SORL1 and SIRT1 mRNA expression and promoter ...

Neurochemistry International 61 (2012) 973–975

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SORL1 and SIRT1 mRNA expression and promoter methylation levels in aging and Alzheimer’s Disease Tatiane Katsue Furuya a, Patrícia Natália Oliveira da Silva a, Spencer Luiz Marques Payão a,b,f, Lucas Trevizani Rasmussen f, Roger Willian de Labio b, Paulo Henrique Ferreira Bertolucci c, Ianna Lacerda Sampaio Braga a, Elizabeth Suchi Chen a, Gustavo Turecki d, Naguib Mechawar d, Jonathan Mill e, Marília de Arruda Cardoso Smith a,⇑ a

Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo (UNIFESP), São Paulo-SP, Brazil Laboratório de Genética, Hemocentro, Faculdade de Medicina de Marília (FAMEMA), Marília-SP, Brazil c Disciplina de Neurologia Clínica, Departamento de Neurologia e Neurocirurgia (UNIFESP), São Paulo-SP, Brazil d Psychiatry Department, Douglas Hospital Research Center, McGill University, Montreal, Canada e Institute of Psychiatry, King’s College, London, United Kingdom f Pró-Reitoria de Pesquisa e Pós-graduação, Universidade Sagrado Coração (USC), Bauru-SP, Brazil b

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Article history: Received 2 June 2012 Received in revised form 7 July 2012 Accepted 13 July 2012 Available online 23 July 2012 Keywords: Alzheimer’s Disease Aging SORL1 and SIRT1 mRNA expression Peripheral blood leukocytes Post mortem brain tissue Promoter DNA methylation

a b s t r a c t Alzheimer’s Disease (AD) is a neurodegenerative disorder and the most common cause of dementia among the elderly. Efforts have been made to understand the genetic and epigenetic mechanisms involved in the development of this disease. As SORL1 (sortilin-related receptor) and SIRT1 (sirtuin 1) genes have been linked to AD pathogenesis, we aimed to investigate their mRNA expression and promoter DNA methylation in post mortem brain tissues (entorhinal and auditory cortices and hippocampus) from healthy elderly subjects and AD patients. We also evaluated these levels in peripheral blood leukocytes from young, healthy elderly and AD patients, investigating whether there was an effect of age on these profiles. The comparative CT method by Real Time PCR and MALDI-TOF mass spectrometry were used to analyze gene expression and DNA methylation, respectively. SORL1 gene was differently expressed in the peripheral blood leukocytes and might act as a marker of aging in this tissue. Furthermore, we found that SORL1 promoter DNA methylation might act as one of the mechanisms responsible for the differences in expression observed between blood and brain for both healthy elderly and AD patients groups. The impact of these studied genes on AD pathogenesis remains to be better clarified. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Alzheimer’s Disease (AD) is the most common cause of dementia and its prevalence increases exponentially with age. It is a heterogeneous disorder and the cause of the sporadic form is still unknown, probably caused by a complex interaction among aging, Abbreviations: ACTB, actin beta; AD, Alzheimer’s Disease; Ab, Amyloid b; APOE, apolipoprotein E; CDR, Clinical Dementia Rating; df, degrees of freedom; DSM-IV, IV Diagnostic and Statistical Manual of Mental Disorders; GLM, General Linear Model; N, number of individuals; NINCDS-ADRDA, National Institute of Neurological and Communicative Disorders and Stroke – Alzheimer’s Disease and Related Disorders Association; MMSE, Mini-Mental State Examination; qRT-PCR, quantitative Reverse Transcription Polymerase Chain Reaction; RQ, relative quantification; SD, Standard deviation; SIRT1, sirtuin 1; SORL1, sortilin-related receptor. ⇑ Corresponding author. Address: Disciplina de Genética, Departamento de Morfologia e Genética, UNIFESP/EPM Rua Botucatu, 740, Edifício Leitão da Cunha – 1° andar, CEP 04023-900 São Paulo/SP, Brazil. Tel.: +55 11 5576 4260; fax: +55 11 5576 4264. E-mail address: [email protected] (M. de Arruda Cardoso Smith). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.07.014

