Yolanda Espinosa-Parrilla

MicroRNA profiling of Parkinson’s disease brains identifies early downregulation of miR-34b/c which modulate mitochondrial function

MicroRNAs (miRNAs) are post-transcriptional gene expression regulators, playing key roles in neuronal development, plasticity and disease. Parkinsons disease (PD) is the second most common neurodegenerative disorder, characterized by the presence of protein inclusions or Lewy bodies and a progressive loss of dopaminergic neurons in the midbrain. Here, we have evaluated miRNA expression deregulation in PD brain samples. MiRNA expression profiling revealed decreased expression of miR-34b and miR-34c in brain areas with variable neuropathological affectation at clinical (motor) stages (Braak stages 4 and 5) of the disease, including the amygdala, frontal cortex, substantia nigra and cerebellum. Furthermore, misregulation of miR-34b/c was detected in pre-motor stages (stages 1-3) of the disease, and thus in cases that did not receive any PD-related treatment during life. Depletion of miR-34b or miR-34c in differentiated SH-SY5Y dopaminergic neuronal cells resulted in a moderate reduction in cell viability that was accompanied by altered mitochondrial function and dynamics, oxidative stress and reduction in total cellular adenosin triphosphate content. MiR-34b/c downregulation was coupled to a decrease in the expression of DJ1 and Parkin, two proteins associated to familial forms of PD that also have a role in idiopathic cases. Accordingly, DJ1 and Parkin expression was reduced in PD brain samples displaying strong miR-34b/c downregulation. We propose that early deregulation of miR-34b/c in PD triggers downstream transcriptome alterations underlying mitochondrial dysfunction and oxidative stress, which ultimately compromise cell viability. A better understanding of the cellular pathways controlling and/or controlled by miR-34b/c should allow identification of targets for development of therapeutic approaches.

Human microRNAs miR-22, miR-138-2, miR-148a, and miR-488 Are Associated with Panic Disorder and Regulate Several Anxiety Candidate Genes and Related Pathways

BACKGROUND The involvement of microRNAs (miRNAs) in neuronal differentiation and synaptic plasticity suggests a role for miRNAs in psychiatric disorders; association analyses and functional approaches were used to evaluate the implication of miRNAs in the susceptibility for panic disorder. METHODS Case-control studies for 712 single-nucleotide polymorphisms (SNPs) tagging 325 human miRNA regions were performed in 203 Spanish patients with panic disorder and 341 control subjects. A sample of 321 anxiety patients and 642 control subjects from Finland and 102 panic disorder patients and 829 control subjects from Estonia was used as a replica. Reporter-gene assays and miRNA overexpression experiments in neuroblastoma cells were used to functionally evaluate the spectrum of genes regulated by the associated miRNAs. RESULTS Two SNPs associated with panic disorder: rs6502892 tagging miR-22 (p < .0002), and rs11763020 tagging miR-339 (p < .00008). Other SNPs tagging miR-138-2, miR-488, miR-491, and miR-148a regions associated with different panic disorder phenotypes. Replication in the north-European sample supported several of these associations, although they did not pass correction for multiple testing. Functional studies revealed that miR-138-2, miR-148a, and miR-488 repress (30%-60%) several candidate genes for panic disorder--GABRA6, CCKBR and POMC, respectively--and that miR-22 regulates four other candidate genes: BDNF, HTR2C, MAOA, and RGS2. Transcriptome analysis of neuroblastoma cells transfected with miR-22 and miR-488 showed altered expression of a subset of predicted target genes for these miRNAs and of genes that might be affecting physiological pathways related to anxiety. CONCLUSIONS This work represents the first report of a possible implication of miRNAs in the etiology of panic disorder.

Allele variants in functional MicroRNA target sites of the neurotrophin-3 receptor gene (NTRK3) as susceptibility factors for anxiety disorders

