Diagnostic accuracy of cerebrospinal fluid biomarkers in genetic prion diseases.

Genetic prion diseases are a rare and diverse group of fatal neurodegenerative disorders caused by pathogenic sequence variations in the prion protein gene, PRNP. Data on CSF biomarkers in patients with genetic prion diseases are limited and conflicting results have been reported for unclear reasons. Here, we aimed to analyse the diagnostic accuracy of CSF biomarkers currently used in prion clinical diagnosis in 302 symptomatic genetic prion disease cases from 11 prion diagnostic centres, encompassing a total of 36 different pathogenic sequence variations within the open reading frame of PRNP. CSF samples were assessed for the surrogate markers of neurodegeneration, 14-3-3 protein (14-3-3), total-tau protein (t-tau) and α-synuclein and for prion seeding activity through the real-time quaking-induced conversion assay. Biomarker results were compared with those obtained in healthy and neurological controls. For the most prevalent PRNP pathogenic sequence variations, biomarker accuracy and associations between biomarkers, demographic and genetic determinants were assessed. Additionally, the prognostic value of biomarkers for predicting total disease duration from symptom onset to death was investigated. High sensitivity of the four biomarkers was detected for genetic Creutzfeldt-Jakob disease associated with the E200K and V210I mutations, but low sensitivity was observed for mutations associated with Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. All biomarkers showed good to excellent specificity using the standard cut-offs often used for sporadic Creutzfeldt-Jakob disease. In genetic prion diseases related to octapeptide repeat insertions, the biomarker sensitivity correlated with the number of repeats. New genetic prion disease-specific cut-offs for 14-3-3, t-tau and α-synuclein were calculated. Disease duration in genetic Creutzfeldt-Jakob disease-E200K, Gerstmann-Sträussler-Scheinker-P102L and fatal familial insomnia was highly dependent on PRNP codon 129 MV polymorphism and was significantly associated with biomarker levels. In a large cohort of genetic prion diseases, the simultaneous analysis of CSF prion disease biomarkers allowed the determination of new mutation-specific cut-offs improving the discrimination of genetic prion disease cases and unveiled genetic prion disease-specific associations with disease duration.

Altered m6A RNA methylation contributes to hippocampal memory deficits in Huntington's disease mice.

N6-methyladenosine (m6A) regulates many aspects of RNA metabolism and is involved in learning and memory processes. Yet, the impact of a dysregulation of post-transcriptional m6A editing on synaptic impairments in neurodegenerative disorders remains unknown. Here we investigated the m6A methylation pattern in the hippocampus of Huntington's disease (HD) mice and the potential role of the m6A RNA modification in HD cognitive symptomatology. m6A modifications were evaluated in HD mice subjected to a hippocampal cognitive training task through m6A immunoprecipitation sequencing (MeRIP-seq) and the relative levels of m6A-modifying proteins (FTO and METTL14) by subcellular fractionation and Western blot analysis. Stereotaxic CA1 hippocampal delivery of AAV-shFTO was performed to investigate the effect of RNA m6A dysregulation in HD memory deficits. Our results reveal a m6A hypermethylation in relevant HD and synaptic related genes in the hippocampal transcriptome of Hdh+/Q111 mice. Conversely, m6A is aberrantly regulated in an experience-dependent manner in the HD hippocampus leading to demethylation of important components of synapse organization. Notably, the levels of RNA demethylase (FTO) and methyltransferase (METTL14) were modulated after training in the hippocampus of WT mice but not in Hdh+/Q111 mice. Finally, inhibition of FTO expression in the hippocampal CA1 region restored memory disturbances in symptomatic Hdh+/Q111 mice. Altogether, our results suggest that a differential RNA methylation landscape contributes to HD cognitive symptoms and uncover a role of m6A as a novel hallmark of HD.

Phospho-RNA-Seq Highlights Specific Small RNA Profiles in Plasma Extracellular Vesicles.

Small RNAs (sRNAs) are bioactive molecules that can be detected in biofluids, reflecting physiological and pathological states. In plasma, sRNAs are found within extracellular vesicles (EVs) and in extravesicular compartments, offering potential sources of highly sensitive biomarkers. Deep sequencing strategies to profile sRNAs favor the detection of microRNAs (miRNAs), the best-known class of sRNAs. Phospho-RNA-seq, through the enzymatic treatment of sRNAs with T4 polynucleotide kinase (T4-PNK), has been recently developed to increase the detection of thousands of previously inaccessible RNAs. In this study, we investigated the value of phospho-RNA-seq on both the EVs and extravesicular plasma subfractions. Phospho-RNA-seq increased the proportion of sRNAs used for alignment and highlighted the diversity of the sRNA transcriptome. Unsupervised clustering analysis using sRNA counts matrices correctly classified the EVs and extravesicular samples only in the T4-PNK treated samples, indicating that phospho-RNA-seq stresses the features of sRNAs in each plasma subfraction. Furthermore, T4-PNK treatment emphasized specific miRNA variants differing in the 5'-end (5'-isomiRs) and certain types of tRNA fragments in each plasma fraction. Phospho-RNA-seq increased the number of tissue-specific messenger RNA (mRNA) fragments in the EVs compared with the extravesicular fraction, suggesting that phospho-RNA-seq favors the discovery of tissue-specific sRNAs in EVs. Overall, the present data emphasizes the value of phospho-RNA-seq in uncovering RNA-based biomarkers in EVs.

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice.

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

In utero and childhood exposure to tobacco smoke and multi-layer molecular signatures in children.

BACKGROUND: The adverse health effects of early life exposure to tobacco smoking have been widely reported. In spite of this, the underlying molecular mechanisms of in utero and postnatal exposure to tobacco smoke are only partially understood. Here, we aimed to identify multi-layer molecular signatures associated with exposure to tobacco smoke in these two exposure windows. METHODS: We investigated the associations of maternal smoking during pregnancy and childhood secondhand smoke (SHS) exposure with molecular features measured in 1203 European children (mean age 8.1 years) from the Human Early Life Exposome (HELIX) project. Molecular features, covering 4 layers, included blood DNA methylation and gene and miRNA transcription, plasma proteins, and sera and urinary metabolites. RESULTS: Maternal smoking during pregnancy was associated with DNA methylation changes at 18 loci in child blood. DNA methylation at 5 of these loci was related to expression of the nearby genes. However, the expression of these genes themselves was only weakly associated with maternal smoking. Conversely, childhood SHS was not associated with blood DNA methylation or transcription patterns, but with reduced levels of several serum metabolites and with increased plasma PAI1 (plasminogen activator inhibitor-1), a protein that inhibits fibrinolysis. Some of the in utero and childhood smoking-related molecular marks showed dose-response trends, with stronger effects with higher dose or longer duration of the exposure. CONCLUSION: In this first study covering multi-layer molecular features, pregnancy and childhood exposure to tobacco smoke were associated with distinct molecular phenotypes in children. The persistent and dose-dependent changes in the methylome make CpGs good candidates to develop biomarkers of past exposure. Moreover, compared to methylation, the weak association of maternal smoking in pregnancy with gene expression suggests different reversal rates and a methylation-based memory to past exposures. Finally, certain metabolites and protein markers evidenced potential early biological effects of postnatal SHS, such as fibrinolysis.

Regional and subtype-dependent miRNA signatures in sporadic Creutzfeldt-Jakob disease are accompanied by alterations in miRNA silencing machinery and biogenesis.

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer's disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation.

The discovery potential of RNA processing profiles.

Small non-coding RNAs (sncRNAs) are highly abundant molecules that regulate essential cellular processes and are classified according to sequence and structure. Here we argue that read profiles from size-selected RNA sequencing capture the post-transcriptional processing specific to each RNA family, thereby providing functional information independently of sequence and structure. We developed SeRPeNT, a new computational method that exploits reproducibility across replicates and uses dynamic time-warping and density-based clustering algorithms to identify, characterize and compare sncRNAs by harnessing the power of read profiles. We applied SeRPeNT to: (i) generate an extended human annotation with 671 new sncRNAs from known classes and 131 from new potential classes, (ii) show pervasive differential processing of sncRNAs between cell compartments and (iii) predict new molecules with miRNA-like behaviour from snoRNA, tRNA and long non-coding RNA precursors, potentially dependent on the miRNA biogenesis pathway. Furthermore, we validated experimentally four predicted novel non-coding RNAs: a miRNA, a snoRNA-derived miRNA, a processed tRNA and a new uncharacterized sncRNA. SeRPeNT facilitates fast and accurate discovery and characterization of sncRNAs at an unprecedented scale. SeRPeNT code is available under the MIT license at https://github.com/comprna/SeRPeNT.

Specific small-RNA signatures in the amygdala at premotor and motor stages of Parkinson's disease revealed by deep sequencing analysis.

