Progenitor Cell Death Research Articles

Acrylamide induces cell death in neural progenitor cells and impairs hippocampal neurogenesis

Acrylamide (ACR) is a well-known neurotoxin in mammalian species that causes neuropathy characterized by ataxia and skeletal muscle weakness. Therefore, ACR-mediated axon damage in the central and peripheral nervous systems is considered to be central-peripheral axonopathy. However, the molecular mechanisms underlying ACR's toxicity to neural progenitor cells are unknown. This study investigated the adverse effects of ACR on mouse multipotent neural progenitor cells and adult hippocampal neurogenesis. ACR significantly reduced the proliferation of neural progenitor cells, and high ACR concentrations induced apoptotic and necrotic cell death. We found that elevated intracellular levels of reactive oxygen species were involved in ACR-mediated cytotoxicity. Interestingly, the administration of ACR to young mice resulted in a significant decrease in the number of newly generated cells in the dentate gyrus of the hippocampus, suggesting an impairment of adult neurogenesis. These results suggest that ACR's deleterious effects on the central nervous system are due to the death of neural progenitor cells and impaired adult neurogenesis.

Lyl1 and Scl Regulate Bcl2 Expression and Control Apoptosis at Different Stages of Hematopoietic Development.

Abstract 569The Lymphoblastic Leukemia gene 1 (Lyl1) encodes a basic-helix-loop-helix (bHLH) transcription factor that is involved in T cell acute lymphoblastic leukemia via chromosomal translocations. Lyl1 has an almost identical bHLH motif to Scl, a known master regulator of hematopoiesis. In our previous work, we used a double knock-out mouse model to demonstrate functional redundancy between Lyl1 and Scl in adult hematopoietic stem cells (HSCs), where in the absence of both genes in adult mice, HSCs and progenitors undergo rapid apoptosis. Despite their common and overlapping functions, the two genes appear to also function at different aspects of hematopoietic development. Lyl1 knock-out mice exhibit a unique defect in B-cell development. This phenotype is recapitulated in vitro, where Lyl1 progenitors grown on a layer of OP9 cells and allowed to differentiate with the addition of cytokines fail to differentiate to B cells as well as wild type progenitors. Rescue assays, where Lyl1 was transduced using a retrovirus into Lyl1 knock-out progenitors and transplanted into lethally irradiated recipients, showed complete rescue of the B cell lineage. Forced expression of Scl into Lyl1 knock-out progenitors failed to rescue, giving rise to only about 5% of peripheral blood B cells. This demonstrates the distinct functions of the two genes in differentiated cells types, despite their functional redundancy in the HSCs. We further sought to dissect the functions and differences between Lyl1 and Scl based on their target genes. Using bioinformatics analysis and a literature search, we identified a number of genes that were candidates for Lyl1 transcriptional regulation; these interactions with Lyl1 and Scl were confirmed by chromatin immunoprecipitation. We found that Bcl2, and genes belonging to this family, are direct target genes of both Lyl1 and Scl in progenitor cells, and that they are downregulated in Lyl1 and Scl single knock-out mouse models. Since Bcl2 is critical for B cell survival, we hypothesized that the B cell deficiency in Lyl1 knock-out mice could be partly due to a loss of Bcl2. We performed rescue assays where Bcl2 was transduced using a retrovirus into Lyl1 knock-out progenitors which were transplanted into lethally irradiated recipient mice. Bcl2 overexpression rescued the B-cell defect in Lyl1 knock-out bone marrow. Since Bcl2 was identified as a target gene for Scl as well, we tested whether Bcl2 could also rescue the rapid apoptosis phenotype of the Lyl1/Scl double knock-out progenitors. Forced expression of Bcl2 in these cells, followed by deletion of the floxed Scl allele, resulted in a delayed onset of apoptosis, but incomplete rescue of the progenitor cell death. These findings distinguish the activities of Lyl1 and Scl in B cells and demonstrate a possible mechanism for their functions, through in part by their activation of the antiapoptotic gene Bcl2. Disclosures:No relevant conflicts of interest to declare.

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis. Methods NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined. Results Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs. Conclusions These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death. General Significance Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.

Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor

Neural progenitor cells (NPCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, and have been used to treat several animal models of CNS disorders. In the present study, we show that the P2X7 purinergic receptor (P2X7R) is present on NPCs. In NPCs, P2X7R activation by the agonists extracellular ATP or benzoyl ATP triggers opening of a non-selective cationic channel. Prolonged activation of P2X7R with these nucleotides leads to caspase independent death of NPCs. P2X7R ligation induces NPC lysis/necrosis demonstrated by cell membrane disruption accompanied with loss of mitochondrial membrane potential. In most cells that express P2X7R, sustained stimulation with ATP leads to the formation of a non-selective pore allowing the entry of solutes up to 900 Da, which are reportedly involved in P2X7R-mediated cell lysis. Surprisingly, activation of P2X7R in NPCs causes cell death in the absence of pore formation. Our data support the notion that high levels of extracellular ATP in inflammatory CNS lesions may delay the successful graft of NPCs used to replace cells and repair CNS damage.

Mathematical modeling supports substantial mouse neural progenitor cell death

BackgroundExisting quantitative models of mouse cerebral cortical development are not fully constrained by experimental data.ResultsHere, we use simple difference equations to model neural progenitor cell fate decisions, incorporating intermediate progenitor cells and initially low rates of neural progenitor cell death. Also, we conduct a sensitivity analysis to investigate possible uncertainty in the fraction of cells that divide, differentiate, and die at each cell cycle.ConclusionWe demonstrate that uniformly low-level neural progenitor cell death, as concluded in previous models, is incompatible with normal mouse cortical development. Levels of neural progenitor cell death up to and exceeding 50% are compatible with normal cortical development and may operate to prevent forebrain overgrowth as observed following cell death attenuation, as occurs in caspase 3-null mutant mice.

Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons

Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

Rapid and selective death of leukemia stem and progenitor cells induced by the compound 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (TDZD-8)

Leukemia is thought to arise from malignant stem cells, which have been described for acute and chronic myeloid leukemia (AML and CML) and for acute lymphoblastic leukemia (ALL). Leukemia stem cells (LSCs) are relatively resistant to current chemotherapy and likely contribute to disease relapse and progression. Consequently, the identification of drugs that can efficiently eradicate LSCs is an important priority. In the present study, we investigated the antileukemia activity of the compound TDZD-8. Analysis of primary AML, blast crisis CML (bcCML), ALL, and chronic lymphoblastic leukemia (CLL) specimens showed rapid induction of cell death upon treatment with TDZD-8. In addition, for myeloid leukemias, cytotoxicity was observed for phenotypically primitive cells, in vitro colony-forming progenitors, and LSCs as defined by xenotransplantation assays. In contrast, no significant toxicity was observed for normal hematopoietic stem and progenitor cells. Notably, cell death was frequently evident within 2 hours or less of TDZD-8 exposure. Cellular and molecular studies indicate that the mechanism by which TDZD-8 induces cell death involves rapid loss of membrane integrity, depletion of free thiols, and inhibition of both the PKC and FLT3 signaling pathways. We conclude that TDZD-8 uses a unique and previously unknown mechanism to rapidly target leukemia cells, including malignant stem and progenitor populations.

