Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations.

Tauopathies, such as Alzheimer's disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development.This work was supported by CurePSP, The Cambridge Newton Trust, The William Scholl Foundation and the NIHR Cambridge Biomedical Research Centre (BRC). The contribution of the NC3Rs CRACK IT- Alzheimer’s Research UK is also acknowledged. S.A. holds a BBSRC studentship. A.G.-R. holds a Michael Foster Studentship. O.P. acknowledges BBSRC support. L.V. is supported by the ERC starting grant relieve-IMDs. S.O. and T.L. acknowledge support by the Irish Institute of Clinical Neuroscience. R.T.K. is supported by the Welcome Trust grant 091543/Z/10/Z and D.G. and M.dC.V.-H. are supported by Wellcome Trust grant #098051.This is the final version. It was first published by OUP at http://dx.doi.org/10.1093/brain/awv22

The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo

SummaryResearch over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo “CDS” or “3′ UTR” develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer

Combinatorial CRISPR screen identifies fitness effects of gene paralogues.

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells

Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants.

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors

Disruption of Mouse Cenpj, a Regulator of Centriole Biogenesis, Phenocopies Seckel Syndrome

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpjtm/tm) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpjtm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpjtm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome

Single-cell sequencing reveals clonal expansions of pro-inflammatory synovial CD8 T cells expressing tissue-homing receptors in psoriatic arthritis

Funder: Kennedy Trust studentshipFunder: Oxford-UCB Prize FellowshipFunder: National Institute of Health Research (NIHR) Newcastle Biomedical Research Centre at Newcastle Hospitals Foundation Trust and Newcastle University and Versus Arthritis Research into Inflammatory Arthritis Centre; ref. 22072).Funder: NIHR Birmingham BRC at the University Hospitals Birmingham NHS Foundation Trust and the University of BirminghamFunder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440Funder: National Institute for Health Research (NIHR) Oxford Biomedical Research CentreFunder: St Baldrick’s FoundationAbstract: Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis

Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors.

Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors

Genome-wide <i>in vivo</i> screen identifies novel host regulators of metastatic colonisation

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.status: publishe

Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications

Superparamagnetic iron oxide nanoparticles can providemultiple benefits for biomedical applications in aqueous environments such asmagnetic separation or magnetic resonance imaging. To increase the colloidal stability and allow subsequent reactions, the introduction of hydrophilic functional groups onto the particles’ surface is essential. During this process, the original coating is exchanged by preferably covalently bonded ligands such as trialkoxysilanes. The duration of the silane exchange reaction, which commonly takes more than 24 h, is an important drawback for this approach. In this paper, we present a novel method, which introduces ultrasonication as an energy source to dramatically accelerate this process, resulting in high-quality waterdispersible nanoparticles around 10 nmin size. To prove the generic character, different functional groups were introduced on the surface including polyethylene glycol chains, carboxylic acid, amine, and thiol groups. Their colloidal stability in various aqueous buffer solutions as well as human plasma and serum was investigated to allow implementation in biomedical and sensing applications.status: publishe

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality