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Neurobiology of Aging 33 (2012) 1485.e9–1485.e15

Role of tau kinases (CDK5R1 and GSK3B) in Parkinson’s disease:A study from India

Gautami Dasa, Amar K. Misrab, Shyamal K. Dasb, Kunal Rayc, Jharna Raya,*a S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, Indiab Movement Disorders Clinic, Bangur Institute of Neurosciences, Kolkata, India

c Molecular & Human Genetics Division, Indian Institute of Chemical Biology (a unit of CSIR), Kolkata, India

Received 27 April 2010; received in revised form 6 September 2010; accepted 23 October 2010

Abstract

Glycogen synthase kinase-3� (GSK3B) and cyclin-dependent kinase 5 (CDK5) are the 2 major protein kinases involved in abnormalhosphorylation of tau. To determine their potential role in the pathogenesis of Parkinson’s disease (PD) we analyzed 2 functional singleucleotide polymorphisms (SNPs) of GSK3B (rs334558 and rs6438552) and rs735555 of CDK5 regulatory subunit 1 (CDK5R1) in 373 PDases and 346 healthy controls of eastern India. The C,C and T,C haplotypes of GSK3B were respectively moderately associated with

increased risk and protection for late onset PD (LOPD) (odds ratio [OR], 1.399; 95% confidence interval [CI], 1.069–1.829; p � 0.015, andOR, 0.436; 95% CI, 0.222–0.853; p � 0.016, respectively). Moreover, moderate to significant interaction between different loci wereobserved for the entire PD cohort or late onset PD only. However, among these interactions, individuals carrying the (C/C) genotype at bothloci (rs6438552 and rs735555) had almost twice the risk of developing PD than those without this genotypic combination (OR, 1.871; 95%CI, 1.181–2.964; p � 0.009). Thus, synergistic effect between the 2 major tau kinases, through these SNPs, appears to determine the riskprofile for PD.© 2012 Elsevier Inc. All rights reserved.

Keywords: CDK5R1; GSK3B; Gene-gene interaction; MAPT; Parkinson’s disease (PD); SNP

www.elsevier.com/locate/neuaging

1. Introduction

Parkinson’s disease (PD) (Mendelian Inheritance in Man[MIM] #168600) is a progressive neurodegenerative disor-der of the central nervous system and is mostly a late onset,complex disorder with multifactorial interaction of genesand environment on an aging background. Accumulation ofproteinaceous aggregates in the form of Lewy bodies is thepathological hallmark of PD (Dauer and Przedborski, 2003).Dopaminergic neurons, which project very long axons tothe striatum of midbrain, are preferentially lost in PD lead-ing to rest tremor, rigidity, bradykinesia, and postural insta-

* Corresponding author at: S.N. Pradhan Centre for Neurosciences, Uni-versity of Calcutta, 35 Ballygunge Circular Road, Kolkata 700 019, India.Tel.: �91 9433210803; fax: �91 33 2223 3260.

E-mail address: jharnaray@gmail.com or thisisjr@rediffmail.com (J.

ay).

0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved.10.1016/j.neurobiolaging.2010.10.016

bility. Axonal transport of these neurons is mediatedthrough microtubules (MTs) that form the neuronal cyto-skeleton. Tau encoded by the MAPT gene is enriched inaxons and is involved in the organization and integrity ofthe MTs, thereby regulating axonal transport (Shahani andBrandt, 2002). The MAPT locus is genetically complexowing to a subchromosomal inversion of �900 kb that tookplace about 3–3.6 million years ago (Stefansson et al.,2005). Within the MAPT gene 2 conserved haplotypes, H1and H2, each comprising of a cluster of markers that aremostly in linkage disequilibrium (LD), have been identifiedwith variable frequency across different ethnic groups (Ev-ans et al., 2004). Several lines of evidence indicate thatMAPT is a strong candidate in PD susceptibility (Pastor etal., 2000; Satake et al., 2009; Simon-Sanchez et al., 2009;Tobin et al., 2008). In our previous study we also reportedthat MAPT contributes significantly in modifying the age at

onset of PD among Indians (Das et al., 2009).

