Eloy Rodríguez

LRRK2 G2019S is a common mutation in Spanish patients with late-onset Parkinson's disease

Mutations in the leucine-rich repat kinase 2 (LRRK2) gene have been shown to cause both autosomal dominant and sporadic Parkinsons disease (PD). The common G2019S mutation shows wide geographical distribution while R1441G has been only reported in Northern Spain. The overall frequency of these mutations remains to be established. To determine the prevalence of G2019S and R1441G mutations in our population of Cantabria (Northern Spain), we recruited 105 consecutive PD patients and 310 controls and conducted genetic analysis of these mutations. G2019S was detected in eight late-onset patients (7.6%). Five of them had no relevant family history. R1441G was not detected in any of our study subjects. The prevalence of G2019S mutation in unselected late-onset PD patients might be higher than previously reported: 3/16 (18.7%) of familial PD and 5/82 (6.1%) of sporadic PD.

Association between Glycogen Synthase Kinase-3β Genetic Polymorphism and Late-Onset Alzheimer’s Disease

Aberrant phosphorylated tau is the major component of the neurofibrillary tangles in Alzheimer’s disease (AD) brains. Glycogen synthase kinase-3β (GSK-3β) phosphorylates tau protein, and increased GSK-3β expression has been associated with neurofibrillary tangles. Saitohin (STH) is a recently identified protein that shares tissue expression pattern with tau, and previous evidence in the Spanish population indicated that a polymorphism at codon 7 (Q7R) of the STH gene was associated with late-onset AD. Since both GSK-3β and STH are related to tau, we examined the association between a polymorphism in the promoter region (–50) of the GSK-3β gene and AD, either through an independent effect or through interaction with the STH (Q7R) polymorphism, in a well-defined group of 333 sporadic AD patients and 307 control subjects from Spain. The current study reveals that GSK-3β (–50) TT genotype is associated with an increased risk (OR 1.99, p = 0.003) for late-onset (after the age of 72 years) AD. Our results indicate that both the GSK-3β (–50) and STH (Q7R) polymorphisms increase the risk of late-onset (subjects >72 years) AD, although they appear to be independent and thus not to interact synergistically.

Cholesteryl ester transfer protein (CETP) polymorphism modifies the Alzheimer's disease risk associated with APOE ε4 allele

Cholesterol regulates the production of amyloid beta (Aβ), which is central to the pathogenesis of Alzheimers disease (AD), with high cellular cholesterol promoting and low cellular cholesterol reducing Aβ in vitro and in vivo. High density lipoprotein (HDL) plays a central role in the removal of excess cholesterol from cells, and cholesteryl ester transfer protein (CETP) is a crucial protein involved in the regulation of HDL levels. Two common polymorphisms in the promoter region (C–629A) and exon 14 I405V of the CETP gene are associated with CETP activity and HDL levels. To investigate if these sequence variants in CETP might be of importance in mediating susceptibility to AD, independently or in concert with apolipoprotein E (APOE) ε4 allele, we studied a sample of 286 Spanish AD patients and 315 healthy controls. In APOE ε4 carriers, homozygous for the CETP (–629) A allele had approximately a three times lower risk of developing AD (odds ratio 2.33, 95% CI 1.01–5.37), than homozygous and heterozygous carriers of the CETP (–629) C allele (odds ratio 7.12, 95% CI 4.51–11.24, P for APOE ε4/CETP (–629) AA genotype interaction < 0.001). Our data suggest that CETP behaves as a modifier gene of the AD risk associated with the APOE ε4 allele, possibly through modulation of brain cholesterol metabolism.

CD14 receptor polymorphism and Alzheimer's disease risk

Activation of microglial cells is involved in the inflammatory component of Alzheimers disease (AD), and it may be triggered by infectious pathogens. CD14, a receptor upregulated in activated microglia, plays a central role in innate immunity through recognition of bacterial lipopolysaccharide and initiation of inflammatory response. A polymorphism in the promoter region (-260) of the CD14 receptor has been found to be related to increased risk of bacterial infections and inflammatory diseases such as atherosclerosis. In a case-control study utilizing a clinically well-defined group of 310 sporadic AD patients and 310 control subjects, we investigated whether the CD14 (-260) polymorphism might be responsible for susceptibility to AD, and we also examined the combined gene effects between CD14 and APOE and several other proinflammatory cytokine genes. The current study does not demonstrate an association between CD14 (-260) polymorphism and AD, neither through an independent effect nor through interaction with APOE epsilon4 allele or interleukin (IL)-1A, IL-6, IL-8, tumor necrosis factor (TNF)-alpha, and intercellular adhesion molecule-1 polymorphisms.

