- Email: [email protected]
LRRK2 variant associated with Alzheimer's disease
Neurobiology of Aging 32 (2011) 1990–1993
LRRK2 variant associated with Alzheimer’s disease Yi Zhao a , P. Ho a , Yuen Yih a , C. Chen d , W.L. Lee b,c , E.K. Tan a,b,c,∗ a
Department of Neurology, Clinical Research and Health Screening, Singapore General Hospital, Singapore b National Neuroscience Institute, Singapore c Duke-NUS Graduate Medical School, Singapore d National University of Singapore, Singapore Received 23 July 2009; received in revised form 29 September 2009; accepted 26 November 2009 Available online 16 December 2009
Abstract Overlapping neurodegenerative pathologies (including Alzheimer’s disease, AD) have been described in Parkinson’s disease (PD) patients with leucine-rich repeat kinase-2 (LRRK2) mutations. We analyzed a LRRK2 PD (R1628P) risk variant in a group of 885 subjects comprising of AD and controls. The frequency of the R1628P allele was higher in AD compared to controls (3.5% vs. 1.6%, OR 2.3, 95 CI 1.2–4.4, p = 0.018). In vitro, the mean percentage of apoptosis and cell death observed for the R1628P transfected human cell lines was higher compared to wild type 21.8 ± 1.9, vs. 17.1 ± 1.3, p < 0.05, 30.2 ± 2.2 vs. 25.7 ± 1.3, p < 0.05). The LRRK2 R1628P variant increases the risk of AD in our population and our in vitro findings suggest that it is a functional variant and predisposes to apoptosis. © 2009 Elsevier Inc. All rights reserved. Keywords: LRRK2; Variants; Alzheimer’s disease
Parkinson’s disease (PD) and Alzheimer’s disease (AD) are the two most common neurodegenerative conditions in the adult population. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been reported to be the most prevalent cause of autosomal dominant PD. A common G2019S mutation accounts for a significant proportion of sporadic PD in some populations (Paisan-Ruiz et al., 2004; Zimprich et al., 2004; Tan et al., 2007). Post-mortem studies of mutation carriers have revealed a spectrum of pathologies suggestive of AD, dementia with Lewy Body, tauopathy, and supranuclear palsy (Zimprich et al., 2004). The location of the LRRK2 gene on chromosome 12q12 is close to a linkage peak for late onset AD (Pericak-Vance et al., 1997) and to the 12q13 risk locus identified in a recent genome wide association study (Beecham et al., 2009). Thus it has been speculated that LRRK2 may be involved in some overlapping neurodegenerative pathways in AD. Even though to date no specific LRRK2 ∗ Corresponding author at: Department of Neurology, Singapore General Hospital, Outram Road, Singapore 169608, Singapore. Tel.: +65 6326 5003; fax: +65 6220 3321. E-mail address: [email protected] (E.K. Tan).
0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.11.019
mutations have been associated with AD patients (Hernandez et al., 2005; Lee et al., 2006; Tedde et al., 2007), comprehensive screening of the gene in AD has yet to be reported. A LRRK2 R1628P (rs33949390) variant has been found to increase the risk of PD in different Asian populations (Ross et al., 2008; Lu et al., 2008; Tan et al., 2008). Since dual PD and AD pathologies can co-exist and the R1628P variant could be a biologically plausible risk variant in AD, we conducted the first case control study to investigate whether the LRRK2 R1628P variant is associated with risk of AD in an Asian population and assessed its pro-apoptotic potential in transfected human cell lines.