several susceptibility genes and environmental risk factors (Blennow et al., 2006). Moreover, the epigenetic variability could affect the AD predisposition and the course of the disease in addition to these factors (Wang et al., 2008). Some findings have linked the lipoprotein receptor SORL1 (sortilin-related receptor; also known as LR11 or SorLA) to AD pathogenesis, based on its reduction in AD brains and its ability to lower Amyloid b (Ab) levels, one of the hallmarks of this disease. SORL1 is known to bind to apolipoprotein E (APOE), the main lipoprotein expressed in the brain. Furthermore, APOE e4 allele is one of the main genetic risk factor associated to the sporadic form of AD (Offe et al., 2006). Another important molecule that has been implicated in the pathogenesis of AD is SIRT1 (sirtuin 1), member of the sirtuins family, which are protein deacetylases, essential for the regulation of various cellular functions. SIRT1 might regulate aging and metabolic processes involved in AD and its loss has been associated with the disease progression (Julien et al., 2009). Furthermore, SIRT1

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might have a neuroprotective role in both healthy aging and AD (Anekonda and Reddy, 2006). We aimed to investigate SORL1 and SIRT1 gene expression and promoter DNA methylation patterns in post mortem brain tissues from AD patients and healthy elderly subjects. We also evaluated these levels in peripheral blood leukocytes from young, healthy elderly and AD patients, investigating whether there was an effect of age on these profiles.

2. Material and methods Post mortem brain tissues derived from the entorhinal and auditory cortices as well as the hippocampus of patients diagnosed with AD (n = 12; Mean Age ± Standard Deviation (SD) = 81.0 ± 6.5 years; Gender (% female/male) = 58.3/41.7) and healthy elderly controls (n = 10; Mean Age ± SD = 78.5 ± 8.9 years; Gender (% female/male) = 60.0/40.0) were provided by Douglas Hospital Research Center Brain Bank (Montreal, Canada). All of the three brain areas were studied for each individual. Both healthy elderly and AD patients had similar age (p > 0.05) and genders did not differ between the groups (p > 0.05). The post mortem delay interval ranged from 7 to 37.5 h. Brains were extracted, sectioned and snap frozen and stored at 80 °C until use. All brain samples (with or without AD) were processed in the same way using the same sampling protocols. The neuropathological characterization of AD was carried out according to well-established clinical and neuropathological criteria. Patients showed at least 3 years of symptoms duration (sporadic form of the disease), with spectrum of the disease varying from severe to terminal. The most prevalent features were extensive plaque and tangle formation, marked neuronal loss, cerebral atrophy, cognitive impairment among others. Peripheral blood leukocytes from young (n = 25; Mean Age ± SD = 21.7 ± 1.6 years; Gender (% female/male) = 76.0/24.0), healthy elderly (n = 23; Mean Age ± SD = 72.1 ± 10.5 years; Gender (% female/male) = 69.4/30.6) and AD patients (n = 36; 75.4 ± 8.9 years; Gender (% female/male) = 73.9/26.1) were collected at the Departamento de Neurologia of Universidade Federal de São Paulo/UNIFESP (São Paulo-SP, Brazil). Age and gender did not differ between healthy elderly and AD patients (p > 0.05) and young was recruited with age ranging between 18 and 28 years old. The healthy elderly group complied with exclusion criteria for depression, stroke, vascular dementia, alcoholism and low cognitive levels. This project included only AD patients with more than 65 years old and at least three years of the sporadic form of the disease and the samples were selected according to NINCDSADRDA criteria and DSM-IV for probable AD. Mini-Mental State Examination (MMSE), Katz index and the Clinical Dementia Rating (CDR) were also evaluated. The Institutional Research Ethics Committee approved this study and all subjects or their legal representatives signed an informed consent according to the Declaration of Helsinki. Total RNA samples were isolated from brain tissues and peripheral blood using RNeasy Lipid Tissue Mini Kit (QIAGEN, Germany) and RiboPure™ Blood Kit (Ambion, USA), respectively, and cDNA synthesis was carried out using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems™, USA), following the protocols provided by the manufacturer. Gene expression was measured by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) on the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems™, USA), using Hs00268342-m1, Hs01009006-m1 and Hs03023943-g1 TaqManÒ Gene Expression Assays (FAM™ dye-labeled MGB probes) for SORL1 and SIRT1 (targets) and ACTB (actin beta; endogenous control) genes, respectively. Relative Quantification (RQ), which means the fold change or how much one gene is more expressed in a group in relation

