Altered human oligodendrocyte heterogeneity in multiple sclerosis

Oligodendrocyte pathology is increasingly implicated in neurodegenerative diseases as oligodendrocytes both myelinate and provide metabolic support to axons. In multiple sclerosis (MS), demyelination in the central nervous system thus leads to neurodegeneration, but the severity of MS between patients is very variable. Disability does not correlate well with the extent of demyelination1, which suggests that other factors contribute to this variability. One such factor may be oligodendrocyte heterogeneity. Not all oligodendrocytes are the same—those from the mouse spinal cord inherently produce longer myelin sheaths than those from the cortex2, and single-cell analysis of the mouse central nervous system identified further differences3,4. However, the extent of human oligodendrocyte heterogeneity and its possible contribution to MS pathology remain unknown. Here we performed single-nucleus RNA sequencing from white matter areas of post-mortem human brain from patients with MS and from unaffected controls. We identified subclusters of oligodendroglia in control human white matter, some with similarities to mouse, and defined new markers for these cell states. Notably, some subclusters were underrepresented in MS tissue, whereas others were more prevalent. These differences in mature oligodendrocyte subclusters may indicate different functional states of oligodendrocytes in MS lesions. We found similar changes in normal-appearing white matter, showing that MS is a more diffuse disease than its focal demyelination suggests. Our findings of an altered oligodendroglial heterogeneity in MS may be important for understanding disease progression and developing therapeutic approaches

Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers.Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.Fil: Gómez Acuña, Luciana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nazer, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rodríguez Seguí, Santiago Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Pozzi, María Berta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Buggiano, Valeria Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Marasco, Luciano Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Agirre, Eneritz. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: He, Cody. University of Chicago; Estados UnidosFil: Alló, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure

Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis

Multiple sclerosis (MS) is characterized by an immune system attack targeting myelin, which is produced by oligodendrocytes (OLs). We performed single-cell transcriptomic analysis of OL lineage cells from the spinal cord of mice induced with experimental autoimmune encephalomyelitis (EAE), which mimics several aspects of MS. We found unique OLs and OL precursor cells (OPCs) in EAE and uncovered several genes specifically alternatively spliced in these cells. Surprisingly, EAE-specific OL lineage populations expressed genes involved in antigen processing and presentation via major histocompatibility complex class I and II (MHC-I and -II), and in immunoprotection, suggesting alternative functions of these cells in a disease context. Importantly, we found that disease-specific oligodendroglia are also present in human MS brains and that a substantial number of genes known to be susceptibility genes for MS, so far mainly associated with immune cells, are expressed in the OL lineage cells. Finally, we demonstrate that OPCs can phagocytose and that MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. Our results suggest that OLs and OPCs are not passive targets but instead active immunomodulators in MS. The disease-specific OL lineage cells, for which we identify several biomarkers, may represent novel direct targets for immunomodulatory therapeutic approaches in MS

Epigenetics in alternative splicing : links between chromatin structure, transcription and non-coding RNA mediated regulation

Generalment s'ha pensat que la regulació de l'splicing alternatiu està controlada principalment per la interacció entre els factors reguladors de l'splicing i la taxa d'elongació de la ARN polimerasa II (RNAPII). Hi ha un evidencia emergent de la complexitat de la regulació de l'splicing alternatiu, que ara també inclou l'activitat d'ARNs no codificants i l'estat de la cromatina. Diverses experiments han demostrat que modificacions en les histones poden regular la inclusió d'exons alternatius, i que la taxa d'elongació de la RNAPII pot estar influenciada pels diferents estats de la cromatina. Els ARNs petits (sRNAs) són una família d'ARNs no codificants associats amb membres de la família de proteïnes Argonauta i són efectors de la via de silenciació gènica. Alguns sRNAs participen en una via alternativa anomenada via de silenciació gènica transcripcional (TGS). Evidències experimentals ha mostrat que els sRNAs interferents que s'uneixen a introns poden promoure l'aparició de modificacions en les histones que alteren la taxa de elongació de la transcripció provocant canvis en l'splicing alternatiu. Aquesta via és coneguda com via de silenciació gènica transcripcional acoplada a splicing alternatiu (TGS-AS). Tenint aixó en compte, nosaltres vam proposar que la proteïna Argonauta 1 (AGO1), podria induir la formació d'heterocromatina i canviar l'splicing alternatiu alterant l'elongació de la RNAPII. Per tal de realitzar una análisi a escala genómica de la regulació de l'splicing alternatiu, hem utilitzat dades provinents de noves tècniques de seqüenciació a gran escala, com ChIP-Seq i RNA-Seq. Hem trobat que hi ha regulació d'splicing alternatiu depenent d'AGO1. Els nostres resultats suggereixen que ARNs interferents endógens podrien estar relacionats amb aquesta regulació. A més, a la part final de la tesi demostrem que hi ha un codi de cromatina que requereix AGO1 que regula l'splicing alternatiu i que és específic per diferents tipus cel·lulars. Adicionalment hem trobat que altres efectors, com CTCF i HP1 alpha, també sòn importants per explicar els canvis en l'splicing dels pre-ARNs. Conjunatment amb altres treballs, aquesta tesis demostra que la regulació de l'splicing alternatiu implica la funció de molts components nuclears i probablement de molts altres que encara han de ser descoberts.The regulation of alternative splicing has been generally thought of being primarily controlled by the interaction of splicing factors with the RNA molecule and by the elongation rate of the RNA polymerase II (RNAPII). There is an emerging understanding of the complexity of how alternative splicing is regulated which now involves the activity of non-coding RNAs and the chromatin state. Different experiments have shown that histone modifications can regulate the inclusion of alternative exons and that the elongation rate of the RNAPII could be influenced by different chromatin states. In this sense, small RNAs (sRNAs), which are a family of non-coding RNAs associated with members of the Argonaute family of proteins, that are effectors of the silencing pathway, which can participate in an alternative pathway known as transcriptional gene silencing (TGS). Experimental evidence shows that siRNAs targeting introns can induce chromatin marks that affect the rate of transcriptional elongation, affecting the splicing of pre-mRNAs, which is called transcriptional gene silencing alternative splicing (TGS-AS) \citep{Allo2009}. Thus, we proposed that the Argonaute protein (AGO1) could trigger heterochromatin formation and affect splicing by affecting RNAPII elongation. In order to perform a genome-wide analysis of the regulation of alternative splicing we used new highthroughput sequencing technologies as ChIP-Seq and RNA-Seq. We found that there is AGO1 dependent alternative splicing regulation, and our results suggest that endogenous sRNAs could be involved. Additionally, in the last part of the thesis we show a cell specific alternative splicing chromatin code, which also involves AGO1. Even though AGO1 regulation of alternative splicing was related to some specific cases, we found that other effectors, CTCF and HP1$\alpha$ were also important for the splicing changes decisions. This thesis and other recent reports show the regulation of alternative splicing as an integrated process