Genetic and functional data indicate that variation in the expression of the neurotrophin‐3 receptor gene (NTRK3) may have an impact on neuronal plasticity, suggesting a role for NTRK3 in the pathophysiology of anxiety disorders. MicroRNA (miRNA) posttranscriptional gene regulators act by base‐pairing to specific sequence sites, usually at the 3′UTR of the target mRNA. Variants at these sites might result in gene expression changes contributing to disease susceptibility. We investigated genetic variation in two different isoforms of NTRK3 as candidate susceptibility factors for anxiety by resequencing their 3′UTRs in patients with panic disorder (PD), obsessive‐compulsive disorder (OCD), and in controls. We have found the C allele of rs28521337, located in a functional target site for miR‐485‐3p in the truncated isoform of NTRK3, to be significantly associated with the hoarding phenotype of OCD. We have also identified two new rare variants in the 3′UTR of NTRK3, ss102661458 and ss102661460, each present only in one chromosome of a patient with PD. The ss102661458 variant is located in a functional target site for miR‐765, and the ss102661460 in functional target sites for two miRNAs, miR‐509 and miR‐128, the latter being a brain‐enriched miRNA involved in neuronal differentiation and synaptic processing. Interestingly, these two variants significantly alter the miRNA‐mediated regulation of NTRK3, resulting in recovery of gene expression. These data implicate miRNAs as key posttranscriptional regulators of NTRK3 and provide a framework for allele‐specific miRNA regulation of NTRK3 in anxiety disorders. Hum Mutat 30:1–10, 2009.

Protein S gene analysis reveals the presence of a cosegregating mutation in most pedigrees with type I but not type III PS deficiency.

DNA sequence analysis of the protein S gene (PROS1) in 22 Spanish probands with type I or III PS deficiency, has allowed the identification of 10 different mutations and 2 new sequence variants in 15 probands. Nine of the mutations, 8 of which are novel, cosegregate with type I or quantitative PS deficiency in 12 of the 13 pedigrees analyzed. One of these mutations (Q238X) also cosegregates with both type I and III PS‐deficient phenotypes coexisting in a type I/III pedigree. Another mutation identified in a pedigree with these two PS phenotypes is the missense mutation R520G, present in the homozygous form in the type I propositus and in the heterozygous form in his type III relatives. By contrast, no cosegregating PROS1 mutation has been found in any of the six families with only type III phenotypes. Three of these families, as well as the two families with type I and I/III phenotypes where no other PROS1 mutation has been identified, segregate the P allele of the S460P variant, although this allele does not always cosegregate with the deficient phenotype. From these results we conclude that while mutations in PROS1 are the main cause of type I PS deficiency, the molecular basis of the type III phenotype is probably more complex, with many cases not being explained by a PROS1 mutation. Hum Mutat 14:30–39, 1999.

Design and evaluation of a panel of single-nucleotide polymorphisms in microRNA genomic regions for association studies in human disease.

MicroRNAs (miRNA) are recognized posttranscriptional gene repressors involved in the control of almost every biological process. Allelic variants in these regions may be an important source of phenotypic diversity and contribute to disease susceptibility. We analyzed the genomic organization of 325 human miRNAs (release 7.1, miRBase) to construct a panel of 768 single-nucleotide polymorphisms (SNPs) covering ∼1 Mb of genomic DNA, including 131 isolated miRNAs (40%) and 194 miRNAs arranged in 48 miRNA clusters, as well as their 5-kb flanking regions. Of these miRNAs, 37% were inside known protein-coding genes, which were significantly associated with biological functions regarding neurological, psychological or nutritional disorders. SNP coverage analysis revealed a lower SNP density in miRNAs compared with the average of the genome, with only 24 SNPs located in the 325 miRNAs studied. Further genotyping of 340 unrelated Spanish individuals showed that more than half of the SNPs in miRNAs were either rare or monomorphic, in agreement with the reported selective constraint on human miRNAs. A comparison of the minor allele frequencies between Spanish and HapMap population samples confirmed the applicability of this SNP panel to the study of complex disorders among the Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 in the CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showing geographical allelic frequency variation among the four HapMap populations, probably because of differences in natural selection. The designed miRNA SNP panel could help to identify still hidden links between miRNAs and human disease.

Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants.

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non‐isotopic Single‐Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single‐stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5′‐upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single‐strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C‐>G, L‐27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co‐segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (‐296C‐>T, 182G‐>C and T57S), were identified in a normal control, two type I/III and two type III PS‐deficient pedigrees. Hum Mutat 15:463–473, 2000.