to another, was calculated using the comparative CT method (2 DDCT). DNA extraction and sodium bisulfite modification were performed using the QIAamp DNA Blood Midi Kit (QIAGEN, Germany) and EZ-96 DNA Methylation™ Kit (Zymo Research, USA), respectively, following the manufacturer’s standard protocol. DNA methylation quantification was performed in the Institute of Psychiatry, King’s College London (London, UK), using the Sequenom EpiTYPER platform (Sequenom, Inc., USA). The target region was PCR amplified in duplicate from bisulfite-treated DNA with primers designed using the Sequenom EpiDEsigner software (www.epidesigner.com). The analysis was based in base-specific cleavage of single-stranded nucleic acids with MALDI-TOF mass spectrometry analysis of the cleavage products (Ehrich et al., 2005). We calculated the mean of CpG island methylation (13 and 22 CpG dinucleotides for SORL1 and SIRT1 genes, respectively) including all the CpG dinucleotides inside the amplicon. Control samples were run in parallel to ensure accurate quantification. Z-score standardized values were used for both 2 DCT and mean of CpG island methylation values once they deviated from the normal distribution. These values were used as dependent variables for the experimental design. Based on the distribution of our data and to detect aberrant values which might comprise the statistical analyses, observations > +2.59 or < 2.59 were considered as outliers and excluded from analyses. The comparison of both expression and promoter DNA methylation values among the three brain regions (entorhinal and auditory cortices and hippocampus) within the healthy elderly group as well as within the AD patients group was performed using one way ANOVA followed by Bonferroni post hoc test. This test was also used to compare these differences in peripheral blood leukocytes from young, healthy elderly and AD patients. Statistical comparisons of each of the three different brain regions between AD patients group and healthy elderly group were performed using Student’s t-test. We also performed this test to identify differences in the mRNA expression values and methylation status between blood and brain for both AD patients and elderly subjects. We used Pearson Correlation to analyze if there was correlation between levels of expression and methylation status in both brain and blood of AD patients and elderly subjects. In the analyses where p values were significant, we performed a General Linear Model (GLM) to calculate both the effect size and observed power. Statistical analyses were performed using SPSSÒ 18.0 (SPSS, Inc, USA). The null hypothesis was rejected at p < 0.05.

3. Results SORL1 gene expression showed significant difference among groups (F2,73 = 7.493; p = 0.001) in the peripheral blood leukocytes. We detected higher expression levels in the healthy elderly (n = 18; 2 DCT Mean ± SD = 0.716 ± 0.426; p = 0.016; RQ = 2.01) and in the AD patients (n = 34; 2 DCT Mean ± SD = 0.749 ± 0.325; p = 0.001; RQ = 1.92) in comparison to the young group (n = 24; 2 DCT Mean ± SD = 0.374 ± 0.421; RQ = 1). The values of effect size and observed power for this analysis were 17.0% and 93.4%, respectively. Moreover, this gene did not show differences in the promoter DNA methylation pattern in blood (p > 0.05). For SIRT1 gene, we did not find differences in the mRNA expression and promoter methylation pattern among the studied groups in the peripheral blood leukocytes (p > 0.05). Regarding the brain tissue, we did not find differences in SORL1 and SIRT1 gene expression and promoter methylation when comparing the healthy elderly and the AD patients groups (p > 0.05). Likewise, no differences were found among the three brain regions

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T.K. Furuya et al. / Neurochemistry International 61 (2012) 973–975 Table 1 Results of Student’s t-test comparing 2 elderly subjects. Analysis

Gene expression (2

DCT

)

Methylation (mean of CpG island)

DCT

and mean of CpG island methylation values of SORL1 gene between blood and brain for both Alzheimer’s Disease patients and healthy

