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P.5.b.004 Methylenetetrahydrofolate reductase c.677C>T polymorphism and risk of Alzheimer's disease in Koreans
P.5.b Dementia and neurological disorders – Dementia disorders (basic) learning session and on memory test compared to the respective day of rats with Scop only. There were no differences in the number of escapes, nor in the number of intertrial crossings in all groups studied. In the step-through passive avoidance test controls increased the latency of reactions (P < 0.05) on learning session and on memory retention tests compared to the first day of learning. The group with Scop decrease the latency of reactions (P < 0.05) in learning as well as in long memory retention tests compared to the controls. Rats treated with Riv increased the latency of reactions (P < 0.05) in memory retention tests compared to the group with Scop only. In step-down passive avoidance rats with Scop-induced amnesia did not change the latency of reactions during learning or in memory retention tests compared to the controls. Groups treated with Scop and Riv increased the latency (P < 0.05) on learning as well as on memory retention tests compared to the respective days of Scop group. The group with Scop decreased (P < 0.05) horizontal and vertical activity compared to the controls. Rats treated with Scop and Riv increased (P < 0.05) horizontal and vertical movements compared to the group with Scop only. Conclusion: Our results permitted us to suggest that Scop impaired learning and memory processes and cholinesterase inhibitor Riv is able to abolish its amnesic effects. References [1] Enz A, Gentsch C, 2004, Co-administration of memantine has no effect on the in vitro or ex vivo determined acetylcholinesterase inhibition of rivastigmine in the rat brain. Neuropharmacology 47, 408–413. [2] Bejar C, Wang RH, Weinstock M, 1999, Effect of rivastigmine on scopolamine-induced memory impairment in rats. Eur J Pharmacol 383, 231–240.
P.5.b.003 Alpha7 nicotinic acetylcholine receptor and apolipoprotein E polymorphisms in Alzheimer’s dementia A. Juh´asz1 ° , A. Riman´oczy1 , A. Feh´er1 , J. K´alm´an1 , M. G´alfi2 , Z. Janka1 . 1 University of Szeged, Department of Psychiatry Faculty of Medicine, Szeged, Hungary; 2 University of Szeged, Department of Biology Faculty of Juh´asz Gyula Teachers Training College, Szeged, Hungary Background: Alzheimer’s disease (AD) is the most common type of dementia characterized by a progressive decline of cognitive functions. Acetylcholine is a major neurotransmitter involved in cognitive functions. The neuronal nicotinic acetylcholine receptor alpha7 (alpha7nAChR) may be involved in cognitive deficits in AD. Amyloid beta (1−42) is a major component of amyloid plaques that binds with high affinity to the alpha7nAChR. The alpha7 subunit modulates the calcium homeostasis and acetylcholine release processes. The human alpha7nAChR gene is found in chromosome 15q13-q14 region. It is known, that the apolipoprotein E (ApoE) 4 allele is a risk factor for AD. In the current study we determined the distribution of partially duplicated alpha7nAChR and ApoE polymorphism in AD and control populations. The aim of this study was to investigate whether the partially duplicated alpha7nAChR polymorphism can be associated with AD and to evaluate the relationship between partially duplicated alpha7nAChR and ApoE polymorphisms in AD. Subjects and Methods: One hundred and twenty one control and 115 AD probands were included in this study. The diagnosis of probable AD was based on NINCDS-ADRDA criteria [1].