T-Cell Receptor Vβ Repertoire Analysis in Patients with Chronic Idiopathic Neutropenia: Evidence for Presence of Predominant T-Cell Clones with Possible Pathogenetic Significance.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by hypoplastic and left-shifted granulocytic series in the bone marrow (BM). It has been shown that CIN patients display increased number of activated T-lymphocytes with myelosuppressive properties in the BM and peripheral blood (PB) that induce Fas-mediated apoptotic death of the BM myeloid progenitor cells by producing interferon-γ and Fas-ligand. Epidemiological data have also shown female predominance and HLA-DRB1*1302 genetic background in CIN patients. The aim of the present study was to characterize T-cell receptor (TCR) Vβ repertoire of CD3+ cells in patients with CIN and explore the presence of dominant T-cell expansions with possible pathogenetic significance. Eighty patients with CIN were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1411±380 neutrophils/μL, range 100-1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease. PB and BM samples from the patients were subjected to flow-cytometric analysis for the quantification of the TCR Vβ repertoire of the CD3+ cells (IOTest Beta Mark kit, Beckman-Coulter). Vβ family expansions were defined as above of 2SD (standard deviation) from the mean values of 85 healthy controls. DNA samples from immunomagnetically sorted PB and BM CD3+ cells were also subjected to multiplex polymerase chain reaction (PCR) analysis using the BIOMED2 protocol that covers all Vβ TCR gene rearrangements. We found that 54 patients, i.e. a proportion of 67.5% displayed one or more prominent clones in PB CD3+ cells. The Vβ16, Vβ12 and, in a lower proportion, the Vβ7.2 were the most frequently expanded subsets in PB, identified in 53.75%, 15% and 6.25% of CIN patients, respectively. Furthermore, CIN patients as a group displayed statistically increased proportion of Vβ16, Vβ12 and Vβ7.2 subsets within the CD3+ PB cells (1.96±1.30, 2.49±2.56 and 2.97±1.72 respectively) compared to controls (0.90±0.29, 1.66±0.54 and 1.47±1.03, respectively) (P<0.001, P<0.01 and P<0.001, respectively). In 20 patients, BM cells were also studied. Sixteen of 20 patients (80%) showed one or more prominent clones in CD3+ BM cells. In accordance with the PB data, the Vβ16, Vβ12 and Vβ7.2 were also the most frequently expanded subsets in the BM, identified in 65%, 10% and 10% of CIN patients, respectively. Further analysis showed that although more than one T-cell subset might be expanded in each patient, an identical expansion pattern was noted individually in BM and PB. Interestingly, a proportion of patients displayed prominent clones only in BM and not in PB but not vice versa, suggesting selective expansion or persistence of these subsets within the BM. None of the patients displayed monoclonal TCR Vβ expansion on multiplex PCR and heteroduplex analysis of BM and PB CD3+ lymphocyte subsets. In conclusion, in vivo dominant T-cell responses are identified in the majority of patients with CIN. These data substantiate further our hypothesis for the T-cell mediated immune nature of CIN and provide novel insights in the pathophysiology of the disease with possible therapeutic implications.

Response of the Erythroid Lineage to Irradiation.

Radiation causes acute marrow damage and anemia, however the relative sensitivity of erythroid progenitors and precursors to radiation damage is unknown. While acute anemia elicits a rapid recovery response, the characteristics of erythroid recovery following irradiation have not been well investigated. Mice display a linear response of radiation dose and red cell damage as measured by micronucleated reticulocytes (MN-RET) up to 1-2Gy. Higher radiation doses lead both to decreased MN-RET frequency and to delayed reticulocyte recovery (Dertinger, et al., Mutation Research, in press). To determine why marrow erythroid toxicity differs at low (< 1Gy) versus high (> 2 Gy) radiation doses, we developed a novel method to quantify erythroid precursors using multispectral imaging flow cytometry. C57Bl/6 mice were irradiated either with 0, 1 or 4 Gy and erythroid progenitors (BFU-E, CFU-E) and precursors (ProE, BasoE, PolyE, OrthoE) in the marrow were analyzed between 1 hr and 5 days later. 1 hr following 1Gy radiation, BFU-E and CFU-E were reduced to 63±5% and 13±1% of unirradiated controls, respectively. 24 hrs after 1Gy, there was no significant change in BFU-E, however, a striking recovery of CFU-E (93±21% of controls) was observed. This CFU-E surge was followed by a wave of recovering erythroid precursors in the marrow beginning at 48 hrs and peaking at 72 hrs post radiation, when BasoE, PolyE, and OrthoE reached > 150% of unirradiated controls. These precursors subsequently fell below 70% and 90% of controls at 4 and 5 days, respectively. We also characterized the kinetics of erythroid progenitors and precursors following high-dose radiation. 1 hr after 4Gy, BFU-E and CFU-E were reduced to 10% and 1% of controls, respectively. Each of these erythroid progenitors reached their nadir (1% of controls) at 48 hrs, and began to modestly recover at 72 hrs. By 5 days after 4Gy, BFU-E were only 16±6% of controls, while CFU-E had variably recovered to 127±62%. Erythroid precursors also reached their nadir at 48 hrs following 4Gy with levels of 1–2% of controls, but then recovered to 50–100% by day 5. We conclude that CFU-E are exquisitely sensitive targets of radiation. While BFU-E are more radioresistant, CFU-E recovered more robustly, consistent with the known cycling kinetics of these progenitors. Our results also indicate that the reticulocyte response following low versus high radiation doses is due to rapid CFU-E recovery and the subsequent generation of a wave of erythroid precursors. It is likely that the decrease in MN-RET seen after high-dose radiation is due to the rapid and nearly complete depletion of erythroid precursors leaving few cells able to repair and complete maturation. Finally, we speculate that the oscillation of CFU-E and erythroid precursor populations following 1Gy is due to the operation of feedback regulation. Compared to BFU-E and late-stage erythroid precursors, CFU-E and ProE exhibited a more complex temporal pattern of recovery, suggesting that they may be focal points of this regulation. Examination of marrow progenitor and precursor cell injury and death following acute irradiation will lead to a better understanding of the regulation of hematopoiesis and lay the foundation for interventional studies to mitigate the effects of acute irradiation.