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1485.e10 G. Das et al. / Neurobiology of Aging 33 (2012) 1485.e9–1485.e15

Aberrant tau phosphorylation can disrupt axonal trans-port, which has been proposed as an underlying mechanismin neurodegeneration (Hernandez and Avila, 2007). Increas-ing phosphorylation of tau by specific kinases results indestabilization of the MT network, cytoskeletal dysfunction,and modification of synaptic plasticity (Iqbal and Grundke-Iqbal, 2006; Mazanetz and Fischer, 2007). Glycogen syn-thase kinase-3� (GSK3B) and cyclin-dependent kinase 5CDK5) are both known to be involved in the abnormalhosphorylation of tau and impair the interaction betweenau and MTs, thereby leading to cytoskeletal destabilizationnd eventually neuronal death. About 80% of sporadic PDases have tau-immunoreactive Lewy bodies (Arima et al.,999; Ishizawa et al., 2003) and both GSK3B and CDK5 arelso reported to be present in Lewy bodies (Brion andouck, 1995; Nagao and Hayashi, 2009).

cdk5 plays a key role in the degeneration of dopaminer-ic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-ine (MPTP)-induced mouse model of PD (Smith et al.,003, 2006). More importantly, elevated CDK5 level haseen found in human postmortem PD brains (Brion andouck, 1995; Nakamura et al., 1997). Monomeric cdk5 isnzymatically inactive unless bound to its brain-specificegulatory subunits, p35 (Ko et al., 2001; Tsai et al., 1994)r p39 (Ko et al., 2001). Inappropriate activation of thedk5/p35 signal leads to neuronal death through pathogenicctivation of calpains, thereby proteolytically cleaving p35o a more stable (2–3-fold greater half-life) and active p25orm (Dhavan and Tsai, 2001). p25 then causes cdk5 to beonstitutively activated, mislocalized, and alters its sub-trate specificity in vivo. Overexpression of cdk5/p25 com-lex induces neuronal death in culture and cytoskeletalbnormalities in transgenic mice (Ahlijanian et al., 2000).ecently rs735555 of CDK5 regulatory subunit 1

CDK5R1) that codes for p35, the rate-limiting factor forDK5 expression (Takahashi et al., 2005), has been shown

o epistatically interact with GSK3B thereby decreasing therisk of Alzheimer’s disease (AD) in a Spanish cohort (Ma-teo et al., 2009). This single nucleotide polymorphism(SNP) (rs735555) has a potential functional significancebecause it is located at the 3= untranslated region (UTR) ofCDK5R1 which has been demonstrated to regulate the ex-pression of the gene in 4 different human cell lines (neuro-blastoma SK-N-BE and SH-SY5Y cells, embryonic kidneyHEK-293 cells, and breast tumor MCF-7 cells) studied(Moncini et al., 2007).

Whereas cdk5 is a candidate for early tau hyperphospho-rylation, GSK3B is associated with the later-stage phos-phorylation of tau (Li et al., 2006). It is a ubiquitous proteinintegrating a number of signaling pathways that are criticalfor neuronal viability and proper function. Several apoptoticstimuli including amyloid beta (A�) peptide, ischemia, andeurotoxins like MPTP and 6-hydroxydopamine appear toe involved in the pathways that activate GSK3B (Lucas et

l., 2001). Phosphorylation of tau by GSK3B occurs sur-

ounding the MT-binding domain and it can also modify 4esidues inside this domain, thereby decreasing tau and MTnteraction and favoring tau self aggregation (Hanger et al.,009). Two functional SNPs of GSK3B (rs334558 ands6438552) are known to be associated with PD (Kwok etl., 2005). The T allele of rs334558 has 1.4-fold increasedranscriptional strength while the T allele of rs6438552 hasncreased ability to phosphorylate the substrates of GSK3BKwok et al., 2005). Therefore, in the present study weade an attempt to dissect the role of these 3 SNPs

rs334558 and rs6438552 of GSK3B, and rs735555 ofDK5R1) in PD pathogenesis among eastern Indians.