MOTIVATION: Most computational tools for small non-coding RNAs (sRNA) sequencing data analysis focus in microRNAs (miRNAs), overlooking other types of sRNAs that show multi-mapping hits. Here, we have developed a pipeline to non-redundantly quantify all types of sRNAs, and extract patterns of expression in biologically defined groups. We have used our tool to characterize and profile sRNAs in post-mortem brain samples of control individuals and Parkinson's disease (PD) cases at early-premotor and late-symptomatic stages. RESULTS: Clusters of co-expressed sRNAs mapping onto tRNAs significantly separated premotor and motor cases from controls. A similar result was obtained using a matrix of miRNAs slightly varying in sequence (isomiRs). The present framework revealed sRNA alterations at premotor stages of PD, which might reflect initial pathogenic perturbations. This tool may be useful to discover sRNA expression patterns linked to different biological conditions. AVAILABILITY AND IMPLEMENTATION: The full code is available at http://github.com/lpantano/seqbuster CONTACT: lpantano@hsph.harvard.edu or eulalia.marti@crg.eu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A pathogenic mechanism in Huntington's disease involves small CAG-repeated RNAs with neurotoxic activity.

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.

Blood expression profiles of fragile X premutation carriers identify candidate genes involved in neurodegenerative and infertility phenotypes.

Male premutation carriers presenting between 55 and 200 CGG repeats in the Fragile-X-associated (FMR1) gene are at risk of developing Fragile X Tremor/Ataxia Syndrome (FXTAS), and females undergo Premature Ovarian Failure (POF1). Here, we have evaluated gene expression profiles from blood in male FMR1 premutation carriers and detected a strong deregulation of genes enriched in FXTAS relevant biological pathways, including inflammation, neuronal homeostasis and viability. Gene expression profiling distinguished between control individuals, carriers with FXTAS and carriers without FXTAS, with levels of expanded FMR1 mRNA being increased in FXTAS patients. In vitro studies in a neuronal cell model indicate that expression levels of expanded FMR1 5'-UTR are relevant in modulating the transcriptome. Thus, perturbations of the transcriptome may be an interplay between the CGG expansion size and FMR1 expression levels. Several deregulated genes (DFFA, BCL2L11, BCL2L1, APP, SOD1, RNF10, HDAC5, KCNC3, ATXN7, ATXN3 and EAP1) were validated in brain samples of a FXTAS mouse model. Downregulation of EAP1, a gene involved in the female reproductive system physiology, was confirmed in female carriers. Decreased levels were detected in female carriers with POF1 compared to those without POF1, suggesting that EAP1 levels contribute to ovarian insufficiency. In summary, gene expression profiling in blood has uncovered mechanisms that may underlie different pathological aspects of the premutation. A better understanding of the transcriptome dynamics in relation with expanded FMR1 mRNA expression levels and CGG expansion size may provide mechanistic insights into the disease process and a more accurate FXTAS diagnosis to the myriad of phenotypes associated with the premutation.

miR-519a-3p, found to regulate cellular prion protein during Alzheimer's disease pathogenesis, as a biomarker of asymptomatic stages.

Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer's disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrPC) during disease progression. That is why, by means of cross studies of miRNAs up-regulated in AD with in silico identification of potential miRNAs-binding to 3'UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3'UTR-PRNP, and second, we analyzed the levels of PrPC expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR-519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non-AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3'UTR in vitro and promotes downregulation of PrPC. Moreover, miR-519a-3p was found to be up-regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.

A highly expressed miR-101 isomiR is a functional silencing small RNA.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5' or 3' of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5'-trimming variant (5'-isomiR-101). RESULTS: The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5'-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5'-isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5'-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5'-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5'-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5'-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5'-isomiR-101. CONCLUSIONS: These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor.