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Genomic DNA was extracted from whole blood samples by Roche kit. The DNA was amplified by PCR. The PCR procedure for alpha7nAChR was performed as previously described [2]. In the case of ApoE, products of the amplifications were digested with CfoI. The fragments were revealed by 8% acrylamide/bis acrylamide gel in the presence of ethidium bromide under UV light. Genotype and allele frequencies in the AD and control subjects were compared by statistical analyses using the c2 test. Results: There were statistically significant differences in the alpha7nAChR wild (71 bp) genotype distribution comparing the control and AD groups (genotype frequencies: Control: 24.8%, AD: 36.5%, df = 2, p = 0.04). In the case of ApoE polymorphism the 3/4 and 4/4 genotypes were found to be more frequent in the AD than in the control group (ApoE 3/4 genotype: Control: 9.1%, AD: 34.8%, ApoE 4/4 genotype: Control: 0.8%, AD: 7.0%). The ApoE 4 allele carriers occurred in significantly higher percentage in the AD than in the control group (ApoE 4 allele: Control: 5.8%, AD: 24.8%, df: 2, p < 0.005). Comparing the alpha7nAChR wild genotype distribution in the presence of ApoE 4 allele, there was no statistically significant difference between the two groups (Control: 41.7%, AD: 34.7%). In the absence of ApoE 4 allele, the alpha7nAChR wild genotype distribution in the control group was 22.9%, and in the AD group was 37.9%. Conclusion: Our data reveal that the polymorphisms of ApoE and partially duplicated alpha7nAChR may be implicated in the susceptibility to AD. There was no association between alpha7nAChR wild genotype and the ApoE 4 carrier occurrence in AD. This work was supported by grants from the Hungarian Ministry of Health (ETT 198 04/ 2006) and the Hungarian Scientific Research Fund (OTKA T 046152/2004 and OTKA K 60589/2006). References [1] McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM, 1984, Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology 34, 939–944. [2] Lai IC, Hong CJ, Tsai SJ, 2001, Association study of a nicotinic receptor variant with schizophrenic disorders. Neuropsychobiology 43, 15−18.
P.5.b.004 Methylenetetrahydrofolate reductase c.677C>T polymorphism and risk of Alzheimer’s disease in Koreans J.M. Kim ° , S.W. Kim, S.J. Yang, I.S. Shin, S.Y. Lee, J.S. Yoon. Chonnam National University Hospital, Psychiatry, Gwangju, South-Korea Purpose: The association between methylenetetrahydrofolate reductase (MTHFR) c.677C>T (A222V) polymorphism and Alzheimer’s disease (AD) is controversial. The objectives of the study were to investigate the association between MTHFR c.677C>T polymorphism and AD in Korean elders and to the extent to which it is modified by the major components of onecarbon metabolism and apolipoprotein E (APOE) genotype. Methods: 732 community residents aged 65 or over were clinically assessed for AD. In order to ascertain dementia and AD, interview measures included the Korean version of the MiniMental State Examination, the Instrumental Activities of Daily Living Scale, and the Clinical Dementia Rating scale. Information on past history and present illness, and family history of dementia was gathered from participants and their family members. A
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P.5.b Dementia and neurological disorders – Dementia disorders (basic)
physical examination was carried out, including blood pressure measurement and neurologic examination. Genotyping was performed for MTHFR c.677C>T and APOE; serum levels of folate, vitamin B12, and homocysteine were assayed. Blood samples were collected in a fasting state and were carried out in the morning where possible. They were drawn into EDTA tubes, and centrifuged, separated into plasma aliquots, and stored at −70ºC within 2 hours of collection. Biochemical assays were carried out after three years. Serum folate and vitamin B12 levels were determined using an immunoassay, and total plasma homocysteine level was measured by high-performance liquid chromatography. MTHFR c.677C>T genotype was determined by a polymerase chain reaction (PCR) and HinFI restriction enzyme digestion. Age, gender and education were included as covariates. Results: In terms of the associations between MTHFR c.677C>T polymorphism and AD, AD was significantly increased with a greater number of T allele and significantly higher in those with TT genotype compared to CC genotype [OR (95% CI): 2.08 (1.01−4.28)] in the univariate analyses. However after adjustment for demographic factors, the associations were reduced in strength [OR (95% CI): 1.73 (0.80−3.74)]. The association between AD and increased number of T allele was significant in the presence of lower vitamin B12 level and in APOE e4 non-carriers (pvalues for linear trend = 0.009 and 0.043, respectively), and bordered significance in the presence of higher homocysteine level (p-value for linear trend = 0.078). Statistical interaction terms reached significance for vitamin B12 and APOE status (p-values for interaction = 0.024 