. Methods

.1. Study subjects

In this study 373 PD cases (mean age at onset being8.07 � 12.26 years; female sex, 27.07%) from Bangurnstitute of Neurosciences, Kolkata, India, and 346 healthyontrols (mean age 48.09 � 8.76 years; female sex,8.32%), with no evidence of any neurodegenerative dis-ases, were recruited. All patients underwent detailed neu-ological examination by movement disorder specialistsSKD and AKM) and only those patients with at least 2arkinsonian symptoms (rest tremor, bradykinesia, rigidity,nd postural instability) were selected for the study. Theetails of the study subjects have been elaborately described inur previous study (Das et al., 2009). PD being an age-relatedisease, we categorized our patients as early onset PD (EOPD)age at onset � 40 years) and late onset PD (LOPD) (age atnset � 40 years), while the controls were grouped as young

(age � 45 years) and old (age � 45 years), deliberatelyeeping age limit a little higher for controls compared with theD patients. The demographic and clinical details of the studyubjects have been presented in Table 1. The study protocoldhered to the tenets of the Declaration of Helsinki and thenstitutional review board on research using human subjectpproved the study after proper review as per regulation of thendian Council of Medical Research.

.2. DNA isolation and genotyping of SNPs

Genomic DNA was prepared from peripheral blood byonventional salting-out method using sodium perchlorateollowed by isopropanol precipitation (Johns and Paulus-homas, 1989). The selected SNPs were genotyped byolymerase chain reaction (PCR)-restriction fragmentength polymorphism (RFLP) method. Both rs334558 ands6438552 of GSK3B were genotyped as reported earlierKwok et al., 2005) while rs735555 of CDK5R1 was PCR-mplified as a 154 bp amplicon using the following primers:DK5R1_3UTR_F: 5=- TTGCATCCACAGGATTCCAG-CG-3= (mismatch base is underlined) and CDK5R1_

3UTR_R: 5=- GGAGAGAACACGACAGGCTTCA-3=.This mismatched forward primer artificially creates a

restriction site for BstU I (New England Biolabs, Beverley,

Lgf1Lb

NMMF

1485.e11G. Das et al. / Neurobiology of Aging 33 (2012) 1485.e9–1485.e15

MA) in the amplicon when the C allele is present while theT allele remains uncut (154 bp). The genotype of the sam-ples were determined by digestion of the PCR product withBstU I followed by polyacrylamide gel (6%) electrophoresisof the restriction digest (fragment sizes: 131 and 23 bp) andstaining with 0.5 �g/mL ethidium bromide solution. About10% of the PCR products were randomly selected andconfirmed by sequencing (data not shown).

2.3. Statistical analyses

Significant deviation of the genotype frequency fromHardy-Weinberg equilibrium (HWE) at each polymorphicvariant was tested by chi square with 1 degree of freedom.Haploview 4.1 (Daly Lab, Broad Institute, Cambridge, MA,USA) was used to test the LD block and predict the haplo-types for the 2 GSK3B markers (Barrett et al., 2005). For theassociation study the data were evaluated for p-value,odds ratio (OR), and 95% confidence interval (CI) byusing the “Online Statistical Calculations” (statpages.org/ctab2x2.html) by John C. Pezzullo, Georgetown Uni-versity, Washington, DC, USA.

3. Results

None of the 3 SNPs showed evidence for deviation fromHWE (p � 0.05). The distribution of allelic and genotypicfrequencies of GSK3B and CDK5R1 polymorphisms did notdiffer significantly between PD cases and controls (data notshown). On comparing the worldwide report of GSK3B onPD (Table 2), we found that similar to our result neither ofthese 2 SNPs were associated in 2 cohorts — British (Cau-casians of UK) and the Chinese (Goris et al., 2007; Kwok etal., 2005). However, studies among Spanish populationshave documented an increased risk among T/T homozy-gotes of rs6438552 (Garcia-Gorostiaga et al., 2009). In 2other Caucasian populations, Australians and Greeks, dif-ferent genotypes of rs334558 were found to show risk andprotection, respectively, and among the Australians this wasevident only in the MAPT H2 haplotype background (Table2). Next, we performed GSK3B haplotype frequency esti-

