Molecular Neuroscience

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Volume 27 Number 2, 2005 ISSN: 0895 8696 JOURNAL OF Molecular Neuroscience Editor-in-Chief: ILLANA GOZES, PhD In This Issue The Biological Clock

1 Volume 27 Number 2, 2005 ISSN: JOURNAL OF Molecular Neuroscience Editor-in-Chief: ILLANA GOZES, PhD In This Issue The Biological Clock Alzheimer s Immunization Signal Transduction/ Neuroprotection/ Polymorphism HumanaJournals.com Search, Read, and Download JMN_27_2_cvr 1 9/12/05, 7:52 AM

2 Journal of Molecular Neuroscience Copyright 2005 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN /05/27: /$30.00 DOI: /JMN:27:02:213 ORIGINAL ARTICLE Monoamine Oxidase A Polymorphism in Brazilian Patients Risk Factor for Late-Onset Alzheimer s Disease? Agnes L. Nishimura, 1 Camila Guindalini, 1 João R. M. Oliveira, 3 Ricardo Nitrini, 2 Valéria S. Bahia, 2 Paulo R. de Brito-Marques, 3 Paulo A. Otto, 1 and Mayana Zatz*,1 1 Human Genome Research Center, Biology Department, Institute of Biosciences, São Paulo University (IBUSP), São Paulo, Brazil; 2 Neurology Department of Medical School, São Paulo University, São Paulo, Brazil; and 3 Behavioral Neurology Unit, Department of Neurology, Faculty of Medical Sciences, University of Pernambuco, Recife, Brazil Received December 8, 2004; Accepted March 30, 2005 Abstract Different studies have attempted to find polymorphisms involved in the serotonergic pathway that could be involved in mood disorders and late-onset Alzheimer s disease (LOAD) symptoms. Here, we compared the frequency of two polymorphisms: monoamine oxidase A(MAOA) and serotonin transporter in LOAD patients versus controls. No evidence of association was observed when these polymorphisms were compared separately; however, the combination of the MAOA allele 1 + the short allele of 5-HTTLPR + ApoE-ε4 was significantly more frequent in patients than in controls. It reinforces the hypothesis that different genes acting together might play a role in AD susceptibility. Based on these data, we suggest replicating these studies in larger samples of LOAD patients belonging to different ethnic groups. DOI: /JMN:27:02:213 Index Entries: Alzheimer s disease; monoamine oxidase A; polymorphisms; serotonergic pathway; Brazilian population. Introduction Alzheimer s disease (AD), the most common mental disorder in the elderly, leads to cognitive impairment, progressive loss of memory, and other symptoms such as depression and mood alteration. It is characterized by two histopathological hallmarks: neurofibrillary tangles, caused by hyperphosphorylation of the Tau protein, and β-amyloid deposits. Mutations in three genes have been associated with the familial form of AD: presenilin 1, presenilin 2, and the amyloid precursor protein (APP), localized at 14q24.3, 1q31 q42, and 21q21, respectively. In addition, the ε4 allele of Apolipo protein E (ApoE-ε4) polymorphic locus is the most important associated with late-onset AD (LOAD) sporadic cases. Because depression and mood disorders are observed in patients with AD, different studies have explored whether genes of the serotonergic pathway might be involved with these symptoms (Collier et al., 1996; Lesch et al.; 1996, Li et al., 1997). Serotonin (5-HT) is an important neurotransmitter in the central and peripheral nervous systems, implicated in the control of mood, appetite, sleep, pain, and behavior. Mutations in this gene *Author to whom all correspondence and reprint requests should be addressed. mayazatz@usp.br Journal of Molecular Neuroscience 213 Volume 27, 2005