Interaction between dopamine β-hydroxylase and interleukin genes increases Alzheimer’s disease risk

Healy and colleagues1 recently reported that dopamine β-hydroxylase (DBH) activity could mediate predisposition to Parkinson’s disease through its role in catalysing the conversion of dopamine to noradrenaline (norepinephrine). By studying a promoter variant (−1021 C/T) in the DBH gene which has been shown to influence plasma DBH activity, they showed that homozygosity for the low DBH expressing T allele was protective against Parkinson’s disease, and proposed that lower levels of DBH protein might lead to increased ratios of dopamine to noradrenaline.1 In Alzheimer’s disease, significant reductions in noradrenergic neurones within locus coeruleus, as well as reduced brain noradrenaline levels, have often been reported.2 In addition, there is a causative link between reduced noradrenaline content and the potentiation of β-amyloid (Aβ) induced cortical inflammation.3 We hypothesised that relative noradrenaline deficiency associated with homozygosity for the (−1021) DBH T allele might lead to increased risk for Alzheimer’s disease, either through an independent effect or through interaction with the proinflammatory interleukin (IL) 1A and IL6 genes. The study involved 266 patients …

MicroRNA Profile in Patients with Alzheimer’s Disease: Analysis of miR-9-5p and miR-598 in Raw and Exosome Enriched Cerebrospinal Fluid Samples

BACKGROUND MicroRNAs have been postulated as potential biomarkers for Alzheimers disease (AD). Exosomes are nanovesicles which transport microRNAs, proteins, and other cargos. It has been hypothesized that the exosome traffic might be increased in neurodegenerative disorders. OBJECTIVE i) To assess the cerebrospinal fluid (CSF) microRNA profile in a group of AD patients and control subjects and to validate a group of microRNAs previously reported by other authors. ii) To compare microRNA levels in whole CSF and in the exosome-enriched fraction in AD patients. METHODS A panel of 760 microRNAs was analyzed in the CSF of 10 AD patients and 10 healthy subjects. Among microRNAs differently expressed, we selected those that had been previously reported by other authors. Candidates were validated in a larger group by individual qPCR assays. MicroRNA expression was also evaluated in exosome-enriched CSF samples of patients with AD and controls. RESULTS Fifteen microRNAs were differently expressed in AD. MiR-9-5p, miR-134, and miR-598 were selected as candidates for further analysis. MiR-9-5p and miR-598 were detected in 50 and 75% of control CSF samples, respectively, while they were not detected in any AD CSF samples. We observed an opposite pattern when we evaluated the microRNA expression in the exosome-enriched CSF AD samples. No pattern variations were noted among healthy subjects. CONCLUSION These data propose miR-9-5p and miR-598 as potential biomarkers for AD. Further studies in plasma and other body fluids will confirm their potential role as easily accessible biomarkers. In addition, our data suggest that exosome trafficking is different between AD and control subjects raising the need to take this phenomenon into consideration in future studies of AD biomarkers.

Genetic interaction between two apolipoprotein E receptors increases Alzheimer's disease risk