1. Methods A total of 897 subjects including 217 AD and 668 controls were recruited for this study. Some of the controls had participated in previous studies of the variant in PD (Ross et al., 2008; Tan et al., 2008). The cases were recruited from the outpatient Neurology clinics. The dementia syndrome was diagnosed using the Diagnostic and Statistical Manual, 4th
Y. Zhao et al. / Neurobiology of Aging 32 (2011) 1990–1993
edition (DSM-IV) criteria, and the diagnosis of AD followed the NINCDS-ADRDA criteria. The controls (of similar race, gender, age) were recruited from the same region as the AD cases and did not have any known neurodegenerative diseases. Subjects with mixed ethnicity were not included. All the study subjects gave informed consent and the institutional ethics committees had approved the AD genetic study. 1.1. Genotyping We utilized the allelic discrimination methods to genotype the variant. As an initial step, primers and probes were designed to detect the R1628P variant using the Primer Express 1.5 software. As per standard protocol, the ABI Prism 7700 Sequence Detection System (Applied Biosystems) was used for the amplification reactions. Polymerase reactions (PCR) were carried out under optimal conditions. After PCR, we measured the allelic specific fluorescence on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) using the Sequence Detection Systems 1.7 software for allelic discrimination. Each analysis was carried out with R1628P positive and negative samples. Sequencing in the forward and reverse directions was undertaken according to the manufacturers’ instructions (BigDye, Applied Biosystems, Warrington, UK). The sequence analysis confirmed the R1628P variant for representative samples with G or C allele. 1.2. R1628P expression plasmid The R1628P LRRK2 variant was generated by PCRmediated site-directed mutagenesis (Stratagene) in the full-length LRRK2 cDNA (Origene). DNA sequencing was carried out to confirm the accuracy of the constructs. We cloned the wild type and variant LRRK2 gene into pEGFP-N1 gene and fused to the N-terminus of EGFP.
1991
1.3. Transfection experiments We followed standardized transfection protocols as previously described (Tan et al., 2007; Tan and Skipper, 2007). Briefly, human embryonic kidney cells (HEK-293T) were maintained at 37 ◦ C in a 95% air/5% CO2 humidified atmosphere incubator and transfected overnight with 3.0 g of each plasmid DNA using LipofectamineTM 2000 reagent (Invitrogen) and OptiMEM culture medium. Cells were further incubated overnight in fresh medium containing oxygen stress reagent H2O2 (0.8 mM). For apoptosis analysis, the cultured cells were harvested by trypsin digestion. 1.4. Measurement of apoptosis Annexin V-PE apoptosis Kit I (BD Biosciences) was used to quantify apoptosis on H2O2 treated transfected cell lines. After re re-suspending the cells in binding buffer, 100 l of cells were transferred to a 5 ml tube and 5 l of Annexin V-PE and 5 l of 7-AAD were added. The reactions were incubated for 15 min and 400 l of binding buffer were added for flow cytometry analysis (FACSCalibur, Becton Dickinson). EGFP positive cells were gated and analyzed with Annexin V and 7-AAD staining for apoptotic cells (Annexin V positive, 7-AAD negative) and late apoptotic or necrotic cells (both Annexin V and 7-ADD positive). The experiments were done in triplicates and the mean of 3 independent sets were taken for analysis. 1.5. Statistical analysis We analyzed categorical and numerical variables using the chi-square and Student’s t-test. Genotype frequencies in the study subjects were assessed for deviation from Hardy–Weinberg equilibrium using chi-square test. We
Table 1 Lrrk2 R1628P allele frequency in AD and controls compared to published reports in Parkinson’s disease. Study
Cases (frequency of C allele)
Controls (frequency of C allele)
Odds ratio
Present study (AD sample) (n = 885)
15 (3.5%)
21 (1.6%)
2.3
PD studies Ross et al. (cohort 1) (n = 825)
33 (3.4%)
11 (1.6%)
2.15
Ross et al. (cohort 2) (n = 661)
21 (3.0%)
14 (2.2%)
1.39
Ross et al. (cohort 3) (n = 500)
13 (2.6%)
6 (1.2%)
2.2
Lu et al. (n = 1377)
64 (3.8%)
20 (0.018%)
2.13
Tan et al. (n = 489)
20 (4.2%)
8 (1.7%)
2.5
1992
Y. Zhao et al. / Neurobiology of Aging 32 (2011) 1990–1993
defined statistical significance at p < 0.05. Even though our study sample size did not have sufficient power to detect an odds ratio of 2 for an allele frequency of 2%, the sample size was comparable to all the previous studies that have detected a significant difference for this allele in PD (Table 1).
