Upregulation of cathepsin D in the caudate nucleus of primates with experimental parkinsonism.

Abstract

In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease.
Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. In vitro overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis.
Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our in vitro studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.

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RESEARCH ARTICLEOpen Access
Upregulation of cathepsin D in the caudate
nucleus of primates with experimental
parkinsonism
Sowmya V Yelamanchili1, Amrita Datta Chaudhuri1, Claudia T Flynn2and Howard S Fox1,3*
Abstract
Background: In Parkinson’s disease there is progressive loss of dopamine containing neurons in the substantia
nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions
of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to
examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination
and cognition, in experimental Parkinson’s disease.
Results: Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model
Parkinson’s disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus
of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of
lysosomes increased concurrently with the increase in Cathepsin D in neurons. In vitro overexpression of Cathepsin
D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such
expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death
associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D
overexpression to apoptosis.
Conclusions: Following damage to the substantia nigra resulting in experimental Parkinson’s disease, we have
identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of
disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our in vitro
studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the
progression of Parkinson’s, and provides a new target for strategies to ameliorate the progression of this disease.
Keywords: Parkinson?’?s, MPTP, striatum, caudate, neurodegeneration, cathepsin, apoptosis, nonhuman primate
Background
Parkinson’s disease (PD) is the second most common
neurodegenerative disease. Apart from its genetic predis-
position, most cases arise sporadically and factors
including drugs and toxic chemicals have been demon-
strated to induce PD [1]. While medical (e.g. L-3,4-dihy-
droxypheylalanine, L-DOPA) and surgical (e.g. deep
brain stimulation) therapies have been effective in the
treatment of PD to a certain extent, nothing to date is
capable of arresting disease progression.
In PD, there is progressive loss of dopamine (DA)
containing neurons in the substantia nigra (SN) pars
compacta. Motor symptoms initially dominate the clini-
cal picture, and as the disease progresses cognitive
abnormalities are often evident [1,2]. While numerous
studies examine damage and neuronal loss in the SN,
other neuronal systems and brain regions are also
affected [3]; and the nature of this additional neuronal
damage is relatively unknown.
Understanding the dysregulation of genes and proteins
involved in neuronal dysfunction and disease progres-
sion can open avenues for new therapeutic targets in
PD. One main outflow tract from the SN is to the stria-
tum where the presynaptic dopaminergic terminals
* Correspondence: hfox@unmc.edu
1Department of Pharmacology and Experimental Neuroscience, University of
Nebraska Medical Center, Omaha, NE 68198, USA
Full list of author information is available at the end of the article
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© 2011 Yelamanchili et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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originating in the SN are lost [4]. To address other PD-
related changes in the striatum, we targeted the caudate
nucleus (CN), a part of the striatum that not only
receives dopaminergic input from the SN as well as the
ventral tegmental area but also glutaminergic projec-
tions from prefrontal associational and anterior cingu-
late limbic areas. Similar to other regions of the
striatum, the CN is involved in motor function and also
has an important role in cognition. Imaging studies have
linked abnormalities in the striatum to altered cognitive
executive functions in those with PD [5-9].
A common cord in many aspects of neuronal survival
is the lysosomal pathways [10]. The lysosomal proteases,
which play a key role in intracellular proteolysis and in
extracellular remodeling [11], are important in maintain-
ing homeostasis by exerting degradation and regulatory
functions. Lysosomal dysfunctions have been specifically
associated with degenerative phenomenon [12] as well
as with age-related neurological diseases [13]. Among
the most powerful hydrolytic enzymes in the lysosomes
are the cathepsins. Cathepsin D (Cat D) is the major
intracellular aspartic protease and is present in relatively
high concentrations within the lysosomes. Previous stu-
dies in a variety of cell systems such as endothelial cells,
T-lymphocytes and fibroblasts have shown that
increases in Cat D leads to apoptosis (reviewed in [14]).
