miR-378-3p Knockdown Recapitulates Many of the Features of Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide range of underlying genetic, epigenetic, and other causes. Despite the myriad origins, a recognizable MDS phenotype has been associated with miRNA aberrant expression. A model of aberrant myeloid maturation that mimics MDS was generated using a stable knockdown of miR-378-3p. This model exhibited a transcriptional profile indicating aberrant maturation and function, immunophenotypic and morphologic dysplasia, and aberrant growth that characterizes MDS. Moreover, aberrant signal transduction in response to stimulation specific to the stage of myeloid maturation as indicated by CyTOF mass cytometry was similar to that found in samples from patients with MDS. The aberrant signaling, immunophenotypic changes, cellular growth, and colony formation ability seen in this myeloid model could be reversed with azacytidine, albeit without significant improvement of neutrophil function.

Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules.

Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG "tomato" or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.

Stress reticulocytes lose transferrin receptors by an extrinsic process involving spleen and macrophages.

As they mature into erythrocytes during normal erythropoiesis, reticulocytes lose surface transferrin receptors before or concurrently with reticulin. Exosome release accounts for most of the loss of transferrin receptors from reticulocytes. During erythropoietic stress, reticulocytes are released early from hematopoietic tissues and have increased reticulin staining and transferrin receptors. Flow cytometry of dually stained erythrocytes of mice recovering from phlebotomy demonstrated delayed loss of reticulin and transferrin receptors during in vitro maturation compared to in vivo maturation, indicating that an in vivo process extrinsic to the reticulocytes facilitates their maturation. Splenectomy or macrophage depletion by liposomal clodronate inhibited in vivo maturation of reticulocytes and increased the numbers of reticulin-negative, transferrin receptor-positive cells during and after recovery from phlebotomy. This reticulin-negative, transferrin receptor-positive population was rarely found in normal mice. Transmission electron microscopy demonstrated that the reticulin-negative, transferrin receptor-positive cells were elongated and discoid erythrocytes, but they had intracellular and surface structures that appeared to be partially degraded organelles. The results indicate that maturation of circulating stress reticulocytes is enhanced by an extrinsic process that occurs in the spleen and involves macrophage activity. Complete loss of reticulin with incomplete loss of surface transferrin receptors in this process produces a reticulin-negative, transferrin receptor-positive erythrocyte population that has potential utility for detecting prior erythropoietic stresses including bleeding, hemolysis and erythropoietin administration, even after recovery has been completed. Am. J. Hematol. 91:875-882, 2016. © 2016 Wiley Periodicals, Inc.

Electrophysiological behavior of neonatal astrocytes in hippocampal stratum radiatum.

BACKGROUND: Neonatal astrocytes are diverse in origin, and undergo dramatic change in gene expression, morphological differentiation and  syncytial networking throughout development. Neonatal astrocytes also play multifaceted roles in neuronal circuitry establishment. However, the extent to which neonatal astrocytes differ from their counterparts in the adult brain remains unknown. RESULTS: Based on ALDH1L1-eGFP expression or sulforhodamine 101 staining, neonatal astrocytes at postnatal day 1-3 can be reliably identified in hippocampal stratum radiatum. They exhibit a more negative resting membrane potential (V M), -85 mV, than mature astrocytes, -80 mV and a variably rectifying whole-cell current profile due to complex expression of voltage-gated outward transient K(+) (IKa), delayed rectifying K(+) (IKd) and inward K(+) (IKin) conductances. Differing from NG2 glia, depolarization-induced inward Na(+) currents (INa) could not be detected in neonatal astrocytes. A quasi-physiological V M of -69 mV was retained when inwardly rectifying Kir4.1 was inhibited by 100 µM Ba(2+) in both wild type and TWIK-1/TREK-1 double gene knockout astrocytes, indicating expression of additional leak K(+) channels yet unknown. In dual patch recording, electrical coupling was detected in 74 % (14/19 pairs) of neonatal astrocytes with largely variable coupling coefficients. The increasing gap junction coupling progressively masked the rectifying K(+) conductances to account for an increasing number of linear voltage-to-current relationship passive astrocytes (PAs). Gap junction inhibition, by 100 µM meclofenamic acid, substantially reduced membrane conductance and converted all the neonatal PAs to variably rectifying astrocytes. The low density expression of leak K(+) conductance in neonatal astrocytes corresponded  to a ~50 % less K(+) uptake capacity compared to adult astrocytes. CONCLUSIONS: Neonatal astrocytes predominantly express a variety of rectifying K(+) conductances, form discrete cell-to-cell gap junction coupling and are deficient in K(+) homeostatic capacity.

Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ.

We have recently shown that a linear current-to-voltage (I-V) relationship of membrane conductance (passive conductance) reflects the intrinsic property of K(+) channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (V M) essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K(+) channels remains elusive. TREK-1 two-pore domain K(+) channel (K2P) is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of heterodimer TWIK-1/TREK-1 channel to the membrane and contributing to astrocyte passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K(+) channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, V M, or membrane input resistance (R in) in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K(+) channels may shed light on this long-standing and important question in astrocyte physiology.

mGluR3 Activation Recruits Cytoplasmic TWIK-1 Channels to Membrane that Enhances Ammonium Uptake in Hippocampal Astrocytes.

