-3
Htt fragment has a molecular weight of 55 kDa (observed 70
kDa). Therefore, the predicted size of the calpain-derived
N-terminal Htt fragments were ?46, 50, and 58 kDa (Fig. 1E,
MWP) (Croall and DeMartino, 1991). Using this information, we
were able to determine potential calpain cleavage sites in Htt
(Fig. 1E), which are based on the fact that calpain cleaves pref-
erentially in the P2 position at a Val, Leu, or Ile and on se-
quenced calpain cleavage sites in calpain-susceptible proteins.
Our predictions show that two cleavage sites in Htt are identical
in sequence to known calpain substrates, most notably, a Leu-
Thr-Ala motif at position 469 and a Ser-Ser-Ser motif at position
536, which is the calpain cleavage site in caspase-12 (Nakagawa
and Yuan, 2000) and protein kinase C (Croall and DeMartino,
Gafni and Ellerby • Cleavage of HuntingtinJ. Neurosci., June 15, 2002, 22(12):4842–4849 4843
1991), respectively. Both of these sites are in close proximity to
our predicted cleavage sites for the largest calpain-derived Htt
fragments. As shown in Figure 1F, a Ser-Ser-Ser motif lies
within the 76 aa sequence in which the caspase cleavage sites
reside in Htt.
The length of the polyglutamine tract modulates
calpain cleavage of Htt
Because production of N-terminal cleavage products plays an
important role in HD pathogenesis (DiFiglia et al., 1997), we
?- and m-calpain produced three products. The cleavage is dependent on enzyme concentrations with low levels (L) of ?- and m-calpain (0.1 and 0.3
?M, respectively) producing 67 and 62 kDa fragments and higher levels (H) of ?- and m-calpain (0.3 and 3.0 ?M, respectively) producing a single 47 kDa
fragment. B, The two larger Htt fragments produced by ?- and m-calpain at lower (L) enzyme concentrations are better visualized with longer exposure
times. C, Calpain cleavage of in vitro-translated Htt (1955-15) is also dependent on Ca2?concentration and is inhibited by the calpain inhibitor known
as calpain inhibitor 1. D, Cleavage of in vitro-translated Htt 3949-15 with caspase-3, m-calpain, caspase-2, and caspase-6. E, Table of potential calpain
cleavage sites. Caspase cleavage sites in Htt are highlighted in red. Caspase-3 cleaves at 513 and 552, caspase-2 cleaves at 552, and caspase-6 cleaves at
586. a, Molecular weight of a fragment on the gel (MWG, in kilodaltons; a, b and c–f values from two separate experiments); b, predicted molecular weight
determined from the relative molecular weight of the caspase-3 cleavage product [MWP, in kilodaltons; MWP? MWG? MWC(caspase-3)/MWG
(caspase-3)]; c, calculated molecular weight (MWC) of a fragment based on predicted cleavage site(s) (in kilodaltons); d, P1 amino acid number and
sequence for predicted calpain cleavage site(s) and known caspase cleavage sites; e, predicted amino acid sequence recognized by calpains (P2, P1, and
P1?) or the amino acid sequence recognized by caspases; f, amino acid sequence of a calpain cleavage site in protein kinase C; g, amino acid sequence
of a calpain cleavage site in caspase-12. F, Htt amino acid sequence from 437 to 586. Caspase sites are highlighted in red, and potential calpain sites are
highlighted in blue.
Htt is cleaved by calpains into four major cleavage products. A, Treatment of the in vitro-translated N-terminal Htt construct Htt 1955-15 with
4844 J. Neurosci., June 15, 2002, 22(12):4842–4849Gafni and Ellerby • Cleavage of Huntingtin
compared the rate of cleavage of normal and expanded Htt by
calpain. The disease form of Htt was more readily cleaved by
calpain (Fig. 2). Cleavage of normal Htt (Htt 3949-15) with
m-calpain produced three calpain-derived N-terminal Htt frag-
ments (62, 67, and 72 kDa) (Fig. 2A). Calpain cleavage of ex-
panded Htt (Htt 3949-138) under identical conditions produced
only small quantities of the repeat-dependent Htt fragments (72,
77, and 92 kDa) (Fig. 2A). The results were similar using either
?-calpain or m-calpain.