Cat D upregulation has been associated with neurode-
generative disorders including Alzheimer’s disease (AD)
and its presence extracellularly in senile plaques was
clearly noted [15]. The upregulation of Cat D was
shown to occur at an early stage in experimental models
of AD leading to slow apoptosis of neurons [16]. There-
fore, we questioned whether deregulation of lysosomal
Cat D is involved in the progressive neuronal damage as
seen in PD, focusing on the CN.
We hypothesized that Cat D can act as a molecular
trigger for neuronal damage in CN. Using a chronic 1-
methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) trea-
ted rhesus monkey model, we assessed the expression
level of Cat D in CN followed by immunohistochemical
analysis on brain sections. Next, we performed in vitro
overexpression of Cat D and subsequently followed the
results by functional and imaging studies to confirm our
hypothesis.
Results
MPTP-treatment of monkeys
Monkeys are sensitive to the PD-like effects of MPTP
and have a good correspondence of brain function, neu-
rochemistry, and neuroanatomy to humans. In monkeys,
both acute and chronic dosing protocols can lead to a
PD-like disease. While the acute effects of high dose
MPTP in experimental animals can be dramatic, the
acute toxicity differs from the course of PD. In addition,
functional recovery can occur in a variety of dosing
protocols.
Chronic MPTP dosing cannot only lead to stable
motor deficits but also cognitive abnormalities as seen
in PD. In order to better mimic the chronic nature of
PD, we utilized a repeated low-dose protocol, in which
animals received a dose of 0.3-0.4 mg/kg MPTP on two
consecutive days, followed by assessment of stable
effects of the treatment at 3-4 weeks post-treatment.
Animals then received repeat dosing in order to achieve
a state of stable, mild functional deficits. Following
treatment, animals displayed behavioral changes (less
grooming and social interaction when housed with
another monkey and aggressiveness to humans) and
spent more time lying down. Some monkeys showed a
dramatically impaired performance on a bimanual
motor skills (BMS) task, with slower times to perform
the test or no interest at all.
Signs of PD-like disease were scored using the Kurlan
scale [17], considered to be the optimal clinical rating
scale for MPTP-induced Parkinsonism in macaques
[18]. Through this dosing and rating system, we induced
a state of mild stable Parkinsonism (Kurlan scores of 3-
6.5 persisting a minimum of 7 weeks following the last
dose, Table 1, Figure 1). Histopathological analysis of
the dorsal striatum clearly showed a profound decrease
in tyrosine hydroxylase (TyH) [Figure 2A (upper panel),
2B (upper panel)] as well as dopamine transporter
(DAT) [Figure 2A (middle panel), 2B (lower panel)]
staining in the MPTP-treated monkeys compared to
untreated controls, revealing persistent damage to the
pre-synaptic dopaminergic terminals from the SN.
Upregulation of Cat D mRNA and protein in MPTP-treated
monkey CN
In order to assess whether neurodegenerative mechan-
isms were ongoing in the CN long after the termination
of MPTP treatment, we assessed the expression of Cat
D. We first analyzed if Cat D is altered at the mRNA
level. We extracted RNA from CN of the MPTP treated
monkeys and quantified Cat D mRNA by quantitative
real-time PCR in comparison to caudate RNA from four
untreated control monkeys. We found a significant
increase (p < 0.05) in Cat D mRNA levels in chronically
MPTP-treated animals (Figure 3A).
Next, we investigated which cell types in CN show
increased production of Cat D protein expression. As
seen in Figure 3B, there is increased Cat D immunor-
eactivity in the neurons of MPTP-treated monkey CN.
Interestingly, there is increased staining in soma and
axonal cones. Intriguingly, this staining pattern has
been previously reported to be present in AD [15].
Furthermore, we examined co-localization of Cat D
and the neuronal marker MAP2 and ascertained that
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its increased expression is indeed in neurons (Figure
4A). Additionally, we examined if Cat D co-localizes
with IBA1, a microglial marker; however we did not
observe distinct expression of Cat D in microglial cells
(Figure 4B).
