[Cell Cycle 6:23, 2970-2981, 1 December 2007]; ©2007 Landes Bioscience

Report

N-Terminal Proteolysis of Full-Length Mutant Huntingtin in an

Inducible PC12 Cell Model of Huntington’s Disease
Tamara Ratovitski1,† ABSTRACT
Masayuki Nakamura1,†,‡ Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis
of Huntington’s disease; however the steps in huntingtin proteolysis are not fully under-
James D’Ambola1

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stood. Huntingtin was shown to be cleaved by caspases and calpains within a region

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Ekaterine Chighladze1 between 460–600 amino acids from the N-terminus. Two smaller N-terminal fragments

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produced by unknown protease have been previously described as cp-A and cp-B. To
Yideng Liang1 further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell

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Wenfei Wang1 model expressing full-length huntingtin with either normal or expanded polyglutamine.
This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated
Rona Graham2 by caspases within the region previously mapped for caspase cleavage, and cleavage

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Michael R. Hayden2 generating two novel N-terminal fragments (cp-1 approximately 90–105 residues long
and cp-2 extending beyond 115–129 epitope of huntingtin). Interestingly, the deletion
David R. Borchelt3 of amino acids 105–114 (mapped previously as a cleavage site for cp-A) failed to

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Ricky R. Hirschhorn1,4 affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments

Christopher A. Ross1,5,*
1Division of Neurobiology; Department of Psychiatry; 5Departments of Neurology
and Neuroscience; Johns Hopkins University School of Medicine; Baltimore,
Maryland USA
2Department of Medical Genetics; Centre for Molecular Medicine and Therapeutics;
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are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and
cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions
prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-
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independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of
calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and
accelerated formation of inclusions. Further delineation of huntingtin cleavage events may
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Child and Family Research Institute; University of British Columbia; Vancouver,

lead to novel therapeutic targets for HD.
British Columbia, Canada
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3Department of Neuroscience; Santa Fe Health Alzheimer’s Disease Center;

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McKnight Brain Institute; University of Florida; Gainesville, Florida USA

4Department of Biology; Hood College; Frederick, Maryland USA
ABBREVIATIONS
HD, Huntington’s disease; Htt, huntingtin; polyQ, polyglutamine; ER, endoplasmic
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†These authors equally contributed to this work.

reticulum; aa, amino acid; Dox, doxycycline; NG108-15, neuroblastoma-glioma; PC12,
‡Current Address: Department of Psychiatry; Kagoshima University Graduate pheochromocytoma; tTA, transactivator; TRE, tet-responsive element; DMEM, Dulbecco’s
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School of Medical and Dental Sciences; Sakuragaoka, Kagoshima, Japan
*Correspondence to: Christopher A. Ross and Tamara Ratovitski; CMSC 8-121; 600
North Wolfe Street; Baltimore, Maryland 21287 USA; Fax: 410.614.0013; Email:
caross@jhu.edu/ tratovi1@jhmi.edu
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modified Eagle’s medium; FBS, fetal bovine serum; NGF, nerve growth factor; HEK,
human embryonic kidney; NER, nuclear extraction reagent; DAPI, 4',6-Diamidino-2-phe-
nylindol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly ADP-ribose
polymerase; FA, formic acid; UPS, ubiquitin-proteasome system; GFP, green fluorescent

Original manuscript submitted: 07/31/07 protein; CMV, cytomegalovirus; NS, non-specific

Revised manuscript submitted: 08/31/07
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Manuscript accepted: 09/02/07

INTRODUCTION
Previously published online as a E-publication:
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http://www.landesbioscience.com/journals/cc/article/4992 Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by CAG

repeat expansion (coding for polyglutamine) within the coding region of the huntingtin
KEY WORDS gene product (Htt). There is a threshold of 36 repeats for HD, and an inverse correla-
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tion between the length of the polyglutamine (polyQ) expansion and the age of onset.1,2
Huntington’s Disease mechanism, proteolysis,
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Clinically HD is characterized by movement disorder and cognitive and behavioral

protein degradation, caspases, calpains
changes, progressing to death within 15–20 years from onset. The symptoms of HD are
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likely to be caused by both neuronal death and dysfunction.

ACKNOWLEDGEMENTS
Htt is an approximately 350 kD ubiquitously expressed protein that has been localized
See page XXX. to several subcellular compartments, including nuclear and cytoplasmic fractions. It has
been found in association with organelles, including mitochondria, ER/Golgi, endosomes,
presynaptic and clathrin-coated vesicles and plasma membrane.3-8 Cellular toxicity in HD
may involve interference of expanded Htt with gene transcription, vesicle transport, mito-
chondrial function, Ca2+ homeostasis, and protein degradation machinery via proteasome
or autophagy.9-15 The nature of cytotoxic species of expanded Htt is unknown, but there is
emerging evidence suggesting that they might be generated via Htt proteolysis. The main

2970 Cell Cycle 2007; Vol. 6 Issue 23

 N-terminal Proteolysis of Huntingtin in PC12 Cell Model

which can be detected with antibodies

to Htt peptides 1–17 and 81–90 but not
115-129.20
Htt can be cleaved between residues
513 and 586 by caspases 2, 3, 6 and 7
(Fig. 1), and this cleavage has been linked
to cell toxicity.33-38 The importance of
caspase 6 mediated cleavage of Htt at
position 586 have been confirmed in
vivo: YAC 128 mice expressing mutant
Htt resistant to cleavage at this site
do not demonstrate a phenotype in
any of the endpoints previously estab-

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lished in the YAC128 model, including

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motor, cognitive and neuropathological
deficits.39 Cleavage of Htt by calpains

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(e.g., at residues 469 and 536, Fig. 1)
has also been reported to contribute
to toxicity.27,40-43 At least two smaller

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N-terminal fragments (cp-A and cp-B)
were previously described in reference 18:

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Cp-A was defined as a fragment produced
by cleavage in the region between resi-