2. Results 2.1. Genotyping DNA samples from a total of 885 subjects comprising of 217 AD and 668 controls were analyzed. The mean (±SD, range) age, age at onset of AD, and age of controls was 75.0 ± 9.0 (44–93), 71.6 ± 8.3 (44–89), and 63.2 ± 12.0 (40–91) years, comprising of 57.0% and 47.0% men. None of the patients gave a positive family history of autosomal dominant dementia. The genotype frequency in both cases and controls followed the Hardy–Weinberg equilibrium. The frequency of the R1628P G allele was higher in AD compared to controls (3.5% vs. 1.6%, OR 2.3, 95 CI 1.2–4.4, p = 0.018). All carriers were heterozygous. As the frequency of the R1628P variant was low, interaction with ApoE could not be meaningfully carried out as a much larger sample size would be required. 2.2. Apoptosis measurements The transfection efficiency in the cell lines was similar for both wild type and variant. After H2O2 exposure, the mean percentage of apoptosis and cell death observed for the R1628P transfected human cell lines was higher compared to wild type 21.8 ± 1.9 vs. 17.1 ± 1.3, p < 0.05, 30.2 ± 2.2 vs. 25.7 ± 1.3, p < 0.05 (Fig. 1). No difference was observed at baseline without H2O2 exposure (data not shown).
Fig. 1. Percentage of transfected cells with apoptosis, necrosis and cell death after 0.4 M of hydrogen peroxide exposure. After H2O2 exposure, the mean percentage of apoptosis and cell death observed for the R1628P transfected human cell lines was higher compared to wild type 21.8 ± 1.9 vs. 17.1 ± 1.3, p < 0.05*, 30.2 ± 2.2 vs. 25.7 ± 1.3, p < 0.05*.
3. Discussion It is interesting to note that LRRK2 mutations have been associated with AD-like pathology (Zimprich et al., 2004), suggesting that there may be a partial overlap between neurodegenerative pathways in both AD and PD. LRRK2 R1628P is located in the COR (C-terminus of Roc [ras of complex proteins]) domain and evolutionarily conserved across species. The substitution of a highly basic polar arginine (R) with a neutral non-polar proline (P) is postulated to result in a conformational change in the LRRK2 secondary structure (Ross et al., 2008). An earlier study had screened several pathogenic LRRK2 mutations, including one in the COR domain (Y1699C) in AD and other patients with dementia but did not find any carriers (Toft et al., 2005). Even though subsequent studies have also confirmed that common LRRK2 mutations (G2019S and I2020T) and the polymorphic G2385R variant have been shown to have no association with AD (Lee et al., 2006; Tan et al., 2007, 2009), R1628P has yet to be examined in the AD population. Furthermore, there have been no reported functional studies on this variant. We showed that the R1628P increases the risk of AD by about two-fold in our population. This magnitude is similar to the risk associated with the variant in PD population across different Asian populations (see summary in Table 1, Ross et al., 2008; Lu et al., 2008; Tan et al., 2008). To further support the functional significance of this variant, we demonstrated that this variant when over-expressed in human cell lines was associated with a higher degree of apoptosis, when subjected to oxidative stress. This risk variant could be a factor in agerelated diseases like AD and PD as aging is associated with a decreased ability of cells to counter-act oxidative stress. While our in vitro observations are considered preliminary, it adds weight to the findings from the association study and those from bioinformatics analysis. The frequency of the R1628P variant in our controls is almost identical to those reported in various Asian populations (Ross et al., 2008; Lu et al., 2008; Tan et al., 2008), suggesting that the association was not confounded by a spuriously low carrier status in our controls. It would be interesting to unravel the exact pathophysiologic mechanism on how this variant exerts its effect in predisposing to cell death for both AD and PD. Oxidative stress and proteasomal impairment are well-recognized hallmarks of neurodegenerative diseases. However, the physiologic function of LRRK2 has not been clarified. Mutations in LRRK2 have been associated with activation of programmed cell death signalling involving the FADD/caspase-8 signaling pathways (Ho et al., 2009). Dysregulated programmed cell death or apoptosis may be involved in AD. Abnormal levels of apoptotic proteins have been reported in AD brains (Engidawork et al., 2001). The R1628P variant could affect the GTP binding capacity, its kinase activity either directly or via its interaction with the different functional domains of LRRK2 or other protein interactors, ultimately leading to dysregulation of apoptosis.
