Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice.

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

Role of Caspase-10-P13tBID axis in erythropoiesis regulation.

Red blood cell production is negatively controlled by the rate of apoptosis at the stage of CFU-E/pro-erythroblast differentiation, depending on the balance between erythropoietin (EPO) levels and activation of the Fas/FasL pathway. At this stage, activation of transient caspases through depolarization via mitochondrial outer membrane permeabilization (MOMP) is also required for terminal erythroid differentiation. Molecular mechanisms regulating the differential levels of MOMP during differentiation and apoptosis, however, remain poorly understood. Here we show a novel and essential role for the caspase-10-P13-tBID axis in erythroid terminal differentiation. Caspase-10 (but not caspase-8, which is activated during apoptosis) is activated at the early stages of erythroid terminal differentiation leading to the cleavage of P22-BID into P18-tBID, and later into P13-tBID. Erythropoietin (EPO) by inducing casein kinase I alpha (CKIα) expression, which in turn phosphorylates P18-tBID, prevents the generation of MYR-P15-tBID (leading to apoptosis) and allows the generation of P13-tBID by caspase-10. Unlike P15-tBID, P13-tBID is not myristoylated and as such, does not irreversibly anchor the mitochondrial membrane resulting in a transient MOMP. Likewise, transduction of a P13-tBID fragment induces rapid and strong erythroid terminal differentiation. Thus, EPO modulates the pattern of BID cleavage to control the level of MOMP and determines the fate of erythroblasts between apoptosis and differentiation. This pathway is impaired in 5q- myelodysplastic syndromes because of CK1α haplo-insufficiency and may contribute to erythroid differentiation arrest and high sensitivity of this disease to lenalidomide (LEN).

Zinc finger protein 191 (ZNF191/Zfp191) is necessary to maintain neural cells as cycling progenitors.

The identification of the factors that allow better monitoring of stem cell renewal and differentiation is of paramount importance for the implementation of new regenerative therapies, especially with regard to the nervous and hematopoietic systems. In this article, we present new information on the function of zinc finger protein 191 (ZNF/Zfp191), a factor isolated in hematopoietic cell lines, within progenitors of the central nervous system (CNS). ZNF/Zfp191 has been found to be principally expressed in progenitors of the developing CNS of humans and mice. Such an overlap of the expression patterns in addition to the high homology of the protein in mammals suggested that ZNF/Zfp191 exerts a conserved function within such progenitors. Indeed, ZNF191 knockdown in human neural progenitors inhibits proliferation and leads to the exit of the cell cycle. Conversely, ZNF191 misexpression maintains progenitors in cycle and exerts negative control on the Notch pathway, which prevents them from differentiating. The present data, together with the fact that the inactivation of Zfp191 leads to embryonic lethality, confirm ZNF191 as an essential factor acting for the promotion of the cell cycle and thus maintenance in the progenitor stage. On the bases of expression data, such a function can be extended to progenitor cells of other tissues such as the hematopoietic system, which emphasizes the important issue of further understanding the molecular events controlled by ZNF/Zfp191.

Maternal serotonin influences cardiac function in adult offspring.

Using the Tph1-invalidated mouse line, in which blood is depleted in serotonin (5-hydroxytryptamine, 5-HT), we have demonstrated previously that maternal 5-HT is required for normal embryonic development. Here, we address the issue of the influence of the maternal 5-HT concentration on the cardiac function of the offspring as adults. We investigated the cardiac phenotype of Tph1-invalidated mice born to Tph1 heterozygous and null mothers. Functionally, all mutants display a significant decrease of cardiac contractility, indicative of impaired left ventricular function. They exhibit progressive dilated cardiomyopathy and are unable to adapt appropriately to a pharmacological stress. Moreover, we show that the cardiopathy is more severe in adult Tph1(-/-) mice born to homozygous mothers than to heterozygous mothers. Importantly, the severity of the cardiac phenotype is inversely correlated with the plasma 5-HT concentration but not the whole-blood 5-HT concentration. Thus, plasma 5-HT concentration may be a useful index of heart failure. These findings show that cardiac function, through the plasma 5-HT concentration, is influenced by the maternal serotonergic status.

Enhanced Renewal of Erythroid Progenitors in Myelodysplastic Anemia by Peripheral Serotonin.

Tryptophan as the precursor of several active compounds, including kynurenine and serotonin, is critical for numerous important metabolic functions. Enhanced tryptophan metabolism toward the kynurenine pathway has been associated with myelodysplastic syndromes (MDSs), which are preleukemic clonal diseases characterized by dysplastic bone marrow and cytopenias. Here, we reveal a fundamental role for tryptophan metabolized along the serotonin pathway in normal erythropoiesis and in the physiopathology of MDS-related anemia. We identify, both in human and murine erythroid progenitors, a functional cell-autonomous serotonergic network with pro-survival and proliferative functions. In vivo studies demonstrate that pharmacological increase of serotonin levels using fluoxetine, a common antidepressant, has the potential to become an important therapeutic strategy in low-risk MDS anemia refractory to erythropoietin.