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dues 82 and 129, which accumulates
within nuclear inclusions in mouse-rat
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stable cells expressing expanded forms
Figure 1. Generation of inducible PC12 cell model. (A) The “tet-off” system was used in which the expres- of Htt. Deletion of amino acids 105–
.D
sion of Htt can be induced by removing doxycycline. This facilitates binding of tTA to the tet-responsive 114, or alteration within this region
element (TRE) controlling Htt expression. The full-length human Htt cDNA with either 23Q or 148Q was
prevented production of the cp-A frag-
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cloned into pTet-splice vector (pTet-splice-FL-Htt-23/148Q). *Stable lines generated with these vectors
expressed Htt with 21Q or 126Q respectively, as confirmed by genotyping. The N-terminal c-myc tag ment. Cp-B fragment is longer (up to
was included for Htt detection. The tTA expression was driven by the cytomegalovirus (CMV) promoter. 214 amino acids) and was found in
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(B) Schematic of Htt proteolysis. Within the sequence indicated are putative cp-A/cp-B (18) and cp-1/cp-2 cytoplasmic inclusions. Cp-A fragment
cleavage sites (hatched pattern); identified caspase and calpain sites (solid boxes). Below are shown was suggested to be produced by an
epitopes recognized by Htt antibodies.
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unknown aspartic protease. Certain lyso-

somal proteases (cathepsin D, B and L)
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pathological feature of all CAG repeat disorders is the formation were found to contribute to Htt degradation and accumulation of
of nuclear and cytoplasmic inclusions in neurons of affected brain stable N-terminal fragments in clonal striatal cells, but it is not clear
regions. Inclusions in brains of HD patients can be stained with anti- whether they mediate Htt clearance, or facilitate generation of toxic
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bodies to ubiquitin, and with antibodies directed at the N-terminal, fragments.44,45

but not C-terminal, epitopes of Htt.16-21 This suggests that inclu- Although data from mouse and cell studies support the role of
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sions contain truncated N-terminal fragments of Htt. Although Htt proteolysis in HD pathogenesis, what steps generate the toxic
the inclusion bodies themselves might be neuroprotective,10,22 the fragments, the lengths of the fragments, and whether fragments
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N-terminal fragments of Htt in cells may be pathogenic. are produced prior to or subsequent to cell toxicity are poorly
Cellular models of HD suggest that short N-terminal fragments understood. There is considerable mounting evidence for the role
accumulate in the nucleus, and are more toxic than longer fragments of specific sized fragments in the pathogenesis of disease. Mouse
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or full-length Htt.18,21-27 Mouse model studies also support a role HD models described above suggest that the 552 amino acid (aa),
of Htt proteolysis in HD pathogenesis. Transgenic mice expressing 513 aa and the 117 aa (“shortstop”) mutant Htt fragments are not
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N-terminal fragments of Htt generally have more severe behavioral toxic. In contrast the 586 aa, the exon 1 (R6/2 mice) and the N171
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and pathologic phenotypes than mice expressing full-length Htt.28-30 (N171-82Q) mutant Htt fragments are toxic. The 586 aa fragment
One exception—the “shortstop” mice, expressing a short N-terminal is produced in vivo from both endogenous and mutant htt. However,
Htt fragment only 117 amino acids long31—do not demonstrate a it is not clear whether cleavage in the caspase and calpain region
phenotype compared to wild type mice. Inclusions are present, but is always necessary and precedes the formation of cp-A and cp-B
these do not correlate with neuronal loss. A recently described induc- - like fragments, or whether these proteolytic pathways may occur
ible mouse model, expressing full-length expanded Htt, accumulate a in parallel. Elucidating the steps in Htt proteolysis may facilitate the
60 kD Htt N-terminal cleavage product in the nucleus,32 and display identification of novel targets for development of HD therapeutics.
relatively severe phenotype. Mice expressing the first 171 amino We have generated a stable inducible cell model of HD, in which
acids of Htt (Htt-N171-82Q) also accumulate nuclear inclusions differentiated rat pheochromocytoma (PC12) cells are induced to