Y. Zhao et al. / Neurobiology of Aging 32 (2011) 1990–1993
Mutations in the Roc/COR domain could lead to dysregulation of kinase activity in vitro (Lu et al., 2008; Lu and Tan, 2008). The COR domain mutant (Y1699C) could interfere with the interaction between LRRK2 and dishevelled family of phosphoproteins (key regulators of Wnt (Wingless/Int) signalling pathways), potentially affecting synaptic transmission and neuronal maintenance (Sancho et al., 2009). Further studies in various in vivo models should provide insights. As the prevalence of this variant is low, it is probable that this risk variant is one of the many factors contributing to disease. It is unclear whether other modulatory genes or environmental factors influence its action. In conclusion, we demonstrated that the LRRK2 R1628P variant is associated with a two-fold risk of AD in our population and our preliminary in vitro findings suggest that it is a functional variant and predisposes to apoptosis. Replication of the findings in other populations would be of interest.
Disclosure statement All the authors certify that they do not have any actual or potential conflicts of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the work submitted that could inappropriately influence (bias) the work. The study has received approval from the relevant ethics committees and informed consent has been taken from every study subject.
Acknowledgements We thank National Medical Research Council, Singapore General Hospital, National Neuroscience Institute and Singapore Millennium Foundation for their support and all the staff who have assisted in this study.
References Beecham, G.W., Martin, E.R., Li, Y.J., Slifer, M.A., Gilbert, J.R., Haines, J.L., Pericak-Vance, M.A., 2009. Genome-wide association study implicates a chromosome 12 risk locus for late-onset Alzheimer disease. Am. J. Hum. Genet. 84 (1), 35–43. Engidawork, E., Gulesserian, T., Yoo, B.C., Cairns, N., Lubec, G., 2001. Alteration of caspases and apoptosis-related proteins in brains of patients with Alzheimer’s disease. Biochem. Biophys. Res. Commun. 281 (1), 84–93. Hernandez, D., Paisan-Ruiz, C., Crawley, A., Malkani, R., Werner, J., Gwinn-Hardy, K., Dickson, D., Wavrant Devrieze, F., Hardy, J., Singleton, A., 2005. The dardarin G 2019 S mutation is a common cause of Parkinson’s disease but not other neurodegenerative diseases. Neurosci. Lett. 389 (3), 137–139.
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Ho, C.C., Rideout, H.J., Ribe, E., Troy, C.M., Dauer, W.T., 2009. The Parkinson disease protein leucine-rich repeat kinase 2 transduces death signals via Fas-associated protein with death domain and caspase-8 in a cellular model of neurodegeneration. J. Neurosci. 29 (4), 1011–1016. Lee, E., Hui, S., Ho, G., Tan, E.K., Chen, C.P., 2006. LRRK2 G2019S and I2020T mutations are not common in Alzheimer’s disease and vascular dementia. Am. J. Med. Genet. B: Neuropsychiatr. Genet. 