Analyse der intranukleären Dynamik und des Exports der ribosomalen 60S-Untereinheit

Die Proteinbiosynthese wird von Ribosomen - großen molekularen Maschinen - durchgeführt, die aus zahlreichen verschiedenen Proteinen und einem ribosomalen RNA-Rückgrat bestehen. Die Biogenese der ribosomalen Untereinheiten von Säugetieren beginnt im Nukleolus mit dem 90S-Vorläuferpartikel, das sukzessive in die prä-40S- und prä-60S-Untereinheiten gespalten wird. In weiteren Prozessierungsschritten werden die Untereinheiten mit Exportrezeptoren bela-den, was ihre Durchquerung der Kernporenkomplexe ins Zytoplasma ermöglicht. Hier erfolgt die Freisetzung der Exportfaktoren und beide Untereinheiten können zusammen das finale Ribosom bilden. Die ribosomale Biogenese ist bisher mit biochemischen, genetischen und elektronenmikroskopi-schen Methoden sehr detailliert untersucht worden, jedoch stellt die Bestimmung der in vivo Kinetik in lebenden Zellen immer noch eine große Herausforderung dar. In dieser Arbeit wurde die Export-Kinetik der großen ribosomalen Untereinheiten ("prä-60S-Partikel") durch einzelne Kernporenkomplexe in lebenden menschlichen Zellen bestimmt. Zur Untersuchung dieses Prozesses in vivo, wurde eine stabile Zelllinie erstellt, welche HaloTag-markiertes eIF6 und GFP-gebundenes NTF2 ko-exprimiert, zur simultanen Beobachtung der großen 60S-Untereinheit (eIF6) und der Kernporen (NTF2) . Durch eine Kombination von hoch-auflösender konfokaler Mikroskopie mit einer schnellen und sensitiven Einzelmolekülverfolgung in einem selbstentwickelten Mikroskop-Aufbau konnte die Dynamik einzelner prä-60S-Partikel während der Interaktion mit und des Exports durch einzelne Kernporen sichtbar gemacht wer-den. Auf diese Weise ergaben sich bisher noch nie gezeigte Einblicke in die Kinetik und Dynamik dieses zellulären Schlüsselprozesses. In dieser Studie zeigte sich, dass für Export-Ereignisse die prä-60S-Partikel vor allem im Zentrum der Kernpore akkumulieren, während nicht-erfolgreiche Exporte innerhalb des nukleären Korbs abbrechen. Der Exportvorgang erfolgt mit nur einem geschwindigkeitsbestimmendem Schritt und einer Translokationszeit von ~24 ms. Nur 1/3 der Exportvorgänge waren erfolg-reich. Unter Berücksichtigung der molekularen Masse der prä-60S-Partikel folgte außerdem, dass der Massenfluss durch eine einzelne Kernpore in vivo bis zu ~125 MDa/s betragen kann.Protein biosynthesis is carried out by ribosomes – large molecular machines – consisting of numerous different proteins and a ribosomal RNA backbone. The biogenesis of mammalian ribosomal subunits begins in the nucleolus with the 90S precursor particle, which is subsequently split into the pre‐40S and pre‐60S subunits. During further processing steps the subunits are loaded with export receptors, which allows for their passage through the nuclear pore complexes into the cytoplasm. Here the export factors are released and both subunits can form the final ribosome. Ribosomal biogenesis has been studied in great detail by biochemical, genetic and electron microscopic approaches, however, determining in vivo kinetics in living cells is still a major challenge. In this work, the export kinetics of large ribosomal subunits (“pre-60S particle”) through single nuclear pore complexes in living human cells was analysed. To study this process in vivo, a stable cell line co-expressing HaloTag-tagged eIF6 and GFP-fused NTF2 was established to simultaneously label ribosomal 60S subunits (eIF6) and NPCs (NTF2). By combining high-resolution confocal microscopy with rapid and sensitive single-molecule tracking in a highly customized microscopic setup, the dynamics of single pre-60S particles during interaction with and export through single nuclear pores could be visualized. In this way, previously unprecedented insights into the kinetics and dynamics of this key cellular process emerged. In this study, it was shown that for export events, pre-60S particles accumulate primarily in the centre of the nuclear pore, while unsuccessful exports terminate within the nuclear basket. The export event occurs with only one rate-determining step and a translocation time of ~24 milliseconds. Only about 1/3 of the export events were successful. Considering the molecular mass of the pre-60S particles, it could be concluded that the mass flow through a single nuclear pore in vivo can be as high as ~125 MDa/s

Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation

The coming decade of digital brain research: a vision for neuroscience at the intersection of technology and computing