Group

Blood

Brain

t

N

Mean ± SD

N

Mean ± SD

AD Elderly

34 18

0.749 ± 0.325 0.716 ± 0.426

27 30

0.142 ± 0.056 0.129 ± 0.054

AD Elderly

30 23

0.017 ± 0.010 0.018 ± 0.011

31 28

0.026 ± 0.010 0.028 ± 0.013

Effect size (%)a

Observed power (%)a

0.19 0.18

60.8 55.0

100 100

– –

18.0 13.9

94.2 78.7

df

p

RQ (2

9.58 7.50

59 46

0.05). Concerning SORL1 gene, we also compared gene expression levels between blood and brain tissues and we detected that both the healthy elderly subjects and the AD patients had lower SORL1 gene expression in the brain than in the blood (p < 0.001; Table 1). Furthermore, we also found DNA methylation differences between blood and brain for these two studied groups (p < 0.05). Brain presented higher methylation levels than blood for both groups (Table 1). The values of effect size and observed power were showed in Table 1. 4. Discussion To the best of our knowledge this is the first study to investigate SORL1 and SIRT1 promoter DNA methylation patterns in brain and blood of AD patients. Regarding SORL1 gene expression in peripheral blood leukocytes, we detected that healthy elderly and AD patients groups showed around twofold higher expression than the young group. Therefore, it might act as a potential systemic marker of the aging process. On the other side, no age-specific methylation changes could be identified in blood for this gene, suggesting that methylation is not the mechanism responsible for these differences in gene expression. A previous study showed lower SORL1 transcript levels in lymphoblasts of sporadic AD patients (Scherzer et al., 2004). Then, further studies should be conducted to clarify the role of both SORL1 gene expression and epigenetic factors controlling it in peripheral tissues of AD patients. In brain tissue, although some studies have reported downregulation of SORL1 and SIRT1 expression in brains of AD patients (Dodson et al., 2006; Julien et al., 2009; Offe et al., 2006; Scherzer et al., 2004), we did not find differences either in their expression or in the methylation of SORL1 and SIRT1 promoter sequences assessed in this study. It is important to highlight that these studies found differences in some different regions than the ones we have investigated here. Consequently, our findings pointed that these mechanisms might not influence in the AD pathogenesis for the brain regions analyzed in this study. Therefore, additional insights into the biological functions of SORL1 and SIRT1 genes are required to identify their precise role and involvement in AD. Our results showed that SORL1 gene is lower expressed in the brain than in blood leukocytes for both healthy elderly and AD patients. Moreover, we found tissue-specific differences in methylation levels, in which brain presented higher methylation than

blood tissue, showing an inverse relationship between promoter activity and methylation levels. Therefore, methylation might be involved in the mechanism that altered gene expression between these tissues. However, it is important to notice that the methylation levels were really low and there might be other mechanisms involved in this regulation, such as methylation changes in regulatory regions other than in the studied ones or other epigenetic mechanisms such as non-coding RNA silencing or histone modifications. 5. Conclusions In conclusion, we found that SORL1 gene could act as a marker of aging in peripheral blood leukocytes. Furthermore, we found that methylation might act as one of the mechanisms responsible for the differences in expression observed between blood and brain for both healthy elderly and AD patients groups. The impact of SORL1 and SIRT1 genes on AD pathogenesis remains to be better clarified. Acknowledgements This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil). References Anekonda, T.S., Reddy, P.H., 2006. Neuronal protection by sirtuins in Alzheimer’s disease. J. Neurochem. 96, 305–313. Blennow, K., de Leon, M.J., Zetterberg, H., 2006. Alzheimer’s disease. Lancet 368, 387–403. Dodson, S.E., Gearing, M., Lippa, C.F., Montine, T.J., Levey, A.I., Lah, J.J., 2006. LR11/ SorLA expression is reduced in sporadic Alzheimer disease but not in familial Alzheimer disease. J. Neuropathol. Exp. Neurol. 65, 866–872. Ehrich, M., Nelson, M.R., Stanssens, P., Zabeau, M., Liloglou, T., Xinarianos, G., Cantor, C.R., Field, J.K., van den Boom, D., 2005. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc. Natl. Acad. Sci. USA 102, 15785–15790. Julien, C., Tremblay, C., Emond, V., Lebbadi, M., Salem Jr., N., Bennett, D.A., Calon, F., 2009. Sirtuin 1 reduction parallels the accumulation of tau in Alzheimer disease. J. Neuropathol. Exp. Neurol. 68, 48–58. Offe, K., Dodson, S.E., Shoemaker, J.T., Fritz, J.J., Gearing, M., Levey, A.I., Lah, J.J., 2006. The lipoprotein receptor LR11 regulates amyloid beta production and amyloid precursor protein traffic in endosomal compartments. J. Neurosci. 26, 1596– 1603. Scherzer, C.R., Offe, K., Gearing, M., Rees, H.D., Fang, G., Heilman, C.J., Schaller, C., Bujo, H., Levey, A.I., Lah, J.J., 2004. Loss of apolipoprotein E receptor LR11 in Alzheimer disease. Arch. Neurol. 61, 1200–1205. Wang, S.C., Oelze, B., Schumacher, A., 2008. Age-specific epigenetic drift in lateonset Alzheimer’s disease. PLoS ONE 3, e2698.