3 214 Nishimura et al. or polymorphisms related to the serotonin pathway, such as the serotonin transporter (5-HTT), could be associated with mood disorders (Wenham et al., 1991; Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Oliveira et al., 1998; Kunugi et al., 2000). The short allele of the 5-HTT gene-linked polymorphic region (5-HTTLPR), involved in the activity of the serotonin transporter gene, has been studied in several mood disorders, including AD, bipolar, and unipolar disease. Some reports suggest that the 5-HT uptake function is reduced in these disorders (Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Kunugi et al., 2000). The search for other polymorphisms associated with AD has been undertaken by many groups of investigators around the world. Several putative candidate loci have been suggested, but most studies have shown controversial results when replicated in different populations, including the 5-HTTLPR polymorphism (Wenham et al., 1991; Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Oliveira et al., 1998; Kunugi et al., 2000). Monoamine oxidase A (MAOA) is a mitochondrial enzyme that catalyzes the degradation of different amines, including neurotransmitters like dopamine, norepinephrine, and serotonin. Several studies have shown that MAOAplays an important role in human behavior and physiology, being associated with mild mental retardation and impulsive aggressive behavior, mood disorders, and alcoholism with antisocial personality (Deckert et al., 1999; Furlong et al., 1999; Kunugi et al., 1999, 2000; Samochowiec et al., 1999; Schuback et al., 1999; Koller et al., 2003; Gutierrez et al., 2004). A functional polymorphism located upstream of this gene was described and consists of a 30-bp repeated sequence and the alleles 2 or 3, which were found to be transcribed 2 10 times more efficiently than others (Sabol et al., 1998). Moreover, the longer alleles (2, 3, and 4) were more frequent in panic disorder patients from German and Italian population than in controls. However, this association was observed only in females, which might be explained because it is an X-linked gene (Deckert et al., 1999). In contrast, no association between any MAOA polymorphic allele and mood disorders was reported by other researchers in the Japanese population, suggesting ethnic differences for association studies with this locus (Kunugi et al., 1999). Taking into account these controversial results, we have undertaken the present study in the Brazilian population, aiming to verify (1) whether MAOA polymorphisms are involved in LOAD, and (2) whether the short allele of 5-HTTLPR associated with MAOA polymorphism increases the risk for developing LOAD. Patients and Methods A total of 128 LOAD patients (46 males and 82 females; mean age, ± 8.99) were selected and diagnosed according to NINCDS-ADRDA criteria. To classify the cognitive impairment, neurological and neuropsychological testing, including Mini- Mental State Exam (MMSE) and Clinical Dementia Rating (CDR), were performed. The age/ethnicmatched control group (45 males and 81 females; mean age, ± 9.08) was selected based on the MMSE and/or Blessed Scale, sociocultural and ethnic background. Informed consent was obtained from all subjects included in this study. Genomic DNAwas isolated from peripheral blood according to standard procedures. Analyses of MAOA, 5-HTTLPR, and ApoE polymorphisms were based on previous studies (Wenham et al., 1991; Lesch et al., 1996; Sabol et al., 1998) comparing LOAD patients and healthy controls. The Hardy-Weinberg equilibrium, as well as genotypic and allelic frequencies comparing LOAD patients and controls, was analyzed using χ 2 tests, Fisher s exact test, and contingency tables. Differences were considered statistically significant at p < Results There were no significant deviations from the Hardy-Weinberg equilibrium for any of the polymorphisms studied. The allele and genotype distribution of the MAOA polymorphism is shown in Table 1. No allele distribution association was observed when the genders were analyzed separately comparing LOAD patients and controls (χ 2 = 6.16, df = 3, p = 0.10 for males, and χ 2 = 8.69, df = 7, p = 0.27 for females). However, a significant association was observed when both sexes were analyzed together (χ 2 = 12.54, df = 4, p = 0.01). Interestingly, allele <1 (2 repeats) and also allele 2 (3.5 repeats) were present only in the LOAD group. For the 5-HTTLPR polymorphism, no association was observed for the allele distribution (p = 0.37), but a borderline association was found for the different genotypes (χ 2 = 5.93, df = 2; p = 0.05) (Table 2), and the

4 MAOA and AD in Brazilian Population 215 Table 1 Allele and Genotype Frequencies of the MAOA Polymorphism in LOAD Patients vs Normal Controls AD Control Statistical analysis MAOA a Genotype Allele Genotype Allele Genotype Allele Male Female Total Male Female Total (n = 46) (n = 82) (n = 210) (n = 45) (n = 81) (n = 207) <1 0-4 (0.02) 0-0 AD male vs control AD total vs control male: χ 2 = 6.16; total: χ 2 = 12.54; df = 3; p = d f =4; p = (0.49) - 84 (0.40) (0.34) AD female vs control female: χ 2_ = 8.69; df = 7; p = (0.06) - 4 (0.02) (0.45) (0.55) (0.63) (0.01) 2-7 (0.03) (0.13) (0.09) (0.01) (0.47) (0.49) (0.34) (0.37) (0.04) (0.01) (0.01) - <11-2 (0.02) <13-2 (0.02) a MAOA alleles: <1 = 2 repeats, 1 = 3 repeats, 2 = 3.5 repeats, 3 = 4 repeats, and 4 = 5 repeats. Table 2 Genotype and Allele Frequencies of 5HTTLPR in LOAD Patients and Controls LOAD (n = 128) Controls (n = 126) Statistical analysis 5-HTTLPR ll 34 (0.26) 48 (0.38) χ 2 = 5.93; df = 2; p = 0.05 ls 72 (0.56) 52 (0.41) ss 22 (0.18) 26 (0.21) l 140 (0.55) 148 (0.57) p = 0.37, OR = 0.84 (95%) CI = s 116 (0.45) 104 (0.43) 5HTTLPR, 5-hydroxytryptamine transporter-linked polymorphic region. l, long variant; s, short variant. ApoE-ε4 allele was significantly more frequent in patients than in controls (p < 0.001). The combination of MAOA allele 1 + the short allele of 5-HTTLPR + ApoE-ε4 was observed in 19 patients and in only 4 controls, which was statistically significant (p = 0.001, OR = 0.18 (95%) CI = ). In contrast, the combination of MAOA allele 3 + the short allele of 5-HTTLPR + ApoE-ε4 did not differ between patients (n = 14) and controls (n = 11) (p = 0.67, OR = 0.77 [95%] CI = ). Discussion With the exception of the APOE-ε4 polymorphism, association studies in different populations have shown controversial results, which could be attributed to differences in ethnic background, founder effects, or environmental factors (Collier et al., 1996; Lesch et al., 1996; Li et al., 1997; Oliveira et al., 1998; Kunugi et al., 2000; Tsai et al., 2001; Farrer et al., 2003; Luchsinger and Mayeux, 2004).