Modulating Cholesterol Levels on Bone Marrow Cells Affects Endothelial and Hematopoietic Cell Mobilization and Differentiation.

A decrease in cholesterol (CH) levels associated with hematological malignancies, such as acute leukemia, correlates with Hematopoietic suppression. Moreover, patients with mevalonate kinase deficiency (which results in hypocholesterolemia) have besides neurological defects, hepatosplenomegaly, thrombocytopenia, anemia and eventually progress into Myelodisplasic Syndromes/Leukemia. For this study, we hypothesized that CH levels might affect hematopoietic differentiation (acting directly on hematopoietic stem cells, HSC) or hematopoietic cell mobilization. For this purpose we started by studying the bone marrow (BM) expression pattern of CD36 and ApoE, 2 proteins involved in CH cellular transport, in BM samples of normal adult mice. Immunofluorescent staining showed that CD36 and ApoE are both expressed within the BM microenvironment, being particularly evident in BM sinusoids and also small subsets of hematopoietic cells. Next, we took advantage of CD36 and ApoEKO models to study their hematological parameters and BM cellular contents. To do so, CD36/ApoEKO mice and their counterparts were euthanized and BM and peripheral blood cells were collected, stained for CD11b (myeloid cells), B220 (B lymphocytes), Sca1 (progenitor cells), Flk1 (endothelial cells) and analyzed by FACS. FACS analysis of BM cells revealed that KO and WT mice had similar progenitor cells and B lymphocytes percentage, but substantially less myeloid cells. Peripheral blood analysis revealed a substantial increase in circulating B lymphocytes, and a decrease in circulating progenitor cells and myeloid cells. Regarding circulating endothelial cells, no differences were detected between KO and WT mice. However, ApoEKO mice had increased levels of BM endothelial cells and progenitor cells. As B lymphocytes are increased in the periphery and diminished in the BM, CD36 deficiency (which results in diminished CH uptake) seems to promote B lymphocyte exit from the BM. An opposing effect seems to occur in the progenitor cell populations, since less percentage in the peripheral blood might imply a failure in their exit from the BM. Alternatively, altered CH levels may affect selectively progenitor cell (subsets) differentiation. In order to investigate the role of CH modulation in hematopoietic differentiation/commitment, progenitor cells (Lin-Sca+) from CD36 WT mice were cultured in a methylcellulose hematopoietic differentiation assay and also cultured in endothelial differentiation conditions in the presence/absence of a CH lowering agent (Pravastatin). At day 9 of differentiation, cells were collected and expression of Sca1, CD11b and Flk1 was analyzed by flow cytometry. Reduced CH availability, as a result of Pravastatin treatment, resulted in substantial lower levels of differentiated endothelial cells, suggesting a blockade in this differentiation process, and in a reduced number of progenitor cells, suggesting progenitor cell death. Notably, the percentage of myeloid cells was not affected by Pravastatin treatment. Taken together, we suggest that CH influences not only trafficking between BM and peripheral blood, influences endothelial cell differentiation, and might also influence BM recovery to injury by diminishing progenitor cell number available for subsequent hematopoiesis. Current studies aim at comparing the BM hematopoietic recovery of CD36, ApoEKO vs control in response to sublethal irradiation, and the contribution of the BM endothelial compartment in this process.