141B (5), 549–550. Lu, C.S., Wu-Chou, Y.H., van Doeselaar, M., Simons, E.J., Chang, H.C., Breedveld, G.J., Di Fonzo, A., Chen, R.S., Weng, Y.H., Lai, S.C., Oostra, B.A., Bonifati, V., 2008. The LRRK2 Arg1628Pro variant is a risk factor for Parkinson’s disease in the Chinese population. Neurogenetics 9 (4), 271–276. Lu, Y.W., Tan, E.K., 2008. Molecular biology changes associated with LRRK2 mutations in Parkinson’s disease. J. Neurosci. Res. 86 (9), 1895–1901. Paisan-Ruiz, C., Jain, S., Evans, E.W., Gilks, W.P., Simón, J., van der Brug, M., López de Munain, A., Aparicio, S., Gil, A.M., Khan, N., Johnson, J., Martinez, J.R., Nicholl, D., Carrera, I.M., Pena, A.S., de Silva, R., Lees, A., Martí-Massó, J.F., Pérez-Tur, J., Wood, N.W., Singleton, A.B., 2004. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 44 (4), 595–600. Pericak-Vance, M.A., Bass, M.P., Yamaoka, L.H., Gaskell, P.C., Scott, W.K., Terwedow, H.A., Menold, M.M., Conneally, P.M., Small, G.W., Vance, J.M., Saunders, A.M., Roses, A.D., Haines, J.L., 1997. Complete genomic screen in late-onset familial Alzheimer disease. Evidence for a new locus on chromosome 12. JAMA 278 (15), 1237–1241. Ross, O.A., Wu, Y.R., Lee, M.C., Funayama, M., Chen, M.L., Soto, A.I., Mata, I.F., Lee-Chen, G.J., Chen, C.M., Tang, M., Zhao, Y., Hattori, N., Farrer, M.J., Tan, E.K., Wu, R.M., 2008. Analysis of Lrrk2 R1628P as a risk factor for Parkinson’s disease. Ann. Neurol. 64 (1), 88–92. Sancho, R.M., Law, B.M., Harvey, K., 2009. Mutations in the LRRK2 Roc-COR tandem domain link Parkinson’s disease to Wnt signalling pathways. Hum. Mol. Genet. 15;18 (20), 3955–3968. Tan, E.K., Skipper, L.M., 2007. Pathogenic mutations in Parkinson disease. Hum. Mutat. 28 (7), 641–653. Tan, E.K., Tan, L.C., Lim, H.Q., Li, R., Tang, M., Yih, Y., Pavanni, R., Prakash, K.M., Fook-Chong, S., Zhao, Y., 2008. LRRK2 R1628P increases risk of Parkinson’s disease: replication evidence. Hum. Genet. 124 (3), 287–288. Tan, E.K., Zhao, Y., Skipper, L., Tan, M.G., Di Fonzo, A., Sun, L., FookChong, S., Tang, S., Chua, E., Yuen, Y., Tan, L., Pavanni, R., Wong, M.C., Kolatkar, P., Lu, C.S., Bonifati, V., Liu, J.J., 2007. The LRRK2 Gly2385Arg variant is associated with Parkinson’s disease: genetic and functional evidence. Hum. Genet. 120 (6), 857–863. Tan, E.K., Lee, J., Chen, C.P., Wong, M.C., Zhao, Y., 2009. Case control analysis of LRRK2 Gly2385Arg in Alzheimer’s disease. Neurobiol. Aging 30 (3), 501–502. Tedde, A., Bagnoli, S., Cellini, E., Nacmias, B., Piacentini, S., Sorbi, S., 2007. No association between the LRRK2 G2019S mutation and Alzheimer’s disease in Italy. Cell. Mol. Neurobiol. 27 (7), 877–881. Toft, M., Sando, S.B., Melquist, S., Ross, O.A., White, L.R., Aasly, J.O., Farrer, M.J., 2005. LRRK2 mutations are not common in Alzheimer’s disease. Mech. Ageing Dev. 126 (11), 1201–1205. Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., Kachergus, J., Hulihan, M., Uitti, R.J., Calne, D.B., Stoessl, A.J., Pfeiffer, R.F., Patenge, N., Carbajal, I.C., Vieregge, P., Asmus, F., MüllerMyhsok, B., Dickson, D.W., Meitinger, T., Strom, T.M., Wszolek, Z.K., Gasser, T., 2004. Mutations in LRRK2 cause Autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44, 601–607.