TWIK-1 two-pore domain K+ channels are highly expressed in mature hippocampal astrocytes. While the TWIK-1 activity is readily detectable on astrocyte membrane, the majority of channels are retained in the intracellular compartments, which raises an intriguing question of whether the membrane TWIK-1 channels could be dynamically regulated for functions yet unknown. Here, the regulation of TWIK-1 membrane expression by Gi/Go-coupled metabotropic glutamate receptor 3 (mGluR3) and its functional impact on ammonium uptake has been studied. Activation of mGluR3 induced a marked translocation of TWIK-1 channels from the cytoplasm to the membrane surface. Consistent with our early observation that membrane TWIK-1 behaves as nonselective monovalent cation channel, mGluR3-mediated TWIK-1 membrane expression was associated with a depolarizing membrane potential (V M). As TWIK-1 exhibits a discernibly high permeability to ammonium (NH4+), a critical substrate in glutamate-glutamine cycle for neurotransmitter replenishment, regulation of NH4+ uptake capacity by TWIK-1 membrane expression was determined by response of astrocyte V M to bath application of 5 mM NH4Cl. Stimulation of mGluR3 potentiated NH4+-induced V M depolarization by ∼30 % in wild type, but not in TWIK-1 knockout astrocytes. Furthermore, activation of mGluR3 mediated a coordinated translocation of TWIK-1 channels with recycling endosomes toward astrocyte membrane and the mGluR3-mediated potentiation of NH4+ uptake required a functional Rab-mediated trafficking pathway. Altogether, we demonstrate that the activation of mGluR3 up-regulates the membrane expression of TWIK-1 that in turn enhances NH4+ uptake in astrocytes, a mechanism potentially important for functional coupling of astrocyte glutamate-glutamine cycle with the replenishment of neurotransmitters in neurons.

LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding.

LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R(320)LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.

Retinoic Acid Signaling Coordinates Macrophage-Dependent Injury and Repair after AKI.

Retinoic acid (RA) has been used therapeutically to reduce injury and fibrosis in models of AKI, but little is known about the regulation of this pathway and what role it has in regulating injury and repair after AKI. In these studies, we show that RA signaling is activated in mouse and zebrafish models of AKI, and that these responses limit the extent of injury and promote normal repair. These effects were mediated through a novel mechanism by which RA signaling coordinated the dynamic equilibrium of inflammatory M1 spectrum versus alternatively activated M2 spectrum macrophages. Our data suggest that locally synthesized RA represses proinflammatory macrophages, thereby reducing macrophage-dependent injury post-AKI, and activates RA signaling in injured tubular epithelium, which in turn promotes alternatively activated M2 spectrum macrophages. Because RA signaling has an essential role in kidney development but is repressed in the adult, these findings provide evidence of an embryonic signaling pathway that is reactivated after AKI and involved in reducing injury and enhancing repair.

Gap junction coupling confers isopotentiality on astrocyte syncytium.

Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K(+) free pipette solutions that alter the K(+) equilibrium potential to non-physiological voltages. We show that an astrocyte's associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte's VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K(+) and thereby maintains a sustained driving force for highly efficient K(+) uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.

Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22.

Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot-Marie-Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT.

Neonatal CD71+ Erythroid Cells Do Not Modify Murine Sepsis Mortality.

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization.

Freshly dissociated mature hippocampal astrocytes exhibit passive membrane conductance and low membrane resistance similarly to syncytial coupled astrocytes.

Mature astrocytes exhibit a linear current-to-voltage K(+) membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K(+) spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K(+) conductance enabled rapid change in the intracellular K(+) concentrations in response to changing K(+) drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes.

Renal fibrosis is not reduced by blocking transforming growth factor-ß signaling in matrix-producing interstitial cells.

Transforming growth factor-ß (TGF-ß) strongly promotes renal tubulointerstitial fibrosis, but the cellular target that mediates its profibrotic actions has not been clearly identified. While in vitro data suggest that TGF-ß-induced matrix production is mediated by renal fibroblasts, the role of these cells in TGF-ß-dependent tubulointerstitial fibrosis following renal injury is not well defined. To address this, we deleted the TGF-ß type II receptor in matrix-producing interstitial cells using two different inducible Cre models: COL1A2-Cre with a mesenchymal enhancer element and tenascin-Cre that targets medullary interstitial cells, and either the mouse unilateral ureteral obstruction or the aristolochic acid renal injury model. Renal interstitial cells lacking the TGF-ß receptor had significantly impaired collagen I production, but, unexpectedly, overall tissue fibrosis was unchanged in the conditional knockouts after renal injury. Thus, abrogating TGF-ß signaling in matrix-producing interstitial cells is not sufficient to reduce fibrosis after renal injury.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling.

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-ß1 (TGF-ß1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-ß1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-ß1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Methylation of promoters of microRNAs and their host genes in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of hematopoietic malignancies characterized by ineffective hematopoiesis. Recently, we identified MDS-associated microRNAs (miRNAs) that are down-regulated in MDS. This study examines possible explanations for that observed down-regulation of miRNA expression in MDS. Since genomic losses are insufficient to explain the down-regulation of all our MDS-associated miRNAs, we explored other avenues. We demonstrate that these miRNAs are predominantly intragenic, and that, in many cases, they and their host genes are expressed in a similar pattern during myeloid maturation, suggesting their co-regulation. This co-regulation is further supported by the down-regulation of several of the host genes in MDS and increased methylation of the shared promoters of several miRNAs and their respective host genes. These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway.

Use of human androgen receptor gene analysis to aid the diagnosis of JMML in female noonan syndrome patients.

Noonan syndrome (NS) patients are at increased risk for developing juvenile myelomonocytic leukemia (JMML), an aggressive clonal disorder of aberrant cell proliferation. Many NS patients exhibit spontaneously remitting monocytosis and transient myeloproliferation. The distinction between bone marrow hyperproliferation due to germline mutation and leukemia resulting from clonal transformation can be difficult in NS patients. The GM-CSF hypersensitivity assay, diagnostic of sporadic JMML, can be positive in NS patients at baseline. In this report, we demonstrate the utility of determining the clonal status of the monocyte population by the HUMARA assay in distinguishing JMML and benign myeloproliferation in female NS patients.