Table 1Clinical and demographic characteristics of subjects recruited in the study

Parkinson’s disease

umber of subjects 373ale/female 272/101ean age (years � SD) 48.07 � 12.26

amilial 52 (13.94%)

Early onseta

Number of subjects 103Mean age (years � SD) 33.02 � 7.12

a Age at onset � 40 years.b Age at onset � 40 years.c Age at blood sample collection � 45 years.d Age at blood sample collection � 45 years.

mation based on genotype at the 2 SNPs (rs334558 and o

rs6438552). The overall haplotype distribution also did notsignificantly differ between cases and controls (data notshown). PD being an age-related disease, we then examinedthe impact of GSK3B haplotypes on the age at onset of thedisease. The C,C haplotype was found to be moderatelyassociated with increased risk of LOPD (OR, 1.399; 95%CI, 1.069 –1.829; p � 0.015) whereas the T,C haplotypeshowed a protective effect against LOPD (OR, 0.436;95% CI, 0.222– 0.853; p � 0.016) suggesting that GSK3Bhas the potential to modulate the age at onset of PD (Table3). No such risk or protective haplotype was identified forearly onset PD (EOPD) patients (data not shown). We thencompared our data with that of the other studies done withrespect to GSK3B haplotype (Table 2). Similar to our resultno risk or protective haplotype was identified in the British(Goris et al., 2007). However, in other studies the T,Thaplotype of GSK3B was shown to be risk or protectivetowards PD and in some populations this association waslargely dependent on the MAPT H2 haplotype background(Table 2).

To analyze the potential interaction between GSK3B andCDK5R1 polymorphisms we stratified the samples accord-ing to their genotypes. A significant synergistic interactionwas identified between C/C genotypes of rs6438552 andrs735555 (OR, 1.871; 95% CI, 1.181–2.964; p � 0.009)suggesting that individuals carrying the C/C genotype forboth the polymorphisms had almost twice the risk of devel-oping PD compared with individuals having other combi-nations (Table 4). Although weak, this interaction was alsoevident among LOPD cases (OR, 1.939; 95% CI, 1.076–3.492; p � 0.034) (Table 4). A moderate interaction wasalso identified between the C/C genotypes of rs334558 andrs735555 (OR, 1.656; 95% CI, 1.094–2.507; p � 0.018) for

OPD (Table 4). Interestingly, individuals with the C/Cenotype of all the 3 markers of GSK3B and CDK5R1 wereound to be at a moderate risk for PD (OR, 1.851; 95% CI,.117–3.067; p � 0.018), whereas it was relatively weak forOPD (OR, 2.039; 95% CI, 1.058–3.928; p � 0.043) (Ta-le 4).

We also attempted to correlate our genotypic data with

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1485.e12 G. Das et al. / Neurobiology of Aging 33 (2012) 1485.e9–1485.e15

activation has a profound influence on tau phosphorylationthrough CDK5 and GSK3B signaling pathways (Lebel etal., 2009), we intended to identify any genotypic correlationwith dyskinesia in our patient pool but did not find any.Eighteen patients developed dementia during the course ofthe disease as diagnosed by Mini Mental State Examination(MMSE) score �24. Because many downstream substratesof GSK3B are involved in synaptic remodeling that is crit-ical for the establishment of proper connections duringmemory formation (Hooper et al., 2008), we examined thedata to find if there was any correlation between the geno-types of the demented and nondemented PD cases but suchcorrelation was not apparent. None of the previous studieshave looked for an association of GSK3B with any specificendophenotype except 1 study where, however, there wasno association with GSK3B alone or with any specific en-dophenotype like age, sex, disease duration, cognitive de-cline, etc. (Goris et al., 2007).

Therefore, our data suggest that both GSK3B andCDK5R1 are likely to contribute to PD pathogenesis amongeastern Indians, and more importantly GSK3B can modulateendophenotypes like that of age at disease onset eitherindividually or in interaction with CDK5R1.