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 N-terminal Proteolysis of Huntingtin in PC12 Cell Model
express full-length Htt with either normal or expanded polygluta-
mine repeats. This cell model reveals several steps of Htt proteolysis:
proteolysis mediated by caspases within the region previously mapped
for caspase cleavage, and cleavage generating two novel N-terminal
fragments cp-1 and cp-2. These short N-terminal fragments accumu-
late within nuclear and cytoplasmic inclusions, and their formation
precedes Htt-induced cell toxicity. We provide evidence that cp-1
and cp-2 fragments generated in differentiated PC12 cells can be
formed by caspase-independent proteolytic cleavage, consistent with
the parallel cleavage model in this cell system. Epitope mapping of
these fragments with antibodies to Htt suggest that they are similar
in length to previously described cp-A and cp-B fragments. However
the deletion of amino acids 105–114 (implicated in generation of
cp-A) failed to affect the production of cp-1 and cp-2 fragments in
this PC12 cell model, suggesting distinct proteolytic events.
MATERIALS AND METHODS
Plasmids and mutagenesis. Full-length constructs for inducible
expression of normal (pTet-splice-FL-Htt-23Q) or expanded (pTet-
splice-FL-Htt-148Q) Htt were described previously in reference 32.
The deletions of residues 105–114 and substitutions of alanines at
positions 109–113 were introduced into pTet-N171-148Q construct
(described in Tanaka et al., 2006) by site-directed mutagenesis using
QuikChange II XL kit (Stratagene) according to manufacturer’s
protocol. The resulting constructs were digested with XhoI and DNA
fragments encoding N-terminal Htt fragments (N171) were ligated
into pTet-splice-FL-23Q after digestion with XhoI. Htt expression
constructs used for transient transfection, including wild-type and
caspase-resistant (quint mutation) forms of normal and expanded
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Htt N-terminal fragment (N1212-15Q and N1212-138Q), were
previously described in references 34 and 40.
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Cell culture and transfection. To establish inducible stable
PC12 cell lines expressing full-length normal or polyQ expanded
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Htt, we used Tet-off PC12 cells (Clontech), stably expressing the
tetracycline transactivator (tTA). Cells were co-transfected with Htt
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constructs and pTK-Hyg vector (Clontech) in the ratio 20:1 using
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
procedure, and were plated on collagen I treated culture dishes
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(Biocoat, BD Biosciences). The stable clones were selected in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
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5% Tet System approved fetal bovine serum (FBS, Clontech), 10%
horse serum (Invitrogen), 100 µg/ml Geneticin (Invitrogen), 200 µg/
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ml hygromycin (Roche), 200 ng/ml doxycycline (Dox, Invitrogen),
100 units/ml penicillin and 100 units/ml streptomycin. After three
to four weeks of selection at 37˚C in a humidified 5% CO2 incu-
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bator, the antibiotic resistant clones were isolated and screened for
Htt expression by Western blot analysis using anti-c-myc antibody
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9E10 (Roche). Stable cell lines were maintained in the same medium.
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The polyQ repeat length in selected lines, confirmed by genotyping,
was 21Q for normal Htt and 126Q for expanded Htt (lines S6
and S6-5). For neuronal differentiation PC12 cells were grown in
DMEM with 1% horse serum, supplemented with nerve growth
factor (NGF, 50 ng/ml, Roche). To study the effects of inhibitors
in PC12 cells, the following inhibitors were added to differentiation
media: caspase pan-specific, Z-VAD, 25 µM (Calbiochem), caspase
2-specific, Z-VDVAD, 10 µM (Calbiochem), caspase 3-specific,
AcDMQD, 25 µM (Calbiochem), caspase 6-specific Z-VEID, 10 µM
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(Calbiochem) and calpain1/2-specific, MDL128170, 10 µM
(Calbiochem), or calpeptin, 50 µM or 75 µM (Calbiochem). Fresh
inhibitors were added every three days. Proteasome inhibitors lacta-
cystin (Calbiochem); MG132 (Calbiochem) or U102 (Biomol) were
added to growth media at indicated concentrations for the last 24 h
of cell incubation. For transient transfection experiments human
embryonic kidney (HEK) 293FT cells (Invitrogen) were grown
in DMEM with 10% FBS, 100 µg/ml Geneticin, 100 units/ml
penicillin and 100 units/ml streptomycin, and transfected with Htt
constructs using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s protocol. Proteasome reporter cell line GFPu-1 was
from American Type Culture Collection (ATCC).
Subcellular fractionation and dissociation of aggregates. PC12
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cells were first fractionated to enrich for cytoplasmic and nuclear
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proteins using 0.5% Nonidet P-40 (protocol 1), or Nuclear and
Cytoplasmic Extraction reagent (NE-PER) from Pierce (protocol 2).
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Nuclear pellets and soluble cytoplasmic fractions C were obtained
after centrifugation at 500 g (protocol 1), or at 15,000 g (protocol 2).
Nuclear pellets were solubilized using SDS buffer (protocol 1) or
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Nuclear Extraction Reagent (NER, Pierce, protocol 2), and super-
natants containing soluble nuclear fractions N and pellets were
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collected after centrifugation at 15,000 g. Cytoplasmic fraction C did
not produce any pellet upon solubilization in SDS buffer. The pellets
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from nuclear fractions were further dissociated using formic acid to
produce formic acid-soluble nuclear fractions F.18,23
ONCell toxicity measurements. Stable PC12 cell lines carrying either
FL-Htt-21Q or FL-Htt-126Q were seeded on collagen I coated
24-well plates (BD Biosciences) at a concentration 105 cells/ml
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in differentiation media either with or without doxycycline. Cell
viability was measured by trypan blue exclusion assay. Results repre-
sent the average of four wells. Staining with annexin V-EGFP and
propidium iodide of PC12 cells was performed using the annexin
V-EGFP apoptosis detection kit (BioVision Research Products)
according to the manufacturer’s protocol, on day 8 after induction of
Htt expression and differentiation. Five fields (200–500 cells each)
were counted blindly for each condition.
Immunofluorescence. PC12 cells, induced to express FL-Htt-126Q
for 8 days, were fixed with 4% paraformaldehyde for 15 min, perme-
alized with 0.5% Triton X-100 (Sigma) for 10 min, blocked in
10% normal goat serum (Sigma) for 30 min, and incubated with
monoclonal anti c-myc antibody, polyclonal antibody to Htt epit-
opes 497–513, or Htt epitopes 55–66 (1 h at room temperature),
followed by goat anti-mouse IgG FITC-conjugated (Vector), or goat
anti-rabbit Cy3-conjugated (Vector) secondary antibodies (1 h at
room temperature). Nuclei were stained with 4', 6-Diamidino-2-ph
enylindol (DAPI, Vector).
Western blot analysis and antibodies. Polyclonal antibodies to
Htt-htt 55–65 and htt 81–90 were described previously in reference
20. Briefly, they were produced in rabbit against a series of synthetic
peptides, the number of each peptide corresponds to the residue
number of wild-type human htt with 23Q (Genbank Accession
#NM-002111).20 Antibody to Htt epitope 497–513 was also gener-
ated in rabbit against the synthetic peptide. Htt 115–129 antibody
(against residues 115–129 of Htt) is from Chemicon International
(cat. # MAB5490), antibody to DLND neoepitope of Htt-htt552
and antibody to DSVD neoepitope of Htt-htt513 are a kind gift
from Sophie Roy (Merck Canada Co. Ltd., Canada). For Western
blotting analysis, PC12 or HEK293 cells were lysed in M-PER buffer
Cell Cycle 2007; Vol. 6 Issue 23
 N-terminal Proteolysis of Huntingtin in PC12 Cell Model

(Pierce) with protease inhibitors (Protease Inhibitor Cocktail III,

Calbiochem), unless otherwise indicated, and protein concentrations
were estimated using BCA method (Biorad). Lysates (60 µg for PC12
cells, and 30 µg for HEK293 cells) were fractionated on NuPAGE
4–12% Bis-Tris polyacrylamide gels (Invitrogen), transferred to
nitrocellulose membranes, and probed with antibodies against Htt,
c-myc (9E10, Roche), and actin (Sigma) or GAPDH (Santa Cruz
Biotechnology) for loading control. β-tubulin (Sigma) and poly
ADP-ribose polymerase (PARP, Biomol) antibodies were used as
fractionation control. Immunoblots were developed with peroxi-
dase-conjugated secondary antibodies (Amersham), and enhanced
chemiluminescence (ECL-Plus detection reagent, Amersham).

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RESULTS

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PC12 cells expressing FL-Htt-126Q produce N-terminal frag-
ments. To study proteolysis of Htt, we have developed several stable

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inducible PC12 cell lines expressing full-length Htt with either
normal (21Q) or expanded (126Q) polyglutamines. We used the

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Tet-off system in which the expression of Htt can be induced by
removing doxycycline (Fig. 1A). PC12 cells were differentiated using
NGF for up to 14 days, and Htt expression was evaluated with either

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anti-c-myc antibodies, or antibodies recognizing the epitope between

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residues 55 and 66 of Htt (Fig. 1B). Differentiated PC12 cells (line
S6) expressed full-length Htt with 126Q, which was detectable after
four days upon induction (Fig. 2A). Two partially resolved fragments
migrating at ~60 kDa (cp-1 and cp-2) were detected at the same time
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with antibody to Htt. The same fragments were also detected with
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antibody to c-myc (data not shown), indicating that they represent
N-terminal Htt cleavage products. These fragments showed gel
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mobility similar to N-terminal fragments identified in transgenic

