[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Aug 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: 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. STEM CELLS 2009;27:1643–1653
[Show abstract][Hide abstract]ABSTRACT: Serotonin (5-HT) is not only the messenger of some thousands of neurons in the
nervous system, but is a ubiquitous substance in peripheral tissues and fluids.
Circulating 5-HT, mainly stored in platelets, regulates hemostasis, vascular tone
and cardiac function. We generated a mouse line defective for the Tph1 gene and,
thus, defective for peripheral 5-HT synthesis. In these Tph1-mutant mice, 5-HT
concentration in blood is 3–15% of that in wild-type mice. We addressed the issue of
the influence of the circulating 5-HT deficiency on the cardiac structure and
function, by testing the hypothesis that there is a correlation between cardiac
dysfunction and individual low circulating 5-HT level. Through cardiac functional
exploration (electrocardiography, echocardiography and conductance catheterization),
we show that 5 months old Tph1-/- male mice, on a C57BL/6 background,
display a significant decrease of cardiac contractility and cardiac output, indicative
of an impaired left ventricular function. They exhibit progressive dilated cardiomyopathy
(DCM), normal valves, and are unable to adapt appropriately to a
pharmacological stress. The Tph1 mutant’s DCM is heterogeneous in its severity
and in the time-course of its progression. Analysis of the circulating 5-HT level in
the platelet and plasma compartments of the Tph1-/- mice reveals that the severity
of the DCM is inversely correlated with the plasma 5-HT concentration but not the
whole-blood 5-HT concentration. Moreover, we show that the cardiopathy is more
severe in adult Tph1-/- mice born to homozygous mothers than to heterozygous
mothers. These findings show that cardiac function, through the plasma 5-HT
concentration, is influenced by the maternal serotonergic status. This work
demonstrates the importance of the fine regulation of the circulating 5-HT levels for
the cardiac function. The regulation of the plasma 5-HT levels, probably determined
during the embryonic development, may depend on modification of the structure
and/or function of blood platelets.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Aug 2008 · The FASEB Journal
[Show abstract][Hide abstract]ABSTRACT: Serotonin (5-HT) is a biogenic amine produced in the brain by cells originating in
the raphe nuclei of the brain stem and released by serotonergic endings. 5-HT is
also synthesized in large quantities in gut enterochromaffin cells. Tryptophan
hydroxylase (TPH), the rate-limiting enzyme of 5-HT synthesis, is found only in
5-HT-producing cells, and exists in two forms, one of which is predominantly in
neuronal cells (TPH2) while the other is non-neuronal (TPH1). The early
appearance of 5-HT, its receptors, and transporter (SERT) during mouse embryonic
development, together with the ability of 5-HT-specific pharmacological agents to
interfere with development, strongly suggest that 5-HT functions as a humoral
morphogen before it becomes a neurotransmitter. Along this line, our recent data
showed that maternal 5-HT is a key factor for normal embryonic development in
mice. We observed that at embryonic day 12.5 (E12.5) tph1-/- embryos from tph1
null mothers either developed normally, suffered growth retardation or died
in utero. To reveal the developmental time point at which differences emerge
between mutant and wild-type embryos, a series of experiments were conducted in
which the various staged null embryos (tph1-/-) were recovered at different time
points, from E7.5 to E18.5 and compared to control embryos obtained from wildtype
females. We performed histological, immuno-histochemical and in situ
hybridization studies. Most interestingly, hematoxylin and eosin staining of the
tph1-/- embryos from 5-HT deficient females, demonstrated abnormal morphogenesis
of organs including brain and liver. Gross anatomical analysis revealed that
beginning at E12.5 and apparent by E13.5, 5-HT deficient embryos were pale and
their overall liver architecture was significantly impaired as compared to wild-type
embryos. Moreover, the livers of E13.5 mutant embryos displayed a lacy
appearance and spaces devoid of cells were also observed. At the brain level, in
addition to morphological abnormalities, the results demonstrate that maternal 5-
HT seems to modulate the expression of SERT. Results will be discussed in terms of
health issues as maintenance of a proper level of 5-HT may be critical for normal
development, for example, although the pathophysiology of autism is unknown,
autism is thought to be a ‘disease of development’.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Feb 2007 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: Serotonin (5-HT) controls multiple biological functions.