To further evaluate the repeat-dependent cleavage of Htt, we
incubated in vitro-translated normal and expanded Htt with in-
creasing concentrations of m-calpain. The 92 kDa fragment de-
rived from expanded Htt was more rapidly produced and de-
graded than the corresponding 72 kDa fragment of wild-type Htt
(Fig. 2B) (n ? 3). Furthermore, incubating normal and expanded
Htt with low levels of m-calpain (30 nM) demonstrated that
expanded Htt is more sensitive to calpain degradation (Fig. 2C)
(n ? 3). After a 120 min incubation at 30°C with m-calpain, a
major portion of the in vitro-translated expanded Htt was cleaved
(41%), whereas normal repeat Htt remained fully intact. Inter-
estingly, previous work using the same in vitro-translated con-
structs demonstrated that cleavage of Htt by caspases is repeat-
independent (Wellington et al., 1998).
Increased calpain expression and Htt fragmentation in
thapsigargin-treated 293T cells
Given our initial in vitro experiments showing cleavage of Htt by
calpains, we investigated whether Htt is cleaved by calpains in
intact cells under conditions of altered Ca2?homeostasis. We
treated 293T cells overexpressing full-length Htt (Htt15) with
thapsigargin, a proapoptotic agent that increases intracellular
Ca2?levels through inhibition of the endoplasmic reticulum
Ca2?/Mg2?ATPase. Treatment increased levels of the 28 kDa
calpain regulatory subunit and converted calpain to a 21 kDa
polypeptide consistent with calpain activation (Fig. 3A). Thapsi-
gargin treatment resulted in a twofold increase in activated cal-
pain (Fig. 3A). Thapsigargin-treated cells generated additional
cleavage products of Htt when compared with untreated cells
(Fig. 3B). These cleavage products are identical in size to those
generated by treating full-length Htt15-transfected cell lysates
with recombinant calpains (Fig. 3B, lane 5). In addition, treatment
of 293T cells overexpressing the caspase-resistant form of full-
length Htt15 (D513A, D552A, D586A) with thapsigargin pro-
duced increased levels of calpain-derived Htt fragments, demon-
strating that the Htt fragments are not attributable to caspase
cleavage (Fig. 3B).
We subsequently investigated whether differential cleavage of
mutant Htt would be observed in a cell culture model by treating
293T cells overexpressing normal and expanded Htt (3949-15 and
3949-138) with thapsigargin. Strikingly, we found increased levels
of expanded Htt cleavage products relative to wild-type (Fig. 3C).
Interestingly, the expression of mutant Htt resulted in depletion of
endogenous full-length normal Htt (Fig. 3C). A 30% reduction in
age of Htt. A, Cleavage of in vitro-translated Htt 3949-15 with m-calpain
produced three distinct cleavage products, whereas the cleavage of Htt
3949-138 produced only a small amount of the largest calpain repeat-
dependent cleavage product and barely detectable levels of the two
smaller repeat-dependent cleavage products. B, Incubating in vitro-
translated Htt (Htt 3949-15 and Htt 3949-138) with increasing concentra-
tions of m-calpain demonstrated that the 92 kDa cleavage product derived
from mutant Htt is more rapidly produced and degraded at lower enzyme
concentrations than the corresponding 72 kDa wild-type Htt cleavage
product (n ? 3). C, In the presence of m-calpain (30 nM), expanded Htt
(Htt 3949-138) is preferentially cleaved relative to the normal protein (Htt
3949-15) (n ? 3).
