40 research outputs found
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Cytokine receptor-like factor 3 (CRLF3): A novel regulator of platelet biogenesis and potential drug target for thrombocythaemia
Get PDFThrombocythaemia is defined as a circulating platelet count above 450x10/L in humans. The major cause of thrombocythaemia is reactive thrombocythaemia which occurs secondary to many conditions such as infection, cancer and inflammation. However, acquired clonal mutations in mainly Janus Kinas 2 , CALR and MPL cause essential thrombocythaemia . ET is a rare disease that leads to an increased risk of cardiovascular thrombotic events. Current treatment of ET uses combination of low dose aspirin to decrease platelet function and cytoreductive agents to decrease thrombopoiesis. The most commonly used cytoreductive agents are hydroxyurea, anagrelide and interferon- and all have unwanted side effects.
Cytokine receptor-like factor 3 is a 2.4kb gene that is ubiquitously expressed throughout the haematopoietic system. Very little is known about the function of CRLF3, with only one peer reviewed journal article in the literature which shows that CRLF3 may negatively regulate the cell cycle at the G0/G1 phase. However, nothing is known about the role of CRLF3 in platelet biology. Using a Crlf3 knockout mouse developed by the Wellcome Trust Sanger Institute we show CRLF3’s role in platelet biogenesis and how it could be used as a novel therapeutic target to treat ET.
Crlf3-/- mice have an isolated and sustained 25-40 decrease in platelet count compared to wildtype controls. Platelet function is unaffected as demonstrated in a range in a range of in vitro assays. The thrombocytopenia is a consequence of abnormalities in hematopoietic cells, as shown by bone marrow transplantations. Megakaryopoiesis is upregulated in Crlf3-/- mice and proplatelet morphology is unaffected, suggesting the thrombocytopenia is due to increased platelet clearance. Indeed, splenectomised Crlf3-/- mice show normalised platelet counts within 7 days, showing rapid splenic removal of platelets is responsible for the thrombocytopenia. Abnormal large platelet structures that resemble proplatelets shafts are abnormally present in the circulation of elderly Crlf3/- mice. Immunohistochemistry showed increased and aberrant tubulin expression in Crlf3-/- platelets compared to WT controls, especially in the preplatelet forms. Cold induced depolymerisation of microtubules was decreased in Crlf3-/- platelets, suggestive of increased tubulin stability, however, the ratio of detyrosinated to tyrosinated tubulin was not altered.
We then crossbred Crlf3-/- mice with JAK2 V617F ET mice, to determine the effect of Crlf3 ablation of thrombocythaemia. Crossbred mice showed restoration of platelet counts to WT values without grossly affecting platelet function or other blood lineages, providing the rational for CRLF3 as a novel therapeutic target for treatment of ET.
Finally, we aimed to resolve the crustal structure of CRLF3 and discover its interactome. To this end, we were able to resolve the crystal structure of a C-terminal portion of the full length protein containing the predicted fibronectin type III domain. To shed light on the interactome of CRLF3, endogenous CRLF3 was tagged with a tandem affinity purification tag using CRISPR/Cas9 technology in induced pluripotent stem cells . We have been able to produce megakaryocytes from these TAP-tagged iPSCs by forward programming. However, as yet we have not been able to generate enough MKs to have adequate material to perform immunoprecipitation assays. Therefore, the interactome of CRLF3 in MKs remains unknown.
In conclusion, we identified a mechanism by which Crlf3 controls platelet biogenesis. Slowed maturation of Crlf3-/- preplatelets in the peripheral circulation potentially due to increased structural stability leads to rapid removal of these immature forms by the spleen and therefore a decrease in platelet count. The isolated effect on platelet numbers and normalisation of platelet count in ET mice deficient of Crlf3 provides the rational for further study on CRLF3 drug targeting as a novel therapeutic strategy for ET.Thesis funded by the British Heart Foundatio
The Salmonella enterica serovar Typhimurium virulence factor STM3169 is a hexuronic acid binding protein component of a TRAP transporter
Get PDFFace valid phenotypes in a mouse model of the most common mutation in EEF1A2 related neurodevelopmental disorder, E122K
Get PDFDe novo heterozygous missense mutations in EEF1A2, encoding neuromuscular translation-elongation factor eEF1A2, are associated with developmental and epileptic encephalopathies. We used CRISPR/Cas9 to recapitulate the most common mutation, E122K, in mice. Although E122K heterozygotes were not observed to have convulsive seizures, they exhibited frequent electrographic seizures and EEG abnormalities, transient early motor deficits and growth defects. Both E122K homozygotes and Eef1a2-null mice developed progressive motor abnormalities, with E122K homozygotes reaching humane endpoints by P31. The null phenotype is driven by progressive spinal neurodegeneration; however, no signs of neurodegeneration were observed in E122K homozygotes. The E122K protein was relatively stable in neurons yet highly unstable in skeletal myocytes, suggesting that the E122K/E122K phenotype is instead driven by loss of function in muscle. Nevertheless, motor abnormalities emerged far earlier in E122K homozygotes than in nulls, suggesting a toxic gain of function and/or a possible dominant-negative effect. This mouse model represents the first animal model of an EEF1A2 missense mutation with face-valid phenotypes and has provided mechanistic insights needed to inform rational treatment design.</p
Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice.