mice expressing full-length Htt with expanded polyglutamine,32
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and are consistent with the size predicted for cp-A and cp-B frag-
ments detected in NG108 cells and clonal striatal cells expressing
Htt.18,44 However, unlike cp-A and cp-B, cp-1 and cp-2 frag-
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ments were readily detected without proteasome inhibition. PC12 Figure 2. PC12 cells expressing FL-Htt-126Q produce N-terminal fragments.
cells expressing full-length Htt with normal polyglutamine repeat PC12 cells (line S6) were differentiated using NGF and induced to express
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(FL-Htt-21Q) produced very low levels of comparable fragments FL-Htt-126Q by removal of doxycycline for indicated time periods. Total cell
(data not shown). extracts (60 µg per lane) were analyzed by Western blotting with antibody to
Since caspase mediated cleavage of Htt has been described previ- residues 55-66 of Htt (A), neoepitope antibody to caspase 2/3/7 cleavage
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ously in references 33–38, we used neoepitope antibodies to caspase sites in Htt (DLND) htt552 (B), neoepitope antibody to caspase 3 site in Htt
(DSVD) htt513 (C), or antibody to GAPDH for loading control. FL-Htt-126Q is
2/3/7 site in Htt (DLND) htt552 to further characterize Htt proteo- indicated by arrowhead. Short N-terminal fragments cp-1/cp-2 (arrows) run
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lytic fragments produced by PC12 cells expressing expanded Htt. 552 as partially resolved doublet. Larger caspase generated fragments (arrows)
aa fragments were visible starting from day 4 upon induction, and are detected with htt552 and htt513 antibodies. Non-specific bands are
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their mobility was consistent with the region previously mapped for marked with NS.
caspase 2 cleavage (Fig. 2B). This antibody also had some reactivity
with uncleaved Htt. Some caspase 3 generated fragments were also
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detected using neoepitope antibody to caspase 3 site of Htt (DSVD)

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htt513 (Fig. 2C), however the signal was low. Thus, we showed that fragments were stabilized, and could be detected without proteasome
our stable PC12 cell model reveals several steps of Htt proteolysis inhibition. To investigate whether our fragments are identical to cp-A
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including caspase-mediated proteolysis within the caspase/calpain and cp-B, we have generated PC12 cell lines expressing FL-Htt-126Q
cleavage domain, and N-terminal proteolysis producing shorter frag- with “cp-A cleavage site” deletions. Using at least three independent
ments (Fig. 1B). cell lines, we demonstrate that deletion of residues 105–114 does not
N-Terminal cp-1/cp-2 fragments may be distinct from cp-A/ affect the formation of 60 kD N-terminal fragments in stable PC12
cp-B. The domain containing the cleavage site for cp-A was previ- lines expressing FL-Htt-126Q (Fig. 3A and data not shown). The
ously mapped to the region between residues 105 and 114 in substitution of alanines at positions 109–113 (CENI to AAAA) also
NG108-1518 and striatal cells,44 expressing expanded Htt. However, did not alter the production of short N-terminal fragments in three
cp-A cleavage product was detected previously only in the presence of independent PC12 lines (data not shown). Therefore, we conclude
proteasome inhibitors, whereas in our PC12 cell model cp-1 and cp-2 that cp-1 and cp-2 fragments are distinct from the previously

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 N-terminal Proteolysis of Huntingtin in PC12 Cell Model

described cp-A and cp-B.

To map cp-1 and cp-2 fragments
we used antibodies recognizing
different Htt epitopes, and detected
the fragments in human embryonic
kidney (HEK) 293 cells transiently
expressing truncated Htt (N1212-15Q
and N1212-138Q), or FL-Htt-148Q
(Fig. 3B, right panel). We found that
HEK293 cells transfected with either
N1212-138Q, or FL-Htt-148Q
produce fragments which co-migrate
with cp-1 and cp-2 fragments from

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PC12 detected with either antibody to

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Htt, or anti-c-myc antibody (Fig. 3B,
left panel). The fragments produced

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from N1212-138Q in HEK293 cells
(Fig. 3B, lanes 6 and 9) migrated
slightly faster than those produced

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from FL-Htt-148Q (lanes 7 and 10),
which is consistent with the different

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number of glutamines. Notably, two
N-terminal fragments produced from

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N1212-15Q construct (lanes 5 and
8) can be easily separated on a gel,
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polyglutamine (138Q) appear as a
single band unresolved in these condi-
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tions. In HEK293 cells both cp-1
and cp-2 were detected with anti-
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bodies recognizing N-terminal c-myc

tag (data not shown) and epitopes
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between residues 55 and 66 of Htt

(Fig. 3B, lanes 5–7), and between
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residues 81 and 90 of Htt (Fig. 3B,

lanes 8–10). However, only cp-2 frag-
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ment was detected with the antibody

against an epitope between residues
Figure 3. N-terminal cp-1/ cp-2 fragments may be distinct from cp-A/cp-B. (A) “Cp-A cleavage site” deletion 115 and 129 of Htt (Fig. 3B, lanes
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(18) does not affect the formation of cp-1/cp-2 fragments in PC12 cells. Western blot of total cell extracts 12 and 13). Since cp-1 and cp-2 frag-
(60 µg per lane) from stable PC12 cells induced to express wild type FL-Htt-126Q (line S6-5, left panel), or ments with expanded polyglutamine
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mutant FL-Htt-153Q with amino acids 105-114 removed (right panel) for the indicated number of days. Htt is
detected with anti-c-myc antibody, FL-Htt is indicated with arrowheads, cp-1/cp-2 fragments are indicated by
can not be resolved on this gel, we
arrows. Left panel was assembled from 2 blots. One representative deletion mutant PC12 cell line out of three only observed a substantial decrease in
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is shown. (B) Epitope mapping of cp-1/cp-2 fragments. Left panel- Western blot of total cell extracts (60 µg signal from detection of cp-2 fragment
per lane) from stable PC12 cells induced to express FL-Htt-126Q for eight days (lanes 2 and 4), or un-induced (lane 13), consistent with the lack
(lanes 1 and 3). Full-length Htt (arrowheads) and cp-1/cp-2 fragments (arrows) are detected with antibody to of recognition of cp-1 by this anti-
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residues 55-66 of Htt (lane 2), or anti-c-myc antibody (lane 4). Right panel- Western blot of total cell extracts
(30 µg per lane) from HEK293 cells transfected with either FL-Htt-148Q (lanes 7 and 10), truncated Htt con-
body. Thus epitope mapping indicate
that cleavage producing cp-1 fragment
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structs (N1212-15Q or N1212-138Q, lanes 5, 8, 12 and lanes 6, 9, 13 respectively), or non-transfected

cells (Cont). Full-length and truncated Htt (arrowheads) and cp-1/cp-2 fragments (arrows) are detected with occurs between the epitopes 81–90,
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antibody to residues 55-66 of Htt (lanes 5–7), with antibody to residues 81–90 of Htt (lanes 8–10), or with and 115–129 of Htt. Based on muta-
antibody to residues 115–129 of Htt (lanes 11–13). HEK293 cells transfected with expanded Htt constructs genesis studies in PC12 cells, described
produce fragments which co-migrated with cp-1/cp-2 fragments detected in PC12. Notably, two N-terminal
fragments produced from N1212-15Q construct (lane 5) can be easily separated on a gel, whereas fragments
above, the cleavage site producing cp-1
with expanded polyglutamine (138Q) appear as a single band unresolved on this gel. Lanes 5–7 are from the is not likely to lie between residues 105
same blot, which is also shown on Figure 6C. Non-specific bands are marked with NS. and 114. Combined, these results map
the position of cleavage generating
cp-1 to be between the epitope 81–90
and residue 105 of Htt. The exact
position of the site of cleavage gener-