Hyperglycemic condition disturbs the proliferation and cell death of neural progenitors in mouse embryonic spinal cord

Spina bifida, which results from failure of fusion in the spinal region of neural tube, is among the most common birth defects associated with diabetic pregnancy. However, the mechanism underlying maternal diabetes-induced congenital malformations including spina bifida is not fully understood. It was hypothesized that hyperglycemic conditions affect the proliferation and apoptosis of neural progenitor cells in the developing spinal neural tube, leading to abnormal neurodevelopment. In the present study, biological processes such as proliferation and apoptosis were investigated in the neuroepithelial cells of the developing spinal neural tube of embryos from diabetic mice, and in embryonic spinal neural tube derived neural progenitor cell cultures exposed to high glucose in vitro. Maternal diabetes caused decreased proliferation and increased apoptosis of the neuroepithelial cells in the developing spinal cord of embryos from diabetic mouse. Decreased proliferation and increased apoptosis were also found in neural progenitor cells exposed to high glucose. In addition, high glucose-induced apoptosis in neural progenitor cells was associated with activation of caspase-3. Thus, high glucose disturbs both proliferation and cell death of neural progenitors in the developing spinal neural tube. This could provide a cellular mechanism by which maternal hyperglycemia induces spina bifida in embryos from diabetic pregnancy.

Alpha 1-adrenoceptor agonists protect against stress-induced death of neural progenitor cells

Here, we show that alpha(1)-adrenoceptor agonists suppress stress-induced death of mouse embryonic brain-derived neural progenitor cells (NPCs). NPCs highly expressed both alpha(1A)- and alpha(1B)-adrenoceptor genes, whereas the gene encoding alpha(1D)-adrenoceptor was expressed at low levels. Application of the alpha(1)-adrenoceptor agonists phenylephrine and cirazoline significantly promoted cell survival of embryonic NPCs that had been exposed to stress, as measured by a lactate dehydrogenase release assay, but had no remarkable effect on differentiation of the NPCs. Both phenylephrine and cirazoline protected NPCs from death induced by growth factor deprivation, N2 nutrient deprivation, tunicamycin treatment or staurosporine treatment. Phenylephrine and cirazoline treatments both maximally reduced stress-induced cell death by approximately 60% but did not change the percentage of undifferentiated cells as measured by nestin staining. Moreover, phenylephrine and cirazoline treatments did not affect the cellular activities of caspase-3 and caspase-7 but markedly reduced propidium iodide penetration into the cytoplasm, suggesting that alpha(1)-adrenoceptor agonists inhibit caspase-3/7-independent death of the embryonic NPCs.

Death effector activation in the subventricular zone subsequent to perinatal hypoxia/ischemia.

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurological injury resulting from birth complications and pre-maturity. Our studies have demonstrated that this injury depletes the subventricular zone (SVZ) of progenitors. In this study, we sought to reveal which cell death pathways are activated within these progenitors after H/I. We found that calpain activity is detected as early as 4 h of reperfusion and is sustained for 48 h, while caspase 3 activation does not occur until 8 h and peaks at 24 h post-insult. Activated calpains and caspase 3 co-localized within precursors situated in the lateral aspects of the SVZ (which coincides with progenitor cell death), whereas neither enzyme was activated in the medial SVZ (which harbors the neural stem cells that are resilient to this insult). These studies reveal targets for neuroprotective agents to protect precursors from cell death towards the goal of restoring normal brain development after H/I.