In recent years, brain research has indisputably entered a new epoch, driven by substantial methodological advances and digitally enabled data integration and modelling at multiple scales— from molecules to the whole brain. Major advances are emerging at the intersection of neuroscience with technology and computing. This new science of the brain combines high-quality research, data integration across multiple scales, a new culture of multidisciplinary large-scale collaboration and translation into applications. As pioneered in Europe’s Human Brain Project (HBP), a systematic approach will be essential for meeting the coming decade’s pressing medical and technological challenges. The aims of this paper are to: develop a concept for the coming decade of digital brain research, discuss this new concept with the research community at large, to identify points of convergence, and derive therefrom scientific common goals; provide a scientific framework for the current and future development of EBRAINS, a research infrastructure resulting from the HBP’s work; inform and engage stakeholders, funding organisations and research institutions regarding future digital brain research; identify and address the transformational potential of comprehensive brain models for artificial intelligence, including machine learning and deep learning; outline a collaborative approach that integrates reflection, dialogues and societal engagement on ethical and societal opportunities and challenges as part of future neuroscience research

Papillary tumor of the pineal region : A distinct molecular entity

Papillary tumor of the pineal region (PTPR) is a neuroepithelial brain tumor, which might pose diagnostic difficulties and recurs often. Little is known about underlying molecular alterations. We therefore investigated chromosomal copy number alterations, DNA methylation patterns and mRNA expression profiles in a series of 24 PTPRs. Losses of chromosome 10 were identified in all 13 PTPRs examined. Losses of chromosomes 3 and 22q (54%) as well as gains of chromosomes 8p (62%) and 12 (46%) were also common. DNA methylation profiling using Illumina 450k arrays reliably distinguished PTPR from ependymomas and pineal parenchymal tumors of intermediate differentiation. PTPR could be divided into two subgroups based on methylation pattern, PTPR group 2 showing higher global methylation and a tendency toward shorter progression-free survival (P = 0.06). Genes overexpressed in PTPR as compared with ependymal tumors included SPDEF, known to be expressed in the rodent subcommissural organ. Notable SPDEF protein expression was encountered in 15/19 PTPRs as compared with only 2/36 ependymal tumors, 2/19 choroid plexus tumors and 0/23 samples of other central nervous system (CNS) tumor entities. In conclusion, PTPRs show typical chromosomal alterations as well as distinct DNA methylation and expression profiles, which might serve as useful diagnostic tools

The coming decade of digital brain research - A vision for neuroscience at the intersection of technology and computing

Brain research has in recent years indisputably entered a new epoch, driven by substantial methodological advances and digitally enabled data integration and modeling at multiple scales – from molecules to the whole system. Major advances are emerging at the intersection of neuroscience with technology and computing. This new science of the brain integrates high-quality basic research, systematic data integration across multiple scales, a new culture of large-scale collaboration and translation into applications. A systematic approach, as pioneered in Europe’s Human Brain Project (HBP), will be essential in meeting the pressing medical and technological challenges of the coming decade. The aims of this paper are To develop a concept for the coming decade of digital brain research To discuss it with the research community at large, with the aim of identifying points of convergence and common goals To provide a scientific framework for current and future development of EBRAINS To inform and engage stakeholders, funding organizations and research institutions regarding future digital brain research To identify and address key ethical and societal issues While we do not claim that there is a ‘one size fits all’ approach to addressing these aspects, we are convinced that discussions around the theme of digital brain research will help drive progress in the broader field of neuroscience

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap-/- mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap-/- mice. Consequently, both Tnfr1-/-Xiap-/- and Tnfr2-/-Xiap-/- mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap-/- mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap-/- mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap-/- mice. Consequently, both Tnfr1-/-Xiap-/- and Tnfr2-/-Xiap-/- mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap-/- mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis

The coming decade of digital brain research - A vision for neuroscience at the intersection of technology and computing

Brain research has in recent years indisputably entered a new epoch, driven by substantial methodological advances and digitally enabled data integration and modeling at multiple scales – from molecules to the whole system. Major advances are emerging at the intersection of neuroscience with technology and computing. This new science of the brain integrates high-quality basic research, systematic data integration across multiple scales, a new culture of large-scale collaboration and translation into applications. A systematic approach, as pioneered in Europe’s Human Brain Project (HBP), will be essential in meeting the pressing medical and technological challenges of the coming decade. The aims of this paper are To develop a concept for the coming decade of digital brain research To discuss it with the research community at large, with the aim of identifying points of convergence and common goals To provide a scientific framework for current and future development of EBRAINS To inform and engage stakeholders, funding organizations and research institutions regarding future digital brain research To identify and address key ethical and societal issues While we do not claim that there is a ‘one size fits all’ approach to addressing these aspects, we are convinced that discussions around the theme of digital brain research will help drive progress in the broader field of neuroscience. Comments on this manuscript are welcome This manuscript is a living document that is being further developed in a participatory process. The work has been initiated by the Science and Infrastructure Board of the Human Brain Project (HBP). Now, the entire research community is invited to contribute to shaping the vision by submitting comments. Comments can be submitted via an online commentary form here. All submitted comments will be considered and discussed. The final decision on whether edits or additions will be made to the next version of the manuscript based on an individual comment will be made by the Science and Infrastructure Board (SIB) of the Human Brain Project (HBP) at regular intervals. New versions of the manuscript will be published every few months on Zenodo. Comments may be submitted until the beginning of 2023. During the Human Brain Project Summit 2023, the manuscript will be adopted by HBP and non-HBP participants, and a final version will be published shortly after