ANALYSIS OF SNAP25 mRNA EXPRESSION AND PROMOTER DNA METHYLATION IN BRAIN AREAS OF ALZHEIMER’S DISEASE PATIENTS T. K. FURUYA, a P. N. O. SILVA, a S. L. M. PAYA ˜ O, a,c,f P. H. F. BERTOLUCCI, b L. T. RASMUSSEN, f R. W. DE LABIO, c I. L. S. BRAGA, a E. S. CHEN, a G. TURECKI, d N. MECHAWAR, d J. MILL e AND M. A. C. SMITH a * a Disciplina de Gene ´tica, Departamento de Morfologia e Gene ´tica, Universidade Federal de Sa ˜o Paulo (UNIFESP), Sa ˜o Paulo-SP, Brazil b Disciplina de Neurologia Clı´nica, Departamento de Neurologia e Neurocirurgia (UNIFESP), Sa ˜o Paulo-SP, Brazil c Laborato ´rio de Gene ´tica, Hemocentro, Faculdade de Medicina de Marı´lia (FAMEMA), Marı´lia-SP, Brazil d Psychiatry Department, Douglas Hospital Research Center, McGill University, Montreal, Canada e Institute of Psychiatry, King’s College, London, United Kingdom f Pro ´-Reitoria de Pesquisa e Po ´ s-graduac ¸a ˜o, Universidade Sagrado Corac ¸a ˜o (USC), Bauru-SP, Brazil Abstract—Alzheimer 0 s Disease (AD) is the most common cause of dementia in elderly people. The presynaptic termi- nal is an important site of pathological changes in AD, lead- ing to synaptic loss in specific brain regions, such as in the cortex and hippocampus. In this study, we investigated syn- aptosomal-associated protein, 25-kDa (SNAP25) mRNA lev- els and promoter DNA methylation in post mortem brain tissues (entorhinal and auditory cortices and hippocampus) from healthy elderly and AD subjects as well as in peripheral blood leukocytes of young, healthy elderly and AD patients. mRNA quantification was performed by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) using the DDC T method and promoter DNA methylation was quan- tified by mass spectrometry using the Sequenom EpiTYPER platform. We observed a significant decrease in SNAP25 expression in AD across all the three brain regions in rela- tion to the healthy elderly subjects, suggesting impairment in synaptic function. The changes in the auditory cortex reflected those observed in the hippocampus and entorhinal cortex, the primary areas affected in AD. However, no AD- associated differences in SNAP25 promoter DNA methyla- tion were observed suggesting that other mechanisms may be involved in mediating the observed gene expression changes. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: aging, Alzheimer’s Disease, promoter DNA meth- ylation, post mortem brain tissue, SNAP25 gene expression, synaptic impairment. INTRODUCTION Alzheimer’s Disease (AD) is a neurodegenerative disor- der and the major cause of cognitive decline among the elderly population (Papassotiropoulos et al., 2006). It is the most common form of dementia, with progressive impairment of episodic memory that becomes more se- vere and, eventually, incapacitating. Other common symptoms include delusions, confusion, poor judgment and decision-making, agitation as well as language distur- bances (Blennow et al., 2006; Bird, 2008). Neuropathologically, AD is characterized by extracellu- lar deposits of b-amyloid (bA) (senile plaques) and the accumulation of hyper-phosphorylated tau protein (neuro- fibrillary tangles) in the brain (Blennow et al., 2006). Fur- thermore, anatomic structural changes are also relevant during the course of the disease. Significant atrophy of some brain regions is observed in the early stages of the disease, mainly in the hippocampal formation and entorhi- nal cortex (Hampel et al., 2008). Because memory forma- tion depends on the presence of an intact entorhinal– hippocampal circuit, it is not surprising that this network is severally impaired in AD (Harris et al., 2010). This heterogeneous and complex disorder can be classified as familial or sporadic. The familial form of the disease is a rare autosomal dominant disorder with onset before age 65 years, resulting from mutations in the amy- loid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes. The e4 allele of apolipopro- tein E (APOE) gene is the most important risk factor in the sporadic form of AD, which might act with several other susceptibility genes, each conferring only a minor increase in risk, in a complex interaction with environmen- tal factors (Blennow et al., 2006). Recent large-scale gen- ome-wide association studies have identified some novel susceptibility loci, such as CLU, PICALM, CR1 and BIN1 0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.06.035 * Corresponding author. Address: Disciplina de Gene´tica, Departa- mento de Morfologia e Gene´ tica, UNIFESP/EPM, Rua Botucatu, 740, Edifı´cio Leita˜o da Cunha, 1° andar, CEP 04023-900, Sa˜o Paulo-SP, Brazil. Tel: +55-11-5576-4260; fax: +55-11-5576-4264. E-mail address: tuty_furuya@yahoo.com.br (M. A. C. Smith). Abbreviations: AD, Alzheimer’s Disease; APOE, apolipoprotein E; APP, amyloid precursor protein; bp, base pair; CDR, clinical dementia rating; C T , cycle threshold; df, degrees of freedom; DSM-IV, IV Diagnostic and Statistical Manual of Mental Disorders; F, female; GLM, general linear model; M, male; MALDI-TOF, matrix-assisted laser desorption ionization time of flight mass spectrometry; MMSE, Mini-Mental State Examination; N, number of individuals; NINCDS–ADRDA, National Institute of Neu- rological and Communicative Disorders and Stroke–Alzheimer’s Dis- ease and Related Disorders Association; PCR–RFLP, Polymerase Chain Reaction–Restriction Fragment Length Polymorphism; PSEN1, presenilin 1; PSEN2, presenilin 2; qRT-PCR, quantitative Reverse Tra- nscription Polymerase Chain Reaction; RQ, relative quantification; SD, standard deviation; SNAP25, synaptosomal-associated protein, 25 kDa; bA, b-amyloid. Neuroscience 220 (2012) 41–46 41