Analysis of the Protein S Gene in Protein S Deficiency

Protein S (PS) is a 71-kDa vitamin K-dependent glycoprotein first identified in human plasma by DiScipio and colleagues in 1977 (1), a year after the discovery of the anticoagulant protein C (PC) (2,3). A few years later, Walker demonstrated that PS acts as a cofactor for activated protein C (APC) in the proteolytic inactivation of the procoagulant factors Va and VIIIa (4,5) and in 1984, the first families with hereditary PS deficiency and venous thrombotic disease were identified (6,7). This demonstrated the physiological importance of PS as an antithrombotic protein, which has been further confirmed by the identification of many other families in which the heterozygotes for PS deficiency have an increased risk of developing venous thrombosis in early adulthood (8-10). PS deficient homozygotes with severe thrombotic events and purpura fulminans in the neonatal period have also been described (11,12). Although the molecular mechanism by which PS enhances APC activity has not yet been completely elucidated (2,3), it has been proposed that PS increases the affinity of APC for the phospholipid membranes where the inactivation complex will form and the inactivation reactions take place (13). PS might also have APC independent anticoagulant properties through direct inhibition of prothrombin and factor X activation (14-16).

Functional Implications of Human-Specific Changes in Great Ape microRNAs.

microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species.

MicroRNA Genetic Variation: From Population Analysis to Functional Implications of Three Allele Variants Associated with Cancer

Nucleotide variants in microRNA regions have been associated with disease; nevertheless, few studies still have addressed the allele‐dependent effect of these changes. We studied microRNA genetic variation in human populations and found that while low‐frequency variants accumulate indistinctly in microRNA regions, the mature and seed regions tend to be depleted of high‐frequency variants, probably as a result of purifying selection. Comparison of pairwise population fixation indexes among regions showed that the seed had higher population fixation indexes than the other regions, suggesting the existence of local adaptation in the seed region. We further performed functional studies of three microRNA variants associated with cancer (rs2910164:C > G in MIR146A, rs11614913:C > T in MIR196A2, and rs3746444:A > G in both MIR499A and MIR499B). We found differences in the expression between alleles and in the regulation of several genes involved in cancer, such as TP53, KIT, CDH1, CLH, and TERT, which may result in changes in regulatory networks related to tumorigenesis. Furthermore, luciferase‐based assays showed that MIR499A could be regulating the cadherin CDH1 and the cell adhesion molecule CLH1 in an allele‐dependent fashion. A better understanding of the effect of microRNA variants associated with disease could be key in our way to a more personalized medicine.

microRNA-Mediated Regulation and the Genetic Susceptibility to Anxiety Disorders

Investigation of how genetic variation within complex gene regulatory networks results in phenotypic alterations may represent a useful approach towards the understanding of human evolution and disease. In this regard, genetic studies can contribute to the identification of genes and pathways underlying the susceptibility to psychiatric disorders including anxiety disorders. However, this has shown to be difficult due to the complexity of both, the genetics and the phenotypes of these disorders. In fact, even though the estimated heritability of psychiatric disorders is high, most genetic risk alleles for these disorders have still not been identified, leading to the conclusion that either major risk alleles are scarce or that they increase risks only marginally, that is to say that their associated Odds Ratio is low. Genetic heterogeneity for complex disorders is widely accepted and, in addition, it has been suggested that non-standard factors, such as epigenetic or regulatory changes or combinations of various of these elements could be involved in the aetiology of psychiatric disorders (Burmeister et al., 2008). Accordingly, increasing evidence at a population and experimental level indicates that genetic variation at regulatory regions underlies differences in gene expression and could be a major contributor to phenotypic diversity in human populations (Buckland et al., 2005; Knight, 2005; Rockman and Wray, 2002). This may be particularly true in the case of psychiatric disorders, where changes in regulatory elements leading to small variations in the dosage of proteins involved in neuronal pathways may disrupt the fine-tuned equilibrium of complex brain functions and contribute to the development of the disease. In this respect, even though the search for susceptibility genes for anxiety disorders has led to the finding of positive associations, most of these studies have produced results that are inconsistent or not clearly replicated, indicating that the genetic basis of anxiety disorders requires further investigation using alternative approaches. Stress has also been shown to have a critical role in the development of anxiety disorders (Lupien et al, 2009), at least partially, through mechanisms related to neural plasticity. Synaptic connections in the brain undergo experience-dependent functional or morphological changes through complex pathways that are not yet fully understood, but for which microRNAs (miRNAs) might have a critical role (Kosik et al., 2006). miRNAs are endogenous small non-coding RNAs that regulate gene expression by means of partial