ZENON, a novel POZ Kruppel-like DNA binding protein associated with differentiation and/or survival of late postmitotic neurons.

The rat tyrosine hydroxylase gene promoter contains an E-box/dyad motif and an octameric and heptameric element that may be recognized by classes of transcription factors highly expressed during nervous system development. In a one-hybrid genetic screen, we used these sites as targets to isolate cDNAs encoding new transcription factors present in the brain. We identified ZENON, a novel rat POZ protein that contains two clusters of Kruppel-like zinc fingers and that presents several features of a transcription factor. ZENON is found in nuclei following transient transfection with the cDNA. The N-terminal zinc finger cluster contains a DNA binding domain that interacts with the E box. Cotranfection experiments revealed that ZENON induces tyrosine hydroxylase promoter activity. Unlike other POZ proteins, the ZENON POZ domain is not required for either activation of transcription or self-association. In the embryonic neural tube, ZENON expression is restricted to neurons that have already achieved mitosis and are engaged in late stages of neuronal differentiation (late postmitotic neurons). ZENON neuronal expression persists in the adult brain; therefore, ZENON can be considered a marker of mature neurons. We propose that ZENON is involved in the maintenance of panneuronal features and/or in the survival of mature neurons.

Serotonin is a key factor for mouse red blood cell survival.

Serotonin (5-HT) is a monoamine originally purified from blood as a vasoactive agent. In nonneuronal tissues, its presence is linked with the expression of tryptophan hydroxylase 1 (TPH1) that catalyzes the rate-limiting step of its synthesis. Targeted disruption in mice of the TPH1 gene results in very low levels of circulating 5-HT. Previous analysis of the TPH1 knockout (TPH1(-/-)) mouse revealed that they develop a phenotype of macrocytic anemia with a reduced half-life of their circulating red blood cells (RBC). In this study, to establish whether the observed reduced half-life of TPH1(-/-) RBC is an intrinsic or an extrinsic characteristic, we compared their survival to RBC isolated from wild-type mice. Both in vivo and in vitro data converge to demonstrate an extrinsic protective effect of 5-HT since presence of 5-HT in the RBC environment protects RBC from senescence. The protective effect played by 5-HT is not mediated through activation of a classical pharmacological pathway as no 5-HT receptors were detected on isolated RBC. Rather, 5-HT acts as an effective antioxidant since reduction of 5-HT circulating levels are associated with a decrease in the plasma antioxidant capacity. We further demonstrate a link between oxidation and the removal of damaged RBC following transfusion, as supplementation with 5-HT improves RBC post-transfusion survival in a mouse model of blood banking.

Maternal serotonin is crucial for murine embryonic development.

The early appearance of serotonin and its receptors during prenatal development, together with the many effects serotonin exerts during CNS morphogenesis, strongly suggest that serotonin influences the development and maturation of the mammalian brain before it becomes a neuromodulator/neurotransmitter. Sites of early serotonin biosynthesis, however, have not been detected in mouse embryos or extraembryonic structures, suggesting that the main source of serotonin could be of maternal origin. This hypothesis was tested by using knockout mice lacking the tph1 gene, which is responsible for the synthesis of peripheral serotonin. Genetic crosses were performed to compare the phenotype of pups born from homozygous and heterozygous mothers. Observations provide the first clear evidence that (i) maternal serotonin is involved in the control of morphogenesis during developmental stages that precede the appearance of serotonergic neurons and (ii) serotonin is critical for normal murine development. Most strikingly, the phenotype of tph1-/- embryos depends more on the maternal genotype than on that of the concepti. Consideration of the maternal genotype may thus help to clarify the influence of other genes in complex diseases, such as mental illness.

Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function.

Serotonin (5-HT) controls a wide range of biological functions. In the brain, its implication as a neurotransmitter and in the control of behavioral traits has been largely documented. At the periphery, its modulatory role in physiological processes, such as the cardiovascular function, is still poorly understood. The rate-limiting enzyme of 5-HT synthesis, tryptophan hydroxylase (TPH), is encoded by two genes, the well characterized tph1 gene and a recently identified tph2 gene. In this article, based on the study of a mutant mouse in which the tph1 gene has been inactivated by replacement with the beta-galactosidase gene, we establish that the neuronal tph2 is expressed in neurons of the raphe nuclei and of the myenteric plexus, whereas the nonneuronal tph1, as detected by beta-galactosidase expression, is in the pineal gland and the enterochromaffin cells. Anatomic examination of the mutant mice revealed larger heart sizes than in wild-type mice. Histological investigation indicates that the primary structure of the heart muscle is not affected. Hemodynamic analyses demonstrate abnormal cardiac activity, which ultimately leads to heart failure of the mutant animals. This report links loss of tph1 gene expression, and thus of peripheral 5-HT, to a cardiac dysfunction phenotype. The tph1-/- mutant may be valuable for investigating cardiovascular dysfunction observed in heart failure in humans.