2974 Cell Cycle 2007; Vol. 6 Issue 23

 N-terminal Proteolysis of Huntingtin in PC12 Cell Model

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Figure 4. N-terminal fragments of Htt are found in nuclear inclusions and accumulate in the aggregate fraction in PC12 cells. (A) Immunofluorescent
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detection of N-terminal fragments in PC12 cells (line S6) induced to express FL-Htt-126Q for 8 days. C-myc epitope is shown in green; epitopes between
residues 497–513 or residues 55–66 of Htt are shown in red; the nuclear staining (DAPI) is shown in blue. The right panel represents the merged image.
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(B–D) Disaggregation of nuclear inclusions in PC12 cells. Two procedures were used in parallel to fractionate and dissociate aggregated Htt fragments
(B, see experimental procedures). Enrichment for cytoplasmic and nuclear proteins in fractions C and N respectively was confirmed by the presence of
β-tubulin and PARP in corresponding fractions obtained using both protocols (C). (D) Western blot analysis of three fractions obtained from PC12 cells (line S6)
differentiated and induced to express FL-Htt-126Q for 8 days. Htt was detected with antibodies to c-myc, or to residues 55–66 of Htt. FL- Htt-126Q is
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indicated with arrowheads, cp-1/cp-2 fragments - with arrows. Fractionation of material obtained with other two independent cell lines produced similar
results (data not shown).
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ating cp-2 fragment (which extends beyond 115–129 epitope of Htt of nuclear inclusions, whereas cytoplasmic aggregates may contain
according to epitope mapping) remains to be identified. diverse mixture of Htt derivatives, including longer fragments.
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Short N-terminal fragments build up nuclear inclusions and To confirm that short N-terminal fragments are incorporated into
accumulate in aggregate fraction. To determine the subcellular nuclear aggregates, we used (previously described in refs. 18 and 23)
20

localization of short N-terminal fragments in PC12 cells expressing biochemical solubilization of aggregates with formic acid (FA, Fig.
expanded Htt, we performed immunofluorescence staining with 4B–D). Using antibodies to c-myc or to Htt (epitope 55–66 aa), we
©

antibodies recognizing different Htt epitopes (Fig. 4A). Our results
indicate that most nuclear inclusions can be labeled with antibodies
recognizing N-terminal epitopes (c-myc and an epitope between
amino acids 55–66), but not with antibodies against further
C-terminal epitope (amino acids 497–513). In contrast, most of the
C-terminal epitopes were detected in cytoplasmic aggregates, or were
diffusely spread around the cytoplasm. This staining pattern is consis-
tent with the idea that N-terminal fragments are main components
detected FL-Htt-126Q protein mostly in SDS soluble cytoplasmic
fraction C, and to a lesser extent in SDS soluble nuclear fraction
N (Fig. 4D). FA-soluble nuclear fraction F was mostly composed
of N-terminal fragments co-migrating with cp-1 and cp-2 frag-
ments detected in the soluble cytoplasmic fraction. Fraction F also
contained small amounts of longer fragments probably generated
by caspases, but did not contain detectable levels of FL-Htt-126Q.
The presence of some FL-Htt-126Q in soluble nuclear fraction N

