mRNA localization is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. To identify mRNAs localized in subcellular compartments of developing neurons, we took an original approach that combines compartmentalized cultures of rat sympathetic neurons and sequential analysis of gene expression (SAGE). Unexpectedly, the most abundant transcript in axons was mRNA for myo-inositol monophosphatase-1 (Impa1), a key enzyme that regulates the inositol cycle and the main target of lithium in neurons. A novel localization element within the 3' untranslated region of Impa1 mRNA specifically targeted Impa1 transcript to sympathetic neuron axons and regulated local IMPA1 translation in response to nerve growth factor (NGF). Selective silencing of IMPA1 synthesis in axons decreased nuclear CREB activation and induced axonal degeneration. These results provide insights into mRNA transport in axons and reveal a new NGF-responsive localization element that directs the targeting and local translation of an axonal transcript.
Down syndrome (DS) is the most common cause of mental retardation. Many neural phenotypes are shared between DS individuals and DS mouse models; however, the common underlying molecular pathogenetic mechanisms remain unclear. Using a transchromosomic model of DS, we show that a 30%-60% reduced expression of Nrsf/Rest (a key regulator of pluripotency and neuronal differentiation) is an alteration that persists in trisomy 21 from undifferentiated embryonic stem (ES) cells to adult brain and is reproducible across several DS models. Using partially trisomic ES cells, we map this effect to a three-gene segment of HSA21, containing DYRK1A. We independently identify the same locus as the most significant eQTL controlling REST expression in the human genome. We show that specifically silencing the third copy of DYRK1A rescues Rest levels, and we demonstrate altered Rest expression in response to inhibition of DYRK1A expression or kinase activity, and in a transgenic Dyrk1A mouse. We reveal that undifferentiated trisomy 21 ES cells show DYRK1A-dose-sensitive reductions in levels of some pluripotency regulators, causing premature expression of transcription factors driving early endodermal and mesodermal differentiation, partially overlapping recently reported downstream effects of Rest +/-. They produce embryoid bodies with elevated levels of the primitive endoderm progenitor marker Gata4 and a strongly reduced neuroectodermal progenitor compartment. Our results suggest that DYRK1A-mediated deregulation of REST is a very early pathological consequence of trisomy 21 with potential to disturb the development of all embryonic lineages, warranting closer research into its contribution to DS pathology and new rationales for therapeutic approaches.
Key Points The Rac effector p21-activated kinase (Pak) regulates hematopoietic engraftment. Pak integrates cytoskeletal changes and proliferation pathways.
SummaryAcquired mutations activating Janus kinase 3 (jak3) have been reported in Down syndrome (DS) and non-DS patients with acute megakaryoblastic leukaemia (AMKL). This highlighted jak3-activation as an important event in the pathogenesis of AMKL, and predicted inhibitors of jak3 as conceptual therapeutics for AMKL. Of 16 DS-transient myeloproliferative disorder (TMD)/AMKL patients tested, seven showed JAK3 mutations. Three mutations deleted the kinase (JH1) domain, abolishing the main function of jak3. Another patient displayed a mutation identical to a previously reported inherited loss-of-function causing severe combined immunodeficiency. Our data suggest that both gain-, and loss-of function mutations of jak3 can be acquired in DS-TMD/AMKL.
Children with Down's syndrome (DS) have 20–50-fold higher incidence of all leukaemias (lymphoid and myeloid), for reasons not understood. As incidence of many solid tumours is much lower in DS, we speculated that disturbed early haematopoietic differentiation could be the cause of increased leukaemia risk. If a common mechanism is behind the risk of both major leukaemia types, it would have to arise before the bifurcation to myeloid and lymphoid lineages. Using the transchromosomic system (mouse embryonic stem cells (ESCs)) bearing an extra human chromosome 21 (HSA21)) we analyzed the early stages of haematopoietic commitment (mesodermal colony formation) in vitro. We observed that trisomy 21 (T21) causes increased production of haemogenic endothelial cells, haematopoietic stem cell precursors and increased colony forming potential, with significantly increased immature progenitors. Transchromosomic colonies showed increased expression of Gata-2, c-Kit and Tie-2. A panel of partial T21 ESCs allowed us to assign these effects to HSA21 sub-regions, mapped by 3.5 kbp-resolution tiling arrays. The Gata-2 increase on one side, and c-Kit and Tie-2 increases on the other, could be attributed to two different, non-overlapping HSA21 regions. Using human-specific small interfering RNA silencing, we could demonstrate that an extra copy of RUNX1, but not ETS-2 or ERG, causes an increase in Tie-2/c-Kit levels. Finally, we detected significantly increased levels of RUNX1, C-KIT and PU.1 in human foetal livers with T21. We conclude that overdose of more than one HSA21 gene contributes to the disturbance of early haematopoiesis in DS, and that one of the contributors is RUNX1. As the observed T21-driven hyperproduction of multipotential immature precursors precedes the bifurcation to lymphoid and myeloid lineages, we speculate that this could create conditions of increased chance for acquisition of pre-leukaemogenic rearrangements/mutations in both lymphoid and myeloid lineages during foetal haematopoiesis, contributing to the increased risk of both leukaemia types in DS.
Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM.
Background The protein kinase ATM (ataxia telangiectasia mutated) mediates cellular response to DNA damage induced by radiation. ATM inhibition decreases DNA damage repair in tumor cells and affects tumor growth. AZD1390 is a novel, highly potent, selective ATM inhibitor designed to cross the blood-brain barrier (BBB) and currently evaluated with radiotherapy in a Phase 1 study in patients with brain malignancies. In the present study, PET was used to measure brain exposure of 11C-labelled AZD1390 after intravenous (i.v.) bolus administration in healthy subjects with an intact BBB. Methods AZD1390 was radiolabeled with carbon-11 and a microdose (mean injected mass 1.21µg) was injected in 8 male subjects (21-65 years). The radioactivity concentration of [ 11C]AZD1390 in brain was measured using a high-resolution PET system. Radioactivity in arterial blood was measured to obtain a metabolite corrected arterial input function for quantitative image analysis. Participants were monitored by laboratory examinations, vital signs, ECG, adverse events (AEs). Results The brain radioactivity concentration of [ 11C]AZD1390 was 0.64 SUV (standard uptake value) and reached maximum 1.00 %ID (percent of injected dose) at Tmax[brain] of 21 min (time of maximum brain radioactivity concentration) after i.v. injection. The whole brain total distribution volume was 5.20 mL*cm -3. No AEs related to [ 11C]AZD1390 were reported. Conclusions This study demonstrates that [ 11C]AZD1390 crosses the intact BBB and supports development of AZD1390 for the treatment of glioblastoma multiforme (GBM) or other brain malignancies. Moreover, it illustrates the potential of PET microdosing in predicting and guiding dose range and schedule for subsequent clinical studies.
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