5 216 Nishimura et al. In the present study, no significant association between the MAOA locus and LOAD was observed when each gender was analyzed separately. However, when both genders were assembled, a statistically significant association was found (p = 0.01). Furthermore, two alleles, allele <1(2 repeats) and allele 2(3.5 repeats) were present only in the LOAD group. In addition, a borderline association was found for the genotype distribution of the 5-HTTLPR locus. In a previous study of this locus in another group of Brazilian patients, a strong association between the short allele and LOAD was found (Oliveira et al., 1998). Because the number of patients is relatively small, these results need to be confirmed in independent populations and in a larger number of patients. The ApoE and 5-HTTLPR polymorphisms were studied previously in the Brazilian population. As expected, in the present analysis, the ApoE-ε4 allele was significantly more frequent in patients than in controls (p < 0.001). It has been shown that MAOA alleles 2, 3, and 4 transcribe 2 10 times more efficiently than others, suggesting that they might represent a risk factor for panic disorder in women (Deckert et al., 1999). Carriers of these isoforms showed higher levels of degradation of several neurotransmitters, resulting in decreasing levels of serotonin as well. It is relevant that the short allele of the 5-HTTLPR seems also to decrease the expression of the serotonin transporter, as well as the 5-HT uptake in lymphoblasts. Finally, we analyzed whether there was a preferential segregation of some alleles in patients as compared with controls. Interestingly, the analysis of the MAOA polymorphic locus showed no preferential association of any allele; however, the combination of the MAOA allele 1 + the short allele of 5-HTTLPR + ApoE-ε4 was significantly more frequent in patients than in controls. The analyses of MAOA and serotonin polymorphisms should be replicated in other population studies. The present data reinforce the hypothesis that different genes acting together might play a role in AD susceptibility, although the underlying mechanism is still unknown. Several drugs have been tested in an attempt to retard the progression of this disease, such as tacrine, donepezil, memantine, rivastigmine, and MAOA inhibitors (Youdim and Weinstock, 2004). Recently, a novel neuroprotective drug, TV3326 (N-propargyl-(3R)-aminoindan-5-yl-ethyl, methyl carbamate), which combines the neuroprotective effects of MAOA s inhibitors and rivastigmine, seems to be useful for AD treatment (Riederer et al., 2004; Youdim and Buccafusco, 2005). It was observed that TV3326 confers neuroprotective activity against cytotoxicity induced by ischemia, reducing neuronal damage (Weinstock et al., 2001; Youdim and Buccafusco, 2005). It has been suggested that this drug acts in APP processing, based on research on rat PC12 and human SH-SY5Y neuroblastoma cells (Youdim and Buccafusco, 2005). It will be of great interest to investigate whether affected AD patients respond differently to potential MAOA inhibitors according to their MAOA genotypes. Acknowledgments We are extremely grateful for the participation and understanding of the patients and controls. We also thank Dr. Maria Rita Passos-Bueno and her group, Dr. Sergio Matioli, Antonia Maria de Cerqueira, and Constância Urbani for their invaluable help. This work was supported by FAPESP- Cepid and CNPq. References Collier D. A., Stober G., Li T., Heils A., Catalano M., Di Bella D., et al. (1996) A novel functional polymorphism within the promoter of the serotonin transporter gene: possible role in susceptibility to affective disorders. Mol. Psychiatry 1, Deckert J., Catalano M., Syagailo Y. V., Bosi M., Okladnova O., Di Bella D., et al. (1999) Excess of high activity monoamine oxidase A gene promoter alleles in female patients with panic disorder. Hum. Mol. Genet. 8, Farrer L. A., Friedland R. P., Bowirrat A., Waraska K., Korczyn A., and Baldwin C. T. (2003) Genetic and environmental epidemiology of Alzheimer s disease in Arabs residing in Israel. J. Mol. Neurosci. 20, Furlong R. A., Ho L., Rubinsztein J. S., Walsh C., Paykel E. S., and Rubinsztein D. C. (1999) Analysis of the monoamine oxidase A(MAOA) gene in bipolar affective disorder by association studies, meta-analyses, and sequencing of the promoter. Am. J. Med. Genet. 88, Gutierrez B., Arias B., Gasto C., Catalan R., Papiol S., Pintor L., and Fananas L. (2004) Association analysis between a functional polymorphism in the monoamine oxidase A gene promoter and severe mood disorders. Psychiatr. Genet. 14, Koller G., Bondy B., Preuss U. W., Bottlender M., and Soyka M. (2003) No association between a polymorphism in the promoter region of the MAOA gene with antiso-

