Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.

International audienceEmbryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19-30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development

ncPRO-seq: a tool for annotation and profiling of ncRNAs in sRNA-seq data

Summary: Non-coding RNA (ncRNA) PROfiling in small RNA (sRNA)-seq (ncPRO-seq) is a stand-alone, comprehensive and flexible ncRNA analysis pipeline. It can interrogate and perform detailed profiling analysis on sRNAs derived from annotated non-coding regions in miRBase, Rfam and RepeatMasker, as well as specific regions defined by users. The ncPRO-seq pipeline performs both gene-based and family-based analyses of sRNAs. It also has a module to identify regions significantly enriched with short reads, which cannot be classified under known ncRNA families, thus enabling the discovery of previously unknown ncRNA- or small interfering RNA (siRNA)-producing regions. The ncPRO-seq pipeline supports input read sequences in fastq, fasta and color space format, as well as alignment results in BAM format, meaning that sRNA raw data from the three current major platforms (Roche-454, Illumina-Solexa and Life technologies-SOLiD) can be analyzed with this pipeline. The ncPRO-seq pipeline can be used to analyze read and alignment data, based on any sequenced genome, including mammals and plants. Availability: Source code, annotation files, manual and online version are available at http://ncpro.curie.fr/. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

A Pragmatic, Data-Driven Method to Determine Cutoffs for CSF Biomarkers of Alzheimer Disease Based on Validation Against PET Imaging

OBJECTIVE: To elaborate a new algorithm to establish a standardized method to define cuff-offs for CSF biomarkers of Alzheimer's disease (AD) by validating the algorithm against CSF classification derived from PET imaging. METHODS: Low and high levels of CSF phosphorylated tau were first identified to establish optimal cut-offs for CSF amyloid-β peptide (Aβ) biomarkers. These Aβ cut-offs were then used to determine cut-offs for CSF tau and phosphorylated tau markers. We compared this algorithm to a reference method, based on tau and amyloid PET imaging status (ADNI study), and then applied the algorithm to 10 large clinical cohorts of patients. RESULTS: A total of 6,922 subjects with CSF biomarkers data were included (mean (SD) age: 70.6 (8.5) years, 51.0% women). In the ADNI study population (n=497), the agreement between classification based on our algorithm and one based on amyloid/tau PET imaging was high with Cohen's kappa coefficient between 0.87 and 0.99. Applying the algorithm to 10 large cohorts of patients (n=6,425), the proportion of persons with AD ranged from 25.9% to 43.5%. DISCUSSION: The proposed novel, pragmatic method to determine CSF biomarkers cut-offs for AD does not require assessment of other biomarkers or assumptions concerning the clinical diagnosis of patients. Use of this standardized algorithm is likely to reduce heterogeneity in AD classification