Sirs: Abnormalities in cholesterolrelated genes appear to increase the risk of Alzheimer’s disease (AD) probably by increasing production of β-amyloid (Aβ) [7, 10]. The apolipoprotein E (APOE) ε4 allele is the main recognised genetic risk factor for sporadic AD [8], but other cholesterol-related genes such as members of the APOE receptor family are also associated with AD [13]. The low density lipoprotein receptor-related protein (LRP) is the most studied member of this family, and a polymorphism in exon 3 (C766T) has been shown to confer increased risk for AD, but other studies failed to replicate such an association [5, 6, 9, 12]. The postulated common pathway of LRP and low density lipoprotein receptor (LDLR) in the neuronal uptake of cholesterolapoE complexes [3] prompted us to examine the combined contribution of both genes to the susceptibility for AD. The study included 274 AD patients (68 % women; mean age at the time of study 75.4 years; SD 8.7; range 50–97 years; mean age at onset 71.6 years; SD 8.6; range 48–94 years) who met NINCDS/ADRDA criteria for probable AD. All AD cases were defined as sporadic because their family history did not mention any first-degree relative with dementia; this information was obtained by direct interviews with relatives. Control subjects consisted of 283 unrelated individuals (71 % women; mean age 80.5 years; SD 7.9; range 63–100) who had complete neurological and medical examinations establishing that they were free of significant illness and all had Mini Mental State Examination scores of 28 or more. Control subjects were randomly selected from a nursing home. The AD and control samples were Caucasians originating from a homogeneous population in a limited geographical area in Northern Spain. All patients and control subjects were ascertained to have parents and grandparents born in Northern Spain to ensure ethnicity; consequently, possible confounding effects of the inclusion in the study of members of different ethnic groups have been minimized. The LRP exon 3 and LDLR exon 8 polymorphisms were determined as described previously [1, 12]. The study was approved by the ethical committee of the University Hospital “Marqués de Valdecilla”. Association between dichotomous variables was analyzed with odds ratio, and 95 % confidence intervals were estimated by the Cornfield method or the exact method. P-values were estimated by chi-square or Fisher exact tests. Interrelations were analyzed by stratification. The distributions of the LRP exon 3 (P = 0.576) and LDLR exon 8 (P = 0.364) genotypes were in Hardy-Weinberg equilibrium. Neither the presence of the LRP exon 3 allele T (C/T and T/T genotypes) (OR 1.39, 95 % CI 0.92–2.10, P = 0.118), nor the presence of the LDLR exon 8 allele A (G/A and A/A genotypes) (OR 0.96, 95 % CI 0.55–1.66, P = 0.877) was associated with AD. However, evaluating the interactive effects of both polymorphisms by stratification (Table 1), the subjects carrying both the LRP exon 3 allele T and the LDLR exon 8 allele A had about a seven times higher risk of developing AD than subjects without these risk genotypes (OR 7.37, 95 % CI 1.47–37.04, P = 0.015), suggesting a gene-gene interaction; the interaction between the two loci remained significant after adjustment for multiple comparisons (P = 0.045). Our analysis showed the expected association between the APOE ε4 allele and AD with an odds ratio of 4.94 (P < 0.001) for carrying one or two copies of ε4. Stratification of the data by APOE genotypes (Table 1) indicated that the risk effect of the interaction of the LRP exon 3 allele T and the LDLR exon 8 allele A was present in the APOE ε4 negative group, while the interaction could not be investigated in the APOE ε4 carriers owing to the low number of subjects in the control group. Glial cells synthesize de novo and secrete cholesterol bound to apoE forming high density lipoprotein complexes, and the apoE receptor protein family modulates the internalization of cholesterolapoE complexes into the neuron [2]. Increases in intracellular cholesterol concentrations lead to stimulation of βand γ-secretase activities and increased Aβ production [7, 10]. It is tempting to speculate that upregulation of LRP and LDLR would cause a massive cellular influx of cholesterol-apoE complexes, induction of Aβ production and increased risk of AD. Several conflicting studies have reported on an association between a polymorphism in exon 3 (C766T) of LRP and AD, some showing positive association with the C allele [6, 9, 12], one showing a positive association with the T allele [5], LETTER TO THE EDITORS

No association between low density lipoprotein receptor genetic variants and Alzheimer's disease risk†

A number of studies suggest that brain cholesterol metabolism may play a role in Alzheimers disease (AD) development, probably through modulation of amyloid beta production. The discovery that apolipoprotein E (APOE) ε4 allele is a risk factor for sporadic AD raises the possibility that the receptors to which APOE binds on the surface of neurons are also involved in the neurodegenerative process. To evaluate the relationship between low density lipoprotein receptor (LDLR) genetic variant and AD, independently or in concert with the APOE ε4 allele, we examined three LDLR polymorphisms located in exons 8 (rs 11669576), 10 (rs 5930), and 13 (rs 5925), in a large group of 322 Spanish AD patients and 314 controls. The current study does not demonstrate an association between LDLR genotypes or haplotypes and AD, neither in the total sample nor when the populations were stratified for the presence or absence of the APOE ε4 allele.

