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Neurobiology of Disease 18 (2005) 152 – 165
Cystatin C prevents degeneration of rat nigral dopaminergic neurons:
in vitro and in vivo studies
Lei Xu,a,b Jiansong Sheng,a,b Zhongshu Tang,a Xuefei Wu,a Yi Yu,a Hong Guo,a Yan Shen,a
Changfu Zhou,b,c Luminita Paraoan,d and Jiawei Zhoua,b,*
a
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghi Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai, 200031, P.R. China
b
Graduate School of the Chinese Academy of Sciences, Shanghai, 200031, P. R. China
c
Research Center of Life Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, P.R. China
d
Department of Medicine, University of Liverpool, Liverpool, L69 3GA, UK
Received 28 December 2003; revised 11 June 2004; accepted 24 August 2004
Available online 24 November 2004
Destruction of nigrostriatal dopaminergic (DA) pathway triggers
various persistent responses, such as inflammation and increased
synthesis of neural growth factors, both in striatum and in substantia
nigra. The pathological processes involved in such responses are poorly
characterized and could contribute to secondary damage and/or
regeneration in the central nervous system (CNS). Cystatin C was
previously implicated in the process of neurodegeneration. However, its
biological role during neurodegeneration is not understood and remains
controversial. The present study identified an increased cystatin C
mRNA level in the DA-depleted rat striatum, starting from the second
week following a 6-OHDA-induced lesion. Immunohistochemical analysis confirmed the increase in cystatin C protein level in the striatum
following DA depletion. Double-labeled fluorescence immunohistochemistry revealed that nigrostriatal neurons, astrocytes, and microglia
contributed to the elevated level of cystatin C. Exposure to 6hydroxydopamine, a DA-specific neurotoxin, resulted in DA neurons
loss in the fetal mesencephalic cultures, an effect which could be partially
reversed by treatment with cystatin C. Moreover, in vivo DA neurons
survival study showed that administration of cystatin C in rats with 6OHDA-induced lesion partially rescued the nigral DA neurons. The
results indicate that the 6-OHDA lesioning induced a relatively slow but
sustained up-regulation of cystatin C expression and suggest that the
inhibitor may exert a neuroprotective action on DA neurons. The
findings raise the possibility that cysteine proteinase inhibitors may be
new candidates for neuroprotective treatment of Parkinson’s disease.
* Corresponding author. Key Laboratory of Proteomics, Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences, Building
23, Room 316, 320 Yueyang Road, Shanghai, 200031, P.R. China. Fax:
+86 21 5492 1073.
E-mail address: [email protected] (J. Zhou).
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2004.08.012
Cystatin C may be useful therapeutically in limiting neuropathy in
Parkinson’s disease.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Cystatin C; Dopaminergic; Cathepsin; Parkinson’s disease; Cell
culture
Introduction
Parkinson’s disease, a common neurodegenerative disease, is
characterized by degeneration of dopaminergic (DA) neurons in
the substantia nigra (SN) (Dawson and Dawson, 2003). The
characteristic features of the disease can be reproduced to some
extent in animals through the administration of various neurotoxic
agents disturbing the DA neurotransmission. For example, intraparenchymal injections of the neurotoxin 6-hydroxydopamine
(6-OHDA) into the medial forebrain bundle in rats destroy
catecholamine-containing neuronal cell bodies and nerve terminals
(Ungerstedt and Arbuthnott, 1970). The 6-OHDA animal model is
widely used to examine the function of neural transplants and the
alterations in the expression of receptors and their sensitivity to
agonist drugs (Betarbet et al., 2002). The lesion causes robust
biochemical and structural changes in the SN and its target striatum
leading to apparent reorganization of the nigrostriatal pathway.
Specifically, the lesion differentially affects mRNA expression
levels of subtypes of DA receptor, cannabinoid CB1 receptor, and
nicotinic acetylcholine receptor subunit (Elliott et al., 1998;
Romero et al., 2000). The lesion also initiates inflammation by
elevating the tumor necrosis factor-a in the DA-denervated
striatum, which can be suppressed by the immunosuppressant
FK506 (Mogi et al., 2000). These processes appear to be
accompanied by neural growth-associated activity as evidenced
by increased expression of a number of factors that are known to
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
stimulate the growth of central neurons in vitro, such as the
neurotrophins brain-derived neurotrophic factor (including its
specific receptor trkB) and members of glial cell line-derived
neurotrophic factor family (Hida et al., 2003; Numan and Seroogy,
1997; Zhou et al., 1996, 2000). Consistent with these changes, it
was shown that the striatal extracts from patients with Parkinson’s
disease promotes growth of DA neurons in fetal mesencephalic
cultures (Carvey et al., 1993), suggesting a trophic activity of the
striatum on DA neurons. Thus, the striatum appears to play a role
in self-neuronal protection when neurodegeneration of midbrain
DA neurons occurs. However, the molecular mechanisms underlying the neural plasticity of the DA-denervated striatum are far
from being elucidated.
Cystatin C, a cysteine protease inhibitor, was recently identified
by an approach based on suppression subtractive hybridization
(SSH) as being differentially expressed in DA-depleted striatum of
the 6-OHDA-lesioned rats (Xu et al., 2000). Cystatin C is a
member of family 2 of the cystatin superfamily (for reviews, see
Akopyan, 1991; Barrett and Kirschke, 1981; Barrett et al., 1984),
widely expressed by various tissues (Abrahamson et al., 1990;
Lofberg et al., 1982, 1983; Paraoan et al., 2001) and present in
various biological fluids including urine, blood, and cerebrospinal
fluid (Abrahamson et al., 1986; Lofberg et al., 1980; Sickmann
et al., 2000). Involvement of cystatin C in various degenerative
processes in the central nervous system (CNS) was only relatively
recently described. Delayed expression of cystatin C by CA1
pyramidal cells and reactive astrocytes of rat hippocampus was
reported in transient forebrain ischemia (Ishimaru et al., 1996;
Palm et al., 1995). However, cystatin C was persistently upregulated in neurons and glia in a rat model for medial temporal
lobe epilepsy (Aronica et al., 2001). In addition, cystatin C protein
level was found increased in neurons cultured in the high oxygen
atmosphere, suggesting that oxidative stress stimulates the expression of cystatin C in cultured neurons and that cystatin C might
therefore have a role in regulation of apoptosis elicited by oxidative
stress (Nishio et al., 2000). Recent studies showed that, in patients
with Alzheimer’s disease (AD), cystatin C is colocalized with
amyloid beta-protein (Abeta) in parenchymal and vascular amyloid
deposits (Levy et al., 2001).
