Modulation of disease risk according to a cathepsin D / apolipoprotein E genotype in Parkinson's disease

Abstract

Aspartyl protease Cathepsin D (CTSD) has been suggested to play a role in the pathogenesis of sporadic Alzheimer's disease (AD) due to interference with protein degradation mechanisms. A C224T (A38V) polymorphism in exon 2 of the CTSD gene is reported to be associated with an increased risk for AD. The partially overlapping pathology between AD and Parkinson's disease (PD) led us to investigate the role of this polymorphism in PD. Using association studies in 457 German PD patients and 340 controls we found no evidence for direct association between the CTSD genotype and PD. However, stratification for the apolipoprotein E (APOE) epsilon4 allele suggests a protective effect of the CTSD T-allele in PD (OR = 0.24, p = 0.002). Our findings suggest interference of CTSD and APOE polymorphisms in the pathogenesis of PD, in the sense of modulating disease risk.

Full text

DOI 10.1007/s00702-003-0832-x
J Neural Transm (2003) 110: 749–755
Modulation of disease risk according to a cathepsin D /
apolipoprotein E genotype in Parkinson’s disease
T. Schulte1,2, S. Böhringer1, L. Schöls2, T. Müller2, C. Fischer1, O. Riess3,
H. Przuntek2, K. Berger4, J. T. Epplen1, and R. Krüger5
Departments of 1Molecular Human Genetics and
2Neurology, Ruhr-University, Bochum,
3Department of Medical Genetics, University of Tübingen,
4Institute of Epidemiology and Social Medicine, University of Münster, and
5Department of Neurology, Neurodegeneration Laboratory, University of Tübingen,
Federal Republic of Germany
Received December 12, 2002; accepted March 6, 2003
Published online May 5, 2003; © Springer-Verlag 2003
Summary. Aspartyl protease Cathepsin D (CTSD) has been suggested to play
a role in the pathogenesis of sporadic Alzheimer’s disease (AD) due to
interference with protein degradation mechanisms. A C224T (A38V) poly-
morphism in exon 2 of the CTSD gene is reported to be associated with an
increased risk for AD. The partially overlapping pathology between AD and
Parkinson’s disease (PD) led us to investigate the role of this polymorphism in
PD. Using association studies in 457 German PD patients and 340 controls we
found no evidence for direct association between the CTSD genotype and PD.
However, stratification for the apolipoprotein E (APOE) ε4 allele suggests a
protective effect of the CTSD T-allele in PD (OR ⫽ 0.24, p ⫽ 0.002). Our
findings suggest interference of CTSD and APOE polymorphisms in the
pathogenesis of PD, in the sense of modulating disease risk.
Keywords: Cathepsin D, apolipoprotein E, Parkinson’s disease, genetics.
Introduction
Aspartyl protease cathepsin D (CTSD) has been implicated in the pathogen-
esis of sporadic Alzheimer’s disease (AD), which is characterized by patho-
logical accumulation of β-amyloid in affected brain regions. In vitro data show
that β-amyloid precursor protein is processed by CTSD and uptake of β-
amyloid results in an increase of CTSD levels leading to lysosomal dysfunc-
tion and cell death (Hoffmann, 1998). Parkinson’s disease is characterized by
the accumulation of α-synuclein in Lewy bodies in affected brain regions
(Spillantini et al., 1998). α-Synuclein has been shown to be digested by CTSD

