DOT1L activity affects neural stem cell division mode and reduces differentiation and ASNS expression.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies.

High grade gliomas are the most common brain tumors in adult. These tumors are characterized by a high infiltration in microglial cells and macrophages. The immunosuppressive tumor environment is known to orient immune cells toward a pro-tumoral and anti-inflammatory phenotype. Therefore, the current challenge for cancer therapy is to find a way to reorient macrophages toward an antitumoral phenotype. Previously, we demonstrated that macrophages secreted antitumoral factors when they were invalidated for the proprotein converstase 1/3 (PC1/3) and treated with LPS. However, achieving an activation of macrophages via LPS/TLR4/Myd88-dependent pathway appears yet unfeasible in cancer patients. On the contrary, the antitumor drug Paclitaxel is also known to activate the TLR4 MyD88-dependent signaling pathway and mimics LPS action. Therefore, we evaluated if PC1/3 knock-down (KD) macrophages could be activated by Paclitaxel and efficient against glioma. We report here that such a treatment of PC1/3 KD macrophages drove to the overexpression of proteins mainly involved in cytoskeleton rearrangement. In support of this finding, we found that these cells exhibited a Ca2+ increase after Paclitaxel treatment. This is indicative of a possible depolymerization of microtubules and may therefore reflect an activation of inflammatory pathways in macrophages. In such a way, we found that PC1/3 KD macrophages displayed a repression of the anti-inflammatory pathway STAT3 and secreted more pro-inflammatory cytokines. Extracellular vesicles isolated from these PC1/3 KD cells inhibited glioma growth. Finally, the supernatant collected from the coculture between glioma cells and PC1/3 KD macrophages contained more antitumoral factors. These findings unravel the potential value of a new therapeutic strategy combining Paclitaxel and PC1/3 inhibition to switch macrophages toward an antitumoral immunophenotype.

Absence of the RING domain in MID1 results in patterning defects in the developing human brain.

The X-linked form of Opitz BBB/G syndrome (OS) is a monogenic disorder in which symptoms are established early during embryonic development. OS is caused by pathogenic variants in the X-linked gene MID1 Disease-associated variants are distributed across the entire gene locus, except for the N-terminal really interesting new gene (RING) domain that encompasses the E3 ubiquitin ligase activity. By using genome-edited human induced pluripotent stem cell lines, we here show that absence of isoforms containing the RING domain of MID1 causes severe patterning defects in human brain organoids. We observed a prominent neurogenic deficit with a reduction in neural tissue and a concomitant increase in choroid plexus-like structures. Transcriptome analyses revealed a deregulation of patterning pathways very early on, even preceding neural induction. Notably, the observed phenotypes starkly contrast with those observed in MID1 full-knockout organoids, indicating the presence of a distinct mechanism that underlies the patterning defects. The severity and early onset of these phenotypes could potentially account for the absence of patients carrying pathogenic variants in exon 1 of the MID1 gene coding for the N-terminal RING domain.

Rbfox1 Is Expressed in the Mouse Brain in the Form of Multiple Transcript Variants and Contains Functional E Boxes in Its Alternative Promoters.

The RNA-binding protein RBFOX1 is an important regulator of neuron development and neuronal excitability. Rbfox1 is a dosage-sensitive gene and in both mice and humans, decreased expression of Rbfox1 has been linked to neurodevelopmental disorders. Alternative promoters drive expression of Rbfox1 transcript isoforms that encode an identical protein. The tissue- and developmental stage-specific expression of these isoforms, as well as the underlying regulatory mechanisms, are, however, unclear. Here, we set out to capture all of the Rbfox1 transcript isoforms and identify transcriptional mechanisms that regulate brain-specific Rbfox1 expression. Isoform sequencing identified multiple alternative Rbfox1 transcript variants in the mouse cerebral cortex, including transcripts with novel first exons, alternatively spliced exons and 3'-truncations. Quantitative RT-PCR determined the expression of the alternative first exons in the developing cerebral cortex and different subregions of the juvenile brain. Alternative first exons were found to be highly stage- and subregion specific in their expression patterns suggesting that they fulfill specific functions during cortex development and in different brain regions. Using reporter assays we found that the promoter regions of the two first exons E1B and E1C/E1C.1 contain several functional E-boxes. Together, we provide an extensive picture of Rbfox1 isoform expression. We further identified important regulatory mechanisms that drive neuron-specific Rbfox1 expression. Thus, our study forms the basis for further research into the mechanisms that ensure physiological Rbfox1 expression in the brain. It also helps to understand why, in patients with neurodevelopmental disorders deletion of individual RBFOX1 transcript isoforms could affect brain function.

Proteomic identification of the heterogeneous nuclear ribonucleoprotein K as irradiation responsive protein related to migration.

Irradiation resistance is a major obstacle of head and neck squamous cell carcinoma (HNSCC) therapy, limiting treatment success and patient survival. The aim of our experiments was to identify irradiation-regulated proteins as potential drug targets. Two established HNSCC cell lines (HNSCCUM-01T and HNSCCUM-02T) were treated with a single 8Gy (Gray) fraction of irradiation. Changes in cellular protein expression were studied after 24h by means of 2D-electrophoresis and MALDI-TOF-mass spectrometry. Ninety-four differentially expressed proteins were identified. The expression levels of four proteins were regulated similarly in both cell lines after irradiation treatment, i.e., GRP78, PRDX, ACTC, and the heterogeneous nuclear ribonucleoprotein K (hnRNPK), suggesting a relevant role during irradiation response. hnRNPK as a p53 interacting protein was verified by Western blotting and immunocytochemical staining as well as functionally analyzed. Knock-down by the use of siRNA resulted in only slightly reduced viability, however, migratory activity was strongly reduced. Combined application of siRNA against hnRNPK and irradiation reduced migration almost completely. We conclude that hnRNPK is potentially implicated in the radiogenic response of HNSCC. The inhibition of hnRNPK might reduce the metastasizing potential of HNSCC especially in combination with irradiation and suggest that this molecule should be further evaluated in this context. BIOLOGICAL SIGNIFICANCE: We showed completely impaired migration of irradiated hnRNPK-knock-out HNSCC cells, suggesting this molecule as a potential drug target in combined treatment schedules.