While its importance in the nervous system is appreciated, its role at the
periphery remains poorly understood. The rate-limiting step in 5-HT
biosynthesis, is catalyzed by two isoforms of tryptophan hydroxylase, TPH1 and
TPH2. Peripheral 5-HT is synthesized by TPH1 in the gastro-intestinal tract, is
stored in blood platelets, participates in blood pressure, coagulation and
hemostasis. Serotonin has also been suggested to regulate cardiovascular
development through the 5-HT2B receptor. In view of the proposed functions of
peripheral serotonin and to better understand the mechanisms by which it acts,
we used a constitutive gene inactivation approach to ablate 5-HT function
specifically in the periphery. The TPH1 mutant mice (TPH1-/-) were generated by
targeting the bacterial lacZ reporter gene into the TPH1 locus under the control
of the TPH1 promoter. Insertion of the marker into this locus made it possible to
follow the activity of the TPH1 promoter in cells lacking the enzyme and to
analyse the fate of these cells during mouse development. The biochemical,
physiological, and histological consequences following disruption of the TPH1
gene include a dramatic reduction in peripheral 5-HT and functional alterations
which can progressively lead to heart failure. Investigation of transverse sections
of the heart showed no primary structural abnormalities indicative of a normal
development. In order to provide a detailed expression analysis of TPH1 and
TPH2 during mouse development, we performed in situ hybridization using
specific non-overlapping probes for both genes. This approach yields novel
information on areas expressing the two TPH isoforms. The expression of TPH2
is neuro-specific and occurs as early as E10.5 in the ventral most neuroepithelial
domain of the hindbrain, while production of peripheral 5-HT has been observed
on E9.5 in the yolk sac. Our data suggest that the low level of circulating 5-HT in
TPH1-/- animals is sufficient for development and viability of the mouse embryo.
Specifically no developmental abnormality was observed either in skeletal or
cardiac muscles. The serotonin level is, however, critical to maintain normal
cardiac function in the adult animal.
[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Dec 2003 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: Microsatellites are common repeated sequences, which are useful as genetic markers and lack any clearly established function. In a previous study we suggested that an intronic polymorphic TCAT repeat in the tyrosine hydroxylase (TH) gene, the microsatellite HUMTH01, may regulate transcription. The TH gene encodes the rate-limiting enzyme in the synthesis of catecholamines, and the microsatellite HUMTH01 has been used in genetic studies of neuropsychiatric and cardiovascular diseases, in which disturbances of catecholaminergic neurotransmission have been implicated. HUMTH01 alleles associated with these diseases act as transcriptional enhancers when linked to a minimal promoter and are recognized by specific nuclear factors. Here we show that allelic variations of HUMTH01 commonly found in humans have a quantitative silencing effect on TH gene expression. Two specific proteins, ZNF191, a zinc finger protein, and HBP1, an HMG box transcription factor, which bind the TCAT motif, were then cloned. Finally, allelic variations of HUMTH01 correlate with quantitative and qualitative changes in the binding by ZNF191. Thus, this repeated sequence may contribute to the control of expression of quantitative genetic traits. As the HUMTH01 core motif is ubiquitous in the genome, this phenomenon may be relevant to the quantitative expression of many genes in addition to TH.
Full-text · Article · Sep 2001 · Human Molecular Genetics
[Show abstract][Hide abstract]ABSTRACT: We have previously shown that the microsatellite HUMTH01, located in the first intron of the tyrosine hydroxylase (TH) gene and characterized by a (TCAT)n motif, is associated both with bipolar disorder and schizophrenia. We have then shown that alleles of this microsatellite are specifically recognized by transacting factors and enhance the transcription of the luciferase reporter gene driven by a tymidine kinase minimal promoter. In order to further characterize the HUMTH01 microsatellite, the sequence encompassing the TH gene from 2Kb upstream the ATG initiation codon up to the third exon was fused in frame with the luciferase gene. In this construct a "cassette" system allowed for inserting either at the orthologous or heterologous positions different alleles of the HUMTH01 microsatellite as well as other unrelated repeated sequences. Transient transfection experiments in different cell lines were performed in order to evaluate the effect of these repeated sequences on transcription. These studies show that the (TCAT)n repeated sequence acts in a specific fashion and is able to differentially modulate TH gene activity according to the length of its alleles. Since this polymorphic tetrarepeat is widespread throughout the genome, these results may also be relevant for other genes either containing or located near this sequence.
No preview · Article · Aug 2000 · American Journal of Medical Genetics
[Show abstract][Hide abstract]ABSTRACT: We have established that the HUMTH01 microsatellite, located in the first intron of the tyrosine hydroxylase (TH) gene and characterized by a (TCAT)n motif, specifically modulates gene activity according to the length of its alleles. In order to identify the proteins that interact with this functional sequence, the (TCAT)n motif was used as a bait for screening a human c-DNA brain library in the one-hybrid yeast system. The protein product of the most represented of the validated c-DNAs among 8 million of independent clones tested, was purified after bacterial expression. Electrophoretic migration shift assays and footprint analysis showed that the recombinant protein, pertaining to a family of transacting factors, exhibits specific affinity for the (TCAT)n sequence. Moreover, the number of sites occupied by this factor within the HUMTH01 microsatellite is correlated with variations in the number of (TCAT)n repetitions and may correspond to variations in its transactivating effects. The (TCAT)n polymorphic sequence is widespread throughout the genome. Thus, the characterization of the transduction pathway impinging on the interaction of this protein with its target may open new perspectives on the regulation of the TH gene as well as that of other genes implicated in normal and pathological complex genetic traits.