750 T. Schulte et al.
in vitro (Hossain et al., 2001) and cellular models of Parkinson’s disease
expressing mutant α-synuclein display characteristics of lysosomal dysfunc-
tion (Stefanis et al., 2001). Therefore CTSD, the major lysosomal/endosomal
aspartic protease may be involved in abnormal protein processing not only in
AD, but also in other neurodegenerative diseases. Indeed, increased CTSD
activity has been found in Huntington’s disease, another neurodegenerative
disease characterized by abnormal protein aggregation (Mantle, 1995).
The involvement of CTSD in the pathogenesis of AD has been supported
by genetic data. For instance, a common C224T polymorphism in the CTSD
gene, leading to an amino acid exchange from alanine to valine, has been
found overrepresented in AD patients compared to control subjects by
Papassotiropoulos and colleagues (2000). The apolipoprotein E (APOE) ε4-
allele is an established susceptibility marker for AD, also modulating the age
of disease onset (Schellenberg, 1995). Interestingly, the combination of the
APOE ε4-allele with the CTSD T-allele may further increase the risk of AD
up to 19-fold (Papassotiropoulos et al., 2000). Based on common clinical and
pathological findings AD and Parkinson’s disease (PD) are thought to result
at least in part from similar pathogenic mechanisms (Perl et al., 1998; Welch
and Gambetti, 1998). APOE analyses in PD, however, have provided contra-
dictory results (Tan et al., 2000). As numerous studies indicate that disturbed
protein degradation is a common feature in neurodegenerative disorders, we
investigated the CTSD locus in a large sample of PD patients and control
individuals of German ancestry.
Material and methods
Patients
In our study we included a large sample of 457 German PD patients (mean age: 67.5 years,
standard deviation (SD): 10.5 years; female: 46%, male: 54%), which were diagnosed with
idiopathic PD based on the UK Parkinson’s Disease Society Brain Bank criteria (Gibb,
1988). A positive family history was reported in 10% of these patients reflecting a repre-
sentative sample of PD as it is observed in the general population. The patients group was
subdivided into early-onset PD (EOPD; age of onset before 50 years) and late-onset PD
(LOPD; age of onset after 50 years). Early-onset PD-subjects with mutations in the parkin
gene were excluded from the investigation. Ethical approval was obtained by the Ethics
Committee of the Ruhr-University Bochum. All subjects gave informed consent.
As controls we included 340 participants of the MEMO study (Memory and Morbid-
ity in Augsburg Elderly, mean age 72 ⫹/⫺ 4.3 years; females: 48% males: 52%). MEMO
is a follow-up project of the second WHO-MONICA (Monitoring Trends and Determi-
nants in Cardiovascular Disease) survey 1989/90 in Augsburg, Germany, examining car-
diovascular risk factors for neurological diseases in an elderly general population (Berger
et al., 2000). All MEMO participants were screened using standardized neurological
examination that included items from the UPDRS-Motor-Scale (Fahn and Elton, 1987).
Those participants with relevant signs of PD (UPDRS score ⬎ 2 for tremor, rigidity or
hypokinesia) were excluded from the controls.
Genotyping
Genotyping of the APOE polymorphism was performed as described previously (Krüger,
1999). The exon 2 polymorphism of CTSD was genotyped by PCR-RFLP according to
Papassotiropoulos and colleagues (Papassotiropoulos et al., 2000).

Cathepsin D in Parkinson’s disease 751
Statistical analyses
Data were evaluated for allele frequencies, genotype frequencies and Hardy-Weinberg
equilibrium using the Genepop program designed by Michael Raymond and Francois
Rousset (1995). Further statistical analyses (χ2, odds ratio (OR) and etiological fraction
(EF)) were performed using standard techniques. Statistical tests were corrected for
multiple testing using a Bonferroni correction, i.e. multiplying p-values with the number
of tests performed (pc). In this context p-values were corrected for the number of tests due
to the stratification according to the APOE e4 allele status and not for the number of all
explorative analyses as discussed by Böhringer and colleagues (Böhringer et al., 2000). A
level of pc ⬍ 0.05 was accepted as statistically significant.
Results
The patient and control groups were in Hardy-Weinberg equilibrium con-
cerning the investigated polymorphisms (data not shown). Analysis of the
C224T polymorphism in the CTSD gene showed no significant differences
in the allelic and genotypic distributions between PD patients and controls
(Table 1). However, individuals carrying the T allele were more frequent in
the control group (9.4% vs. 7.4%). The distribution of the APOE alleles and
genotypes are depicted in Tables 1 and 2. Regarding the allele frequencies, no
Table 1. CTSD polymorphism: distribution of alleles and genotypes in PD patients
and controls
CTSD Allele frequencies Genotype frequencies
nC T C/C C/T T/T
PD 457 0.926 0.074 0.851 0.149 0
Controls 340 0.906 0.094 0.815 0.182 0.003
EOPD 128 0.930 0.070 0.859 0.141 0
LOPD 329 0.924 0.076 0.848 0.152 0
PD Parkinson’s disease; CTSD cathepsin D; APOE apolipoprotein E; EOPD early
onset Parkinson’s disease; LOPD late onset Parkinson’s disease
Table 2. APOE polymorphism: distribution of alleles and genotypes in PD patients and
controls
APOE Allele frequencies Genotype frequencies
n2 3 4 2/2 2/3 2/4 3/3 3/4 4/4
PD 382 0.088 0.762 0.151 0 0.134 0.042a0.602 0.186 0.037
Controls 306 0.077 0.779 0.144 0.003 0.131 0.016a0.601 0.225 0.023
EOPD 94 0.080 0.750 0.170 0 0.128 0.032 0.585 0.202 0.053
LOPD 288 0.090 0.766 0.144 0 0.135 0.045 0.608 0.181 0.031
ap ⫽ 0.053; χ2 ⫽ 3.747; OR ⫽ 2.63; 95%CI ⫽ 0.95–7.27; EF ⫽ 0.026. PD Parkinson’s
disease; CTSD cathepsin D; APOE apolipoprotein E; EOPD early onset Parkinson’s
disease; LOPD late onset Parkinson’s disease; OR odds ratio; CI confidence interval;
EF etiological fraction