The role of cystatin C in the neurodegeneration associated with
PD is unknown. The present study demonstrates that the expression
of cystatin C is up-regulated, both at mRNA and protein level, in
the DA-depleted striatum following 6-OHDA treatment. Moreover,
the evidence provided by in vitro and in vivo studies suggests a
role of cystatin C in the prevention of degeneration of mesencephalic DA neurons, thus implying that cystatin C might be a
potential neuroprotectant of nigral DA neurons.
Materials and methods
Animals
All animal experiments were carried out in accordance with the
U.S. National Institutes of Health Guide for the Care and Use of
Laboratory Animals. Female Sprague–Dawley rats (180–220 g)
provided by the Animal House, Shanghai Institutes for Biological
Sciences, were caged in groups of three with food and water given
ad libitum. The animals were kept in a temperature controlled
environment at 218C on a 12:12-h light–dark cycle. Timed
pregnant rats were used as the cell culture source.
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Induction of unilateral lesion in medial forebrain bundle (MFB) by
6-OHDA and animal behavioral tests
All surgery was performed under Equithesin anesthesia (0.3 ml/
100 g) and adequate measures were taken to minimize pain or
discomfort. Unilateral DA degeneration of the striatum was
achieved by stereotaxic injections of 6-OHDA (Sigma, St. Louis,
MO, USA) into the ascending medial forebrain bundle as described
previously (Ungerstedt and Arbuthnott, 1970). Briefly, 4 Al of 6OHDA (2.5 Ag/Al in 0.2 mg/ml ascorbate–saline) were injected at
4.4 mm caudal to bregma, 1.2 mm lateral to midline, 7.8 mm below
dura. Sham-lesioned animals received administration of 4 Al of
ascorbate vehicle. The lesion parameters used in the present study
result in the selective destruction of virtually all dopaminergic
neurons in the SN (N95%) and the ventral tegmental area (N80%),
as indicated by reduction in mRNA level of tyrosine hydroxylase
(TH) (Zhu et al., 1993), DA contents, and TH immunoreactivity
(Zhou et al., 1996). One week after lesioning, rotational behavior
was assessed with apomorphine (0.05 mg/kg). Animals that
exhibited adequate turning (i.e., at least six full body turns per
min for rats and three full body turns per min for mice contralateral
to the lesion side) were used in the study.
Isolation of total RNA and Northern blot analysis
The animals were sacrificed by decapitation at various time
points after the lesion was induced and the brains were removed
within 5 min postmortem. For each time point, four to five 6OHDA-lesioned animals were used. The lesion-side striata were
dissected and stored at 808C until used. The tissues were
homogenized and total RNA was isolated using a Totally RNA
isolation kit (Ambion, Austin, TX). Polyadenylated RNA was
further purified with Oligotex mRNA Mini Kit (Qiagen,
Germany).
Northern blot analysis followed standard procedures (Sambrock
et al., 1989) with a few modifications. Thirty micrograms of total
RNA isolated from DA-depleted and sham-lesioned striata of rats
at 1, 2, and 5 weeks postlesion were size fractionated by 1.0%
formalin-denatured agarose gel electrophoresis and transferred to a
nylon membrane (Amersham). A 32P-labeled cystatin C-specific
cDNA probe was produced using random primer DNA Labeling
Kit (TaKaRa Biotechnology, Dalian, China) followed by EcoRI
digestion. The specific activity of the probe was approximately 109
dpm/Ag DNA. After hybridization with the labeled probes at 428C,
the filters were sequentially washed with 2 SSC, 0.1% SDS at
room temperature followed by 0.2 SSC, 0.1% SDS at 428C, and
0.1 SSC, 0.1% SDS at 658C. Autoradiography was performed
using an intensifying screen at 808C and the exposure time was
varied so that the band intensity was kept within the linear range.
Densitometric scanning of blots allowed the calculation of the ratio
of signal obtained with cystatin C transcript and 28S ribosome in
both denervated striatum and control. The results were expressed
as percentage of the control (100%).
Semiquantitative reverse transcriptase (RT)-PCR
Single strand (ss) cDNA was synthesized from 1 Ag total RNA
in a volume of 20 Al containing 50 pmol random hexamers (Gibco
BRL, UK), 0.2 mM each dATP, dCTP, dGTP, and dTTP (Promega,
Madison, WI, USA), 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM
MgCl2, 50 mM DTT, 0.75 U RNasin (Promega), and either 200 U
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L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
or no MMLV reverse transcriptase (Promega). Reactions were
incubated for 1 h at 378C, terminated by heating 5 min at 958C, and
stored at 808C.
PCR primers were designed to specifically amplify a 394-bp
fragment of cathepsin H (between nucleotides 505–958, GenBank
accession number NM_012939; forward primer: 5V-GTGGATTGTGCCCAGAACTT-3V; reverse primer: 5V-TGTTCTTTCCACGCTCAATG-3V), and a 405-bp fragment of cathepsin L (between
nucleotides 196–600, GenBank accession number NM_013156;
forward primer: 5V-ATGGCACGAATGAGGAAGAG-3V; reverse
primer: 5V-TGCCTTGATCGTGAGAACAG-3V). Primers were also
designed to amplify regions of coding sequence from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Negative controls containing no template were performed for each set of PCR
reactions. Amplification was performed with Mastercycler
GradientR (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany)
in a volume of 20 Al containing 1–3 Al template from the ss cDNA
synthesis reaction, 25 pmol each primer, 0.2 mM each dATP,
dGTP, dCTP, and dTTP, 1.5 mM MgCl2, 0.5 ACi {a-32P}-dATP,
and 1.25 U Taq polymerase (Promega). PCR reactions were
subjected to 5 min denaturation at 948C and 25 cycles of (948C for
3 min, 948C for 1 min, 558C for 1 min, 728C for 1 min), followed
by a final amplification at 728C for 7 min. The concentration of
GAPDH cDNA of each sample was adjusted to the same level
before PCR amplification.
Seven-microliter aliquots from the amplification reactions was
analyzed on 1.5% agarose gel electrophoresis. The gels were fixed
with 6% trichloric acid for 30 min, vacuum-dried, and then
exposed to X-ray film at 808C for 5 h. All PCR products
obtained were sequenced using automated PCR sequencer (ABI
Prism System, Perkin-Elmer, Shelton, CT, USA).
In situ hybridization
Cystatin C mRNA in situ hybridization was performed
according to the procedure described previously (Ying et al.,
2002). A pBluescript II SK( ) construct containing a 670-bp fulllength cDNA of mouse cystatin C (GenBank accession number
M59470) was linealized and digoxigenin-labeled antisense and
sense riboprobes were synthesized using an in vitro transcription
kit (Roche Diagnostics GmbH, Mannheim, Germany).