LRRK2 variant associated with Alzheimer’s disease Yi Zhao a , P. Ho a , Yuen Yih a , C. Chen d , W.L. Lee b,c , E.K. Tan a,b,c,∗ a
Department of Neurology, Clinical Research and Health Screening, Singapore General Hospital, Singapore b National Neuroscience Institute, Singapore c Duke-NUS Graduate Medical School, Singapore d National University of Singapore, Singapore Received 23 July 2009; received in revised form 29 September 2009; accepted 26 November 2009 Available online 16 December 2009
Abstract Overlapping neurodegenerative pathologies (including Alzheimer’s disease, AD) have been described in Parkinson’s disease (PD) patients with leucine-rich repeat kinase-2 (LRRK2) mutations. We analyzed a LRRK2 PD (R1628P) risk variant in a group of 885 subjects comprising of AD and controls. The frequency of the R1628P allele was higher in AD compared to controls (3.5% vs. 1.6%, OR 2.3, 95 CI 1.2–4.4, p = 0.018). In vitro, the mean percentage of apoptosis and cell death observed for the R1628P transfected human cell lines was higher compared to wild type 21.8 ± 1.9, vs. 17.1 ± 1.3, p < 0.05, 30.2 ± 2.2 vs. 25.7 ± 1.3, p < 0.05). The LRRK2 R1628P variant increases the risk of AD in our population and our in vitro findings suggest that it is a functional variant and predisposes to apoptosis. © 2009 Elsevier Inc. All rights reserved. Keywords: LRRK2; Variants; Alzheimer’s disease
Parkinson’s disease (PD) and Alzheimer’s disease (AD) are the two most common neurodegenerative conditions in the adult population. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been reported to be the most prevalent cause of autosomal dominant PD. A common G2019S mutation accounts for a significant proportion of sporadic PD in some populations (Paisan-Ruiz et al., 2004; Zimprich et al., 2004; Tan et al., 2007). Post-mortem studies of mutation carriers have revealed a spectrum of pathologies suggestive of AD, dementia with Lewy Body, tauopathy, and supranuclear palsy (Zimprich et al., 2004). The location of the LRRK2 gene on chromosome 12q12 is close to a linkage peak for late onset AD (Pericak-Vance et al., 1997) and to the 12q13 risk locus identified in a recent genome wide association study (Beecham et al., 2009). Thus it has been speculated that LRRK2 may be involved in some overlapping neurodegenerative pathways in AD. Even though to date no specific LRRK2 ∗ Corresponding author at: Department of Neurology, Singapore General Hospital, Outram Road, Singapore 169608, Singapore. Tel.: +65 6326 5003; fax: +65 6220 3321. E-mail address: [email protected] (E.K. Tan).
0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.11.019
mutations have been associated with AD patients (Hernandez et al., 2005; Lee et al., 2006; Tedde et al., 2007), comprehensive screening of the gene in AD has yet to be reported. A LRRK2 R1628P (rs33949390) variant has been found to increase the risk of PD in different Asian populations (Ross et al., 2008; Lu et al., 2008; Tan et al., 2008). Since dual PD and AD pathologies can co-exist and the R1628P variant could be a biologically plausible risk variant in AD, we conducted the first case control study to investigate whether the LRRK2 R1628P variant is associated with risk of AD in an Asian population and assessed its pro-apoptotic potential in transfected human cell lines.
1. Methods A total of 897 subjects including 217 AD and 668 controls were recruited for this study. Some of the controls had participated in previous studies of the variant in PD (Ross et al., 2008; Tan et al., 2008). The cases were recruited from the outpatient Neurology clinics. The dementia syndrome was diagnosed using the Diagnostic and Statistical Manual, 4th
Y. Zhao et al. / Neurobiology of Aging 32 (2011) 1990–1993
edition (DSM-IV) criteria, and the diagnosis of AD followed the NINCDS-ADRDA criteria. The controls (of similar race, gender, age) were recruited from the same region as the AD cases and did not have any known neurodegenerative diseases. Subjects with mixed ethnicity were not included. All the study subjects gave informed consent and the institutional ethics committees had approved the AD genetic study. 1.1. Genotyping We utilized the allelic discrimination methods to genotype the variant. As an initial step, primers and probes were designed to detect the R1628P variant using the Primer Express 1.5 software. As per standard protocol, the ABI Prism 7700 Sequence Detection System (Applied Biosystems) was used for the amplification reactions. Polymerase reactions (PCR) were carried out under optimal conditions. After PCR, we measured the allelic specific fluorescence on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) using the Sequence Detection Systems 1.7 software for allelic discrimination. Each analysis was carried out with R1628P positive and negative samples. Sequencing in the forward and reverse directions was undertaken according to the manufacturers’ instructions (BigDye, Applied Biosystems, Warrington, UK). The sequence analysis confirmed the R1628P variant for representative samples with G or C allele. 1.2. R1628P expression plasmid The R1628P LRRK2 variant was generated by PCRmediated site-directed mutagenesis (Stratagene) in the full-length LRRK2 cDNA (Origene). DNA sequencing was carried out to confirm the accuracy of the constructs. We cloned the wild type and variant LRRK2 gene into pEGFP-N1 gene and fused to the N-terminus of EGFP.