White matter lesions in CADASIL

CADASIL est une forme héréditaire, autosomique dominante, de maladie des petits vaisseaux cérébraux dans laquelle surviennent précocement des lésions de la substance blanche cérébrale qui progressent avec le temps, mais dont la nature histopathologique demeure très mal connue. La maladie est causée par des mutations très stéréotypées du récepteur Notch3. Une des signatures de CADASIL est la présence, dans les vaisseaux, d’une accumulation du domaine extracellulaire de NOTCH3 (Notch3ECD). Un faisceau d’arguments suggère que le processus pathogène de CADASIL résulte d’un effet toxique de ces dépôts de Notch3ECD, qui passerait par l’accumulation avec le NOTCH3ECD d’autres protéines de la matrice extracellulaire. Il a cependant été montré que des mutations CADASIL affectent les capacités de signalisation du récepteur, de manière constitutive ou avec le temps, ce qui a conduit à formuler l’hypothèse qu’une perte de fonction Notch3 pourrait également constituer un déterminant important du processus pathogène.Nous avons réalisé une analyse détaillée des lésions de la substance blanche dans un modèle murin de la maladie CADASIL obtenu par surexpression d’un allèle Notch3 avec la mutation R169C / R170C, qui en récapitule les stades précliniques (TgPACNotch3R169C). Ceci a permis de mettre en évidence aux stades précoces un oedème intramyélinique associé à une dégradation / décompaction de la myéline détectable en immunohistochimie dès l’âge de 6 mois. L’analyse de l’intégrité axonale au sein des lésions de la myéline suggère une perte secondaire. Une méthode de quantification semi-automatisée des débris myélinique a été élaborée.Nous avons ensuite testé l’hypothèse qu’une perte de fonction Notch3 pourrait constituer un déterminant majeur dans le processus pathogène de CADASIL. Nous avons pour cela identifié un set de gènes dont l’expression est sensible à la quantité de Notch3, capable de détecter une diminution de moitié de la dose de Notch3. La mesure de l’expression de ces gènes chez des souris Knock-in pour la mutation R170C, hétérozygotes ou homozygotes, a montré que l’activité Notch3 n’était pas diminuée dans ce modèle. Nous avons ensuite étudié l’impact de la suppression des copies endogènes de Notch3 sur les lésions de la substance blanche chez les souris TgPACNotch3R169C, qui n’apparaissent pas aggravées. Ces résultats plaident contre un effet hypomorphe commun à toutes les mutations CADASIL et suggèrent que les lésions de la substance blanche ne sont pas secondaires à un tel effet.Nous avons enfin étudié le rôle pathogène de l’excès de TIMP3 et vitronectine, deux protéines dont il a été démontré qu’elles s’accumulent précocement avec le NOTCH3ECD. En utilisant des approches d’interaction génétique (diminution et/ou augmentation de la quantité de TIMP3 et vitronectine chez les souris TgPACNotch3R169C), nous avons observé un effet différent de l’excès des deux protéines sur les anomalies de la réactivité cérébrovasculaire et celles de la substance blanche. En effet, la réduction de la quantité de vitronectine limite les lésions de la substance blanche sans effet sur la réactivité cérébrovasculaire alors que la réduction de TIMP3 corrige les anomalies vasculaires fonctionnelles sans effet sur la substance blanche. Ces résultats apportent la preuve de concept du rôle pathogène de l’accumulation des protéines TIMP3 et vitronectine dans le processus pathogène de CADASIL et remettent en question les dogmes faisant de l’hypoperfusion le facteur promoteur des lésions de la substance blanche dans la maladie.CADASIL is an autosomal dominant, hereditary, small vessel disease of the brain causing early and progressive white matter lesions. The histopathological characteristics of these lesions remain poorly known. The disease is caused by stereotyped mutations in the gene coding for the NOTCH3 receptor. One of CADASIL hallmarks is the presence in vessels of an abnormal accumulation of NOTCH3 extracellular domain (NOTCH3ECD). Data suggest that CADASIL pathophysiological process may be caused by a toxic effect resulting from NOTCH3ECD deposits, due to an abnormal recruitment of other extracellular matrix components. However, it has been shown that CADASIL mutations differentially affect Notch3 signaling, constitutively or progressively. The latter observations led scientists to propose the hypothesis that Notch3 loss of function may play an important role in CADASIL pathogenesis.We conducted a detailed white matter analysis in a CADASIL mouse model that overexpresses a Notch3 allele with the R169C/R170C mutation and that recapitulates the preclinical stages of the disease (TgPACNotch3R169C). In this model, we observed intramyelinic edema associated with myelin degradation / decompaction detectable by immunochemistry in the brain of mice as young as 6 months of age. Axonal integrity analysis in myelin lesions suggested that axonal loss may appear secondarily. A semi-quantitative method for the quantification of myelin debris has been developed.Next, we tested the hypothesis that Notch3 loss of function might play a key role in CADASIL pathophysiology. We first identified a set of genes that are sensitive to a reduction in Notch3 dosage by half. Quantification of these genes expression in both heterozygous and homozygous mice Knock-in for the R170C mutation showed that Notch3 activity was not lowered in this model. In addition, we analyzed the effect of a suppression of endogenous Notch3 copies on white matter lesions observed in TgPACNotch3R169C mice and observed no worsening of these lesions. Together these results suggest that hypomorphism is not a feature common to all CADASIL mutations, and that white matter lesions in CADASIL do no result from Notch3 loss of function.Finally, we studied the pathogenic effect of Timp3 and vitronectine accumulation, both proteins having been shown to accumulate with NOTCH3ECD early in the course of the disease. By the use of genetic interaction approaches (lowering and increase in Timp3 and vitronectine in TgPACNotch3R169C mice), we observed differential effects of the proteins on white matter lesions and cerebrovascular reactivity impairment. Indeed, vitronectine lowering improves white matter lesions without any effect on cerebrovascular reactivity while Timp3 diminution restores cerebrovascular reactivity without any effect on white matter lesions. These results provide proof of concept for the implication of TIMP3 and vitronectin excess in CADASIL pathogenesis and questions the dogma that make hypoperfusion the main determinant of white matter lesions in CADASIL