The animals were sacrificed 4 weeks postlesion. Following the
pretreatments, the brain sections were prehybridized for 3 h at
508C in the hybridization buffer containing 50% deionized
formamide, 10% dextran sulfate, 10 mM Tris–HCl (pH 7.5),
600 mM NaCl, 0.25% SDS, 1 Denhardt’s solution and 400 Ag/ml
salmon sperm DNA, and hybridized in the same buffer containing
0.2 Ag/ml of either antisense or sense cystatin C probe at 508C
overnight. Following hybridization, sections were washed and the
hybridization signals were detected by incubation with antidigoxigenin antibody conjugated with alkaline phosphatase using
4-nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
as substrates.
In vitro DA neuron survival assay
Fetal ventral mesencephalons were obtained at day 14 (E0 =
day of vaginal plug) of gestation immediately after the rats were
sacrificed by decapitation. The rat embryos were stored in icecold Hank’s balanced saline solution (Ca2+- and Mg2+-free,
HBSS) until dissection. After removal of meninges, the embryos
were dissected out in HBSS and cut into 0.5 mm sections.
Following washing, the tissue was incubated in HBSS containing 1 mg/ml trypsin and 0.5 mg/ml DNase at 378C for 10 min.
Tissue was then sequentially washed in HBSS containing
DNase. Rat mesencephalic cells were seeded into 96-well plates
(Nunc, Denmark) precoated with poly-l-lysine (10 Ag/ml) as
described previously (Zhou et al., 1994). Cell viability in
suspensions of dissociated cells was determined by the ability
of viable neurons to exclude the dye trypan blue. The cells were
plated at 105 cells/cm2 in Dulbecco’s modified Eagle’s medium
(DMEM, Gibco BRL) and Ham’s F12 (1:1), supplemented with
10% fetal calf serum. Four hours later, cultures were switched to
serum-free conditions, that is, DMEM/Ham’s F12 (1:1) with
addition of B27 (1: 50) and streptomycin/penicillin. Human
cystatin C (Calbiochem, San Diego, CA, USA) was added
immediately after the medium was changed. Cells were
incubated at 378C in a 95% air/5% CO2 humidified atmosphere.
Twenty-four hours after switching to serum-free medium, the
cultures were fixed in 4% paraformaldehyde and immunostained
for TH.
In vivo DA neurons survival study
Retrograde labeling and unilateral lesions induced by nigral 6OHDA infusion were performed as described previously (Rosenblad et al., 2000; Sauer and Oertel, 1994). Briefly, under
Equithesin anesthesia (0.3 ml/100 g), 21 rats were injected
bilaterally with 0.2 Al of a 2% saline solution of the retrograde
tracer fluorogold (Fluorochrome, Inc., Englewood, CO, USA).
Injections were made at the following coordinates: AP, +0.5 mm;
ML, F3.4 mm relative to bregma; DV, 5.0 mm relative to the
dura and incisor bar set to 0.0 mm. One week after the fluorogold
injections, the animals were randomly divided into three groups (I,
II, and III) with seven animals in each group and reanesthetized for
the placement of 6-OHDA lesioning in the SN of right side. The
administration of cystatin C or saline containing 0.1% bovine
serum albumin as vehicle was performed 6 h before the 6-OHDA
challenge.
Group I received the vehicle to test whether the vehicle was
protective in itself. Groups II and III received 5 or 15 Ag human
cystatin C in 0.1% BSA containing PBS, respectively. The
injections of vehicle, cystatin C, or 6-OHDA were administered
to animals anesthetized as described above, placed in a stereotaxic frame (ASI Instrument, USA) with the incisor bar set at
3.3 mm, directly into the right substantia nigra pars compacta
(SNc) using the following coordinates: AP, 5.4 mm; L, 2.2
mm; and V, 8.5 mm from skull (Kearns et al., 1997; Paxinos
and Watson, 1986). All injections were performed using a
Hamilton 10-Al syringe at a rate of 0.4 Al/min. At the completion
of each injection, the needle was left in place for 5 min and then
withdrawn at a rate of 1 mm/min. One animal from each group
died after the surgery because of infection. Four weeks after
surgery, the rats were anesthetized with an overdose of sodium
pentobarbital and perfused with precooled saline followed by 4%
paraformaldehyde.
Immunohistochemistry
Immunohistochemistry was used to characterize the cystatin Cpositive cell population in the brain of 6-OHDA-treated rats. From
each experimental animal, five sets of 30 Am thick, frozen brain
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
sections were collected in cryoprotectant solution (0.1 M PBS,
30% sucrose, and 30% ethylene glycol) and stored at 208C until
stained.
Immunohistochemical staining of cell type-specific markers
was performed on sequential sections using the following
antibodies: mouse anti-RIP (1:1000; kind gift of Drs. S. R.
Whittemore and X. M. Xu), mouse anti-GFAP (glial fibrillary
acidic protein, 1:400, Sigma), and mouse anti-CD11b (1:100,
Chemicon, Temecula, CA, USA). After washing with PBS
containing 0.1% Triton X-100, the sections were incubated for
1–3 h with appropriate secondary antibodies (1:300; FITCconjugated horse anti-mouse or goat anti-rabbit IgG; Vector
Laboratories, USA). Following fixation in 4% paraformaldehyde,
cystatin C immunoreactivity was assayed using an anti-cystatin C
antibody (Dako, Denmark) and visualized using a Texas redconjugated goat anti-rabbit IgG antibody (1:500; Molecular
Probe, USA). Our Western blot analysis on lysis of cultured
293 cells transfected with pcDNA3.1-cystatin C-eGFP showed a
single band at right size. This suggests that the antibody we used
has high specificity. Moreover, the same antibody has been used
to characterize distribution of cystatin C in the retinal pigmental
cells (Paraoan et al., 2001).
Every fifth section was incubated with the primary antibody
against TH (1:5,000; Sigma) using an ABC kit (Vector Laboratories). TH immunoreactivity was visualized using 3,3V-diaminobenzidine as the chromogen.
155
Morphometric analysis
Cell numbers in vitro
The number of TH-positive neurons in fetal mesencephalic cell
cultures treated with or without cystatin C protein was counted on
the entire surface area of a culture well. The results were expressed
as either real numbers or a percentage of TH-positive cells counted
in a paired culture treated with H2O.