752 T. Schulte et al.
significant difference was observed between cases and controls. Notably, indi-
viduals with the APOE ε2/ε4-genotype were more frequent in the PD group
compared to non-parkinsonian subjects (χ2 ⫽ 3.747, p ⫽ 0.053, OR ⫽ 2.63).
Subdividing our patients into EOPD and LOPD, the APOE ε4-allele was
observed more frequently in younger patients (17% vs. 14.4%), although the
p-value did not reach significance (Table 2).
According to Papassotiropoulos and colleagues (2000), we stratified the
subjects according to their APOE ε4-carrier status. This revealed a significant
difference between PD cases and controls: The risk to develop PD was de-
creased in carriers of the CTSD T-allele according to the APOE ε4-carrier
status (χ2 ⫽ 10.5, p ⫽ 0.001; pc ⫽ 0.002, OR ⫽ 0.24), indicating a possible
protective effect of this allelic combination. A protective effect is supported
by the finding, that, no patient in the EOPD group carrying the APOE ε4-
allele shared a CTSD T-allele at all. As expected, the combination of the
APOE ε4-allele without the CTSD T-allele, in turn, is overrepresented in PD
patients compared to controls (92.2% vs. 73.7%, OR ⫽ 4.22, Table 3).
Discussion
This is the first study investigating a genetic contribution of CTSD in the
pathogenesis of PD based on a large sample of German PD patients. Our
results argue against a direct role of the C224T polymorphism in the CTSD
gene in neurodegeneration in PD. Since PD represents a multifactorial
disorder, it can be expected that different genes are involved in the same
pathogenic pathway and act synergistically. CTSD is a key enzyme of the
endosomal-lysosomal (E-L) pathway, representing one of two major protein
degradation pathways. Protein degradation mechanisms have been shown
to be disturbed in PD as well as in AD (Welch and Gambetti, 1998). The
ubiquitin proteasome pathway is widely accepted to play an important role of
in the pathogenesis of PD, because all currently identified genes in the patho-
genesis of PD are implicated in ubiquitin mediated protein degradation
(Krüger et al., 2002). In addition a recent study observed lysosomal dysfunc-
Table 3. Distribution of combined APOE ε4- and CTSD T-alleles in PD patients and
controls
PD patients Controls EOPD LOPD
APOE ε4-allele non-carriers 248 220 58 190
No CTSD T-allele 205 (0.827) 188 (0.851) 46 (0.793) 159 (0.837)
CTSD T-allele present 43 (0.173) 32 (0.149) 12 (0.207) 31 (0.163)
APOE ε4-allele carriers 90 80 25 65
No CTSD T-allele 83 (0.922)a,b 59 (0.737) 25 (1.0) 58 (0.892)
CTSD T-allele presenta7 (0.078)a,c 21 (0.263) 0 (0) 7 (0.108)
ap ⫽ 0.001; pc ⫽ 0.002; χ2 ⫽ 10.5; bOR ⫽ 4.22; 95%CI ⫽ 1.68–10.57; cOR ⫽ 0.24;
95%CI ⫽ 0.095–0.59. PD Parkinson’s disease; CTSD cathepsin D; APOE apolipoprotein
E; EOPD early onset Parkinson’s disease; LOPD late onset Parkinson’s disease; OR odds
ratio; CI confidence interval