www.landesbioscience.com Cell Cycle 2975

 N-terminal Proteolysis of Huntingtin in PC12 Cell Model
Figure 5. Increased cell death in PC12 cells, expressing FL Htt 126Q.
(A) Cell toxicity measured by trypan blue exclusion assay. Cells expressing
FL-Htt-126Q (line S6) show significant toxicity on day 12 after induction of
Htt expression (n = 4, *p < 0.05, FL-Htt-126Q versus FL-Htt-21Q). (B) Cell
toxicity and apoptosis measured by staining with annexin V-EGFP and prop-
CE
idium iodide. On day 8 after induction of FL-Htt-126Q expression, live PC12
cells (line S6-5) were double stained with annexinV-EGFP and propidium
IEN
iodide. About 12% of the cells are positive for either annexin V alone, or
for both dyes compared to ~2% in control. Five fields (200–500 cells each)
were counted for each condition (n = 5, **p < 0.005, dox- versus dox+).
SC
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might be a result of slight contamination of the nuclear fraction
with cytoplasmic proteins, as revealed by detection of low levels of
ES
β-tubulin in the nuclear fraction (Fig. 4C). Since most of the cyto-
plasmic inclusions had perinuclear localization (Fig. 3 and Fig. 8B),
ND
it is possible that these aggregates were co-purified with the nuclear
pellet (see experimental procedures), and thus FA-soluble fraction F
LA
may contain proteins from both nuclear and cytoplasmic inclusions.
In contrast, PC12 cells expressing Htt with normal repeat length
did not accumulate any Htt species in FA-soluble fraction (data not
07
shown).
FL-Htt-126Q induces moderate toxicity in PC12 cells. The effect
20
of expanded Htt expression on PC12 cell viability was measured by
©
trypan blue exclusion assay. Cells expressing FL-Htt-126Q (line
S6) begin to show cell toxicity on day 12 after induction of expres-
sion and differentiation (Fig. 5A). Cells expressing Htt with normal
repeat length (21Q) did not show toxicity greater than baseline.
Because of only a moderate degree of cell toxicity observed on day
12, an apoptotic marker, annexin V, was used to evaluate earlier
changes in cells expressing expanded Htt (Line S6-5, Fig. 5B). On
day 8 after induction of Htt expression, live cells were double stained
with annexinV-EGFP and propidium iodine, the latter penetrates
2976
cells that have lost membrane integrity. About 12% of the cells
were positive for either annexin V alone, or for both dyes on day 8
after induction of expression of expanded Htt, compared to ~2% in
control (Fig. 5B).
N-terminal fragments can be formed independently of caspase
cleavage. Differentiated PC12 cells accumulate two kinds of expanded
Htt cleavage products: approximately 100 kD caspase generated frag-
ments, and shorter N-terminal fragments potentially produced by
some unknown protease. The results of the time-course experiment
(Fig. 2) are consistent with both sequential and parallel models of Htt
cleavage. To further investigate whether the initial cleavage of Htt by
caspases /calpains is necessary for generation of cp-1 and cp-2 frag-
ments, differentiated PC12 cells, induced to express FL-Htt-126Q,
E .
were treated with caspase or calpain inhibitors (Fig. 6A). Specific
UT
inhibitors to caspase 2, 3, and 6, as well as pan-specific caspase inhib-
itor and specific calpain 1/2 inhibitor (MDL 28170) were added to
RIB
PC12 cells on day 2 after induction of Htt expression. On day 8
upon induction the cells were harvested and Htt fragments were visu-
alized with either anti-c-myc antibody, or antibodies to Htt (data not
IST
shown). Our data indicate that inhibition of caspases 2, 3 or 6 fails
to affect the levels of N-terminal fragments, however treatment of
D
PC12 cells with calpain 1/2 inhibitor results in a significant increase
in levels of cp-1 and cp-2 fragments (Fig. 6A, lane 5). To verify that
OT
caspase inhibitors efficiently blocked caspase-mediated cleavage of
Htt, neoepitope antibodies to caspase 2/3/7 cleavage site in Htt
ON
(DLND) htt552 were used (Fig. 6B, lanes 6–10). The production
of 552 aa caspase generated fragments was inhibited by pan-specific
caspase inhibitor (Fig. 6B, lane 6), or caspase 2 specific inhibitor
.D
(Fig. 6, lane 7), which confirms that in this system it is caspase 2 that
is cleaving Htt at residue 552, not caspase 3. The levels of N-terminal
fragments remained unchanged (Fig. 6B, lanes 1 and 2).
Independent evidence in favor of this hypothesis came from
transient transfection experiments with HEK293 cells using Htt
N1212-Quint constructs with deleted cleavage sites for caspases 2,
3 and 6.34 As described in the previous section, cp-1 and cp-2 frag-
ments are generated from Htt N1212 constructs with either 15Q or
138Q in HEK293 cells (Fig. 3B and Fig. 6C, upper panel). Caspase
2 fragments of Htt, visualized with neoepitope antibody htt552,
were only produced from the wild type Htt constructs, but not from
constructs with deleted caspase sites (compare lanes 1 and 2, with
lanes 3 and 4, Fig. 6C, lower panel). However, generation of cp-1 and
cp-2 fragments was not affected by deletions of caspase cleavage sites
in Htt (Fig. 6C, upper panel, lanes 3 and 4). These results, combined
with the data obtained with caspase inhibitors in PC12 cells are
highly suggestive of the N-terminal cleavage of Htt occurring via a
caspase-independent pathway in these cell systems.
Inhibition of calpain increases accumulation of N-terminal frag-
ments and accelerates aggregation. Treatment of PC12 cells with
calpain inhibitor MDL28170 leads to an increase in accumulation of
cp-1 and cp-2 fragments (Fig. 6A). Since some of calpain inhibitors
may also inhibit the proteasome activity, we further investigated the
effects of both classes of inhibitors on the production of cp-1 and cp-2
fragments. When PC12 cells, induced to express FL-Htt-126Q, were
treated with two different concentrations of calpain inhibitor MDL
28170, or another calpain inhibitor, calpeptin, levels of accumulated
cp-1 and cp-2 fragments increased in a dose-dependent manner (Fig.
7A, lanes 2–4 and 11–13, respectively). Proteasome inhibitors lacta-
cystin and MG132 also elevated levels of cp-1 and cp-2 fragments
Cell Cycle 2007; Vol. 6 Issue 23
 N-terminal Proteolysis of Huntingtin in PC12 Cell Model
CE
IEN
SC
BIO
ES
(Fig. 7A, lanes 5 and 6); however U102, an inhibitor of caspase-like
ND
(PGPH) activity of the proteasome, failed to do so (Fig. 7A, lane 7).
It should be noted that accumulation of larger (~100 kD) Htt frag-
LA
ments (possibly generated by caspase or calpain cleavage), was also
increased with both calpain and proteasome inhibition.
Since both calpain inhibitors and proteasome inhibitors affect
07
cp-1 and cp-2 fragments in a similar way, it was essential to rule out
the possibility that these effects of calpain inhibitors may be due to
20
a non-specific inhibition of the ubiquitin-proteasome system (UPS).
©
This was achieved by using the green fluorescent protein (GFP)
proteasome reporter cell line GFPu-1.46 GFPu-1 HEK293 cells
express a reporter consisting of a short degron, CL1,47 fused to the
C-terminus of GFP. GFPu is unstable compared to GFP, however in
conditions of inhibition or impairment of UPS, GFPu is stabilized,
and therefore GFPu fluorescence can be used as a measure of UPS
activity.46 We found that, in fact, treatment of GFPu-1 cells with
proteasome inhibitors (lactacystin and MG132) leads to impairment
of UPS, measured by a robust increase in fluorescence, compared
www.landesbioscience.com
Figure 6. N-terminal fragments are formed
independently of caspase cleavage in PC12
and HEK293 cells. (A) Western blot analysis
of total cell extracts (60 µg per lane) from
PC12 cells induced to express FL-Htt-126Q
(line S6-5) for eight days in the presence of
the following inhibitors: caspase pan-specific,
Z-VAD, 25 µM (lane 1), caspase 2-specific,
Z-VDVAD, 10 µM (lane 2), caspase 3-specific,
AcDMQD, 25 µM (lane 3), caspase 6-specific
Z-VEID, 10 µM (lane 4), and calpain1/2-spe-
cific, MDL128170, 10 µM (lane 5). Control
extract was loaded in lane 6. Htt proteins are
detected with anti-c-myc antibody. Blot was re-
probed with the antibody to GAPDH for load-
.
ing control. (B) Total cell extracts (60 µg per
E
lane) from PC12 cells treated as described
UT
in (A) (lanes 1–4), and control extract (lane
5), analyzed by Western blot with anti-c-myc
RIB
antibody. Blot was re-probed with neoepitope
antibodies to caspase 2/3/7 cleavage site
in Htt (DLND) htt552 (lanes 6–10), and with
IST
antibody to actin for loading control. (C)
Western blot analysis of total cell extracts (30
µg per lane) from HEK293 cells transfected
D
with either wild type FL-Htt construct (lane
5), truncated Htt-N1210 constructs (lane 1
OT
and 2) or mutant Htt-N1212-Quint constructs
with deleted cleavage sites for caspases 2,
3, and 6 -D513A, D530A, D552A, D586A,
ON and D589A (lanes 3 and 4). Htt proteins are
detected with antibody to Htt (upper panel),
or htt552 antibody (lower panel). FL-Htt and
.D
Htt-N1212 are indicated with arrowheads,
cp-1/cp-2 fragments- with arrows. Non-spe-
cific bands are marked with NS. Parts of blot
(C) are also shown on Figure 3B.
to non-treated cells. However, calpain inhibitor MDL28170
(25 µM) does not increase GFPu fluorescence, and thus it does not
efficiently inhibit proteasome activity (Fig. 7B). Therefore the effect
of MDL28170 on the accumulation of Htt fragments is likely due to
a specific inhibition of calpains.
Increased accumulation of cp-1 and cp-2 fragments in PC12
cells treated with calpain inhibitors may reflect increased production
of these fragments, or reduced degradation. To evaluate these two
possibilities, PC12 cells expressing FL-Htt-126Q were treated with
protein synthesis inhibitor cycloheximide for 17 hours. As expected,
the basal levels of Htt were reduced by cycloheximide. Notably, a
proportional increase in cp-1/cp-2 fragments accumulation with
calpain inhibition was still observed in the absence of protein
synthesis in cells treated with cyclohexamide (data not shown).
Thus, the accumulation of cp-1 and cp-2 fragments with calpain
inhibition does not seem to be caused by increased Htt synthesis,
and therefore it may result from altered clearance of cp-1 and cp-2
fragments. To test the effect of calpain inhibitors on cp-1 and cp-2
Cell Cycle 2977
 N-terminal Proteolysis of Huntingtin in PC12 Cell Model