4. Discussion

The main purpose of the present study was to analyze therole of GSK3B and CDK5R1 polymorphisms and also theirnteraction in PD susceptibility. Similar to a recent genome-ide association study (GWAS) among Asians we did not

dentify association with any of the SNPs of the genestudied (Satake et al., 2009). Moreover, 2 other associationtudies on British and Chinese populations also did not findny association of the 2 GSK3B SNPs with PD (Goris et al.,

2007; Kwok et al., 2005). A closer look into the studiesdone on GSK3B so far does not show a common pattern ofassociation. For example within the Caucasian populationthere is variation — a study on British population did notshow any sort of association with the disease, but interest-ingly, the C/C genotype of rs334558 showed a protectiveeffect in the Greek population (Kalinderi et al., 2009)whereas among the Australians the T/T homozygotes hav-ing at least 1 copy of MAPT H2 had increased risk of PD(Kwok et al., 2005). More importantly, in the Greek popu-lation individuals with the T,T haplotype (highly expressedwith enhanced ability to phosphorylate substrates) ofGSK3B had an increased risk of PD (Kalinderi et al., 2009)but this was only evident on the H2 haplotype backgroundof MAPT among the Spanish and Australian populations(Garcia-Gorostiaga et al., 2009; Kwok et al., 2005) clearlyindicative of an interaction between MAPT and GSK3B andis consistent with the fact that hyperphosphorylation of taucan lead to neurodegeneration. On the other hand, tau over-expression causes MTs to bundle and this impedes mito-

chondrial movement, leading to mitochondrial degenera-T

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1485.e13G. Das et al. / Neurobiology of Aging 33 (2012) 1485.e9–1485.e15

tion, loss of adenosine triphosphate (ATP) and synapticdegeneration (Gendron and Petrucelli, 2009). With respectto the H1-specific Chinese and the Indian population (hav-ing a very high frequency of MAPT H1) the expression oftau is high compared with the western population (lower H1frequency) and therefore greater level of GSK3B would berequired to maintain normal structure and function of theneuronal cytoskeleton. This is evident from the report thatT,T haplotype of GSK3B provides protection in the Chinesepopulation (Kwok et al., 2005). Among Indians, the C,Chaplotype (decreased expression with reduced ability tophosphorylate substrates) of GSK3B probably cannot ade-quately phosphorylate tau to maintain the neuronal cyto-skeletal integrity and this over time contributes to increasedsusceptibility to LOPD. Our study therefore suggests thatgenetic background is a major determinant whether thecontribution of GSK3B would be independent of MAPT ornot in PD pathogenesis.

The fast anterograde (kinesin-driven) axonal transportpathway involves enzymatic activities of CDK5, GSK3B,and protein phosphatase 1 (PP1) and several lines of evi-dence indicate that these proteins can physically interact invivo (Morfini et al., 2004). While GSK3B is the key in tauphosphorylation, CDK5 only acts as a modulator of GSK3Bby inhibiting its regulation through protein phosphatase 1(PP1). Although the individual effect of rs735555 has notyet been studied, the T/T genotype is implicated in de-creased expression of CDK5R1 (Mateo et al., 2009). In thatcase, C/C individuals of rs735555 would have increasedlevel of p35. However, increased expression of CDK5 in-hibits GSK3B activity. Therefore the C/C genotypes at

Table 3Distribution of GSK3B haplotypes in LOPD cases and old controls

Haplotype Samples

LOPD (540) Old controls (3

C,C 44.44% (240) 36.72% (139)C,T 29.81% (161) 33.59% (129)T,T 23.16% (125) 23.96% (92)T,C 2.59% (14) 5.73% (22)

Haplotypes were constructed using alleles of 2 SNPs (rs334558 and rs643and were calculated from the haplotype frequency. p-value � 0.05 was cKey: CI, confidence interval; LOPD, late onset PD; OR, odds ratio; PD, P