and 0.035, respectively) and bordered on significance for homocysteine level (p-value for interaction = 0.082). Conclusions: These findings suggest gene–environment and gene–gene interactions on the risk of AD in Koreans. Analyses described here were, in part, exploratory and clearly require replication and confirmation. As discussed above, this may be best achieved through research in other Asian populations where a potential effect on AD may be greatest. The high T allele frequency of MTHFR c.677C>T (0.55−0.57) in Koreans, may indicate some public health relevance for this gene as a risk factor for AD. The relatively high prevalence of AD in Koreans compared to other ethnic populations might be explained, at least in part, by high T allele frequency of MTHFR c.677C>T. Further potential public health importance is derived from the fact that, although the genotype is innate, modifying nutritional factors are themselves potentially modifiable through measures such as vitamin supplementation and exercise. Further observational and experimental research is indicated. References [1] Kim JM, Stewart R, Shin IS, Jung JS, Yoon JS, 2004, Assessment of association between mitochondrial aldehyde dehydrogenase polymorphism and Alzheimer’s disease in an older Korean population. Neurobiol Aging 25, 295–301. [2] Nishiyama M, Kato Y, Hashimoto M, Yukawa S, Omori K, 2000, Apolipoprotein E, methylenetetrahydrofolate reductase (MTHFR) mutation and the risk of senile dementia – an epidemiological study using the polymerase chain reaction (PCR) method. J Epidemiol 10, 163−72.
P.5.b.005 Vesicular glutamate transporters in pathologies of the central nervous system E. Lepicard1 ° , O. Poirel1 , A. Kashani1 , B. Amilhon1 , E. Hirsch2 , J. Epelbaum3 , J.P. David4 , J. Delacourte5 , B. Giros1 , C. Betancur1 , S. El Mestikawy1 . 1 INSERM U513, Neurobiologie et Psychiatrie,
Creteil, France; 2 INSERM U679, CHU Piti´e, Salpˆetri`ere, Paris, France; 3 INSERM U549, Centre Broca, Paris, France; 4 INSERM U422, Bˆat. INSERM G´erard Biserte, Lille, France; 5 G´erontologie 3, Hˆopital Emile-Roux, Limeil-Br´evannes, France Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS). Before its release, glutamate is accumulated into synaptic vesicle by three recently identified transporters (named VGLUT1−3). VGLUT1 and VGLUT2, which are expressed by “classical” glutamatergic neurons, have a complementary distribution in the CNS. VGLUT3 is expressed within cholinergic interneurons from the striatum and the accumbens, serotoninergic neurons from the raphe nucleus and in a subpopulation of GABAergic interneurons of the cerebral cortex and the hippocampus. These transporters are functional and anatomical markers of the glutamatergic transmission. Numerous data of the literature suggest the implication of glutamatergic systems in Parkinson disease (PD) and Alzheimer disease (AD), as well as in several psychiatric disorders. To better understand the involvement of excitatory transmission in these diseases, we developed specific antiserums to carry out a quantitative and qualitative study of the VGLUTs expression in human post-mortem brain. First, we quantified VGLUT1&2 in different brain areas of controls or patients with PD using western blot and immunoautoradiography. We found a highly significant 24% increase (p < 0.001) in the putamen and a 50% decrease of VGLUT1 expression in the prefrontal and temporal cortex of PD patients (p < 0.01 and p < 0.001 respectively). VGLUT2 expression was increased in the putamen by 29% in PD patients (p < 0.01). Thus changes in glutamatergic transmission occurring in PD are underlied by profound alterations of both glutamatergic systems. Furthermore, these alterations are structure – as well as system-dependent. Second, VGLUT1&2 expression was measured in the prefrontal cortex (A9 area) of 17 individuals at different stages of AD using western blot and immunoautoradiography. A dramatic decrease of VGLUT1 and VGLUT2 expression (51%, p < 0.001 and 57%, p < 0.01) was observed in the prefrontal cortex (A9) of patients with AD. Moreover, VGLUT1 decrease was highly correlated to the cognitive status of the patients (Clinical Dementia Rating scale, CDR, r2= 0.81, p < 0.0001). The progressive reduction of VGLUT1 expression and its high correlation to CDR suggest that this marker could be used as a biological index of dementia. To conclude, we have unraveled major dysregulations of VGLUT1 and VGLUT2 systems in brain of patients with PD or AD. These data suggest that synaptic alterations of glutamatergic pathways in the CNS may lead to pathological processes such as cognitive and executive deficits. In this context, the recently discovered vesicular glutamate transporters are invaluable functional biomarkers of glutamatergic transmission. They may be useful to gain insight into the neurobiological processes underlying symptoms of both neurological and psychiatric pathologies. This work was supported by F´ed´eration pour la Recherche sur le Cerveau (FRC), Association France Parkinson and Association France Alzheimer.
