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  1. Article ; Online: Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease.

    Piwecka, Monika / Rajewsky, Nikolaus / Rybak-Wolf, Agnieszka

    Nature reviews. Neurology

    2023  Volume 19, Issue 6, Page(s) 346–362

    Abstract: In the past decade, single-cell technologies have proliferated and improved from their technically challenging beginnings to become common laboratory methods capable of determining the expression of thousands of genes in thousands of cells simultaneously. ...

    Abstract In the past decade, single-cell technologies have proliferated and improved from their technically challenging beginnings to become common laboratory methods capable of determining the expression of thousands of genes in thousands of cells simultaneously. The field has progressed by taking the CNS as a primary research subject - the cellular complexity and multiplicity of neuronal cell types provide fertile ground for the increasing power of single-cell methods. Current single-cell RNA sequencing methods can quantify gene expression with sufficient accuracy to finely resolve even subtle differences between cell types and states, thus providing a great tool for studying the molecular and cellular repertoire of the CNS and its disorders. However, single-cell RNA sequencing requires the dissociation of tissue samples, which means that the interrelationships between cells are lost. Spatial transcriptomic methods bypass tissue dissociation and retain this spatial information, thereby allowing gene expression to be assessed across thousands of cells within the context of tissue structural organization. Here, we discuss how single-cell and spatially resolved transcriptomics have been contributing to unravelling the pathomechanisms underlying brain disorders. We focus on three areas where we feel these new technologies have provided particularly useful insights: selective neuronal vulnerability, neuroimmune dysfunction and cell-type-specific treatment response. We also discuss the limitations and future directions of single-cell and spatial RNA sequencing technologies.
    MeSH term(s) Humans ; Transcriptome/genetics ; Brain ; Gene Expression Profiling ; Brain Diseases/genetics
    Language English
    Publishing date 2023-05-17
    Publishing country England
    Document type Journal Article ; Review ; Research Support, Non-U.S. Gov't
    ZDB-ID 2491514-2
    ISSN 1759-4766 ; 1759-4758
    ISSN (online) 1759-4766
    ISSN 1759-4758
    DOI 10.1038/s41582-023-00809-y
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: RNA Dynamics in Alzheimer's Disease.

    Rybak-Wolf, Agnieszka / Plass, Mireya

    Molecules (Basel, Switzerland)

    2021  Volume 26, Issue 17

    Abstract: Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder that heavily burdens healthcare systems worldwide. There is a significant requirement to understand the still unknown molecular mechanisms underlying AD. Current evidence ... ...

    Abstract Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder that heavily burdens healthcare systems worldwide. There is a significant requirement to understand the still unknown molecular mechanisms underlying AD. Current evidence shows that two of the major features of AD are transcriptome dysregulation and altered function of RNA binding proteins (RBPs), both of which lead to changes in the expression of different RNA species, including microRNAs (miRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs). In this review, we will conduct a comprehensive overview of how RNA dynamics are altered in AD and how this leads to the differential expression of both short and long RNA species. We will describe how RBP expression and function are altered in AD and how this impacts the expression of different RNA species. Furthermore, we will also show how changes in the abundance of specific RNA species are linked to the pathology of AD.
    MeSH term(s) Alzheimer Disease/genetics ; Animals ; Humans ; MicroRNAs/genetics ; RNA/genetics ; RNA, Circular/genetics ; RNA, Long Noncoding/genetics ; RNA, Messenger/genetics ; Transcriptome/genetics
    Chemical Substances MicroRNAs ; RNA, Circular ; RNA, Long Noncoding ; RNA, Messenger ; RNA (63231-63-0)
    Language English
    Publishing date 2021-08-24
    Publishing country Switzerland
    Document type Journal Article ; Review
    ZDB-ID 1413402-0
    ISSN 1420-3049 ; 1431-5165 ; 1420-3049
    ISSN (online) 1420-3049
    ISSN 1431-5165 ; 1420-3049
    DOI 10.3390/molecules26175113
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids.

    Wandres, Miriam / Aigner, Denise / Kastelic, Nicolai / Boltengagen, Anastasiya / Rybak-Wolf, Agnieszka / Rajewsky, Nikolaus

    Journal of visualized experiments : JoVE

    2024  , Issue 205

    Abstract: Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to ... ...