Databases and resources for human small non-coding RNAs

Recent advances in high-throughput sequencing have facilitated the genome-wide studies of small non-coding RNAs (sRNAs). Numerous studies have highlighted the role of various classes of sRNAs at different levels of gene regulation and disease. The fast growth of sequence data and the diversity of sRNA species have prompted the need to organise them in annotation databases. There are currently several databases that collect sRNA data. Various tools are provided for access, with special emphasis on the well-characterised family of micro-RNAs. The striking heterogeneity of the new classes of sRNAs and the lack of sufficient functional annotation, however, make integration of these datasets a difficult task. This review describes the currently available databases for human sRNAs that are accessible via the internet, and some of the large datasets for human sRNAs from high-throughput sequencing experiments that are so far only available as supplementary data in publications. Some of the main issues related to the integration and annotation of sRNA datasets are also discussed

Databases and resources for human small non-coding RNAs

Recent advances in high-throughput sequencing have facilitated the genome-wide studies of small non-coding RNAs (sRNAs). Numerous studies have highlighted the role of various classes of sRNAs at different levels of gene regulation and disease. The fast growth of sequence data and the diversity of sRNA species have prompted the need to organise them in annotation databases. There are currently several databases that collect sRNA data. Various tools are provided for access, with special emphasis on the well-characterised family of micro-RNAs. The striking heterogeneity of the new classes of sRNAs and the lack of sufficient functional annotation, however, make integration of these datasets a difficult task. This review describes the currently available databases for human sRNAs that are accessible via the internet, and some of the large datasets for human sRNAs from high-throughput sequencing experiments that are so far only available as supplementary data in publications. Some of the main issues related to the integration and annotation of sRNA datasets are also discussed

Methods to Study Splicing from RNA-Seq

<p>Graphical representation of methods to study splicing from RNA-Seq. Methods are divided according to whether they perform Mapping, Reconstruction of events or isoforms, Quantification of events and/or isoforms and whether they can perform a Comparison between two or more conditions of event or isoform relative abundances, or of isoform expression. We only list the Mapping methods that are spliced-mappers or the ones that use some heuristics to map to known exon and junctions. Methods for Reconstruction (blue), Quantification (green) and Comparison (red) are divided according to whether they work with isoforms (lighter color) or with events (darker color). Methods that work at both levels, are overlapped by the two color tones. Some methods perform reconstruction and quantification and are grouped with those that only perform reconstruction. Some mapping methods also perform quantification and are repeated in two levels. Methods that require an annotation are indicated. Quantification methods that work with or without annotation are in different groups. Solid arrows connect Mapping methods to the tools in the other three levels; since, in principle, any Mapping method producing BAM as output could be fed to methods reading BAM as input. Some methods perform Mapping and Quantification or Mapping and Differential Splicing, and are connected with a solid arrow too. We indicate with dashed gray arrows those cases when a Comparison method can use the output from a Quantification method.</p

An Atlas of Vagal Sensory Neurons and Their Molecular Specialization

Summary: Sensory functions of the vagus nerve are critical for conscious perceptions and for monitoring visceral functions in the cardio-pulmonary and gastrointestinal systems. Here, we present a comprehensive identification, classification, and validation of the neuron types in the neural crest (jugular) and placode (nodose) derived vagal ganglia by single-cell RNA sequencing (scRNA-seq) transcriptomic analysis. Our results reveal major differences between neurons derived from different embryonic origins. Jugular neurons exhibit fundamental similarities to the somatosensory spinal neurons, including major types, such as C-low threshold mechanoreceptors (C-LTMRs), A-LTMRs, Aδ-nociceptors, and cold-, and mechano-heat C-nociceptors. In contrast, the nodose ganglion contains 18 distinct types dedicated to surveying the physiological state of the internal body. Our results reveal a vast diversity of vagal neuron types, including many previously unanticipated types, as well as proposed types that are consistent with chemoreceptors, nutrient detectors, baroreceptors, and stretch and volume mechanoreceptors of the respiratory, gastrointestinal, and cardiovascular systems. : Visceral sensory neurons are necessary for the control of organ functions, but knowledge on the complexity of neuron types involved is missing. Kupari et al. molecularly identify jugular and nodose ganglion neurons and find a large diversity of neuron types that are consistent with the numerous sensory functions of the vagus nerve. Keywords: single cell RNA-sequencing, vagus nerve, sensory neurons, viscerosensory, somatosensory, transcriptome, jugular ganglion, nodose ganglion, mechanoreceptor, nocicepto