Cell numbers in vivo
The numbers of fluorogold-labeled and TH-positive neurons in
the SNc were assessed as described previously (Rosenblad et al.,
2000; Sauer and Oertel, 1994). In brief, three consecutive sections
centered around the level of the medial terminal nucleus of the
accessory optic tract (MTN; 5.3 mm in the atlas of Paxinos and
Watson, 1986) were used and all labeled/stained neurons lateral to
the MTN were counted at 400 magnification. Fluorogold-labeled
neurons were included if they were brightly fluorescent under epiillumination at 330 nm, displayed a neuronal profile, and extended
at least one neuritic process. TH-positive neurons (excited at
530 nm) were counted when displaying a nucleus surrounded by
TH-positive cytoplasm. The localization of the oculomotor nerve
rootlets was the criteria for delineating the SN from the ventral
tegmental area. The ventral tegmental area was considered to be
within and medial to the rootlets, whereas the SN was considered
to be located laterally. The extent of DA neuronal loss was
Fig. 1. The 6-OHDA lesion increased expression of cystatin C mRNA in the denervated striatum (A, C) and the lesioned substantia nigra (B, D). Total RNA
from 6-OHDA-lesioned nigrostriata and sham-operated rats, 1, 2, and 5 weeks postsurgery, was extracted and subjected to Northern blot analysis using a cDNA
fragment of cystatin C as a probe. The cystatin C mRNA band at 0.7–0.8 kb is indicated with the arrow. (A, B) Northern blots show that the mRNA levels of
cystatin C were significantly increased in both the striatum (A) and the substantia nigra (B) of the lesioned side compared to that in sham-operated group at 1,
2 and 5 weeks following the lesion. CTL, control (sham operated); LES, 6-OHDA lesioned. (C, D) The optical density of the bands (A, B) was expressed as a
percentage of that of the control group at each time point (mean F SEM). *P b 0.05 compared with the control group. Two to five samples were analyzed at
each time points.
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L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
estimated by the loss of TH-positive SN neurons on the lesioned
side with respect to the control side of the brain.
immunostained for both cystatin C and TH and visualized as
described above.
Semiquantitation of immunoreactive signals in the
caudate–putamen and the globus pallidus
For the purpose of a general survey of the expression of
cystatin C in disparate regions of the rat brain, a semiquantitative
analysis was performed. The relative levels of signals in all the
sections immunostained were visually inspected and assigned a
grade, between b+++Q (strongest) and bFQ (weakest) immunoreactivity.
Statistical analysis
Terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL) staining
Some of the brain sections at both striatal and nigral levels from
the 6-OHDA-lesioned rats were analyzed for evidence of apoptosis
by using in situ cell death detection kit (Roche, USA). The staining
was performed according to manufacturer’s protocol. The sections
were analyzed under a fluorescence microscope using an excitation
wavelength in the range of 450–500 nm and detection in the range
of 515–565 nm. The brain sections were then sequentially
Statistical analysis used the GraphPad Prism v4.0 software
(GraphPad Software Inc., USA). The data were submitted to either
a one- or two-way analysis of variance (ANOVA) as indicated in
the Results section. Either the Dunnet test or the Student–
Newman–Keul’s test (as a post hoc test) was used to compare
data samples from the control group with the different treatment
groups or between pairs of groups. Differences were considered
significant when P values were less than 0.05.
Results
Up-regulation of cystatin C mRNA level in the denervated striatum
and SN following 6-OHDA lesioning
Up-regulation of a variety of genes by DA-depletion in the
striatum might contribute to the neural plasticity of the striatal
Fig. 2. In situ hybridization of cystatin C mRNA in the rat brain following 6-hydroxydopamine lesioning. The brain sections were prepared from the lesioned
animals 4 weeks postsurgery and were hybridized with a digoxigenin-labeled cystatin C cRNA probe. More prominent dotlike hybridization products in the
caudate–putamen (B), the globus pallidus (D), and the subventricle zone (F) of the lesioned side were seen compared to those in the contralateral side (A, C,
and E). LV, lateral ventricle. Scale bar, 100 Am.
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
157
Table 1
Distribution of cystatin C immunoreactivity in both the striatum and the substantia nigra at various time points following 6-OHDA lesioning
Time (weeks postlesion)
1
2
5
CPu
Pallidum
SNc
SNr
Contralateral
side
Lesion
side
Contralateral
side
Lesion
side
Contralateral
side
Lesion
side
Contralateral
side
Lesion
side
+
+
+
+
++
+++
F
+
+
F
++
++
+
+
+
+
++
++
+
+
+
+
++
++
Data were from at least three animals in each group. +++, ++, +, and F indicate strong, moderate, weak, and very weak immunoreactive signals, respectively.
CPu, caudate putamen; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.
cells. A previous suppressional subtractive hybridization screening
for genes with up-regulated expression induced by DA neurons
depletion revealed cystatin C as one of the most differentially
expressed genes in injured striatum (Xu et al., 2000). The level of
cystatin C mRNA in rat striatum denervated as a consequence of a
6-OHDA-induced lesion was investigated in this study using
Northern blot analysis and in situ hybridization. The denervated
striatum were taken for Northern blot analysis at 1, 2, and 5 weeks
following induction of lesion. The rat cystatin C gene transcript
was identified as a single transcript of approximately 0.7–0.8 kb
(Fig. 1A). Quantitative analysis of the signals in lesioned and
sham-operated striatum revealed the level of cystatin C mRNA was
moderately increased at 1 week postlesion in the denervated
striatum compared to the sham control (1.2-fold; P b 0.05), and by
5 weeks postlesion the increase became more prominent (1.57-fold
of the control; P b 0.05) (Figs. 1A and C).
In situ hybridization analysis revealed that cystatin C mRNA
was expressed in vast regions of the rat adult brain, such as the
cerebrocortex, the lateral septum, the hippocampus, and the
striatum (data not shown). In the striatum, the hybridization
signals were scattered in the entire nucleus, in particular the head
of the caudate–putamen nucleus. Following the 6-OHDA
injection, however, the density of hybridization signals in the
denervated caudate–putamen nucleus appeared elevated at 4
weeks postlesion compared to that in the striatum of contralateral
side (Figs. 2A and B). Increased hybridization signals were also
detected in the globus pallidus (Figs. 2C and D) and subventricular zone of the lesion side (Figs. 2E and F). The expression
levels of cystatin C mRNA in other brain regions, such as the
cerebrocortex, the lateral septum, were not significantly altered
following the lesion. In parallel control experiments, brain
sections hybridized with the sense probe were completely devoid
of signals at all samples (data not shown).
The change of cystatin C mRNA in the SN following
lesioning was also examined using Northern blot analysis (Figs.
1B and D). Modest increase in the level of cystatin C mRNA was
observed at 1 week postlesion in the lesioned SN compared to the
sham control (1.26-fold; P b 0.05). The levels were further
elevated in 2 and 5 weeks postlesion (1.4- and 1.6-fold, respectively, P b 0.05).
Fig. 3. Photomicrographs showing expression of cystatin C protein in the caudate–putamen following 6-OHDA lesioning. The rats were sacrificed 5 weeks
after the lesion, their brains were cryosectioned, and immunohistochemistry using cystatin C antibody was performed as described in Materials and methods.