Table 4Interaction between GSK3B and CDK5R1 SNPs with increased risk of PD

GSK3B genotype CDK5R1 genotype Sample

rs6438552 (C/C) rs735555 (C/C) 58 of 3rs6438552 (C/C) rs735555 (C/C) 43 of 2rs334558 (C/C) rs735555 (C/C) 93 of 2rs334558 (C/C) rs6438552 (C/C) rs735555 (C/C) 47 of 3rs334558 (C/C) rs6438552 (C/C) rs735555 (C/C) 35 of 2

Only those interactions determined to be statistically significant are show

Key: CI, confidence interval; OR, odds ratio; PD, Parkinson’s disease; SNPs, sin

rs735555 of CDK5R1 in combination with that ofrs6438552 alone or together with rs334558 of GSK3B fur-ther lowers GSK3B activity to phosphorylate the increasedlevel of tau in the Indian population causing decreasedstability of the MT network ultimately leading to PD. Thesynergistic interactions that have been observed in LOPDcan be explained by the fact that high levels of tau caninterfere with the normal trafficking of cargos (includingmitochondria) through the MTs, decrease ATP levelsthereby rendering neurons to be more susceptible to age-related stressor, and ultimately resulting in neurodegenera-tion (Gendron and Petrucelli, 2009). Therefore, our study isin agreement with the fact that hypophosphorylation of taucould also be a cause of PD and this is especially true forpopulations that are H1-only or with high frequency of H1(Kwok et al., 2005).

However, it should also be mentioned here that GSK3Bis involved in a number of signaling pathways and CDK5affects at multiple levels during the progression of PD(Smith et al., 2004). Therefore, it is important to study thegenes that are involved in these pathways to identify theother risk variants. For example, in a recent study on aSpanish PD cohort, GSK3B has been shown to synergisti-ally interact with Heme oxygenase-1 (HO-1) that codes forcellular stress protein (Infante et al., 2010).The present study is of biological interest since both

SK3B and CDK5 are now being targeted for treating PD.owever, prior knowledge about the pattern of variation in

he frequencies of functional SNPs of CDK5R1, GSK3B,nd MAPT haplotype in different ethnic populations mighte helpful in predicting the degree of effectiveness of the

Significance

p-value OR 95% CI

0.015 1.399 1.069–1.8290.221 0.833 0.629–1.1030.753 0.949 0.698–1.2910.016 0.436 0.222–0.853

of GSK3B. Numbers in parentheses indicate the number of chromosomesd statistically significant and represented in bold.n’s disease.

Significance

p-value OR 95% CI

31 of 346 controls 0.009 1.871 1.181–2.964PD 17 of 191 old controls 0.034 1.939 1.076–3.492PD 46 of 191 old controls 0.018 1.656 1.094–2.507

25 of 346 controls 0.018 1.851 1.117–3.067PD 13 of 191 old controls 0.043 2.039 1.058–3.928

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1485.e14 G. Das et al. / Neurobiology of Aging 33 (2012) 1485.e9–1485.e15

drug molecule in specific ethnic groups. Although a largenumber of association studies have been done on MAPT indifferent ethnic populations, only a handful have been per-formed on GSK3B with respect to PD and more importantly,most were done on a relatively modest number of samples.To our knowledge this is the first study that emphasizes therole of GSK3B in modulating the age at onset of PD andgene-gene interaction between GSK3B and CDK5R1. How-ver, replicate studies in additional and larger cohorts woulde necessary in support of the observation followed byunctional analysis using the variant proteins to unravel theolecular basis for their role in PD pathogenesis.

isclosure statement

The authors disclose no conflicts of interest.The study protocol adhered to the tenets of the Declara-

ion of Helsinki and the institutional review board on re-earch using human subject approved the study after propereview as per regulation of the Indian Council of Medicalesearch.

cknowledgements

The authors thank all the subjects who have participatedn this study and the Institute of Blood Transfusion Medi-ine and Immunology (IBTMI), Kolkata, India, for helpingn procurement of control samples. We also thank Mr.umanta Kumar Ghosh for technical help. This study haseen partially supported by the University Grant Commis-ion (UGC), India [Grant No. F.14-38/2007 (Inno/ASIST)],ouncil of Scientific and Industrial Research (CSIR), India,nd a predoctoral fellowship to GD by CSIR.

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