Lorena de Mena

Profile of microRNAs in the plasma of Parkinson's disease patients and healthy controls

The pathological hallmark of Parkinson’s disease (PD) is the loss of dopaminergic neurons and the presence of Lewy bodies and Lewy neurites in the substantia nigra pars compacta, corpus striatum and brain cortex [1]. PD is a complex disease caused by the interaction of genetic/ inherited and environmental/acquired risk factors [2]. MicroRNAs (miRNAs) are small RNAs that control gene expression by binding to the 30 UTRs of mRNAs [3]. Postmortem analysis of brain tissues and in vitro studies have identified several miRNAs implicated in PD. MiR-133b was shown to be downregulated in PD brains and to promote the survival of dopaminergic neurons [4]. MiR-433 was related with PD through targeting the FGF20, which in turn regulates the expression of a-synuclein [5]. MiR-7 targets a-synuclein and could regulate oxidative stress and cell death, while miR-184 and let-7 regulate dopaminergic neurons survival and activity [6, 7]. Recently, a miRNA profiling of PD brains identified early downregulation of miR-34b/c, modulating mitochondrial function in areas with pathological affectation [8]. Plasma (circulating) miRNAs have been proposed as biomarkers for several diseases and aging [9, 10]. Our aim was to characterize the plasma miRNA profile in PD patients and healthy controls, to determine its usefulness as a biomarker for PD. The study was approved by the Ethical Committee of Hospital Universitario Central de Asturias (HUCA) in accordance with the ethical standards of the Declaration of Helsinki and all the participants signed an informed consent. The study cohort consisted of sexand age-matched healthy controls (n = 25; mean age 67.6; 52 % males) and patients (n = 31; mean age 63.9; 55 % males) who fulfilled the PD-clinical diagnosis criteria [11]. None of the patients were receiving drugs for PD-treatment or had a diagnosis of cardiovascular or tumor disease. Full details of the experimental procedure are available as supplementary material. Briefly, blood was collected in tubes with EDTA, centrifuged and plasma was aliquoted (350 ll). A total of 2 pg of a synthetic Arabidopsis thaliana miRNA (Ath-miR-159a; 5 ll of a 0.4 pg/ll dilution) was immediately added and each aliquot stored at -80 C until use. Ath-miR-159a was used as control of the extraction process (supplementary material). Total plasma RNA was extracted (TRIzol LS Reagent, Ambion) and resuspended in 25 ll of RNAse-free water. Five ll of each sample were retrotranscribed (RT) with the Megaplex RT primers Human pool A and TaqMan microRNA Reverse transcription kit (Applied Biosystems). Three ll of the RT product were pre-amplified with the Megaplex Preamp primers Human pool A and TaqMan Universal Master Mix no AmpErase UNG (Applied Biosystems). All the preamplifications were assayed with a custom Ath-miR-159a Taqman assay in an ABI 7500 Real-Time PCR (Applied Electronic supplementary material The online version of this article (doi:10.1007/s00415-013-6900-8) contains supplementary material, which is available to authorized users.

Replication of MAPT and SNCA, but not PARK16-18, as susceptibility genes for Parkinson's disease.

Recent genome‐wide association studies of Parkinsons disease have nominated 3 new susceptibility loci (PARK16‐18) and confirmed 2 known risk genes (MAPT and SNCA) in populations of European ancestry. We sought to replicate these findings. We genotyped single‐nucleotide polymorphisms in each of these genes/loci in 1445 Parkinsons disease patients and 1161 controls from northern Spain. Logistic regression was used to test for association between genotype and Parkinsons disease under an additive model, adjusting for sex, age, and site. We also performed analyses stratified by age at onset. Single‐nucleotide polymorphisms in MAPT (rs1800547; P = 3.1 × 10−4) and SNCA (rs356219; P = 5.5 × 10−4) were significantly associated with Parkinsons disease. However, none of the markers in PARK16‐18 associated with Parkinsons disease in the overall sample, or in any age stratum, with P values ranging from .09 to .88. Although our data further validate MAPT and SNCA as Parkinsons disease susceptibility genes, we failed to replicate PARK16, PARK17, and PARK18. Potential reasons for the discordance between our study and previous genome‐wide association studies include effects of population structure, power, and population‐specific environmental interactions. Our findings suggest that additional studies of PARK16‐18 are necessary to establish the role of these loci in modifying risk for Parkinsons disease in European‐derived populations.

Analysis of the Micro-RNA-133 and PITX3 Genes in Parkinson's Disease

MicroRNAs are small RNA sequences that negatively regulate gene expression by binding to the 3′ untranslated regions of mRNAs. MiR‐133b has been implicated in Parkinsons disease (PD) by a mechanism that involves the regulation of the transcription factor PITX3. The variation in these genes could contribute to the risk of developing PD. We searched for DNA variants in miR‐133 and PITX3 genes in PD patients and healthy controls from Spain. We found common DNA variants in the three miR‐133 genes. Genotyping of a first set of patients (n = 777) and controls (n = 650) showed a higher frequency of homozygous for a miR‐133b variant (−90 del A) in PD‐patients (6/575; 1%) than in healthy controls (0/650) (P = 0.03). However, this association was not confirmed in a second set of patients (1/250; 0.4%) and controls (2/210; 1%). No common PITX3 variants were associated with PD, although a rare missense change (G32S) was found in only one patient and none of the controls. In conclusion, we report the variation in genes of a pathway that has been involved in dopaminergic neuron differentiation and survival. Our work suggests that miR‐133 and PITX3 gene variants did not contribute to the risk for PD.