1991
1.3. Transfection experiments We followed standardized transfection protocols as previously described (Tan et al., 2007; Tan and Skipper, 2007). Briefly, human embryonic kidney cells (HEK-293T) were maintained at 37 ◦ C in a 95% air/5% CO2 humidified atmosphere incubator and transfected overnight with 3.0 g of each plasmid DNA using LipofectamineTM 2000 reagent (Invitrogen) and OptiMEM culture medium. Cells were further incubated overnight in fresh medium containing oxygen stress reagent H2O2 (0.8 mM). For apoptosis analysis, the cultured cells were harvested by trypsin digestion. 1.4. Measurement of apoptosis Annexin V-PE apoptosis Kit I (BD Biosciences) was used to quantify apoptosis on H2O2 treated transfected cell lines. After re re-suspending the cells in binding buffer, 100 l of cells were transferred to a 5 ml tube and 5 l of Annexin V-PE and 5 l of 7-AAD were added. The reactions were incubated for 15 min and 400 l of binding buffer were added for flow cytometry analysis (FACSCalibur, Becton Dickinson). EGFP positive cells were gated and analyzed with Annexin V and 7-AAD staining for apoptotic cells (Annexin V positive, 7-AAD negative) and late apoptotic or necrotic cells (both Annexin V and 7-ADD positive). The experiments were done in triplicates and the mean of 3 independent sets were taken for analysis. 1.5. Statistical analysis We analyzed categorical and numerical variables using the chi-square and Student’s t-test. Genotype frequencies in the study subjects were assessed for deviation from Hardy–Weinberg equilibrium using chi-square test. We
Table 1 Lrrk2 R1628P allele frequency in AD and controls compared to published reports in Parkinson’s disease. Study
Cases (frequency of C allele)
Controls (frequency of C allele)
Odds ratio
Present study (AD sample) (n = 885)
15 (3.5%)
21 (1.6%)
2.3
PD studies Ross et al. (cohort 1) (n = 825)
33 (3.4%)
11 (1.6%)
2.15
Ross et al. (cohort 2) (n = 661)
21 (3.0%)
14 (2.2%)
1.39
Ross et al. (cohort 3) (n = 500)
13 (2.6%)
6 (1.2%)
2.2
Lu et al. (n = 1377)
64 (3.8%)
20 (0.018%)
2.13
Tan et al. (n = 489)
20 (4.2%)
8 (1.7%)
2.5
1992
Y. Zhao et al. / Neurobiology of Aging 32 (2011) 1990–1993
defined statistical significance at p < 0.05. Even though our study sample size did not have sufficient power to detect an odds ratio of 2 for an allele frequency of 2%, the sample size was comparable to all the previous studies that have detected a significant difference for this allele in PD (Table 1).
2. Results 2.1. Genotyping DNA samples from a total of 885 subjects comprising of 217 AD and 668 controls were analyzed. The mean (±SD, range) age, age at onset of AD, and age of controls was 75.0 ± 9.0 (44–93), 71.6 ± 8.3 (44–89), and 63.2 ± 12.0 (40–91) years, comprising of 57.0% and 47.0% men. None of the patients gave a positive family history of autosomal dominant dementia. The genotype frequency in both cases and controls followed the Hardy–Weinberg equilibrium. The frequency of the R1628P G allele was higher in AD compared to controls (3.5% vs. 1.6%, OR 2.3, 95 CI 1.2–4.4, p = 0.018). All carriers were heterozygous. As the frequency of the R1628P variant was low, interaction with ApoE could not be meaningfully carried out as a much larger sample size would be required. 2.2. Apoptosis measurements The transfection efficiency in the cell lines was similar for both wild type and variant. After H2O2 exposure, the mean percentage of apoptosis and cell death observed for the R1628P transfected human cell lines was higher compared to wild type 21.8 ± 1.9 vs. 17.1 ± 1.3, p < 0.05, 30.2 ± 2.2 vs. 25.7 ± 1.3, p < 0.05 (Fig. 1). No difference was observed at baseline without H2O2 exposure (data not shown).