The length of the polyglutamine tract modulates calpain cleav-
fragmentation are observed in thapsigargin-treated 293T cells overex-
pressing Htt. A, 293T cells overexpressing full-length Htt15 treated with
thapsigargin (Thaps) and probed with calpain (Cp) regulatory subunit
antibody. B, 293T cells overexpressing full-length Htt15 and caspase-
resistant full-length Htt15 D513A, D552A, D586A treated with thapsi-
gargin (Thaps) and probed with anti-Htt antibody 2166. The controls in B
are lysates from 293T cells overexpressing full-length Htt15, which are
subsequently treated with m-calpain. C, 293T cells overexpressing normal
Htt 3949-15 and expanded Htt 3949-138 treated with thapsigargin (Thaps)
and probed with anti-Htt antibody 2166.
According to Western blot analysis, calpain activation and Htt
Gafni and Ellerby • Cleavage of HuntingtinJ. Neurosci., June 15, 2002, 22(12):4842–4849 4845
endogenous Htt was observed in the presence of the mutant Htt
fragment when compared with the normal Htt fragment (Fig. 3C).
Increased calpain expression and Htt fragmentation in
the human HD caudate
Increased calpain activation and Htt fragmentation was observed
in the caudate of human HD patients (Fig. 4) (n ? 3). The age,
sex, and postmortem interval of the HD and control caudate
tissue used for these studies are shown in Table 1. The 21 kDa
active form of calpain was detected in HD tissue and not in
controls (Fig. 4A) (n ? 3). Interestingly, both the catalytically
inactive precursor of the calpain regulatory subunit and the
activated forms are increased in the HD patients relative to
controls. Also noteworthy was the finding that a patient with
earlier disease onset has higher levels of calpain expression (Fig.
4A,B). The expression of the 21 kDa active form of calpain
regulatory subunit was on average eightfold higher in the HD
group when compared with controls, and the total calpain levels
were 2.5-fold higher in the HD group relative to controls (Fig.
4B) (n ? 3). Furthermore, total levels of the large subunit of
?-calpain were increased in HD relative to control tissue (Fig.
4C) (n ? 3).
HD patients also had a decrease in full-length Htt (data not
shown), along with a degradation of Htt into smaller fragments,
despite the increase in length of the polyglutamine tract (Fig. 4D)
(n ? 3) when compared with age-matched controls. The different
pattern of fragmentation observed is likely attributable to activa-
tion of distinct proteolytic pathways. Because calpains are so
dramatically activated in the HD caudate, it follows that this
pathway contributes to the cleavage pattern observed in HD and
not in control tissue. Additional experiments demonstrate that all
three of the Htt cleavage products in the human HD caudate
contain the N terminus (data not shown). Cleavage of full-length
Htt15 and Htt44 (approximate size of repeat in HD patients)
with m-calpain showed that the largest N-terminal cleavage prod-
uct in HD patients is identical in size to one of the calpain-
derived N-terminal Htt fragments (Fig. 4D, see asterisk), suggest-
ing that at least one of the HD fragments may be calpain-derived.
Increased expression of calpain and altered
subcellular localization in the caudate of HD patients
Given our finding that Htt is a substrate for calpains, immuno-
histochemical analysis was performed on human HD caudate and
age-matched controls (Fig. 5) (n ? 3). The antibodies used were
specific to the large catalytic subunits of ?-calpain and m-calpain
as well as to the small calpain regulatory subunit, as shown by
Western blot analysis (data not shown). In HD patients, immu-
fragmentation are observed in the HD caudate. A, Human HD and
control caudate lysates probed with calpain (Cp) regulatory subunit anti-
body (n ? 3). B, Quantification of calpain regulatory subunit expression in
the human HD and control caudate (n ? 3). C, Quantification of
?-calpain large subunit expression in the HD and control caudate (n ? 3).
D, Human HD and control caudate lysates probed with anti-Htt antibody
2166 (n ? 3). The controls are lysates from 293T cells overexpressing
full-length Htt15 and Htt44, which are subsequently treated with
m-calpain. The cleavage product labeled with an asterisk represents an
Htt44 N-terminal calpain cleavage product equivalent in size to the
largest N-terminal cleavage product found in HD lysates.