Embryonic Cerebral Cortical Progenitors Are Resistant to Apoptosis, but Increase Expression of Suicide Receptor DISC‐Complex Genes and Suppress Autophagy Following Ethanol Exposure

In utero exposure to ethanol can result in severe fetal brain defects. Previous studies showed that ethanol induces apoptosis in differentiated cortical neurons. However, we know little about ethanol's effects on proliferating embryonic cortical progenitors. This study investigated the impact of ethanol exposure on the Fas/Apo-1/CD95 suicide receptor pathway, and on the survival of proliferating cortical neuroepithelial progenitors. Murine embryonic-derived primary cortical neuroepithelial cells were maintained as neurosphere cultures and exposed to a dose range of ethanol for periods ranging from 1 to 5 days. Programmed cell death was measured by 4 independent means (Annexin-V staining, caspase activation, DNA fragmentation, and autophagic vacuole formation). Surface Fas/Apo-1 suicide receptor expression was measured by flow cytometry. Expression of Fas/Apo-1-associated DISC-complex genes was measured by quantitative polymerase chain reaction. Ethanol exposure did not substantially increase apoptosis, necrosis, or surface Fas/Apo-1 expression. Moreover, ethanol significantly decreased caspase activation and autophagic activity. Finally, ethanol exposure induced mRNA expression of genes that constitute the death receptor complex. This study provides surprising evidence that ethanol does not induce either programmed cell death or necrosis of immature progenitors during neurogenesis, although ethanol may render neural progenitors susceptible to future apoptotic insults. Furthermore, our novel observation that ethanol suppresses autophagy is consistent with a hypothesis that ethanol promotes premature neural progenitor maturation. Taken together with our previous data regarding the role of the Fas/Apo-1 receptor in neural development, we conclude that ethanol disrupts basic proliferation and differentiation machinery rather than initiating cell death per se.

Computational Models of Neocortical Neuronogenesis and Programmed Cell Death in the Developing Mouse, Monkey, and Human

This paper presents a computational model allowing quantitative simulations of acquisition of neocortical neuronal number across mammalian species. When extrapolating scientific findings from rodents to humans, it is particularly pertinent to acknowledge the importance of the accelerated enlargement of the neocortex during human evolution. Neocortex development is marked by discrete stages of neural progenitor cell proliferation and death, neuronal differentiation, and neuronal programmed cell death. We have developed computational models of human and rhesus monkey neocortical neuronal cell acquisition based on experimentally derived parameters of cell cycle length, commitment to cell cycle exit, and cell death. Our model results agree with independent stereological studies estimating neocortical neuron number in adult and developing rhesus monkey and human. Comparisons of our primate models with previously developed rodent models suggest correlations between the lengthening of the duration of the neuronogenesis period and a lengthening of the cellular processes of cell cycle progression and death can account for the vast increase in size of the primate neocortex. Furthermore, when compared with rodents, we predict that cell death may play a larger role in shaping the primate neocortex. Our mathematical models of the development and evolution of the neocortex provide a quantitative, biologically based construct for extrapolation between rodent and humans. These models can assist in focusing future experimental research on the differing mechanisms of rodent versus human neocortical development.

MTOR Inhibition Induces Endothelial Progenitor Cell Death

Immunosuppressants are necessary to prevent graft rejection after solid organ transplantation. However, they are also known to have significant side effects, including endothelial toxicity. Endothelial progenitor cells originate in the bone marrow and are recognized by their angiogenic and endothelial reparative properties. The effects of the immunosuppressants cyclosporine A (CyA), tacrolimus and rapamycin were analyzed on endothelial progenitor-like cells. Rapamycin induced rapid cell death, even at concentrations much lower than those used clinically, in peripheral blood mononuclear cells (PBMC) cultured to favor outgrowth of endothelial progenitors. Cyclosporine A and tacrolimus had no significant effects at clinical concentrations. The effect of rapamycin was specific to endothelial progenitor cells, in particular to the early stages of differentiation, as a lesser effect was observed in late outgrowth endothelial progenitors, mature aortic endothelial cells, and macrophages derived from the same PBMCs. The mechanism of cell death appeared to be apoptosis; however, its induction was probably multifactorial and did not depend on caspase or cathepsin activation. In conclusion, rapamycin induces endothelial progenitor cell death, possibly because it blocks survival signals given by growth factors critically required by these cells.