P.5.b.003 Alpha7 nicotinic acetylcholine receptor and apolipoprotein E polymorphisms in Alzheimer’s dementia A. Juh´asz1 ° , A. Riman´oczy1 , A. Feh´er1 , J. K´alm´an1 , M. G´alfi2 , Z. Janka1 . 1 University of Szeged, Department of Psychiatry Faculty of Medicine, Szeged, Hungary; 2 University of Szeged, Department of Biology Faculty of Juh´asz Gyula Teachers Training College, Szeged, Hungary Background: Alzheimer’s disease (AD) is the most common type of dementia characterized by a progressive decline of cognitive functions. Acetylcholine is a major neurotransmitter involved in cognitive functions. The neuronal nicotinic acetylcholine receptor alpha7 (alpha7nAChR) may be involved in cognitive deficits in AD. Amyloid beta (1−42) is a major component of amyloid plaques that binds with high affinity to the alpha7nAChR. The alpha7 subunit modulates the calcium homeostasis and acetylcholine release processes. The human alpha7nAChR gene is found in chromosome 15q13-q14 region. It is known, that the apolipoprotein E (ApoE) 4 allele is a risk factor for AD. In the current study we determined the distribution of partially duplicated alpha7nAChR and ApoE polymorphism in AD and control populations. The aim of this study was to investigate whether the partially duplicated alpha7nAChR polymorphism can be associated with AD and to evaluate the relationship between partially duplicated alpha7nAChR and ApoE polymorphisms in AD. Subjects and Methods: One hundred and twenty one control and 115 AD probands were included in this study. The diagnosis of probable AD was based on NINCDS-ADRDA criteria [1].
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Genomic DNA was extracted from whole blood samples by Roche kit. The DNA was amplified by PCR. The PCR procedure for alpha7nAChR was performed as previously described [2]. In the case of ApoE, products of the amplifications were digested with CfoI. The fragments were revealed by 8% acrylamide/bis acrylamide gel in the presence of ethidium bromide under UV light. Genotype and allele frequencies in the AD and control subjects were compared by statistical analyses using the c2 test. Results: There were statistically significant differences in the alpha7nAChR wild (71 bp) genotype distribution comparing the control and AD groups (genotype frequencies: Control: 24.8%, AD: 36.5%, df = 2, p = 0.04). In the case of ApoE polymorphism the 3/4 and 4/4 genotypes were found to be more frequent in the AD than in the control group (ApoE 3/4 genotype: Control: 9.1%, AD: 34.8%, ApoE 4/4 genotype: Control: 0.8%, AD: 7.0%). The ApoE 4 allele carriers occurred in significantly higher percentage in the AD than in the control group (ApoE 4 allele: Control: 5.8%, AD: 24.8%, df: 2, p < 0.005). Comparing the alpha7nAChR wild genotype distribution in the presence of ApoE 4 allele, there was no statistically significant difference between the two groups (Control: 41.7%, AD: 34.7%). In the absence of ApoE 4 allele, the alpha7nAChR wild genotype distribution in the control group was 22.9%, and in the AD group was 37.9%. Conclusion: Our data reveal that the polymorphisms of ApoE and partially duplicated alpha7nAChR may be implicated in the susceptibility to AD. There was no association between alpha7nAChR wild genotype and the ApoE 4 carrier occurrence in AD. This work was supported by grants from the Hungarian Ministry of Health (ETT 198 04/ 2006) and the Hungarian Scientific Research Fund (OTKA T 046152/2004 and OTKA K 60589/2006). References [1] McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM, 1984, Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology 34, 939–944. [2] Lai IC, Hong CJ, Tsai SJ, 2001, Association study of a nicotinic receptor variant with schizophrenic disorders. Neuropsychobiology 43, 15−18.