Since our data suggest that cp-1 and

cp-2 fragments may be substrates for
calpains, we next tested whether incuba-
tion of PC12 lysates with calpain1 and
2 will facilitate cleavage of expanded Htt
fragments in vitro (Fig. 8C). Clearly, both
full-length Htt and its cp-1 and cp-2
fragments are efficiently digested upon
addition of calpain 1 or calpain 2 at higher
concentration, evidenced by a decrease
in FL-Htt and fragments levels, and by
appearance of lower molecular weight
bands representing Htt cleavage products.

E .
This increase of degradation was blocked

UT
by calpain inhibitors. Notably, the longer
fragments (~100 kD) potentially gener-

RIB
ated by caspase/calpain cleavage were also
affected by calpains in vitro.
Overall, inhibition of calpains leads to

IST
abnormal degradation of cp-1 and cp-2
fragments, and, as a result, to a dramatic

D
increase in accumulated fragments, and
accelerated formation of inclusions. Thus,

OT
calpain mediated cleavage may be a first
step in the clearance of cp-1/cp-2, followed
ON by their complete breakdown by protea-
some machinery.
.D
DISCUSSION
CE

Numerous reports indicate the important

role of Htt proteolysis in the pathogenesis
IEN

of HD.15-31 Studies of Htt cleavage have

Figure 7. Calpain and proteasome inhibitors increase accumulation of Htt N-terminal fragments in PC12 been focused on two domains within the
cells. (A) Western blot analysis of total cell extracts (60 µg per lane) from PC12 cells induced to express
SC

Htt sequence that are highly susceptible to

FL-Htt-126Q (line S6-5) for eight days, and treated with the following inhibitors for 24 h: calpain inhibi-
proteolysis: cleavage by caspases or calpains
tor MDL128170, 10 µM (lane 3) or 25 µM (lanes 4 and 8); calpeptin, 50 µM (lane 13) or 75 µM
BIO

(lane 12); lactacystin, 10 µM (lane 5); MG132, 0.5 µM (lane 6); U102, 10 µM (lane 7), non-treated within a region between about 460 and
control cells (lanes 2, 9, 11), or non-induced cells (lanes 1 and 10). Lanes 1–5 are from the same blot. 600 amino acids from the N-terminus,
Htt proteins are detected with anti-c-myc antibody. Blots were re-probed with antibody to actin for load- and cleavage by unknown proteases near
ing control. FL-Htt is indicated by arrowheads, caspase fragments—by arrows, cp-1/cp-2 fragments—by the N-terminus (Fig. 1B). The application
ES

double arrows. (B) GFPu fluorescence of GFPu-1 HEK293 cells treated with lactacystin (10 µM), MG132
of previously described stable cell models
(0.5 µM), or MDL28170 (25 µM).
ND

of HD to the study of Htt proteolysis has

been limited, because most of these cell
degradation, we determined fragments levels during 21 h in the lines express truncated forms of Htt, which do not contain both
LA

presence of cycloheximide (Fig. 8A). When the protein synthesis is Htt domains prone to proteolysis.24-26,48 In two reports focused on
blocked, a decline in the steady state levels of a protein reflects the the characterization of the N-terminal cleavage step, the mapping
07

rate of its degradation. Our time course experiments demonstrate of the cleavage sites were accomplished using either truncated Htt
that the rate of cp-1 and cp-2 clearance is markedly diminished in constructs, or transient overexpression of full-length Htt in condi-
20

the presence of calpain inhibitors, and is decreased even more in the tions involving proteasome inhibition.18,44
presence of lactacysin (Fig. 8A). To further investigate the Htt proteolytic pathway and the
©

As described above, cp-1 and cp-2 fragments mostly accumulate possible relationships among different cleavage events, an inducible
in the aggregate fraction (Fig. 4). To determine whether reduced PC12 cell model expressing full-length expanded Htt was developed.
degradation of the fragments caused by calpain inhibition will result In our model both caspase-derived fragments and short N-terminal
in more aggregation, we performed immunofluorescent staining of fragments cp-1 and cp-2 accumulate prior to formation of inclu-
PC12 cells, treated with calpain inhibitor (Fig. 8B). We found that sions and Htt induced toxicity. Unlike previous reports, the novel
these cells form much more aggregates than untreated cells, most of N-terminal fragments cp-1 and cp-2 described here are detectable
the inclusions being localized to nuclear and perinuclear compartments. without proteasome inhibition, which may be more relevant to

2978 Cell Cycle 2007; Vol. 6 Issue 23

 N-terminal Proteolysis of Huntingtin in PC12 Cell Model

cell physiology. The generation of

these cp-1 and cp-2 fragments is not
affected by the deletion of residues
105–114. Therefore, our cp-1 and
cp-2 fragments appear to be distinct
from the cp-A and cp-B fragments
described by Trottier’s group.18 They
appear very similar to the short
N-terminal fragments observed in
our inducible mouse model of HD
(described previously, ref. 32).
Caspase- mediated cleavage of
Htt has been described in vitro,

E .
and fragments derived from caspase

UT
cleavage accumulate in brains of HD
patients.33,36 In the YAC 128 trans-

RIB
genic mouse model of HD, alteration
of the Htt caspase 6 cleavage site at
residue 586 completely ameliorate