Proper regulation of cathepsin levels is important in
neurons, and upregulation as well as absence of cathe-
psins have considerable consequences on the mainte-
nance and function of nervous system. Lysosomal
proteases are rarely secreted outside the cells under
Table 1 MPTP-treated animal subjects
Animal Total MPTP (mg/kg)Weeks since first treatmentWeeks since last dose Final Kurlan score
543
547
549
4.1
4.1
2.7
31
31
23
7
7
8
3
3
6.5
Animals received intermittent low-dose MPTP as indicated in Figure 1. The total dose of MPTP received by each animal, total time since first treatment, the time
between the last dose and animal sacrifice, and the clinical score at sacrifice is indicated.
Figure 1 MPTP treatments and clinical score of monkeys. The rhesus monkeys were treated with MPTP at the time points indicated by the
color-coded asterisks. Doses were given on two consecutive days at the amounts (in mg/kg) indicated under the asterisks. The Kurlan rating
score (rating the animal’s posture, gait, tremor, general mobility, hand movements, climbing, holding food, eating, balance, gross motor skills,
and defense reaction) is given for each monkey over time. Untreated control animals and animals before MPTP treatment had values of 0
(normal), higher values indicate increased deficiency. Daggers indicate time of sacrifice of each animal.
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normal conditions. Normal levels of Cat D have the
ability to digest > 95% of total brain protein within 24
hr under in vitro conditions [19], implicating that the
leakage of lysosomal compartments can have profound
deleterious effects on the cell itself and if released out-
side the cells would effect the surrounding brain milieu.
To investigate the relationship of Cat D to lysosomes in
the brains, we performed double immunofluorescence
for Cat D and LAMP-2, a marker for lysosomes. As
seen in Figure 5A (upper), normal neurons contain a
few lysosomes in the neuronal cell body and Cat D is
localized with the lysosomes. However in the MPTP-
treated monkey CN sections (Figure 5A lower), we see a
drastic increase in the number of lysosomes throughout
the neuronal cell body as well as in Cat D immunoreac-
tivity. Quantification reveals an average of four-fold
increase in lysosomes (Figure 5B). Given this increase,
we next questioned whether increased lysosomal biogen-
esis could arise from overexpression of Cat D.
Changes in Cat D and lysosomes due to Cat D
overexpression
To investigate the effect of increased Cat D in neurons,
we overexpressed human C-terminal GFP-tagged Cat D
(Cat D-GFP) or a control GFP (green fluorescent pro-
tein) construct in the BE-2 (M17) human neuroblastoma
cell line. Using lysotracker red as an indicator of lyso-
somes in control BE-2 cells, we observed the presence
of normal intracellular lysosomes (Figure 6A). In Cat D
transfected cells, Cat D-GFP largely co-localizes with
lysosomes, and the number of lysosomes was signifi-
cantly increased relative to controls (Figure 6A, B), con-
sistent with the findings in our in vivo model.
Cat D is present extralysosomaly and extracellularly
We next asked if we could detect Cat D enzyme activity
in the cytosol and extracellularly. We performed cytoso-
lic fractionation from cellular organelles (Additional file
1) using BE-2 cells, either non-transfected, transfected
with GFP alone, or transfected with Cat D-GFP. The
Cat D expression as well as activity were measured in
cytosolic extracts and in the culture supernatants of the
Cat D transfected cells was indeed significantly greater
when compared to controls. The active/mature form of
Cat D was detected in cytosol (Figure 6C upper panel).