6 MAOA and AD in Brazilian Population 217 cial personality traits in alcoholics. Alcohol Alcohol 38, Kunugi H., Ishida S., Kato T., Tatsumi M., Sakai T., Hattori M., et al. (1999) A functional polymorphism in the promoter region of monoamine oxidase-agene and mood disorders. Mol. Psychiatry 4, Kunugi H., Ueki A., Otsuka M., Isse K., Hirasawa H., Kato N., et al. (2000) Alzheimer s disease and 5-HTTLPR polymorphism of the serotonin transporter gene: no evidence for an association. Am. J. Med. Genet. 96, Lesch K. P., Bengel D., Heils A., Sabol S. Z., Greenberg B. D., Petri S., et al. (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, Li T., Holmes C., Sham P. C., Vallada H., Birkett J., Kirov G., et al. (1997) Allelic functional variation of serotonin transporter expression is a susceptibility factor for late onset Alzheimer s disease. Neuroreport 8, Luchsinger J. A. and Mayeux R. (2004) Dietary factors and Alzheimer s disease. Lancet Neurol. 3, Oliveira J. R., Gallindo R. M., Maia L. G., Brito-Marques P. R., Otto P. A., Passos-Bueno M. R., et al. (1998) The short variant of the polymorphism within the promoter region of the serotonin transporter gene is a risk factor for late onset Alzheimer s disease. Mol. Psychiatry 3, Riederer P., Danielczyk W., and Grunblatt E. (2004) Monoamine oxidase-b inhibition in Alzheimer s disease. Neurotoxicology 25, Sabol S. Z. Hu S., and Hamer D. (1998) A functional polymorphism in the monoamine oxidase Agene promoter. Hum. Genet. 103, Samochowiec J., Lesch K. P., Rottmann M., Smolka M., Syagailo Y. V., Okladnova O., et al. (1999) Association of a regulatory polymorphism in the promoter region of the monoamine oxidase A gene with antisocial alcoholism. Psychiatry Res. 86, Schuback D. E., Mulligan E. L., Sims K. B., Tivol E. A., Greenberg B. D., Chang S. F., et al. (1999) Screen for MAOA mutations in target human groups. Am. J. Med. Genet. 88, Tsai S. J., Hong C. J., Liu T. Y., Cheng C. Y., and Liu H. C. (2001) Association study for a functional serotonin transporter gene polymorphism and late-onset Alzheimer s disease for Chinese patients. Neuropsychobiology 44, Weinstock M., Kirschbaum-Slager N., Lazarovici P., Bejar C., Youdim M. B., and Shoham S. (2001) Neuroprotective effects of novel cholinesterase inhibitors derived from rasagiline as potential anti-alzheimer drugs. Ann. N. Y. Acad. Sci. 939, Wenham P. R., Price W. H., and Blandell G. (1991) Apolipoprotein E genotyping by one-stage PCR. Lancet 337, Youdim M. B. and Buccafusco J. J. (2005) CNS Targets for multi-functional drugs in the treatment of Alzheimer s and Parkinson s diseases. J. Neural Transm. 112, Youdim M. B. and Weinstock M. (2004) Therapeutic applications of selective and non-selective inhibitors of monoamine oxidase A and B that do not cause significant tyramine potentiation. Neurotoxicology 25,

specific alleles in a2 [OR = 0.65 ( ), P = 0.009], a5 [OR = 1.44 ( ), P = 0.03], and a6 [OR = 1.41 ( ), P = 0.004].

specific alleles in a2 [OR = 0.65 ( ), P = 0.009], a5 [OR = 1.44 ( ), P = 0.03], and a6 [OR = 1.41 ( ), P = 0.004]. Original article 1 Meta-analysis of the association between the monoamine oxidase-a gene and mood disorders Ming Fan, Bing Liu, Tianzi Jiang, Xingpeng Jiang, Huizhi Zhao and Jing Zhang Objective To evaluate