Semiquantitative evaluation of cystatin C expression showed prominent increase in 6-OHDA-treated rats compared with control. Panels A and B illustrate low
magnification views, while panels C and D represent high magnification views of the same respective fields. A and C, contralateral side; B and D, lesioned
side. Scale bars: A and C, 400 Am; C and D, 50 Am.
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L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
Elevated levels of cystatin C protein in the striatum and the SN
Immunohistochemical staining was performed in order to assess
the level of cystatin C protein in the nigrostriatum of lesioned
animals at various time point postlesion. At 2 weeks postlesion, the
number of cystatin C-immunoreactive cells, evaluated semiquantitatively, in both the caudate–putamen (Table 1, Fig. 3) and the
globus pallidus (Table 1) of the lesion side was elevated compared
with that in the contralateral side. The immunoreactivity of cells in
the two areas of the lesion side appeared unchanged at 1 week
postlesion (Table 1). By 5 weeks postlesion, the cystatin Cimmunoreactivity was further elevated in the two brain areas
investigated compared to that at 2 weeks postlesion (Table 1). The
immunohistological results were thus largely consistent with the
observations made by Northern blot analysis (Fig. 1). In addition,
the 6-OHDA-induced lesion led to a significant change in the
distribution pattern of cystatin C within the striatum, as the
majority of cystatin C-positive cells at 5 weeks postlesion appeared
in the globus pallidus and the dorsal part of the caudate–putamen.
This pattern was very similar to that demonstrated by the in situ
hybridization analysis illustrated in Fig. 2. The up-regulations in
these particular regions may be a consequence of the denervation
of the topographic dopaminergic projection in the striatum.
The level of cystatin C was also altered in the SN following the
6-OHDA lesioning. The intensity of cystatin C immunoreactivity
in both the pars compacta and reticulata of SN of the lesion side
was increased at 2 and 5 weeks postlesion compared with that in
the contralateral side (Table 1). In the contralateral side of the SN,
the cystatin C expression was localized in microglial- and
neuronal-like cells. The latter appeared to include some nigral
DA neurons as evidenced by colocalization of TH and cystatin C
staining (Figs. 4A–C). Since administration of 6-OHDA primarily
induces cell death of nigral DA neurons, it is likely that the cystatin
C-expressing cells were degenerating DA neurons.
As a step towards examining the relevance of cystatin C upregulation in the process of 6-OHDA-induced degeneration of the
Fig. 4. Photomicrographs of brain sections in the midbrain of the rats following 6-OHDA-induced lesion. The rats were sacrificed 2 weeks postlesion and the
brain tissue was fixed and double immunostained for cystatin C and either tyrosine hydroxylase (TH), glial fibrillary acid protein (GFAP), or CD11b. Some
brain sections were also TUNEL stained. Colocalization of cystatin C and TH was observed in nigral DA neurons of contralateral side (A–C). The 6-OHDA
treatment resulted in sustained up-regulation of cystatin C in the SNc where apoptotic cells were detected (E–G) and no TH immunoreactivity was observed
(H), while no TUNEL-positive cells were observed in the SN of contralateral side (D). (A, E, I, and L) Immunostaining using anti-cystatin C antibody; (B, F, J,
and M) same fields immunostained with anti-TH (B), TUNEL (F), anti-GFAP (J), and anti-CD11b (M) antibodies, respectively. Colocalization is illustrated in
merged images shown in C, G, K, and N. Micrographs A–D were from the substantia nigra of contralateral side; the rest of micrographs were from the
substantia nigra of lesion side. SNc, substantia nigra pars compacta. Arrows indicate double-labeled cells. Scale bars: A–C: 10 Am; D: 120 Am; E–H, 60 Am; I–
K, 30 Am; L–N: 10 Am.
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
nigrostriatum, TUNEL staining was performed on animals with
unilateral nigrostriatal lesion. At 1 and 2 weeks postlesion,
numerous TUNEL-stained cells were detected in the SNc of the
lesioned side (Fig. 4F), whereas no staining in the SN of
contralateral side was observed (Fig. 4D). The striatum of both
sides was completely devoid of TUNEL-positive signals (data not
shown). To determine whether the degenerating nigral cells
expressed cystatin C, TUNEL staining was combined with double
immunohistochemical labeling for cystatin C and TH. A significant
portion of cystatin C-expressing cells in the lesion side of SNc were
labeled with TUNEL 1 week postlesion (data not shown). By 2
weeks after the lesion, the majority of cystatin C-positive cells in the
SNc were TUNEL-positive (Figs. 4E–G) and they were completely
devoid of TH-like immunoreactivity (Fig. 4H), indicating that
degenerating DA neurons expressed cystatin C. In contrast,
TUNEL-positive staining was not observed in the contralateral
side where numerous TH-positive neurons were detected (Figs. 4A–
D). Since administration of 6-OHDA is known to specifically
induce cell death of nigral DA neurons, these results indicate that
up-regulation of cystatin C is associated with degeneration of nigral
159
DA neurons following the injury. Whether cystatin C contributed to
apoptosis as a secondary action following 6-OHDA exposure needs
to be determined.
The cystatin C-immunoreactive cells contributing to the
increased immunoreactivity described above appeared morphologically to be of both neuronal and glial cell origin. In order to
determine precisely the identities of cystatin C-positive cells,
double-labeling immunohistochemical staining of brain sections of
striatum and SN was performed. A significant proportion of striatal
neurons (35.6%, n = 200) showed strong cystatin C immunoreactivity (Fig. 5A). GFAP colocalized with cystatin C in the
striatum of both contralateral and ipsilateral sides (Fig. 5B), and
more than 49.7% of cystatin C-positive cells were GFAP-positive
cells. Moreover, microglial cells identified with CD11b antibody
also presented strong cystatin C-immunoreactivity in the same
brain area (14.7%, n = 200) where cystatin C-expressing astrocytes
were found (Figs. 5E and F). However, no RIP-positive, mature
oligodendrocytes (Yan et al., 2003) exhibited cystatin C immunoreactivity (Figs. 5C and D). Conversely, no cystatin C-expressing
astrocytes were observed in the SN of the lesion side (Figs. 4I–K).
Fig. 5. Characterization of cystatin C-expressing cells in the DA-depleted striatum following 6-OHDA-induced lesion. The rats were sacrificed 2 weeks
postlesion and the brain tissue was fixed and double immunostained for cystatin C and cell markers, that is, glial fibrillary acid protein (GFAP), RIP, and
CD11b. (A, C, and E) Immunostaining using anti-cystatin C antibody; (B, D, and F) immunostaining of the same field with anti-GFAP (B), anti-RIP (D), and
anti-CD11b (F) antibodies. A typical neuronal-like cystatin C-expressing cell is marked with an arrowhead in panel A. There was no colocalization of cystatin
C and RIP (C and D). Arrows indicate double-labeled cells.