Cat D activity assay showed an increase in Cat D activity
in the cytosolic fractions as well (Figure 7A, B). Not
only can the presence of cytosolic Cat D be deleterious
Figure 2 Loss of dopaminamergic nerve terminal markers in the striatum of MPTP treated monkeys. (A) Immunohistochemical staining
reveals strong staining of striatal sections of control brains (left panel: top and middle) for tyrosine hydroxylase and dopamine transporter;
whereas, MPTP administered monkey striatal sections (right panel: top and middle) show a minimal staining. The bottom panels are sections
stained with luxol fast blue. The images are unmagnified scans of the slides. (B) Immunohistochemical staining reveals strong staining of striatal
sections a control brains (left) for TyH and DAT; whereas, in an MPTP administered monkey, striatal sections (right) show minimal staining,
Bar = 20 μm. The images are representative of the monkeys in each group.
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to the cell itself but the presence of extracellular Cat D
could be lethal to the surrounding cells. Thus, we subse-
quently questioned if Cat D expression triggered cell
death.
Overexpression of Cat D leads to caspase activation and
apoptosis
To investigate whether Cat D overexpression induces
pathways leading to neuronal death, we first performed
a lactate dehydrogenase (LDH) assay on the culture
supernatant, assessing loss of plasma membrane integ-
rity. Figure 8A shows that there is a significant amount
of LDH-release caused by overexpressing Cat D in rat
striatal neurons when compared to controls. We next
performed a fluorescence live imaging assay using the
cell-permeable fluorescein isothiocyanate (FITC) conju-
gate of the caspase inhibitor VAD-FMK. This inhibitor
irreversibly binds to the activated caspase, allowing for
the in situ labeling of cells in which the caspase activa-
tion cascade has been initiated. As seen in Figure 8B,
when compared to control, the Cat D transfected rat
striatal neurons show a greater labeling for caspases,
likely due to initiation of apoptosis. Interestingly, cas-
pase activation is also seen along the neurites in Cat D
transfected cells (see Figure 8B) indicating neuronal
damage.
To evaluate the presence of apoptotic cells, Cat D-
GFP transfected and control BE-2 cells were stained
with Hoechst stain (Figure 9A). The fragmentation of
nuclei was clearly observed in Cat D transfected cells
when compared to controls. To further strengthen our
observation, we performed an in situ TUNEL assay. As
seen in Figure 9B, fragmented DNA in Cat D trans-
fected cells was clearly labeled. Also, since we found
caspase activation, we looked for cytosolic release of
Cytochrome C (Cyto-C), an activator for apoptosis. As
seen in Figure 9C, there was a clear cytosolic mobiliza-
tion of Cyto-C in Cat D overexpressing cells confirming
its release, which is strongly linked to activation of the
apoptotic pathway.
Discussion
In human PD a major unmet challenge has been to curb
the progression of disease that can affect the higher cog-
nitive functions of the patient. The molecular triggers
that induce the progression of disease to areas like the
CN and that contribute to associative and executive cog-
nitive functions are currently unknown [2,20,21]. Using
a nonhuman primate model of PD, we report for the
first time that lysosomal instability and alterations in a
lysosomal protease, Cat D, in CN can lead to degenera-
tion and dysfunction of neurons (Figure 10).
In the chronic MPTP model of PD, we first found a
significant upregulation of Cat D in the CN. Importantly,
Figure 3 Cat D is upregulated in MPTP caudate nucleus. (A)
Taqman qRTPCR for Cat D mRNA was performed on RNA from
caudate samples. A significant upregulation in Cat D mRNA is seen.
The delta Ct (dCt) method was performed to determine relative
concentrations, using the average of the Ct of 18S and GAPDH as
the normalizing value. Mean and standard error of the mean shown,
significance is indicated by *p < 0.05 (Student’s t-test). (B)
Photomicrographs of representative sections of caudate of two
animals (#1, #2) from each group of the control and MPTP monkeys.
Caudate sections were immunohistochemically stained with anti-Cat
D. Minimal staining is seen in sections from control animals (bar =
50 μm) and in corresponding increased magnification (40×, bar =
10 μm); whereas, in MPTP treated animal, there is increased staining.
Increased magnification (40×, bar = 10 μm) reveals that the staining
is seen within neurons in the cell body, axon hillock (bottom right).
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