Mechanisms of cell death of neural progenitor cells caused by trophic support deprivation

Cell death of neural progenitor cells is the primary problem limiting the value of neural progenitor cell-based therapy for central nervous system disorders. However, little is known about the mechanism of cell death of neural progenitor cells. In this study, we investigated the mechanisms of cell death of a multipotent cell line, MEB5, caused by deprivation of epidermal growth factor (EGF). When EGF was removed from the culture medium, the total number of viable MEB5 cells reduced, and nuclear condensation and elevation of caspase-3-like enzyme activity were observed in MEB5 cells. Treatment with a broad-range caspase inhibitor reduced cell death in a concentration-dependent manner, indicating that MEB5 cells undergo caspase-mediated apoptotic cell death caused by EGF deprivation. We also investigated the effects of glutamate receptor antagonists, antioxidants and nitric oxide synthase inhibitor on EGF deprivation-induced cell death. N-methyl- d-aspartate (NMDA) glutamate receptor antagonists, α-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor antagonist and nitric oxide synthase inhibitor failed to reduce cell death. In contrast, two antioxidants with different chemical structures reduced cell death in a concentration-dependent manner. The production of reactive oxygen species was detected in MEB5 cells after EGF deprivation by monitoring dichlorodihydrofluorescein fluorescence as a marker of reactive oxygen species-related radicals. Our results suggest that oxidative stress triggers caspase-mediated apoptosis of neural progenitor cells by trophic support deprivation.

MEK-ERK Signaling Is Involved in Interferon-γ-induced Death of Oligodendroglial Progenitor Cells*♦

Oligodendrocytes are exposed to various cytokines in inflammatory lesions in the central nervous system. In this study, we focused on the direct effects of interferon-gamma (IFNG) on highly purified rat oligodendroglial cultures at different developmental stages. Among the three stages tested, IFNG had direct cytotoxic effects on actively proliferating oligodendrocyte progenitors but much less on immature oligodendrocytes and none on mature oligodendrocytes. This stage-specific susceptibility of progenitors to IFNG-induced cytotoxicity consisted of two components, delay in the G(1)/S transition of the cell cycle and increased cell death at least partly mediated by apoptosis, suggesting that progression of the cell cycle was tightly linked to this toxic mechanism. There was no functional difference in the signal transducers and activators of transcription (STAT) pathways between progenitors and mature oligodendrocytes as determined by induction of IRF1 mRNA in response to IFNG. We found that partial inhibition of the MEK-ERK pathway, one of the mitogen-activated protein kinase phosphorelay modules, by U0126 partially reversed the IFNG-induced cytotoxicity in progenitors. In addition, ERK activity was quickly down-regulated after in vitro differentiation of progenitors to immature oligodendrocytes. Therefore, we concluded that simultaneous activation of the STAT pathway by IFNG and of the ERK pathway by exogenous trophic factors played a role in the stage-specific IFNG-induced cytotoxicity in oligodendroglial progenitors. Our study has implications with respect to the mechanisms of periventricular leukomalacia in infants and of persistent demyelination in multiple sclerosis lesions in adults.