160
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
However, microglial cells in the SN expressed cystatin C (Figs.
4L–N), suggesting that elevated cystatin C in the microglia may
modulate interaction between neurons and microglia in the SN
following lesioning.
Increased mRNA levels of cathepsin H but not cathepsin L in the
SN following 6-OHDA lesion
Cathepsins H and L are among the target cysteine proteinases
for the inhibitory action of cystatin C (Anastasi et al., 1983). To
investigate a potential link between cathepsins H and L and
degenerative events of nigrostriatal cells following administration
of 6-OHDA, their expression level in the SN following the 6OHDA treatment was analyzed using RT-PCR. The analysis
indicated that the mRNA level of cathepsin H, but not cathepsin
L, was increased at all time points examined (1, 2, and 5 weeks
postlesion), compared to the contralateral side (Fig. 6). The
increase coincided with the up-regulation of cystatin C mRNA in
the SN following the lesion (Figs. 1B and D).
Cystatin C promoted the survival of the mesencephalic DA neurons
in cultures
Our findings in the cystatin C immunohistochemical analysis of
the present study indicated that the expressions of cystatin C were
up-regulated in both the striatum and the SN, the two brain areas
were at two distinct states. The striatum had no apparent cell
apoptosis in this model, whereas the SN underwent severe apoptosis
following lesioning. Thus, the question was raised whether the
cystatin C was beneficial or deleterious to nigral DA neurons. To
investigate the potential roles of cystatin C in this setting, we
examined the effect of cystatin C on the survival of rat fetal
mesencephalic neurons in cultures. Addition of cystatin C into the
cultures markedly increased the number of surviving TH-positive
neurons in the rat mesencephalic cultures in a dose-dependent
manner (Fig. 7). The number of TH-positive neurons in the cultures
was increased up to 2.2-fold in the presence of cystatin C at
20 ng/ml, compared with the untreated (Fig. 7A). The effect of
leupeptin, another cysteine protease inhibitor, on the survival of DA
neurons in vitro was also examined. As shown in Fig. 7B, treatment
with leupeptin promoted the survival of DA neurons in dosedependent manner yielding similar magnitude as cystatin C. These
results indicate the survival-promoting effect of cystatin C on DA
neurons. The scale of survival was similar to that promoted by
leupeptin.
The protective contribution of cystatin C on DA neurons against
apoptosis triggered by the 6-OHDA, a DA-specific neurotoxin, was
investigated in primary cultures of fetal ventral mesencephalic
neurons. The cultures were treated with 6-OHDA for 24 h in the
presence or absence of cystatin C. The number of TH-positive
neurons was quantified in at least three independent, entire wells of
96-well plates. Treatment with 6-OHDA (0.3–100 Am) reduced the
number of TH-positive neurons in a dose-dependent manner (Fig.
7C). The number of TH-positive neurons was reduced by 56.7%
following addition of 30 AM 6-OHDA (136.3 F 8.1 TH-positive
neurons/well) compared with the untreated cultures (315.0 F 16.1
TH-positive neurons/well, P b 0.05). However, the 6-OHDA
toxicity could be reversed by addition of cystatin C, as coadministration of 6-OHDA and cystatin C significantly reduced the death of
TH-positive neurons (Fig. 7C). In the presence of 20 ng/ml cystatin
C, the average number of TH-positive neurons was reduced by
30.0% when the cultures were treated with 30 AM 6-OHDA (i.e.,
from 315.0 F 16.1 per well to 220.7 F 10.3 per well). These results
strongly suggest a neuroprotective action of cystatin C on DA
neurons against toxicity of 6-OHDA.
Nigral infusion of cystatin C reduced the cell lose induced by
striatal injection of 6-OHDA
In order to investigate the neuroprotective action of cystatin C
in vivo, the experimental animals received nigral infusion of
cystatin C followed 6 h later by a selective lesion of nigral DA
neurons with intrastriatal injection of 6-OHDA. To evaluate the
effect of the treatment independent of TH expression, nigral DA
neurons were retrogradely labeled by bilateral injection with
fluorogold 1 week prior to the lesion. Control 6-OHDA-lesioned
animals received nigral infusion of BSA.
The lesioning induced a marked loss of both retrogradely
labeled and TH-positive neurons in the SNc, with only 7.4% of
fluorogold-labeled neurons and 6.9% of TH-positive neurons
remaining undegenerated, in comparison with the contralateral
side (Figs. 8C–D, respectively, and Table 2). This observation was
consistent with a previous report by Kearns et al. (1997), which
demonstrated that approximately 94% of nigral TH-positive
neurons degenerated in the same model. The degenerating
fluorogold-labeled neurons appeared surrounded by small brightly
fluorescent dots, which may represent phagocytic cells that
engulfed fluorogold-positive debris (Crews and Wigston, 1990;
Rosenblad et al., 2000). In contrast, in rats that had received an
infusion of 5 Ag cystatin C prior to lesioning, 28% of fluorogoldpositive neurons and 27.7% of TH-positive neurons in the SNc
were observed (Figs. 8E and F, respectively). The quantification
of the results is presented in Table 2. Similarly, the treatment with
15 Ag cystatin C significantly increased the number of both
Fig. 6. Semiquantitative RT-PCR analysis of cathepsin H and cathepsin L mRNA in the substantia nigra ipsilateral to the lesion side following 6-OHDA
infusion. Specific PCR products were amplified as described in Materials and methods.
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
161
Discussion
Fig. 7. Neuroprotective action of cystatin C on nigral dopaminergic neurons
in vitro. The ventral mesencephalic cells plated at cell density of 105/cm2
were either untreated or treated with various concentrations of cystatin C or
leupeptin and were maintained in serum-free conditions for 24 h. Dose–
response curves to cystatin C (A) and to leupeptin (B) on the survival of
mesencephalic DA neurons in vitro. (C) The cultures were treated with 6hydroxydopamine ranging from 0.3 to 100 AM. The treatment led to a
decline in the number of TH-positive neurons. Coadministration of cystatin
C (20 ng/ml) and 6-hydroxydopamine partially restored the number of THpositive neurons. Data represent the mean F SEM of at least triplicates
from three independent experiments. *P b 0.05, compared with control (A
and B). *P b 0.05, compared with the corresponding cultures treated with
6-OHDA only (C).
fluorogold- and TH-positive neurons in the SNc (27.1% and
28.2% of that in the contralateral side, respectively, P b 0.01;
Table 2) (Figs. 8G and H). These in vivo findings, together with
the results of the in vitro dopamine neuron assay, suggest that
cystatin C is a neuroprotective agent capable of rescuing DA
neurons from neurotoxicity.