Get PDFNBEAL2 encodes a multidomain scaffolding protein with a putative role in granule ontogeny in human platelets. Mutations in NBEAL2 underlie gray platelet syndrome (GPS), a rare inherited bleeding disorder characterized by a lack of α-granules within blood platelets and progressive bone marrow fibrosis. We present here a novel Nbeal2(-/-) murine model of GPS and demonstrate that the lack of α-granules is due to their loss from platelets/mature megakaryocytes (MKs), and not by initial impaired formation. We show that the lack of Nbeal2 confers a proinflammatory phenotype to the bone marrow MKs, which in combination with the loss of proteins from α-granules drives the development of bone marrow fibrosis. In addition, we demonstrate that α-granule deficiency impairs platelet function beyond their purely hemostatic role and that Nbeal2 deficiency has a protective effect against cancer metastasis.This work was funded by the British Heart
Foundation to CG (FS09/039) and WHO and AR (RG/09/12/28096); NHSBT to CB and HM;
Wellcome Trust (WT098051) to ZM, ELC, JE, HWJ and AOS.This is the accepted manuscript. The final published version is available from Blood at http://www.bloodjournal.org/content/early/2014/09/25/blood-2014-04-566760
Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.
Get PDFSomatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF
Platelet-Associated Matrix Metalloproteinases Regulate Thrombus Formation and Exert Local Collagenolytic Activity
Get PDFObjective Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, such as vascular remodeling. Members of the matrix metalloproteinase (MMP) family not only remodel the extracellular matrix but also modulate platelet function. Here, we made a systematic comparison of the roles of MMP family members in acute thrombus formation under flow conditions and assessed platelet-dependent collagenolytic activity over time. Approach and Results Pharmacological inhibition of MMP-1 or MMP-2 (human) or deficiency in MMP-2 (mouse) suppressed collagen-dependent platelet activation and thrombus formation under flow, whereas MMP-9 inhibition/deficiency stimulated these processes. The absence of MMP-3 was without effect. Interestingly, MMP-14 inhibition led to the formation of larger thrombi, which occurred independently of its capacity to activate MMP-2. Platelet thrombi exerted local collagenolytic activity capable of cleaving immobilized dye-quenched collagen and fibrillar collagen fibers within hours, with loss of the majority of the platelet adhesive properties of collagen as a consequence. This collagenolytic activity was redundantly mediated by platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 but occurred independently of platelet -granule release (Nbeal2(-/-) mice). The latter was in line with subcellular localization experiments, which indicated a granular distribution of MMP-1 and MMP-2 in platelets, distinct from -granules. Whereas MMP-9 protein could not be detected inside platelets, activated platelets did bind plasma-derived MMP-9 to their plasma membrane. Overall, platelet MMP activity was predominantly membrane-associated and influenced by platelet activation status. Conclusions Platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 differentially modulate acute thrombus formation and at later time points limit thrombus formation by exerting collagenolytic activity
Effects of iron supplements and iron-containing micronutrient powders on the gut microbiome in Bangladeshi infants: a randomized controlled trial
Get PDFAnemia is highly prevalent globally, especially in young children in low-income countries, where it often overlaps with a high burden of diarrheal disease. Distribution of iron interventions (as supplements or iron-containing multiple micronutrient powders, MNPs) is a key anemia reduction strategy. Small studies in Africa indicate iron may reprofile the gut microbiome towards pathogenic species. We seek to evaluate the safety of iron and MNPs based on their effects on diversity, composition, and function of the gut microbiome in children in rural Bangladesh as part of a large placebo-controlled randomized controlled trial of iron or MNPs given for 3 months (ACTRN12617000660381). In 923 infants, we evaluate the microbiome before, immediately following, and nine months after interventions, using 16S rRNA gene sequencing and shotgun metagenomics in a subset. We identify no increase in diarrhea with either treatment. In our primary analysis, neither iron nor MNPs alter gut microbiome diversity or composition. However, when not adjusting for multiple comparisons, compared to placebo, children receiving iron and MNPs exhibit reductions in commensal species (e.g., Bifidobacterium, Lactobacillus) and increases in potential pathogens, including Clostridium. These increases are most evident in children with baseline iron repletion and are further supported by trend-based statistical analyses
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CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.