Anemia in Chronic Heart Failure

Anemia occurs commonly in patients with chronic heart failure (CHF) and has been proposed as a novel therapeutic target in this patient population. The present review will summarize the current knowledge of the prevalence, causative factors, and pathophysiological correlates of anemia in CHF. Potential risks and benefits of therapy with erythropoietic agents for the treatment of anemia in CHF will also be discussed. Prevalence of Anemia in CHF Estimates of the prevalence of anemia in patients with CHF and low ejection fraction range widely from 4% to 61% (median 18%).1–15 Variability in estimated prevalence is partly attributable to use of inconsistent definitions of anemia in individual reports. The World Health Organization definition of anemia (hemoglobin concentration 13.0 g/dL in men and 12.0 g/dL in women) takes into account known gender differences in distribution of hemoglobin values, 16 whereas the National Kidney Foundation defines anemia as hemoglobin 12 g/dL in men and postmenopausal women. 17 These standard definitions of anemia are not based on wellestablished physiological or population norms. Published reports in CHF populations have used these and other study-specific definitions of anemia (including other arbitrary or statistically defined hemoglobin and hematocrit categories and administrative diagnostic codes from hospital records). Despite these inconsistencies in the definition of anemia cases, most studies indicate that the prevalence of anemia is increased in CHF populations with comorbid kidney disease, advanced age, and more severe symptoms (range, 30% to 61%) when compared with less symptomatic ambulatory populations (range, 4% to 23%). In patients with CHF and preserved ejection fraction, the few published reports indicate that anemia is also highly prevalent in this group.18–20 Underlying Cause of Anemia in CHF Anemia occurs when there is a deficiency in new erythrocyte production relative to the rate of removal of aged erythrocytes. Erythropoietin, a 30.4-kDa glycoprotein growth factor produced primarily by kidney, is the key component of the homeostatic system for regulation of red blood cell mass and tissue oxygen delivery.21–24 Erythropoietin prevents the programmed cell death of erythrocyte progenitor cells and thereby stimulates their proliferation, maturation, and terminal differentiation. 23 Any abnormality that reduces renal secretion of or bone marrow response to erythropoietin may result in anemia. Iron deficiency is present in 30% of anemic patients with CHF, so the majority of observed anemia is normocytic, often classified as anemia of chronic disease. Clinical characteristics commonly associated with increased risk of anemia in CHF populations are listed in the Table. Although risk factors for anemia identified in cross-sectional studies do not provide evidence of a causal link, these observations suggest that several distinct mechanisms may commonly contribute to anemia in patients with CHF. Several of the most important potential causal pathways will be discussed briefly below and are summarized in Figure 1. Chronic kidney disease is a common comorbidity in patients with CHF and is a strong independent predictor of increased risk of anemia in several studies. In chronic kidney disease populations without heart failure, moderate to severe kidney disease (defined as glomerular filtration rate [GFR] 60 mL/min) is associated with diminished erythropoietin production and a progressive decrease in hemoglobin values that is linearly related to reduction in GFR.25 The estimated prevalence of at least moderate chronic kidney disease (defined as GFR 60 mL/min) in CHF populations is 20% to 40%. 4,9,26,27 Anemia is frequently associated with decreased body mass index in published reports, a finding that suggests that patients with cachexia are at greater risk for anemia. Serum levels of proinflammatory cytokines are increased in cachectic patients with CHF and may contribute to development of anemia by several mechanisms. Proinflammatory cytokines including tumor necrosis factor- (TNF-), interleukin-1, and interleukin-6 have been shown to disrupt multiple aspects of erythropoiesis, including reduction of renal erythropoietin secretion, suppression of erythropoietin activity in red blood cell precursors in the bone marrow level, and reduction of bioavailability of iron stores for hemoglobin synthesis.28–31 Proinflammatory cytokines also increase levels of the liverderived peptide hormone, hepcidin.32,33 Hepcidin interacts with ferroportin and other iron transport proteins in the enterocyte to inhibit gut iron absorption and thereby reduces iron bioavailability for hemoglobin synthesis. 32,33 In a mouse

Stochastic modeling of oligodendrocyte generation in cell culture: model validation with time-lapse data.

BackgroundThe purpose of this paper is two-fold. The first objective is to validate the assumptions behind a stochastic model developed earlier by these authors to describe oligodendrocyte generation in cell culture. The second is to generate time-lapse data that may help biomathematicians to build stochastic models of cell proliferation and differentiation under other experimental scenarios.ResultsUsing time-lapse video recording it is possible to follow the individual evolutions of different cells within each clone. This experimental technique is very laborious and cannot replace model-based quantitative inference from clonal data. However, it is unrivalled in validating the structure of a stochastic model intended to describe cell proliferation and differentiation at the clonal level. In this paper, such data are reported and analyzed for oligodendrocyte precursor cells cultured in vitro.ConclusionThe results strongly support the validity of the most basic assumptions underpinning the previously proposed model of oligodendrocyte development in cell culture. However, there are some discrepancies; the most important is that the contribution of progenitor cell death to cell kinetics in this experimental system has been underestimated.