IST
pathology, indicating the impor-
tance of caspase 6 mediated cleavage

D
of Htt.39 However it is not clear
whether initial cleavage of Htt by

OT
caspases is a necessary first step
in a sequential proteolytic pathway
ON generating short N-terminal frag-
ments, or whether these fragments
can be produced independently from
.D
caspase cleavage. Our new data indi-
cate that in PC12 cells Htt can be
CE

cleaved by caspase 2 at residue 552,

as well as caspase 3 at residue 513.
IEN

This cleavage event does not seem

to affect the production of shorter
SC

N-terminal fragments of Htt cp-1

and cp-2, consistent with the parallel
BIO

cleavage model in these system. This

suggests that in PC12 cells caspase 2
derived Htt fragments (552 aa long)
ES

and shorter cp-1 and cp-2 frag-

Figure 8. Inhibition of calpain increases stability of cp1/2 fragments and accelerates aggregation in PC12 ments may have diverse functional
ND

cells expressing FL-Htt-126Q. (A) Cp-1/cp-2 levels determined by Western blotting with anti-c-myc antibody of roles, and may be generated, active,
proteins from PC12 cells (line S6-5) treated with calpain inhibitor MDL28170 or lactacystin in the presence
of cycloheximide, or cells treated with cycloheximide alone (control) for indicated time periods. Cells were
or cleared in different sub-cellular
LA

harvested on day 8 post induction. One representative blot out of three is shown. Equal loading was con- compartments. Experiments with
firmed by staining of the blots after transfer with Ponceau-S-red (not shown). The graph shows quantification of HEK293 cells, which overexpress
cp-1/cp-2 protein levels from 3 experiments described above (n = 3). Results are means ± standard deviation Htt N1212-Quint constructs with
07

and expressed as % of initial level. (B) Immunofluorescent detection of Htt aggregates in PC12 cells induced to deleted cleavage sites for caspases 2,
express FL-Htt-126Q (line S6-5) for eight days in the absence or presence of calpain inhibitor (MDL28170, 10
3 and 6, also failed to demonstrate a
20

µM). Htt expression is detected with anti-c-myc antibody (green), nuclei are stained with DAPI (blue). Aggregates
are indicated with arrows. One representative field is shown for each condition. The graph shows quantification correlation between cleavage of Htt
©

of aggregation. Five fields combined containing 200–400 cells were counted on each of three different plates by caspases and the formation of
(n = 3, ***p < 0.005 with calpain inhibitor versus control). (C) Cleavage of FL-Htt-126Q and cp-1/cp-2 frag- cp-1 and cp-2 fragments. However,
ments by calpain-1 and -2 in vitro. PC12 cell lysates (100 µg) from cells expressing FL-Htt-126Q (line S6-5) for it should be noted that blocking
eight days were incubated with the indicated amounts of calpain 1 and 2 for 30 min at 30˚C in the absence or
presence of calpain inhibitor (MDL28170, 25 µM). Htt proteins are detected with anti-c-myc antibody. Note the
caspase cleavage sites might facilitate
decrease in FL-Htt (arrowhead) and cp-1/cp-2 fragments (arrows), and the appearance of low molecular weight cleavage at other sites (generating
bands with higher concentration of calpains. shorter N-terminal fragments),
which are normally not favorable
within the context of normal Htt in
vivo. Notably, we found that caspase

www.landesbioscience.com Cell Cycle 2979

 N-terminal Proteolysis of Huntingtin in PC12 Cell Model
6 is expressed at very low levels by PC12 cells (our unpublished
observations), therefore the possible connection between caspase 6
mediated cleavage and the N-terminal proteolysis of Htt remains to
be evaluated in more physiologically relevant cell systems and mouse
models.
Two calpain cleavage sites have been identified at amino acids 469
and 536 of Htt.40 Alteration of these sites reduced Htt toxicity and
aggregation in cells, suggesting that calpains may generate toxic Htt
fragments.40 Our data indicate that calpain may be also involved in
other steps of Htt proteolysis and clearance. We suggest that short
N-terminal Htt fragments cp-1 and cp-2 are degraded via calpain
dependent pathway in PC12 cells, and that inhibition of calpains
results in increased accumulation of the cp-1 and cp-2 fragments,
and increased aggregation. Because two specific calpain inhibitors
caused similar changes in the levels of cp-1 and cp-2, and these
inhibitors did not affect the UPS, their effect on cp-1 and cp-2 frag-
ments accumulation is likely due to a specific inhibition of calpains,
rather than possible non-specific effects on other proteases or protea-
some. This raises the possibility that cp-1/cp-2 fragments may be
processed by calpains before subsequent clearance by the proteasome.
A similar multi-step degradation pathway has been described for a
number of transcription factors, including p53.49,50 Toxic mutant
Htt fragments resistant to degradation by proteasome were found to
be accumulated in HD neurons.51-53 Thus our findings that calpains
may mediate mutant Htt clearance are consistent with the idea that
activation of calpains in HD neurons27 may represent a cellular
protective mechanism that facilitates the clearance of toxic mutant
Htt fragments.
The proteases that generate cp-1 and cp-2 fragments of Htt in
PC12 cells remain to be identified. The involvement of an aspartyl
CE
protease in the generation of cp-A fragment have been suggested
previously in ref. 18. However, in our PC12 cells, expressing
IEN
FL-Htt-126Q, levels of cp-1/cp-2 fragments were not affected by
aspartyl protease inhibitor pepstatin A at 5 or 100 µM (data not
shown), indicating that cp-1/cp-2 are formed via distinct proteolytic
SC
pathway.
In our current model, expression of mutant full-length Htt
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induces relatively modest toxicity in PC12 cells, compared with
greater cell death in our previously described inducible PC12 cell
model expressing a short N-terminal (N63) Htt fragment.25 This
ES
is consistent with the observations that N-terminal Htt fragments
are usually more toxic than full-length Htt. In the PC12 cell model
ND
described here, cp-1 and cp-2 fragments are generated prior to
toxicity, suggesting the possibility of causal relationship. Calpain
LA
inhibitors had a profound effect on cell toxicity even in uninduced
PC12 cells, so their effects on toxicity in relation to levels of cp-1
and cp-2 were not interpretable. Future studies will be focused on the
07
mapping of the exact sites of the cleavage events producing cp-1 and
20
cp-2 fragments, and testing the effects of alterations of these sites on
mutant Htt induced toxicity. This will make it possible to evaluate
©
whether proteolytic cleavage of Htt producing short N-terminal
fragments (cp-1 and cp-2) is a critical step in a pathogenic pathway
leading to HD.
In conclusion, Htt cleavage is emerging as a complex pathogenic
process, generating several toxic fragments potentially relevant to HD
pathogenesis. The novel inducible PC12 cell model described here
will be used for further elucidation of these cleavage events, which
may provide potential therapeutic targets for HD within the pathway
of Htt proteolysis.
2980
Acknowledgements
This work was supported by HDSA Coalition for the Cure, High
Q Foundation, NINDS 16375, NIH NS038144-08, and Hood
College Board of Associates Grant to RRH. We thank Lisa Ellerby
(the Buck Institute for Age Research, Novato, CA) for constructs
and discussions, and greatly appreciate a gift of htt552 and htt513
antibodies from Sophie Roy (Merck Canada Co. Ltd., Canada)
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