P.5.b.004 Methylenetetrahydrofolate reductase c.677C>T polymorphism and risk of Alzheimer’s disease in Koreans J.M. Kim ° , S.W. Kim, S.J. Yang, I.S. Shin, S.Y. Lee, J.S. Yoon. Chonnam National University Hospital, Psychiatry, Gwangju, South-Korea Purpose: The association between methylenetetrahydrofolate reductase (MTHFR) c.677C>T (A222V) polymorphism and Alzheimer’s disease (AD) is controversial. The objectives of the study were to investigate the association between MTHFR c.677C>T polymorphism and AD in Korean elders and to the extent to which it is modified by the major components of onecarbon metabolism and apolipoprotein E (APOE) genotype. Methods: 732 community residents aged 65 or over were clinically assessed for AD. In order to ascertain dementia and AD, interview measures included the Korean version of the MiniMental State Examination, the Instrumental Activities of Daily Living Scale, and the Clinical Dementia Rating scale. Information on past history and present illness, and family history of dementia was gathered from participants and their family members. A
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P.5.b Dementia and neurological disorders – Dementia disorders (basic)
physical examination was carried out, including blood pressure measurement and neurologic examination. Genotyping was performed for MTHFR c.677C>T and APOE; serum levels of folate, vitamin B12, and homocysteine were assayed. Blood samples were collected in a fasting state and were carried out in the morning where possible. They were drawn into EDTA tubes, and centrifuged, separated into plasma aliquots, and stored at −70ºC within 2 hours of collection. Biochemical assays were carried out after three years. Serum folate and vitamin B12 levels were determined using an immunoassay, and total plasma homocysteine level was measured by high-performance liquid chromatography. MTHFR c.677C>T genotype was determined by a polymerase chain reaction (PCR) and HinFI restriction enzyme digestion. Age, gender and education were included as covariates. Results: In terms of the associations between MTHFR c.677C>T polymorphism and AD, AD was significantly increased with a greater number of T allele and significantly higher in those with TT genotype compared to CC genotype [OR (95% CI): 2.08 (1.01−4.28)] in the univariate analyses. However after adjustment for demographic factors, the associations were reduced in strength [OR (95% CI): 1.73 (0.80−3.74)]. The association between AD and increased number of T allele was significant in the presence of lower vitamin B12 level and in APOE e4 non-carriers (pvalues for linear trend = 0.009 and 0.043, respectively), and bordered significance in the presence of higher homocysteine level (p-value for linear trend = 0.078). Statistical interaction terms reached significance for vitamin B12 and APOE status (p-values for interaction = 0.024 and 0.035, respectively) and bordered on significance for homocysteine level (p-value for interaction = 0.082). Conclusions: These findings suggest gene–environment and gene–gene interactions on the risk of AD in Koreans. Analyses described here were, in part, exploratory and clearly require replication and confirmation. As discussed above, this may be best achieved through research in other Asian populations where a potential effect on AD may be greatest. The high T allele frequency of MTHFR c.677C>T (0.55−0.57) in Koreans, may indicate some public health relevance for this gene as a risk factor for AD. The relatively high prevalence of AD in Koreans compared to other ethnic populations might be explained, at least in part, by high T allele frequency of MTHFR c.677C>T. Further potential public health importance is derived from the fact that, although the genotype is innate, modifying nutritional factors are themselves potentially modifiable through measures such as vitamin supplementation and exercise. Further observational and experimental research is indicated. References [1] Kim JM, Stewart R, Shin IS, Jung JS, Yoon JS, 2004, Assessment of association between mitochondrial aldehyde dehydrogenase polymorphism and Alzheimer’s disease in an older Korean population. Neurobiol Aging 25, 295–301. [2] Nishiyama M, Kato Y, Hashimoto M, Yukawa S, Omori K, 2000, Apolipoprotein E, methylenetetrahydrofolate reductase (MTHFR) mutation and the risk of senile dementia – an epidemiological study using the polymerase chain reaction (PCR) method. J Epidemiol 10, 163−72.