The present study revealed that the expression of cystatin C, a
cysteine protease inhibitor, was up-regulated in response to 6OHDA lesions of nigrostriatal pathway. The spatiotemporal
characteristics of the expression indicate that cystatin C could be
part of the cascades of degeneration and/or regeneration triggered
by the 6-OHDA-induced lesion. The finding that cystatin C has a
significant survival promoting effect on mesencephalic DA
neurons, both in vitro and in vivo, suggests that cystatin C may
play an important role in brain self-protection following injury.
The up-regulation of cystatin C mRNA and protein in the
striatum was observed at 2 and 5 weeks postsurgery, suggesting
that 6-OHDA-induced lesions can change gene expression in
denervated target tissue, and that mRNA levels of cystatin C in
striatal cells may be modulated by afferent dopaminergic input in a
slow-rising and long-lasting fashion. The latter characteristic is
similar to cystatin C spatial and temporal distribution in other
disease models. For instance, increased cystatin C expression in the
hippocampus and entorhinal cortex is detectable 24 h after onset of
status epilepticus and persists for at least 3 months thereafter
(Aronica et al., 2001). In transient forebrain ischemia, a delayed
expression of cystatin C was reported in rat hippocampus (Palm
et al., 1995).
Cystatin C expression was previously correlated with diverse
CNS diseases or injuries and it was documented in degenerating
neural cells. In transient forebrain ischemia, expression of cystatin
C was reported in degenerating pyramidal neurons of the CA1
region except for reactive astrocytes of rat hippocampus (Palm
et al., 1995). Lee et al. (2002) assumed that cystatin C may
contribute to apoptosis since following toxicity induced by 50 AM
6-OHDA cystatin C immunoreactivity, cathepsin B immunoreactivity, and cathepsin D immunoreactivity were localized in
degenerating PC12, while TUNEL-positive staining was also
observed in the same cells at 24, 48, and 72 h. In agreement with
these findings, we observed in the present study that cystatin C was
localized in apoptotic nigral neurons in the 6-OHDA-treated
animals. These observations raised the possibility that the elevated
expression of cystatin C may contribute to apoptosis following
exposure to deleterious stimuli. The results presented in this study,
however, demonstrated that cystatin C is able to counteract
progression of apoptosis of nigral DA neurons induced by specific
neurotoxins 6-OHDA. Moreover, elevated expression of cystatin C
was observed in other regions, such as subventricle subventricular
zone (Fig. 2) and lateral septum, apart from the striatum. The fact
that these brain areas, where cell apoptosis is not evident, are not
directly involved in degeneration of nigrostriatal pathway suggests
that up-regulation of cystatin C in the adult brain after lesion does
not necessarily lead to cell death. Indeed, overexpression of
cystatin C in mammalian cells does not induce cell apoptosis
(Paraoan et al., 2001).
The most important finding of the present study is the
protective action of cystatin C on the nigral DA neurons both in
vitro and in vivo. In the in vitro DA neuron survival assay using
dissociated cultures of fetal ventral mesencephalons, it was found
that cystatin C (20 Ag/ml) increased the number of surviving THpositive neurons by more than twofold. In the in vivo study, it was
observed that the infusion of cystatin C in the SN prior to lesioning
provided partial protection of the nigral DA neurons from cell
death induced by intrastriatally administered 6-OHDA. Up to 27–
28% of either the retrogradely labeled or the TH-positive nigral
162
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
Fig. 8. In vivo neuroprotective action of cystatin C on nigral dopaminergic neurons. The infusion of cystatin C protein, unilateral nigrostriatal lesioning, and
immunohistochemistry were performed as described in the Materials and methods. (A, C, E, and G) Coronal sections through the midbrain showing
retrogradely labeled (fluorogold-positive) nigral neurons. (B, D, F, and H) The same sections stained with anti-TH antibody. Compared to contralateral side (A
and B), the majority of fluorogold-positive and TH-immunoreactive neurons in the lesioned side were lost in the BSA-only-treated control animals (C and D).
In contrast, cystatin C-treated animals (in both doses of 5 and 15 Ag) had significantly more remaining fluorogold-positive or TH-immunoreactive neurons in
the substantia nigra in the lesioned side. Scale bar, 200 Am.
neurons survived by 4 weeks postlesion compared to only 7.4%
and 6.9%, respectively, in the control group. The extent of
protection in the cystatin C-treated animals was considerably
lower than that in the GDNF-treated animals in which more than
80% of nigral DA neurons were rescued from cell death induced by
neurotoxin (Kearns et al., 1997; Rosenblad et al., 2000). This
difference indicates that although cystatin C has a potent survivalpromoting action in vitro, its protective effect in vivo is limited to
some extent, at least in our experimental system. Since the exact
mechanism of the neuroprotective effect of cystatin C on nigral
neurons is not yet understood, the mode of administration of the
protein to the animal models may not have been optimal for
demonstrating its maximum protective capacity. Alternative
delivery procedures, such as delayed infusion or virus-based gene
therapy, may result in a better protective effect and should be
studied in the future. Also treatment in combination with other
protease inhibitors may achieve better effect in combating
neurodegeneration.
At present there is significant experimental evidence for the
involvement of cysteine protease inhibitors (including inhibitors of
cathepsins and calpains) as well of other protease inhibitors in
neuroprotection against brain injury (Lee et al., 1991; Ohe et al.,
1996; Rami and Krieglstein, 1993; Yamashima et al., 1998).
Various cell-permeable calpain inhibitors have been synthesized
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
163
Table 2
The number of retrogradely fluorogold-labeled neurons and TH-immunoreactive neurons in the substantia nigraa
Group
n
Controlb
5 Ag
15 Ag
6
6
6
Fluorogold labeled
TH immunoreactive
Contralateral
Lesioned
Percentage
Contralateral
Lesioned
Percentage
226.3 F 11.6
224.8 F 7.5
239.3 F 6.3
16.7 F 2.3
63.0 F 5.9c
65.2 F 6.1c
7.4 F 0.9
28.0 F 2.4c
27.1 F 2.1c
572.2 F 15.9
558.0 F 15.1
563.0 F 10.4
39.8 F 4.2
154.4 F 12.5c
158.5 F 12.7c
6.9 F 0.6
27.7 F 2.2c
28.2 F 2.4c
a
The table presents the average number of labeled neurons counted in three consecutive sections from six animals. Only the neurons localized laterally from
the medial terminal nucleus of the accessory optic tract were counted (see Materials and methods). Percentages indicate the proportion of neurons remaining in
the substantia nigra of lesioned side compared to respective contralateral side and are presented as mean F SEM.
b
Control group was treated with bovine serum albumin (as described in Materials and methods).
c
Values for cystatin C-treated groups are statistically different from control group (P b 0.001, ANOVA followed by post hoc Newman–Keul’s test).
and have shown significant neuroprotection in animal models of
the CNS injuries and diseases (reviewed in Ray and Banik, 2003).