According to Western blot analysis, calpain activation and Htt
Table 1. Human caudate tissue samples used for Western blot analysis
AgeSex PMI (hr) Use
HD (grade 3):
PMI, Postmortem interval; WB, Western blot; IHC, immunohistochemistry; F,
female; M, male.
4846 J. Neurosci., June 15, 2002, 22(12):4842–4849Gafni and Ellerby • Cleavage of Huntingtin
noreactivity to the calpain regulatory subunit was detected in
perinuclear vesicular structures (Fig. 5C–F), whereas age-
matched controls showed much lower levels of calpain staining
(Fig. 5A,B). In addition, we saw a number of cells in deeper layers
of the HD caudate exhibiting intense perinuclear vesicular stain-
ing, including medium spiny neurons (Fig. 5D,F). The enhanced
immunoreactivity of calpains in the HD caudate correlates with
the higher levels of calpain found by Western blot analysis (Fig.
4). The pattern of calpain staining is distinct from the diffuse
pattern of calpain staining reported in other studies. The intensity
of staining in HD tissue is not only attributable to increased
calpain levels in HD tissue but also to the concentration of
calpains in Htt-containing vesicular structures.
Immunohistochemical analysis with antibodies to ?-calpain
and m-calpain showed intense staining in a large number of cells
surrounding the ventricle; this staining appeared to colocalize
with Htt in aggregates in cytosolic and possibly nuclear compart-
ments (Fig. 6). Based on staining with specific cell-type markers,
it was determined that these cells are both neurons and glia (Figs.
5C,E and Fig. 6B,C,E,F,H,I) (data not shown). Calpain and Htt
aggregation was observed in age-matched control caudate, al-
though at greatly reduced levels in all regions (Fig. 6A,D,G).
Interestingly, the two calpain isoforms were differentially ex-
pressed in Lewy bodies within the HD caudate. Although the
?-calpain catalytic subunit and calpain regulatory subunit were
expressed at high levels with Htt within Lewy body structures,
expression of the m-calpain catalytic subunit was not detected in
these structures (Fig. 5C).
One pathological mechanism proposed for HD is that the pro-
duction of a toxic fragment(s) containing the polyglutamine tract
amplifies pathways leading to neuronal dysfunction and cell death
(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). Caspases represent one class
of proteases that may initiate the cleavage of Htt in HD (Gold-
berg et al., 1996). Evidence for caspase activation has been
observed in the HD brain, and expression of the expanded
polyglutamine form of Htt in cell culture promotes cell death
(Goldberg et al., 1996). These initial results do not exclude the
are upregulated in the HD caudate. Immu-
nohistochemical analysis shows increased
expression of the ?- and m-calpain catalytic
subunit, as well as Htt, in the caudate from
HD patients (A–I). Intense perinuclear ve-
sicular staining of all three proteins is ob-
served in HD caudate tissue surrounding
the lateral ventricle (A–I) as well as in cells
deeper within the caudate, including me-
dium spiny neurons. Staining with calpain
isoform-specific antibodies also indicates
that only ?-calpain and Htt proteins (not
m-calpain) are associated with Lewy bodies
(data not shown). Arrowheads indicate pe-
rinuclear vesicles, and intranuclear staining
is indicated by an asterisk. In A–I, the lateral
ventricle is toward the bottom of the picture.
Insets in B, E, and H represent boundaries of
the 100? image in C, F, and I, respectively.
Both ?-calpain and m-calpain
HD caudate. Immunohistochemical analysis shows that high levels of
calpain are localized to perinuclear vesicles in cells near the lateral
ventricle (A, C, E) as well as to medium spiny neurons deeper within the
caudate (B, D, F) in HD patients relative to controls (n ? 3). In addition,
calpain is expressed at high levels in the Lewy bodies of HD patients (C).
Arrowheads indicate perinuclear vesicles. Lewy bodies are to the right of
the asterisk. In A, C, and E, the lateral ventricle is toward the bottom of the
picture. Insets in C and D represent boundaries of the 100? image in E
and F, respectively.