P.5.b.005 Vesicular glutamate transporters in pathologies of the central nervous system E. Lepicard1 ° , O. Poirel1 , A. Kashani1 , B. Amilhon1 , E. Hirsch2 , J. Epelbaum3 , J.P. David4 , J. Delacourte5 , B. Giros1 , C. Betancur1 , S. El Mestikawy1 . 1 INSERM U513, Neurobiologie et Psychiatrie,
Creteil, France; 2 INSERM U679, CHU Piti´e, Salpˆetri`ere, Paris, France; 3 INSERM U549, Centre Broca, Paris, France; 4 INSERM U422, Bˆat. INSERM G´erard Biserte, Lille, France; 5 G´erontologie 3, Hˆopital Emile-Roux, Limeil-Br´evannes, France Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS). Before its release, glutamate is accumulated into synaptic vesicle by three recently identified transporters (named VGLUT1−3). VGLUT1 and VGLUT2, which are expressed by “classical” glutamatergic neurons, have a complementary distribution in the CNS. VGLUT3 is expressed within cholinergic interneurons from the striatum and the accumbens, serotoninergic neurons from the raphe nucleus and in a subpopulation of GABAergic interneurons of the cerebral cortex and the hippocampus. These transporters are functional and anatomical markers of the glutamatergic transmission. Numerous data of the literature suggest the implication of glutamatergic systems in Parkinson disease (PD) and Alzheimer disease (AD), as well as in several psychiatric disorders. To better understand the involvement of excitatory transmission in these diseases, we developed specific antiserums to carry out a quantitative and qualitative study of the VGLUTs expression in human post-mortem brain. First, we quantified VGLUT1&2 in different brain areas of controls or patients with PD using western blot and immunoautoradiography. We found a highly significant 24% increase (p < 0.001) in the putamen and a 50% decrease of VGLUT1 expression in the prefrontal and temporal cortex of PD patients (p < 0.01 and p < 0.001 respectively). VGLUT2 expression was increased in the putamen by 29% in PD patients (p < 0.01). Thus changes in glutamatergic transmission occurring in PD are underlied by profound alterations of both glutamatergic systems. Furthermore, these alterations are structure – as well as system-dependent. Second, VGLUT1&2 expression was measured in the prefrontal cortex (A9 area) of 17 individuals at different stages of AD using western blot and immunoautoradiography. A dramatic decrease of VGLUT1 and VGLUT2 expression (51%, p < 0.001 and 57%, p < 0.01) was observed in the prefrontal cortex (A9) of patients with AD. Moreover, VGLUT1 decrease was highly correlated to the cognitive status of the patients (Clinical Dementia Rating scale, CDR, r2= 0.81, p < 0.0001). The progressive reduction of VGLUT1 expression and its high correlation to CDR suggest that this marker could be used as a biological index of dementia. To conclude, we have unraveled major dysregulations of VGLUT1 and VGLUT2 systems in brain of patients with PD or AD. These data suggest that synaptic alterations of glutamatergic pathways in the CNS may lead to pathological processes such as cognitive and executive deficits. In this context, the recently discovered vesicular glutamate transporters are invaluable functional biomarkers of glutamatergic transmission. They may be useful to gain insight into the neurobiological processes underlying symptoms of both neurological and psychiatric pathologies. This work was supported by F´ed´eration pour la Recherche sur le Cerveau (FRC), Association France Parkinson and Association France Alzheimer.