A search for SNCA 3' UTR variants identified SNP rs356165 as a determinant of disease risk and onset age in Parkinson's disease.

Alpha-synuclein gene (SNCA) polymorphisms have been associated with the common sporadic form of Parkinson’s disease (PD). We searched for DNA variants at the SNCA 3′ UTR through single strand conformation analysis and direct sequencing in a cohort of Spanish PD patients and controls. We have genotyped the rs356165 SNCA 3′ UTR polymorphism in a total of 1,135 PD patients and 772 healthy controls from two Spanish cohorts (Asturias and Navarre). We identified six SNCA 3′ UTR variants. Single nucleotide polymorphism (SNP) rs356165 was significantly associated with PD risk in the Spanish cohort (p = 0.0001; odd ratio = 1.37, 95%CI = 1.19–1.58). This SNP was also significantly associated with early age at onset of PD. Our work highlights rs356165 as an important determinant of the risk of developing PD and early age at onset and encourages future research to identify a functional effect on SNCA expression.

Mitochondrial transcription factor A (TFAM) gene variation in Parkinson's disease.

Mitochondrial function is necessary to supply the energy required for cell metabolism. Mutations/polymorphisms in mitochondrial DNA (mtDNA) have been implicated in Parkinsons disease (PD). The mitochondrial transcription factor A (TFAM) controls the transcription of mtDNA and regulates the mtDNA-copy number, thus being important for maintaining ATP production. TFAM dysfunction may also be involved in PD, and TFAM gene mutations/polymorphisms could contribute to the risk of developing PD. We searched for gene variants in the seven TFAM-exons in a total of 250 PD-patients. We found five common polymorphisms, and only one was a missense change (S12T in exon 1). Genotype and allele frequencies did not differ between patients and healthy controls (n=225) for the five polymorphisms. Our work suggests that TFAM-variants did not contribute to the risk of developing PD.

Mitochondrial Transcription Factor A (TFAM) Gene Variation and Risk of Late-Onset Alzheimer's Disease

Impaired mitochondrial function and an increased number of mutations in mitochondrial DNA (mtDNA) has been found in brains of patients with late-onset Alzheimers disease (LOAD). The TFAM-gene encodes the mitochondrial transcription factor A, a protein that controls the transcription, replication, damage sensing, and repair of mtDNA. TFAM is on human chromosome region 10q21.1, where a locus for LOAD has been mapped. Our objective was to determine the role of TFAM-gene variation in the risk of LOAD. The seven TFAM coding exons were analysed through single strand conformation analysis and direct sequencing in a cohort of Spanish LOAD-patients and healthy controls. We found four common polymorphisms, two in the flanquing intronic and two in the coding sequences. Polymorphism rs1937 (+35 G/C) was the only missense change (S12T). Genotyping of this polymorphism in 300 LOAD-patients and 183 healthy controls showed a significantly higher frequency of GG-homozygotes in the patients (92% vs. 86%; p=0.04; OR=1.91, 95%CI=1.02-3.50). This suggests that S12 is a risk factor for LOAD in our population. In conclusion, rare variants (mutations) in the TFAM gene were not found in LOAD-patients, but the S12T polymorphism was a moderate risk factor for LOAD in our population.

Alpha-synuclein transcript isoforms in three different brain regions from Parkinson's disease and healthy subjects in relation to the SNCA rs356165/rs11931074 polymorphisms

Mutations in the alpha-synuclein (SNCA) gene cause autosomal dominant Parkinsons disease (PD). Common SNCA polymorphisms have been associated with the risk of developing PD. Abnormal expression and post-translational modification of SNCA has been found in PD-brains. In addition to a full length transcript (SNCA-140) there are three short isoforms (SNCA-98, -112, and -126) that could be prone to aggregation. The association between SNCA polymorphisms and PD could be explained through an increased expression of these alternative transcripts. Our aim was to measure the different SNCA transcripts in the substantia nigra (SN), cerebellum (CB), and occipital cortex (OC) from PD-patients (n=9) and healthy subjects (n=6). In addition, we determined whether two SNCA polymorphisms (SNPs rs356165 and rs11931074) were related to differences in transcript isoform expression. PD brain tissues showed higher levels of the three short transcripts in the SN, but only SNCA-112 and SNCA-98 were significantly increased in the CB of patients vs. controls (p=0.02, p=0.03). The genotyping of a large cohort of PD-patients and controls showed that haplotype rs356165-A+rs11931074-G had a protective effect (OR=0.71; CI=0.59-0.83), while the G-T haplotype increased the risk for PD (OR=1.44; CI=1.06-1.96). We did not find significant differences for the SNCA levels between the haplotypes. In conclusion, we found statistically significant higher levels of the SNCA-112 and SNCA-98 transcripts in the CB of PD brains, and a trend toward higher levels of the short transcript isoforms in the SN of PD brains.