However, the information on the neuroprotective effects of
cysteine protease inhibitors directly on the DA neurons is still
limited. Recent studies provided evidence to support a role for the
involvement of the calpains in the loss of DA neurons in a mouse
model of PD. Inhibition of calpain proteolysis using either a
calpain inhibitor MDL-28170 or adenovirus-mediated overexpression of the endogenous calpain inhibitor protein calpastatin
significantly attenuated 1-methyl-4-pheynyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced loss of nigral DA neurons (Crocker et al.,
2003). In addition, we have previously demonstrated that amyloidprecursor-like protein 2 (APLP2) modified with chondroitin
sulphate, which has structural/functional homology to the
Kunitz-type serine protease inhibitors, but not the form lacking
the modification, is able to promote the survival of fetal
mesencephalic DA neurons in culture (Shen et al., 2002); the
study indicated that the serine protease inhibitory characteristic of
APLP2 plays an important role in the process. The present findings
provide evidence supporting the role of cysteine proteinase
inhibitor cystatin C in combating negative impact of neurotoxicity
on nigral DA neurons.
How might the cysteine protease inhibitory activity of cystatin
C protect DA neurons? The cysteine protease class comprises
cathepsins B, H, L, K, S, and O (Barrett and Kirschke, 1981). Most
cathepsins are lysosomal and are involved in normal cellular
metabolism, participating in various processes such as peptide
biosynthesis and protein degradation. Cathepsins may also cleave
some protein precursors, thereby releasing regulatory peptides. It
has been shown that cathepsins could leak out of lysosomes in
neurodegenerative conditions, for example, in ischemia (Nitatori et
al., 1995; Sun, 1989). Members of this cathepsin family, for
example, cathepsin B, are able to induce neuronal apoptosis when
secreted by microglia (Kingham and Pocock, 2001; Yamashima,
2000). Controlled proteolysis is crucial in the plasticity of the CNS
(Lynch and Baudry, 1984), whereas excessive proteolysis contributes to various neuropathologies (Crocker et al., 2003; SierraParedes et al., 1999). Taken together, these observations suggest
that cysteine proteases are involved in neurodegeneration-associated pathology and that their inhibitors may be instrumental in
regulating their deleterious effects. Noteworthy, in the present
study, not only cystatin C was up-regulated but also one of its
substrates, cathepsin H. It is therefore possible that cystatin C
protects nigral neurons against apoptosis by inhibiting the activity
of cathepsin H. Inhibition of other cysteine proteinases present at
sites of injury in the brain could also not be excluded. Moreover,
cystatin C may inhibit activities of caspases that have a cysteine
residue at their active site and cleave the substrate after aspartate
residues (Yamashima, 2000), which were also shown to participate
in the process of neuronal death. Prevention of procathepsin B
processing by cystatin C, for example, may be beneficial in
neuronal death protection (Ryan et al., 1995).
There is evidence suggesting that activity and/or distribution
of the lysosomal proteases cathepsins B and D may be
implicated in abnormal protein processing resulting in formation
of the neurotoxic amyloid A4 peptide, which is associated with
Alzheimer’s disease. Comparative studies of cathepsin protease
activities in normal frontal cortex versus frontal cortex from
patients with Alzheimer’s disease, Lewy body dementia, PD,
and Huntington’s disease revealed a significant increase in the
activity of cathepsins H, D, and dipeptidyl aminopeptidase II,
specifically in cases with Huntington’s disease, but not those
with PD (Mantle et al., 1995). However, the potential role of
cathepsins in the pathogenesis of PD could not be ruled out as
the two regions that are severely affected in PD, the SN and the
striatum, were not examined by the abovementioned study.
Nevertheless, the data from the present study indicate that the
level of cathepsin H mRNA was elevated in the lesioned SN,
and moreover the presence of a specific cysteine protease
inhibitor (cystatin C) effectively reduced cell death of nigral DA
neurons. The inferred link between cathepsins and nigral DA
neurons death raises the possibility for novel strategies for
protection of nigral DA neurons in PD.
Oxidative stress contributes to DA cell death in PD and the
neurotoxin 6-OHDA, which is easily oxidized to reactive
oxygen species (ROS), appears to induce neuronal death by a
free radical-mediated mechanism (Ferger et al., 2001; Lotharius
et al., 1999). The 6-OHDA toxicity may be primarily mediated
by apoptotic pathways (Lotharius et al., 1999). The neuroprotective effect of cystatin C on nigral DA neurons challenged
with 6-OHDA demonstrated by this study suggests that release
of cysteine proteases could be one of feature of the toxicity
induced by 6-OHDA. This step leading to neuronal cell death
could be blocked, at least in part, by treatment with cystatin C
protein.
In the present study, cystatin C, a cysteine protease inhibitor,
was able to partially protect DA neurons. This could be due to fact
that multiple death pathways are involved in degeneration of nigral
DA neurons following treatment of 6-OHDA and cysteine
proteases are only part of these pathways. Moreover, the upregulation of endogenous cystatin C protein level was elicited in
relatively late stage (1 week after lesioning). Thus, the increased
level of cystatin C, which occurred in delayed manner, cannot
completely reverse the degenerative process that have already
164
L. Xu et al. / Neurobiology of Disease 18 (2005) 152–165
started. This may explain why cystatin C-expressing cells in the
nigra are also TUNEL-positive after 6-OHDA treatment. Our result
that earlier administration of cystatin C partially protected nigral
DA neurons in vivo is consistent with this notion.
In summary, the robust changes in the expression of cystatin
C following 6-OHDA/-lesioning suggest that an imbalance of
cysteine proteases and their inhibitors may contribute to the
pathophysiological processes leading to cell death during the
neurodegeneration in the nigrostriatum. The protective action of
cystatin C on DA neurons against 6-OHDA-induced neurotoxicity suggests that cystatin C is one of the contributors to
neuronal survival. Cystatin C may therefore be useful therapeutically in limiting neuropathy in Parkinson’s disease.
Acknowledgments
We thank Drs. S.R. Whittemore and XM Xu, Louisville
University, for kindly providing RIP antibody. This work was
supported by grants from the Chinese Academy of Sciences (No.
KSCX2-SW-209; KSCX1-SW-11), the National Natural Science
Foundation of China (No. 30000047 to L.X.), the National Basic
Research Program of China (G1999054000), the Chinese Ministry
of Science and Technology (No. 2001AA221221), and Shanghai
Metropolitan Fund for Research and Development (04DZ14005).
The support of the Royal Society, UK, is also gratefully acknowledged (grant no. 15429 to L.P.).
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