Interaction between prion protein and interleukin-1A genes increases early-onset Alzheimer’s disease risk

Sirs: Individuals homozygous for a common polymorphism (methionine [M]/valine [V]) at codon 129 in the gene (PRNP) encoding the prion protein (PrP) are at increased risk of developing Creutzfeldt-Jakob disease [1] and other neurodegenerative processes including Alzheimer’s disease (AD) [6, 8, 15]. Given the extensive heterogeneity of PRNP 129 polymorphism frequencies across ethnic groups [11], homozygosity at PRNP 129 seems to be a risk factor for AD in some European populations (Dutch [6], German [15] and Polish [8]) but this association was not confirmed by studies in Spanish [3], Italian [2]and Japanese [12] populations. The postulated common pathway of PrP and interleukin-1A (IL-1A) in the cytokine-mediated inflammation occurring in the experimentally-induced prion disease [13, 17], prompted us to investigate the interaction between PRNP 129 and IL-1A ()889) polymorphisms in determining the risk of early-onset and late-onset AD. The study included 258 AD patients (68% women; mean age at the time of study 75.4 years; SD 9.3; range 50–98 ; mean age at onset 71.6 years; SD 9.2; range 48–95 ) who met NINCDS/ADRDA criteria for probable AD. As assessed by Mini Mental State Examination (MMSE), 24% of AD cases had scores between 24 and 18, 38% had scores between 18 and 12, and 38% of patients had scores lower than 12. All AD cases were defined as sporadic because their family history did not mention any first-degree relative with dementia; this information was obtained by direct interviews with relatives. Control subjects consisted of 249 unrelated individuals (70% women; mean age 80.0 years; SD 7.9; range 63–98 years) who had complete neurological and medical examinations establishing that they were free of significant illness and all had MMSE scores of 28 or more. Control subjects were randomly selected from a nursing home, and they arose from the same base population as the cases. Cases and controls were stratified into two age groups: according to the median age at onset of AD patients and age of controls at sampling together (76 years) as a cut-off point, early age at onset was defined as £ 76 years and late age at onset as > 76 years. The AD and control samples were Caucasians originating from a homogeneous population in a limited geographical area in Northern Spain. All patients and control subjects were ascertained to have parents and grandparents born in Northern Spain to ensure ethnicity; consequently, possible confounding effects of the inclusion in the study of members of different ethnic groups have been minimized. Blood samples were taken after written informed consent had been obtained from the subjects or their representatives. The study was approved by the ethical committee of the University Hospital ‘‘Marques de Valdecilla’’. The PRNP 129 and IL-1A ()889) polymorphisms were determined as described previously [3, 4]. Association between dichotomous variables was analyzed with odds ratio, and 95% confidence intervals were estimated by the Cornfield method or the exact method. P-values were estimated by chi-square or Fisher exact tests. Interrelations were analyzed by multiple logistic regression. All statistical analysis were performed with the package Stata Intercooled, version 8/SE (Stata Corporation, College Station, Texas, USA). The distributions of the PRNP 129 (p = 0.090) and IL-1A ()889) (p = 0.060) genotypes were in Hardy-Weinberg equilibrium. Stratification of the data by age groups (Table 1), indicated that in the early-onset (£ 76 years) group the adjusted OR in the subjects who only carried the PRNP 129 M/ M genotype was 0.80 (95% CI 0.322.00, p = 0.632) and in those with only the IL-1A ()889) allele 2 (1/2 and 2/2 genotypes) it was 1.71 (95% CI 0.68-4.31, p = 0.254); however, the subjects carrying both the PRNP 129 M/M genotype and the IL-1A ()889) allele 2 (1/2 and 2/2 genotypes) had about a O. Combarros (&) AE P. Sanchez-Juan I. Mateo AE J. Infante AE E. Rodriguez C. Sanchez-Quintana AE J. Berciano Neurology Service ‘‘Marques de Valdecilla’’ University Hospital University of Cantabria 39008 Santander, Spain Fax: +34-942/202655 E-Mail: combarro@unican.es