Get PDFThe process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia
Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study
Get PDFTranslation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.</p
Investigating the impact of EEF1A2 missense mutations on protein synthesis in neurodevelopmental disorders
No full textMore than 50 independent de novo missense mutations in EEF1A2 have been identified in several hundred individuals with neurodevelopmental disorders, predominantly epilepsy, intellectual disability, and autism. EEF1A2 encodes a muscle and neuron-specific elongation factor, responsible for the delivery of aminoacyl-tRNAs to the ribosome during mRNA translation, though various secondary functions have been reported. There is therefore a large scope for mutations to impact on protein synthesis and neuronal homeostasis, consistent with growing evidence for the role of dysregulated proteostasis in neurological disorders.
Current evidence suggests that the eEF1A2 missense mutations act through a combination of loss and gain of function mechanisms. To gain a deeper insight into the mechanistic basis of this disorder, I used computational methods to model the impact of each variant on eEF1A2 stability and oligomeric state. FoldX analysis predicted that some mutations disrupt eEF1A2 stability, whilst others act by disturbing dimerization. Whilst binding to aminoacylated tRNAs is mutually exclusive to dimerization, eEF1A2 dimers have been observed to interact with the actin cytoskeleton, so the ratio of monomers to dimers may play a key role in elucidating how certain mutations influence protein function.
Investigating the functional consequences of eEF1A2 mutations is challenging due to the confounding expression of an independently encoded isoform, eEF1A1, in all cultured cell lines. Despite previous research finding translational errors in yeast, a dual-fluorescence assay observed no increase in amino acid misincorporation in HEK293T cells expressing EEF1A2ᴱ¹²²ᴷ cDNA. Mistranslation events however could have been masked by endogenous expression of eEF1A1. With the aim of overcoming this confound, an shRNA-based system was established for the inducible knockdown of eEF1A1 in vitro. Induction of shRNA demonstrated that eEF1A1 knockdown compromised cell viability, a phenotype that could be rescued by expression of eEF1A1 cDNA, but not eEF1A2 cDNA. This suggests a cellular role unique to eEF1A1, and demonstrates the challenges of establishing genotype-phenotype relationships for eEF1A2 mutations.
Knowledge of the cellular consequences of these mutations, and their implications for neuronal and brain function, is limited. In order to characterise the effect of mutant eEF1A2 on the neuronal translatome, a translating ribosome affinity purification (TRAP) strategy was used to isolate ribosome-associated mRNAs. TRAP utilises a cell-specific ribosomal tag, enabling the removal of unwanted, eEF1A1-expressing cell types. Coupled with RNA sequencing (TRAP-seq), this methodology was used to identify mis-translating mRNAs in the hippocampus of an EEF1A2ᴱ¹²²ᴷ/+ mouse model. The data shows that the E122K mutation leads to changes primarily in translation, not transcription, and longer transcripts, and those encoded by autism-associated SFARI genes, are under-translated. Functional analysis indicates that mutants may be impaired in neuronal and synaptic development. This can be compared with quantitative proteomic analysis caried out on eEF1A2ᴱ¹²²ᴷ/ᴱ¹²²ᴷ and eEF1A2ᴰ²⁵²ᴴ/ᴰ²⁵²ᴴ whole mouse brain.
The findings from the mass spectrometry data showed a significant overlap with the affected pathways identified in the TRAP-seq, notably in synaptic organisation, and axon guidance. The altered regulation of translational machinery is also observed in mutant samples, including the upregulation of proteins responsible for translation elongation and aminoacyl tRNA biosynthesis.
In summary, this project has identified several pathways for further investigation, and suggested potential mechanisms underlying the neurodevelopmental disorder-causing EEF1A2 missense mutations. Understanding the pathophysiology of the mutations will be key for informing future therapies, and has implications for other disorders relating to neurodevelopment and proteostasis