Fig. 1. Percentage of transfected cells with apoptosis, necrosis and cell death after 0.4 M of hydrogen peroxide exposure. After H2O2 exposure, the mean percentage of apoptosis and cell death observed for the R1628P transfected human cell lines was higher compared to wild type 21.8 ± 1.9 vs. 17.1 ± 1.3, p < 0.05*, 30.2 ± 2.2 vs. 25.7 ± 1.3, p < 0.05*.
3. Discussion It is interesting to note that LRRK2 mutations have been associated with AD-like pathology (Zimprich et al., 2004), suggesting that there may be a partial overlap between neurodegenerative pathways in both AD and PD. LRRK2 R1628P is located in the COR (C-terminus of Roc [ras of complex proteins]) domain and evolutionarily conserved across species. The substitution of a highly basic polar arginine (R) with a neutral non-polar proline (P) is postulated to result in a conformational change in the LRRK2 secondary structure (Ross et al., 2008). An earlier study had screened several pathogenic LRRK2 mutations, including one in the COR domain (Y1699C) in AD and other patients with dementia but did not find any carriers (Toft et al., 2005). Even though subsequent studies have also confirmed that common LRRK2 mutations (G2019S and I2020T) and the polymorphic G2385R variant have been shown to have no association with AD (Lee et al., 2006; Tan et al., 2007, 2009), R1628P has yet to be examined in the AD population. Furthermore, there have been no reported functional studies on this variant. We showed that the R1628P increases the risk of AD by about two-fold in our population. This magnitude is similar to the risk associated with the variant in PD population across different Asian populations (see summary in Table 1, Ross et al., 2008; Lu et al., 2008; Tan et al., 2008). To further support the functional significance of this variant, we demonstrated that this variant when over-expressed in human cell lines was associated with a higher degree of apoptosis, when subjected to oxidative stress. This risk variant could be a factor in agerelated diseases like AD and PD as aging is associated with a decreased ability of cells to counter-act oxidative stress. While our in vitro observations are considered preliminary, it adds weight to the findings from the association study and those from bioinformatics analysis. The frequency of the R1628P variant in our controls is almost identical to those reported in various Asian populations (Ross et al., 2008; Lu et al., 2008; Tan et al., 2008), suggesting that the association was not confounded by a spuriously low carrier status in our controls. It would be interesting to unravel the exact pathophysiologic mechanism on how this variant exerts its effect in predisposing to cell death for both AD and PD. Oxidative stress and proteasomal impairment are well-recognized hallmarks of neurodegenerative diseases. However, the physiologic function of LRRK2 has not been clarified. Mutations in LRRK2 have been associated with activation of programmed cell death signalling involving the FADD/caspase-8 signaling pathways (Ho et al., 2009). Dysregulated programmed cell death or apoptosis may be involved in AD. Abnormal levels of apoptotic proteins have been reported in AD brains (Engidawork et al., 2001). The R1628P variant could affect the GTP binding capacity, its kinase activity either directly or via its interaction with the different functional domains of LRRK2 or other protein interactors, ultimately leading to dysregulation of apoptosis.
Y. Zhao et al. / Neurobiology of Aging 32 (2011) 1990–1993
Mutations in the Roc/COR domain could lead to dysregulation of kinase activity in vitro (Lu et al., 2008; Lu and Tan, 2008). The COR domain mutant (Y1699C) could interfere with the interaction between LRRK2 and dishevelled family of phosphoproteins (key regulators of Wnt (Wingless/Int) signalling pathways), potentially affecting synaptic transmission and neuronal maintenance (Sancho et al., 2009). Further studies in various in vivo models should provide insights. As the prevalence of this variant is low, it is probable that this risk variant is one of the many factors contributing to disease. It is unclear whether other modulatory genes or environmental factors influence its action. In conclusion, we demonstrated that the LRRK2 R1628P variant is associated with a two-fold risk of AD in our population and our preliminary in vitro findings suggest that it is a functional variant and predisposes to apoptosis. Replication of the findings in other populations would be of interest.
Disclosure statement All the authors certify that they do not have any actual or potential conflicts of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the work submitted that could inappropriately influence (bias) the work. The study has received approval from the relevant ethics committees and informed consent has been taken from every study subject.
Acknowledgements We thank National Medical Research Council, Singapore General Hospital, National Neuroscience Institute and Singapore Millennium Foundation for their support and all the staff who have assisted in this study.
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