Cathepsin D in Parkinson’s disease 753
tion in a neuronal cell culture model of PD, suggesting a role in protein
aggregation and cellular, particularly dopaminergic, dysfunction (Stefanis et
al., 2001). Thus current concepts of PD center on the disturbed protein degra-
dation pathways. Apart from its role in protein degradation there is increasing
evidence for an involvement of lysosomal protease CTSD as a mediator of
apoptosis (Kagedal et al., 2001). Therefore CTSD represents an interesting
candidate gene in PD.
Concerning APOE the ε4-allele is supposed to play a modifying role in the
development of several neurodegenerative disorders, but its role in the patho-
genesis of PD remains controversial (Tan et al., 2000). Recent interest fo-
cussed on the ε2-allele and its association with PD, since several studies found
the ε2-allele more frequent in PD patients compared to controls (Harhangi et
al., 2000). Applied to our dataset, the higher ε2-allele frequency in patients
compared to controls is in line with these results. The APOE ε4-allele is more
frequent in EOPD, but was not associated with PD overall, a fact that is in
agreement with an earlier study on a smaller scale overlapping in part with the
study population (Krüger et al., 1999).
An intriguing result of our study appeared when individuals were strati-
fied for the combined APOE ε4- and CTSD T-alleles, respectively. This allelic
combination decreased the risk to develop PD significantly. These findings
are counter intuitive since a recent association study in AD patients by
Papassotiropoulos and co-workers identified the same allelic combination as
a risk factor for neurodegeneration in AD (Papassotiropoulos et al., 2000).
Carriers of both, the T-allele for CTSD and at least one ε4-allele at the APOE
locus, were 19 times more likely to have AD than non-carriers of these alleles
(Papassotiropoulos et al., 2000). This observation, however, has not been
confirmed by others (Menzer et al., 2001; Bertram et al., 2001).
APOE intake is increased in neurons of AD brains and it is a protein
internalised and degraded within the lysosome by CTSD (Cataldo et al.,
1997). According to findings in APOE knockout mice over expressing human
mutant amyloid precursor protein (Val717Phe), APOE is suggested to act
as an isoform specific molecular chaperone influencing pathological protein
deposition (Holzman et al., 2000), featured in neurodegenerative disorders
like AD and PD (Welch and Gambetti, 1998). The CTSD polymorphism has
been associated with increased procathepsin D secretion and altered intracel-
lular maturation in one study (Toitou et al., 1994). This finding has not been
validated yet, however, it supports the hypothesis, that enhanced CTSD activ-
ity is followed by increased or altered processing of APOE via the E-L
pathway. This would imply a possible link for a synergistic action of CTSD
exon 2 and APOE polymorphisms, in sense of modifying disease risk in PD
and AD.
In summary our study based on the analysis of genes involved in protein
degradation and/or aggregation argues against a direct role of CTSD in the
pathogenesis of PD. However stratification of patients and controls for the
APOE ε4-carrier status revealed a risk modulating effect of the C224T poly-
morphism in the CTSD gene in Parkinson’s disease. Thus our findings support
the hypothesis of a certain genetic background dictating the degree of vulner-

754 T. Schulte et al.
ability of the different neuronal subtypes reflecting the concept of genetic
susceptibility in PD. The identification of genetic modifiers may be relevant
for future studies aiming at the treatment of neurodegenerative diseases with
abnormal protein aggregation. Extended studies based on different ethnic
backgrounds and different analytic approaches (i.e. case-control vs. family-
based) and functional studies investigating the interaction of CTSD and
APOE on the protein level are warranted to draw firm conclusions about this
issue.
Acknowledgements
This study has been supported in part by the German Research Society (Deutsche
Forschungs Gemeinschaft, grant: Scho754/2-1 to L.S. and R.K.). The MEMO-Study is
supported by the German Research Society (Deutsche Forschungs Gemeinschaft, grant:
BE1996/1-1).
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Authors’ address: Dr. R. Krüger, Department of Neurology, Neurodegeneration
Laboratory, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076 Tübingen,
Germany, e-mail: rejko.krueger@uni-tuebingen.de