The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt–Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS

ETMR-05: Single-cell transcriptomics of ETMR reveals developmental cellular programs and tumor-pericyte communications in the microenvironment [Abstract]

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are pediatric brain tumors bearing a grim prognosis, despite intensive multimodal therapeutic approaches. Insights into cellular heterogeneity and cellular communication of tumor cells with cells of the tumor microenvironment (TME), by applying single-cell (sc) techniques, potentially identify mechanisms of therapy resistance and target-directed treatment approaches. MATERIAL AND METHODS: To explore ETMR cell diversity, we used single-cell RNA sequencing (scRNA-seq) in human (n=2) and murine ETMR (transgenic mode; n=4) samples, spatial transcriptomics, 2D and 3D cultures (including co-cultures with TME cells), multiplex immunohistochemistry and drug screens. RESULTS: ETMR microenvironment is composed of tumor and non-tumor cell types. The ETMR malignant compartment harbour cells representing distinct transcriptional metaprograms, (NSC-like, NProg-like and Neuroblast-like), mirroring embryonic neurogenic cell states and fuelled by neurogenic pathways (WNT, SHH, Hippo). The ETMR TME is composed of oligodendrocyte and neuronal progenitor cells, neuroblasts, microglia, and pericytes. Tumor-specific ligand-receptor interaction analysis showed enrichment of intercellular communication between NProg-like ETMR cells and pericytes (PC). Functional network analyses reveal ETMR-PC interactions related to stem-cell signalling and extracellular matrix (ECM) organization, involving factors of the WNT, BMP, and CxCl12 networks. Results from ETMR-PC co-culture and spatial transcriptomics pointed to a pivotal role of pericytes in keeping ETMR in a germinal neurogenic state, enriched in stem-cell signalling. Drug screening considering cellular heterogeneity and cellular communication suggested novel therapeutic approaches. CONCLUSION: ETMR demonstrated diversity in the microenvironment, with enrichment of cell-cell communications with pericytes, supporting stem-cell signalling and interfering in the organization of the tumor extracellular matrix. Targeting ETMR-PC interactions might bring new opportunities for target-directed therapy

Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4

Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented

Mouse models of pediatric high-grade gliomas with MYCN amplification reveal intratumoral heterogeneity and lineage signatures

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Simultaneous Brg1 Knockout and MYCN Overexpression in Cerebellar Granule Neuron Precursors Is Insufficient to Drive Tumor Formation but Temporarily Enhances their Proliferation and Delays their Migration

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. According to the World Health Organization (WHO) classification of central nervous system (CNS) tumors, this embryonal tumor is divided into a wingless (WNT)-activated, Sonic hedgehog (SHH)-activated, and non-WNT/non-SHH entity. The latter is poorly defined but frequently carries mutations in Brahma-related gene 1 (BRG1) or amplifications of MYCN. Here, we investigated whether a combination of a Brg1 knockout and an overexpression of MYCN in cerebellar granule neuron precursors or multipotent neural stem cells is sufficient to drive brain tumor formation in mice. To this end, we generated Math1-creE

Malignant gliomas with H3F3A G34R mutation or MYCN amplification in pediatric patients with Li Fraumeni syndrome

Subependymomas are benign tumors characteristically encountered in the posterior fossa of adults that show distinct epigenetic profiles assigned to the molecular group 'subependymoma, posterior fossa' (PFSE) of the recently established DNA methylation-based classification of central nervous system tumors. In contrast, most posterior fossa ependymomas exhibit a more aggressive biological behavior and are allocated to the molecular subgroups PFA or PFB. A subset of ependymomas shows epigenetic similarities with subependymomas, but the precise biology of these tumors and their potential relationships remain unknown. We therefore set out to characterize epigenetic traits, mutational profiles, and clinical outcomes of 50 posterior fossa ependymal tumors of the PFSE group. On histo-morphology, these tumors comprised 12 ependymomas, 14 subependymomas and 24 tumors with mixed ependymoma-subependymoma morphology. Mixed ependymoma-subependymoma tumors varied in their extent of ependymoma differentiation (2-95%) but consistently exhibited global epigenetic profiles of the PFSE group. Selective methylome analysis of microdissected tumor components revealed CpG signatures in mixed tumors that coalesce with their pure counterparts. Loss of chr6 (20/50 cases), as well as TERT mutations (21/50 cases), were frequent events enriched in tumors with pure ependymoma morphology (p < 0.001) and confined to areas with ependymoma differentiation in mixed tumors. Clinically, pure ependymoma phenotype, chr6 loss, and TERT mutations were associated with shorter progression-free survival (each p < 0.001). In conclusion, our results suggest that subependymomas may acquire genetic and epigenetic changes throughout tumor evolution giving rise to subclones with ependymoma morphology (resulting in mixed tumors) that eventually overpopulate the subependymoma component (pure PFSE ependymomas)