Increased expression of the calpain regulatory subunit in the
Gafni and Ellerby • Cleavage of HuntingtinJ. Neurosci., June 15, 2002, 22(12):4842–4849 4847
possibility that other proteases may contribute to the initiation or
further truncation of Htt. To continue our investigation of the
proteolytic pathways that contribute to the generation of toxic
fragments in HD, we investigated the role of calpains in the
cleavage of Htt. In the present work, we demonstrate that Htt is
cleaved by calpains, but more importantly that the cleavage is
modulated by CAG repeat length. In addition, cleavage of Htt by
calpains occurs under conditions that modulate Ca2?homeosta-
sis and not necessarily all conditions that induce cell death
(Wellington et al., 2000).
It is interesting to note that although potential calpain sites in
Htt encompass a range of locations within the Htt protein, one site
is tightly clustered in the region containing the caspase cleavage
sites. In some cases, calpains play an upstream role in activating
caspases, whereas in other cases, they act in parallel with caspases
to promote cell death or shuttle the cell toward a necrotic death by
rendering caspases inactive (Pike et al., 1998; Chua et al., 2000;
Lankiewicz et al., 2000; Wang, 2000; Blomgren et al., 2001). Our
current work demonstrates that Htt is cleaved by calpains indepen-
dently of caspases. Future work will address how these two families
of cysteine proteases interact in HD pathogenesis.
One particularly important finding is that some of the frag-
ments generated by calpains are small enough to diffuse into the
nucleus. Larger N-terminal Htt fragments form strictly perinu-
clear aggregates, whereas smaller Htt fragments (?60 kDa) can
also translocate to the nucleus (Hackam et al., 1998). In a number
of transgenic mouse models expressing full-length Htt, the
N-terminal fragments redistribute to the nucleus and cleavage of
Htt is believed to precede entry of Htt into the nucleus (Hodgson
et al., 1999). The sizes of caspase cleavage products range from 70
to 80 kDa and therefore are found in perinuclear aggregates in
the cytoplasm. In addition, caspases produce single cleavage
products that are not further truncated. In contrast, calpains
generate a cascade of fragments and intermediates derived from
full-length Htt. One might predict from our results that trunca-
tion by calpains may contribute to the redistribution of Htt to the
Uncontrolled calpain activity or activation may contribute to
ischemic brain injury, Alzheimer’s disease, multiple sclerosis, and
Parkinson’s disease (for review, see Wang, 2000). In models of
ischemia, calpain activation serves as a link between initial ionic
disturbances and apoptotic pathways. We have shown that cal-
pains are aberrantly activated in the HD caudate. Saito et al.
(1993) previously have shown activation of the large subunit of
?-calpain with no net increase in total levels of calpain in the HD
putamen. Our results demonstrate a dramatic increase in the
levels of both the precursor and active forms of calpain in the
caudate, which is more severely affected in HD.
Currently, the physiological function(s) of calpains is un-
known. However, it should be noted that calpain activity is
essential for a number of cellular functions unrelated to cell
death. Therefore, cleavage of Htt by calpains under normal phys-
iological conditions may modulate important cellular events. Un-
like many cysteine proteases, calpains tend to cleave substrates at
interdomain boundaries, thereby modulating the function of their
substrates rather than inactivating them. It is possible that calpain
cleavage of normal Htt modulates events related to the potential
role of Htt in vesicular trafficking (DiFiglia et al., 1995) and/or
the control of BDNF levels by Htt (Zuccato et al., 2001). This
would be in contrast to the cleavage of expanded Htt by calpain,
which generates products that are toxic to the cell. Furthermore,
increased levels of Htt fragments lead to the depletion of normal
full-length Htt, which could also impede normal cell function.
Our work suggests that calpain cleavage of Htt may play an
important role in the pathogenesis of HD and compliments a
recent report that Htt can be cleaved by calpains (Kim et al.,
2001). Further work will be directed at evaluating the relative
contribution of caspase and calpain cleavage in the natural history
of disease pathology and progression in the HD transgenic mouse
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