Visibility of blood flow on optical coherence tomography angiography in a case of branch retinal artery occlusion

Purpose: We report the variability in flow angiogram during the course of branch retinal artery occlusion (BRAO) in a case imaged by optical coherence tomography angiography (OCTA). Case Report: OCTA was performed in a patient with BRAO at initial examination and 6 hours later. Initially, the occluded retinal artery and its branches were not detected on OCTA whereas a slow perfusion was present on fluorescein angiography. Six hours after initial examination, flow was detected on OCTA image in the previously occluded artery. Conclusion: This case confirmed the relevance of using OCTA in monitoring BRAO and showed that capillaries with a very slow flow are not visible on OCTA angiograms. It emphasizes that non-perfusion on OCTA should be interpreted with caution

Mission d'expertise du projet Inovaccess 2013 Grenoble: Troisième rapport de l'évaluation & Rapport sur la capitalisation

Inovaccess est un dispositif expérimental de prise en compte de la problématique du handicap par le monde économique. Il s'agit, à la suite de la loi de 2005, d'initier un projet permettant d'avancer dans la réflexion et la mise en œuvre d'actions liées à l'emploi des personnes en situation de handicap et d'être, in fine, force de proposition pour compléter le dispositif réglementaire à l'échelle nationale. La Ville de Grenoble, pilote en matière d'accessibilité, a été retenue par l'AGEFIPH (Association de GEstion du Fonds pour l'Insertion professionnelle des Personnes Handicapées) pour initier cette expérience dans trois quartiers marqués par leur emprise économique et leur faible taux de logements. Parallèlement, il a été demandé à l'équipe de recherche de procéder à un suivi en " continu " du projet, pour être à même de " ré-ajuster " certaines actions si nécessaire, et de procéder à un travail de capitalisation de cette expérience unique, en vue de son éventuelle reproduction, voire extension à d'autres villes

Mission d'expertise du projet Inovaccess 2013 Grenoble: Troisième rapport de l'évaluation & Rapport sur la capitalisation

Inovaccess est un dispositif expérimental de prise en compte de la problématique du handicap par le monde économique. Il s'agit, à la suite de la loi de 2005, d'initier un projet permettant d'avancer dans la réflexion et la mise en œuvre d'actions liées à l'emploi des personnes en situation de handicap et d'être, in fine, force de proposition pour compléter le dispositif réglementaire à l'échelle nationale. La Ville de Grenoble, pilote en matière d'accessibilité, a été retenue par l'AGEFIPH (Association de GEstion du Fonds pour l'Insertion professionnelle des Personnes Handicapées) pour initier cette expérience dans trois quartiers marqués par leur emprise économique et leur faible taux de logements. Parallèlement, il a été demandé à l'équipe de recherche de procéder à un suivi en " continu " du projet, pour être à même de " ré-ajuster " certaines actions si nécessaire, et de procéder à un travail de capitalisation de cette expérience unique, en vue de son éventuelle reproduction, voire extension à d'autres villes

Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model

International audienceIntroductionSmall vessel disease (SVD) of the brain is a leading cause of age- and hypertension-related cognitive decline and disability. Cerebral white matter changes are a consistent manifestation of SVD on neuroimaging, progressing silently for many years before becoming clinically evident. The pathogenesis of these changes remains poorly understood, despite their importance. In particular, their pathological correlate at early stages remains largely undefined. Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), caused by dominant mutations of the NOTCH3 receptor, is regarded as a paradigm for the most common form of sporadic SVD. In this study, we used immunohistochemistry, confocal microscopy and electron microscopy, together with qualitative and quantitative analyses to assess oligodendroglial, axon and myelin damage in TgPAC-Notch3R169C mice, a model of preclinical CADASIL.ResultsThe principal cerebral white matter changes in TgPAC-Notch3R169C mice are microvacuoles (≤1 μm diameter) in the myelin sheaths associated with focal myelin degradation and occurring in the absence of oligodendrocyte loss. Half the damaged myelin sheaths still contain an apparently intact axon. Clearance of myelin debris appears inefficient, as demonstrated by the significant but mild microglial reaction, with occasional myelin debris either contacted or internalized by microglial cells.ConclusionOur findings suggest that segmental intramyelinic oedema is an early, conspicuous white matter change in CADASIL. Brain white matter intramyelinic oedema is consistently found in patients and mouse models with compromised ion and water homeostasis. These data provide a starting point for novel mechanistic studies to investigate the pathogenesis of SVD-related white matter changes