    Abstract Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.
    MeSH term(s) Humans ; Transcriptome ; Brain ; Organoids ; Gene Expression Profiling ; Cell Nucleus
    Language English
    Publishing date 2024-03-29
    Publishing country United States
    Document type Journal Article ; Video-Audio Media
    ZDB-ID 2259946-0
    ISSN 1940-087X ; 1940-087X
    ISSN (online) 1940-087X
    ISSN 1940-087X
    DOI 10.3791/66225
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Sites of transcription initiation drive mRNA isoform selection

    Alfonso-Gonzalez, Carlos / Legnini, Ivano / Holec, Sarah / Arrigoni, Laura / Ozbulut, Hasan Can / Mateos, Fernando / Koppstein, David / Rybak-Wolf, Agnieszka / Bönisch, Ulrike / Rajewsky, Nikolaus / Hilgers, Valérie

    Cell. 2023 May, v. 186, no. 11 p.2438-2455.e22

    2023  

    Abstract: The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, ... ...

    Abstract The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3′ end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3′ end site choice is globally influenced by the site of transcription initiation (TSS). “Dominant promoters,” characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3′ end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.
    Keywords Drosophila ; epigenetics ; humans ; messenger RNA ; nervous system ; organoids ; transcription initiation ; transcription ; mRNA isoform ; 5ʹ-3ʹ coupling ; transcription start site ; alternative polyadenylation ; long-read sequencing ; human brain organoids ; p300/CBP
    Language English
    Dates of publication 2023-05
    Size p. 2438-2455.e22.
    Publishing place Elsevier Inc.
    Document type Article ; Online
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2023.04.012
    Database NAL-Catalogue (AGRICOLA)

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  5. Article ; Online: Spatiotemporal, optogenetic control of gene expression in organoids.

    Legnini, Ivano / Emmenegger, Lisa / Zappulo, Alessandra / Rybak-Wolf, Agnieszka / Wurmus, Ricardo / Martinez, Anna Oliveras / Jara, Cledi Cerda / Boltengagen, Anastasiya / Hessler, Talé / Mastrobuoni, Guido / Kempa, Stefan / Zinzen, Robert / Woehler, Andrew / Rajewsky, Nikolaus

    Nature methods

    2023  Volume 20, Issue 10, Page(s) 1544–1552

    Abstract: Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we ... ...

    Abstract Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.
    MeSH term(s) Humans ; Optogenetics ; Hedgehog Proteins/metabolism ; Organoids/metabolism ; Cell Differentiation ; Gene Expression
    Chemical Substances Hedgehog Proteins
    Language English
    Publishing date 2023-09-21
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2169522-2
    ISSN 1548-7105 ; 1548-7091
    ISSN (online) 1548-7105
    ISSN 1548-7091
    DOI 10.1038/s41592-023-01986-w
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

    Rybak-Wolf, Agnieszka / Wyler, Emanuel / Pentimalli, Tancredi Massimo / Legnini, Ivano / Oliveras Martinez, Anna / Glažar, Petar / Loewa, Anna / Kim, Seung Joon / Kaufer, Benedikt B / Woehler, Andrew / Landthaler, Markus / Rajewsky, Nikolaus

    Nature microbiology

    2023  Volume 8, Issue 7, Page(s) 1252–1266

    Abstract: Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. ... ...

    Abstract Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.
    MeSH term(s) Humans ; Herpesvirus 1, Human/genetics ; Herpes Simplex/complications ; Herpes Simplex/drug therapy ; Acyclovir/pharmacology ; Acyclovir/therapeutic use ; Antiviral Agents/pharmacology ; Antiviral Agents/therapeutic use ; Encephalitis, Viral/drug therapy ; Organoids
    Chemical Substances Acyclovir (X4HES1O11F) ; Antiviral Agents
    Language English
    Publishing date 2023-06-22
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 2058-5276
    ISSN (online) 2058-5276
    DOI 10.1038/s41564-023-01405-y
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Whole-mount in situ hybridization using DIG-labeled probes in planarian.

    Rybak-Wolf, Agnieszka / Solana, Jordi

    Methods in molecular biology (Clifton, N.J.)

