Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.     

Mutations in USP9X Are Associated with X-Linked Intellectual Disability and Disrupt Neuronal Cell Migration and Growth

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual''s phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved.     

Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans

Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26‐mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.     

Evidence of Notch-Hesr-Nrf2 Axis in Muscle Stem Cells,but Absence of Nrf2 Has No Effect on Their Quiescent and Undifferentiated State

Nrf2 is a master regulator of oxidative stresses through the induction of anti-oxidative genes. Nrf2 plays roles in maintaining murine hematopoietic stem cells and fly intestinal stem cells. The canonical Notch signaling pathway is also crucial for maintaining several types of adult stem cells including muscle stem cells (satellite cells). Here, we show that Dll1 induced Nrf2 expression in myogenic cells. In addition, primary targets of Notch signaling, Hesr1 and Hesr3, were involved in the up-regulation of Nrf2 mRNA and expression of its target genes. In vitro, Nrf2 had anti-myogenic and anti-proliferative effects on primary myoblasts. In vivo, although Nrf2-knockout mice showed decreased expression of its target genes in muscle stem cells, adult muscle stem cells of Nrf2-knockout mice did not exhibit the phenotype. Taken together, in muscle stem cells, the Notch-Hesr-Nrf2 axis is a pathway potentially inducing anti-oxidative genes, but muscle stem cells either do not require Nrf2-mediated anti-oxidative gene expression or they have a complementary system compensating for the loss of Nrf2.     

Loss of Usp9x Disrupts Cortical Architecture,Hippocampal Development and TGFβ-Mediated Axonogenesis

The deubiquitylating enzyme Usp9x is highly expressed in the developing mouse brain, and increased Usp9x expression enhances the self-renewal of neural progenitors in vitro. USP9X is a candidate gene for human neurodevelopmental disorders, including lissencephaly, epilepsy and X-linked intellectual disability. To determine if Usp9x is critical to mammalian brain development we conditionally deleted the gene from neural progenitors, and their subsequent progeny. Mating Usp9x^loxP/loxP mice with mice expressing Cre recombinase from the Nestin promoter deleted Usp9x throughout the entire brain, and resulted in early postnatal lethality. Although the overall brain architecture was intact, loss of Usp9x disrupted the cellular organization of the ventricular and sub-ventricular zones, and cortical plate. Usp9x absence also led to dramatic reductions in axonal length, in vivo and in vitro, which could in part be explained by a failure in Tgf-β signaling. Deletion of Usp9x from the dorsal telencephalon only, by mating with Emx1-cre mice, was compatible with survival to adulthood but resulted in reduction or loss of the corpus callosum, a dramatic decrease in hippocampal size, and disorganization of the hippocampal CA3 region. This latter phenotypic aspect resembled that observed in Doublecortin knock-out mice, which is an Usp9x interacting protein. This study establishes that Usp9x is critical for several aspects of CNS development, and suggests that its regulation of Tgf-β signaling extends to neurons.     

hCD2-iCre and Vav-iCre Mediated Gene Recombination Patterns in Murine Hematopoietic Cells

Cre-recombinase mediated conditional deletion of Lox-P site flanked ("floxed") genes is widely used for functional gene annotation in mice. Many different Cre-transgenic mouse lines have been developed for cell-type specific gene disruption. But often, the precise tissue-patterns of Cre activity remain incompletely characterized. Two widely used transgenes for conditional gene recombination in hematopoietic cells are Vav-iCre driven from the murine Vav1 promotor, and hCD2-iCre driven from the human CD2 promotor. Vav-iCre expresses active Cre in fetal and adult hematopoietic stem cells and all descendants, hCD2-iCre in immature and mature B and T lymphocytes. To better characterize which hematopoietic cells contain hCD2-iCre activity, we compared EYFP fluorescence in hCD2-iCre^+/- R26-stop-EYFP^+/- and Vav-iCre^+/- R26-stop-EYFP^+/-mice. R26-stop-EYFP ubiquitously encodes EYFP preceded by a floxed stop cassette. By removing it, Cre activity induces measurable EYFP expression. Our results confirm the known activity patterns for both Cre transgenes and unveil additional hCD2-iCre mediated reporter gene recombination in common lymphoid progenitors, in natural killer cells and their progenitors, and in plasmacytoid and conventional dendritic cells. This supports previously proposed common lymphoid origins for natural killer cells and subsets of dendritic cells, and indicates the need to consider pleiotropic effects when studying hCD2-iCre mediated conditional knockout mice. Vav-iCre^+/- R26-stop-EYFP^+/-mice did not show the non-hematopoietic recombination in vascular endothelial cells seen in other Vav-Cre mouse lines, but displayed an unexpected Vav-iCre mediated recombination in a bone cell subset lacking hematopoietic markers. This pinpoints the need to consider stromal cell contributions to phenotypes of Vav-iCre mediated conditional knockout mice. Altogether, our data provide the first detailed assessment of hCD2-iCre and Vav-iCre mediated deletion of floxed genes during lymphocyte development from hematopoietic stem cells and open up novel applications for either Cre-transgenic mouse line.     

GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis

Ferroptosis, a newly defined mode of regulated cell death caused by unbalanced lipid redox metabolism, is implicated in various tissue injuries and tumorigenesis. However, the role of ferroptosis in stem cells has not yet been investigated. Glutathione peroxidase 4 (GPX4) is a critical suppressor of lipid peroxidation and ferroptosis. Here, we study the function of GPX4 and ferroptosis in hematopoietic stem and progenitor cells (HSPCs) in mice with Gpx4 deficiency in the hematopoietic system. We find that Gpx4 deletion solely in the hematopoietic system has no significant effect on the number and function of HSPCs in mice. Notably, hematopoietic stem cells (HSCs) and hematopoietic progenitor cells lacking Gpx4 accumulated lipid peroxidation and underwent ferroptosis in vitro. α-Tocopherol, the main component of vitamin E, was shown to rescue the Gpx4-deficient HSPCs from ferroptosis in vitro. When Gpx4 knockout mice were fed a vitamin E-depleted diet, a reduced number of HSPCs and impaired function of HSCs were found. Furthermore, increased levels of lipid peroxidation and cell death indicated that HSPCs undergo ferroptosis. Collectively, we demonstrate that GPX4 and vitamin E cooperatively maintain lipid redox balance and prevent ferroptosis in HSPCs.Subject terms: Cell biology, Physiology, Stem-cell research     

Inactivation of Uaf1 Causes Defective Homologous Recombination and Early Embryonic Lethality in Mice

The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1^+/− mice were small at birth and exhibited reduced fertility, thus resembling Usp1^−/− mice. Unexpectedly, homozygous Uaf1^−/− embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1^+/− murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1^−/− mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.     

ARAP3 Functions in Hematopoietic Stem Cells

ARAP3 is a GTPase-activating protein (GAP) that inactivates Arf6 and RhoA small GTPases. ARAP3 deficiency in mice causes a sprouting angiogenic defect resulting in embryonic lethality by E11. Mice with an ARAP3 R302,303A mutation (Arap3^KI/KI) that prevents activation by phosphoinositide-3-kinase (PI3K) have a similar angiogenic phenotype, although some animals survive to adulthood. Here, we report that hematopoietic stem cells (HSCs) from rare adult Arap3^KI/KI bone marrow are compromised in their ability to reconstitute recipient mice and to self-renew. To elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in hematopoiesis, we conditionally deleted Arap3 in hematopoietic cells and in several cell types within the HSC niche. Excision of Arap3 in hematopoietic cells using Vav1-Cre does not alter the ability of ARAP3-deficient progenitor cells to proliferate and differentiate in vitro or ARAP3-deficient HSCs to provide multi-lineage reconstitution and to undergo self-renewal in vivo. Thus, our data suggest that ARAP3 does not play a cell-autonomous role in HSPCs. Deletion of Arap3 in osteoblasts and mesenchymal stromal cells using Prx1-Cre resulted in no discernable phenotypes in hematopoietic development or HSC homeostasis in adult mice. In contrast, deletion of Arap3 using vascular endothelial cadherin (VEC or Cdh5)-driven Cre resulted in embryonic lethality, however HSCs from surviving adult mice were largely normal. Reverse transplantations into VEC-driven Arap3 conditional knockout mice revealed no discernable difference in HSC frequencies or function in comparison to control mice. Taken together, our investigation suggests that despite a critical role for ARAP3 in embryonic vascular development, its loss in endothelial cells minimally impacts HSCs in adult bone marrow.     

Early Lineage Priming by Trisomy of Erg Leads to Myeloproliferation in a Down Syndrome Model

Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(17¹⁶)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(17¹⁶)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(17¹⁶)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL.     

SOCS5 is expressed in primary B and T lymphoid cells but is dispensable for lymphocyte production and function

Suppressors of cytokine signaling (SOCSs) are key regulators of cytokine-induced responses in hematopoietic as well as nonhematopoietic cells. SOCS1 and SOCS3 have been shown to modulate T-cell responses, whereas the roles of other SOCS family members in the regulation of lymphocyte function are less clear. Here, we report the generation of mice with a targeted disruption of the Socs5 gene. Socs5^−/− mice were born in a normal Mendelian ratio and were healthy and fertile. We found that SOCS5 is expressed in primary B and T cells in wild-type mice. However, no abnormalities in the lymphocyte compartment were seen in SOCS5-deficient mice. We examined antigen- and cytokine-induced proliferative responses in B and T cells in the absence of SOCS5 and found no deviations from the responses seen in wild-type cells. Because SOCS5 has been implicated in Th1 differentiation, we also investigated the importance of SOCS5 in T helper cell responses. Unexpectedly, SOCS5-deficient CD4 T cells showed no abnormalities in Th1/Th2 differentiation and Socs5^−/− mice showed normal resistance to infection with Leishmania major. Therefore, although SOCS5 is expressed in primary B and T cells, it appears to be dispensable for the regulation of lymphocyte function.     

SRSF2 Is Essential for Hematopoiesis,and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing

Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2^flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage⁻ Sca⁺ c-Kit⁺ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.     

Transcription Factor GATA1 Is Dispensable for Mast Cell Differentiation in Adult Mice

Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1^−/y), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1^−/y bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5′ end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit⁺ FcεRIα⁺ mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.