BACKGROUND: Epidermal Growth Factor (EGF) plays an important function in the regulation of cell growth, proliferation, and differentiation by binding to its receptor (EGFR) and providing cancer cells with increased survival responsiveness. Signal transduction carried out by EGF has been extensively studied at both transcriptional and post-transcriptional levels. Little is known about the involvement of microRNAs (miRNAs) in the EGF signaling pathway. miRNAs have emerged as major players in the complex networks of gene regulation, and cancer miRNA expression studies have evidenced a direct involvement of miRNAs in cancer progression. RESULTS: In this study, we have used an integrative high content analysis approach to identify the specific miRNAs implicated in EGF signaling in HeLa cells as potential mediators of cancer mediated functions. We have used microarray and deep-sequencing technologies in order to obtain a global view of the EGF miRNA transcriptome with a robust experimental cross-validation. By applying a procedure based on Rankprod tests, we have delimited a solid set of EGF-regulated miRNAs. After validating regulated miRNAs by reverse transcription quantitative PCR, we have derived protein networks and biological functions from the predicted targets of the regulated miRNAs to gain insight into the potential role of miRNAs in EGF-treated cells. In addition, we have analyzed sequence heterogeneity due to editing relative to the reference sequence (isomiRs) among regulated miRNAs. CONCLUSIONS: We propose that the use of global genomic miRNA cross-validation derived from high throughput technologies can be used to generate more reliable datasets inferring more robust networks of co-regulated predicted miRNA target genes.

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. Parkinson's 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.

A common 56-kilobase deletion in a primate-specific segmental duplication creates a novel butyrophilin-like protein.

BACKGROUND: The Butyrophilin-like (BTNL) proteins are likely to play an important role in inflammation and immune response. Like the B7 protein family, many human and murine BTNL members have been shown to control T lymphocytes response, and polymorphisms in human BTNL2 have been linked to several inflammatory diseases, such as pulmonary sarcoidosis, inflammatory bowel disease and neonatal lupus. RESULTS: In this study we provide a comprehensive population, genomic and transcriptomic analysis of a 56-kb deletion copy number variant (CNV), located within two segmental duplications of two genes belonging to the BTNL family, namely BTNL8 and BTNL3. We confirm the presence of a novel BTNL8*3 fusion-protein product, and show an influence of the deletion variant on the expression level of several genes involved in immune function, including BTNL9, another member of the same family. Moreover, by genotyping HapMap and human diversity panel (HGDP) samples, we demonstrate a clear difference in the stratification of the BTNL8_BTNL3-del allele frequency between major continental human populations. CONCLUSION: Despite tremendous progress in the field of structural variation, rather few CNVs have been functionally characterized so far. Here, we show clear functional consequences of a new deletion CNV (BTNL8_BTNL3-del) with potentially important implication in the human immune system and in inflammatory and proliferative disorders. In addition, the marked population differences found of BTNL8_BTNL3-del frequencies suggest that this deletion CNV might have evolved under positive selection due to environmental conditions in some populations, with potential phenotypic consequences.

Upregulation of a small vault RNA (svtRNA2-1a) is an early event in Parkinson disease and induces neuronal dysfunction.

MicroRNAs (miRNAs) and other small non-coding RNAs (sncRNAs) are post-transcriptional regulators of gene expression, playing key roles in neuronal development, plasticity, and disease. Transcriptome deregulation caused by miRNA dysfunction has been associated to neurodegenerative diseases. Parkinson disease (PD) is the second most common neurodegenerative disease showing deregulation of the coding and small non-coding transcriptome. On profiling sncRNA in PD brain areas differently affected, we found that upregulation of a small vault RNA (svtRNA2-1a) is widespread in PD brains, occurring early in the course of the disease (at pre-motor stages). SvtRNA2-1a biogenesis was dependent on Dicer activity on its precursor (vtRNA2-1) but independent of Drosha endonuclease, unlike the canonical miRNAs. Although endogenous svtRNA2-1a was enriched in Ago-2 immunoprecipitates in differentiated SH-SY5Y neuronal cells, overexpression of svtRNA2-1a induced subtle transcriptomic changes, suggesting that gene expression regulation may involve other mechanisms than mRNA decay only. Function enrichment analysis of the genes deregulated by svtRNA2-1a overexpression or svtRNA2-1a predicted targets identified pathways related to nervous system development and cell type specification. The expression pattern of svtRNA2-1a during development and aging of the human brain and the detrimental consequences of a svtRNA2-1a mimic overexpression in neuronal cells further indicate that low svtRNA2-1a levels may be important for the maintenance of neurons. Our results suggest that early svtRNA2-1a upregulation in PD may contribute to perturbations of gene expression networks, underlying metabolic impairment and cell dysfunction. A better understanding of the pathways regulated by svtRNA2-a, and also the mechanisms regulating its expression should facilitate the identification of new targets for therapeutic approaches in PD.

Neuron-derived extracellular vesicles contain synaptic proteins, promote spine formation, activate TrkB-mediated signalling and preserve neuronal complexity.