    2014  Volume 1211, Page(s) 41–51

    Abstract: In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used ... ...

    Abstract In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used techniques to determine and visualize gene expression patterns in planaria. Here, we present the established version of whole-mount in situ hybridization (WISH) and whole-mount fluorescence in situ hybridization (WFISH) protocol optimized over the last years by several labs from the rapidly growing planaria field and give an overview of recently introduced modifications which can be critical in the study of low abundant transcripts.
    MeSH term(s) Animals ; Base Sequence ; Digoxigenin/analysis ; In Situ Hybridization, Fluorescence/methods ; Planarians/genetics ; RNA Probes/analysis ; RNA Probes/genetics ; RNA, Helminth/analysis ; RNA, Helminth/genetics
    Chemical Substances RNA Probes ; RNA, Helminth ; Digoxigenin (NQ1SX9LNAU)
    Language English
    Publishing date 2014
    Publishing country United States
    Document type Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-1459-3_4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Sites of transcription initiation drive mRNA isoform selection.

    Alfonso-Gonzalez, Carlos / Legnini, Ivano / Holec, Sarah / Arrigoni, Laura / Ozbulut, Hasan Can / Mateos, Fernando / Koppstein, David / Rybak-Wolf, Agnieszka / Bönisch, Ulrike / Rajewsky, Nikolaus / Hilgers, Valérie

    Cell

    2023  Volume 186, Issue 11, Page(s) 2438–2455.e22

    Abstract: The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, ... ...

    Abstract The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.
    MeSH term(s) Humans ; Alternative Splicing ; Polyadenylation ; Promoter Regions, Genetic ; RNA Isoforms/metabolism ; RNA, Messenger/metabolism ; Transcription Initiation Site
    Chemical Substances RNA Isoforms ; RNA, Messenger
    Language English
    Publishing date 2023-05-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2023.04.012
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article: Generation of human brain organoids for mitochondrial disease modeling

    Le, Stephanie / Petersilie, Laura / Inak, Gizem / Menacho-Pando, Carmen / Kafitz, Karl W. / Rybak-Wolf, Agnieszka / Rajewsky, Nikolaus / Rose, Christine R. / Prigione, Alessandro

    Journal of visualized experiments. 2021 June 21, , no. 172

    2021  

    Abstract: Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective ...

    Abstract Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.
    Keywords brain ; humans ; mitochondria ; neurons ; organoids
    Language English
    Dates of publication 2021-0621
    Size p. e62756.
    Publishing place Journal of Visualized Experiments
    Document type Article
    ZDB-ID 2259946-0
    ISSN 1940-087X
    ISSN 1940-087X
    DOI 10.3791/62756
    Database NAL-Catalogue (AGRICOLA)

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  10. Article ; Online: A Circular RNA Expressed from the FAT3 Locus Regulates Neural Development.

    Seeler, Sabine / Andersen, Maria Schertz / Sztanka-Toth, Tamas / Rybiczka-Tešulov, Mateja / van den Munkhof, Marleen H / Chang, Chi-Chih / Maimaitili, Muyesier / Venø, Morten Trillingsgaard / Hansen, Thomas Birkballe / Pasterkamp, R Jeroen / Rybak-Wolf, Agnieszka / Denham, Mark / Rajewsky, Nikolaus / Kristensen, Lasse Sommer / Kjems, Jørgen

    Molecular neurobiology

    2023  Volume 60, Issue 6, Page(s) 3239–3260

    Abstract: Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, ...

    Abstract Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, or migration.
    MeSH term(s) Mice ; Animals ; Humans ; RNA, Circular/genetics ; Cell Differentiation/genetics ; Neurogenesis/genetics ; Neural Stem Cells ; Neocortex ; Epidermal Growth Factor ; Cadherins
    Chemical Substances RNA, Circular ; FAT3 protein, human ; Epidermal Growth Factor (62229-50-9) ; Cadherins
    Language English
    Publishing date 2023-02-25
    Publishing country United States
    Document type Journal Article
    ZDB-ID 645020-9
    ISSN 1559-1182 ; 0893-7648
    ISSN (online) 1559-1182
    ISSN 0893-7648
    DOI 10.1007/s12035-023-03253-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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