Extracellular vesicles (EVs) play an important role in intercellular communication as carriers of signalling molecules such as bioactive miRNAs, proteins and lipids. EVs are key players in the functioning of the central nervous system (CNS) by influencing synaptic events and modulating recipient neurons. However, the specific role of neuron-to-neuron communication via EVs is still not well understood. Here, we provide evidence that primary neurons uptake neuron-derived EVs in the soma, dendrites, and even in the dendritic spines, and carry synaptic proteins. Neuron-derived EVs increased spine density and promoted the phosphorylation of Akt and ribosomal protein S6 (RPS6), via TrkB-signalling, without impairing the neuronal network activity. Strikingly, EVs exerted a trophic effect on challenged nutrient-deprived neurons. Altogether, our results place EVs in the spotlight for synaptic plasticity modulation as well as a possible therapeutic tool to fight neurodegeneration.

A non-biased framework for the annotation and classification of the non-miRNA small RNA transcriptome.

MOTIVATION: Recent progress in high-throughput sequencing technologies has largely contributed to reveal a highly complex landscape of small non-coding RNAs (sRNAs), including novel non-canonical sRNAs derived from long non-coding RNA, repeated elements, transcription start sites and splicing site regions among others. The published frameworks for sRNA data analysis are focused on miRNA detection and prediction, ignoring further information in the dataset. As a consequence, tools for the identification and classification of the sRNAs not belonging to miRNA family are currently lacking. RESULTS: Here, we present, SeqCluster, an extension of the currently available SeqBuster tool to identify and analyze at different levels the sRNAs not annotated or predicted as miRNAs. This new module deals with sequences mapping onto multiple locations and permits a highly versatile and user-friendly interaction with the data in order to easily classify sRNA sequences with a putative functional importance. We were able to detect all known classes of sRNAs described to date using SeqCluster with different sRNA datasets.

SeqBuster, a bioinformatic tool for the processing and analysis of small RNAs datasets, reveals ubiquitous miRNA modifications in human embryonic cells.

High-throughput sequencing technologies enable direct approaches to catalog and analyze snapshots of the total small RNA content of living cells. Characterization of high-throughput sequencing data requires bioinformatic tools offering a wide perspective of the small RNA transcriptome. Here we present SeqBuster, a highly versatile and reliable web-based toolkit to process and analyze large-scale small RNA datasets. The high flexibility of this tool is illustrated by the multiple choices offered in the pre-analysis for mapping purposes and in the different analysis modules for data manipulation. To overcome the storage capacity limitations of the web-based tool, SeqBuster offers a stand-alone version that permits the annotation against any custom database. SeqBuster integrates multiple analyses modules in a unique platform and constitutes the first bioinformatic tool offering a deep characterization of miRNA variants (isomiRs). The application of SeqBuster to small-RNA datasets of human embryonic stem cells revealed that most miRNAs present different types of isomiRs, some of them being associated to stem cell differentiation. The exhaustive description of the isomiRs provided by SeqBuster could help to identify miRNA-variants that are relevant in physiological and pathological processes. SeqBuster is available at http://estivill_lab.crg.es/seqbuster.

A myriad of miRNA variants in control and Huntington's disease brain regions detected by massively parallel sequencing.

Huntington disease (HD) is a neurodegenerative disorder that predominantly affects neurons of the forebrain. We have applied the Illumina massively parallel sequencing to deeply analyze the small RNA populations of two different forebrain areas, the frontal cortex (FC) and the striatum (ST) of healthy individuals and individuals with HD. More than 80% of the small-RNAs were annotated as microRNAs (miRNAs) in all samples. Deep sequencing revealed length and sequence heterogeneity (IsomiRs) for the vast majority of miRNAs. Around 80-90% of the miRNAs presented modifications in the 3'-terminus mainly in the form of trimming and/or as nucleotide addition variants, while the 5'-terminus of the miRNAs was specially protected from changes. Expression profiling showed strong miRNA and isomiR expression deregulation in HD, most being common to both FC and ST. The analysis of the upstream regulatory regions in co-regulated miRNAs suggests a role for RE1-Silencing Transcription Factor (REST) and P53 in miRNAs downregulation in HD. The putative targets of deregulated miRNAs and seed-region IsomiRs strongly suggest that their altered expression contributes to the aberrant gene expression in HD. Our results show that miRNA variability is a ubiquitous phenomenon in the adult human brain, which may influence gene expression in physiological and pathological conditions.