Adult neural stem cell fate is determined by thyroid hormone activation of mitochondrial metabolism.

OBJECTIVE: In the adult brain, neural stem cells (NSCs) located in the subventricular zone (SVZ) produce both neuronal and glial cells. Thyroid hormones (THs) regulate adult NSC differentiation towards a neuronal phenotype, but also have major roles in mitochondrial metabolism. As NSC metabolism relies mainly on glycolysis, whereas mature cells preferentially use oxidative phosphorylation, we studied how THs and mitochondrial metabolism interact on NSC fate determination. METHODS: We used a mitochondrial membrane potential marker in vivo to analyze mitochondrial activity in the different cell types in the SVZ of euthyroid and hypothyroid mice. Using primary adult NSC cultures, we analyzed ROS production, SIRT1 expression, and phosphorylation of DRP1 (a mitochondrial fission mediator) as a function of TH availability. RESULTS: We observed significantly higher mitochondrial activity in cells adopting a neuronal phenotype in vivo in euthyroid mice. However, prolonged hypothyroidism reduced not only neuroblast numbers but also their mitochondrial activity. In vitro studies showed that TH availability favored a neuronal phenotype and that blocking mitochondrial respiration abrogated TH-induced neuronal fate determination. DRP1 phosphorylation was preferentially activated in cells within the neuronal lineage and was stimulated by TH availability. CONCLUSIONS: These results indicate that THs favor NSC fate choice towards a neuronal phenotype in the adult mouse SVZ through effects on mitochondrial metabolism.

Differential thyroid hormone sensitivity of fast cycling progenitors in the neurogenic niches of tadpoles and juvenile frogs.

Adult neurogenesis occurs in neural stem cell (NSC) niches where slow cycling stem cells give rise to faster cycling progenitors. In the adult mouse NSC niche thyroid hormone, T3, and its receptor TRα act as a neurogenic switch promoting progenitor cell cycle completion and neuronal differentiation. Little is known about whether and how T3 controls proliferation of differentially cycling cells during xenopus neurogenesis. To address this question, we first used Sox3 as a marker of stem cell and progenitor populations and then applied pulse-chase EdU/IdU incorporation experiments to identify Sox3-expressing slow cycling (NSC) and fast cycling progenitor cells. We focused on the lateral ventricle of Xenopus laevis and two distinct stages of development: late embryonic development (pre-metamorphic) and juvenile frogs (post-metamorphic). These stages were selected for their relatively stable thyroid hormone availability, either side of the major dynamic phase represented by metamorphosis. TRα expression was found in both pre and post-metamorphic neurogenic regions. However, exogenous T3 treatment only increased proliferation of the fast cycling Sox3+ cell population in post-metamorphic juveniles, having no detectable effect on proliferation in pre-metamorphic tadpoles. We hypothesised that the resistance of proliferative cells to exogenous T3 in pre-metamorphic tadpoles could be related to T3 inactivation by the inactivating Deiodinase 3 enzyme. Expression of dio3 was widespread in the tadpole neurogenic niche, but not in the juvenile neurogenic niche. Use of a T3-reporter transgenic line showed that in juveniles, T3 had a direct transcriptional effect on rapid cycling progenitors. Thus, the fast cycling progenitor cells in the neurogenic niche of tadpoles and juvenile frogs respond differentially to T3 as a function of developmental stage.

Human longevity is characterised by high thyroid stimulating hormone secretion without altered energy metabolism.

Few studies have included subjects with the propensity to reach old age in good health, with the aim to disentangle mechanisms contributing to staying healthier for longer. The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating levels of thyroid stimulating hormone (TSH) and thyroid hormone (TH) in an inverse relationship. Greater longevity has been associated with higher TSH and lower TH levels, but mechanisms underlying TSH/TH differences and longevity remain unknown. The HPT axis plays a pivotal role in growth, development and energy metabolism. We report that offspring of nonagenarians with at least one nonagenarian sibling have increased TSH secretion but similar bioactivity of TSH and similar TH levels compared to controls. Healthy offspring and spousal controls had similar resting metabolic rate and core body temperature. We propose that pleiotropic effects of the HPT axis may favour longevity without altering energy metabolism.

Thyroid hormone signaling and homeostasis during aging.

Studies in humans and in animal models show negative correlations between thyroid hormone (TH) levels and longevity. TH signaling is implicated in maintaining and integrating metabolic homeostasis at multiple levels, notably centrally in the hypothalamus but also in peripheral tissues. The question is thus raised of how TH signaling is modulated during aging in different tissues. Classically, TH actions on mitochondria and heat production are obvious candidates to link negative effects of TH to aging. Mitochondrial effects of excess TH include reactive oxygen species and DNA damage, 2 factors often considered as aging accelerators. Inversely, caloric restriction, which can retard aging from nematodes to primates, causes a rapid reduction of circulating TH, reducing metabolism in birds and mammals. However, many other factors could link TH to aging, and it is these potentially subtler and less explored areas that are highlighted here. For example, effects of TH on membrane composition, inflammatory responses, stem cell renewal and synchronization of physiological responses to light could each contribute to TH regulation of maintenance of homeostasis during aging. We propose the hypothesis that constraints on TH signaling at certain life stages, notably during maturity, are advantageous for optimal aging.

Towards a humanized PPARγ reporter system for in vivo screening of obesogens.

Overeating and lack of exercise are major contributors to the current obesity epidemic, but environmental contaminants, or obesogens, are also considered to be potential actors. A common obesogen target is the Peroxisome Proliferator Activated Receptor Gamma (PPARγ). Screening for exogenous obesogens requires in vivo systems as many xenobiotics exert their effects through metabolites. We thus developed a humanized in vivo PPARγ reporter model, using Xenopus laevis larvae, a species possessing metabolic capacities comparable to mammals. A somatic transgenesis approach was used to co-express an expression vector for the human PPARγ protein simultaneously with one of a series of reporter vectors, each containing a PPARγ Response Element (PPRE)-eGFP sequence. Treatment of tadpoles with PPARγ agonists, antagonists or candidate obesogens, significantly modulated eGFP expression. Thus, the system provides a promising proof of principle for a sensitive and reliable humanized in vivo tool to screen both novel PPARγ drug ligands and potential endocrine disruptors or obesogens targeting this receptor.

Is thyroid hormone signaling relevant for vertebrate embryogenesis?

Classically, thyroid hormones (THs) have been primarily associated with postembryonic development (Tata, 1968), notably metamorphosis in anuran amphibians and flat fish. This period is parallel to the perinatal period in man and many marked developmental transitions in other species. As amply described in other chapters, metamorphosis is characterized by a peak of thyroxine (T(4)) and triiodothyronine (T(3)) that is synchronous with the metamorphic climax. In contrast, the developmental period that characterizes embryonic development prior to the significant production of TH by the endogenous thyroid gland has received little attention. Furthermore, the prevailing concepts of TH physiology during this period have been framed by two observations in amphibians and mammals: first, TRs are expressed, while circulating TH levels are much lower than those during metamorphosis and, second, extrapolating from the knowledge largely obtained from in vitro models, in the absence of TH, the aporeceptor represses target gene transcription during premetamorphic development. We propose to revisit both concepts in the light of accumulating data, first, on TH availability both in eggs and in embryos and, second, on the increasing knowledge of the complexity of TR and TH control of transcription.

Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians--screening for estrogen, androgen and thyroid hormone disruption.

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption.

Thyroid hormone (TH) is essential for vertebrate brain development. Most research on TH and neuronal development focuses on late development, mainly the perinatal period in mammals. However, in human infants neuromotor development correlates best with maternal TH levels in the first trimester of pregnancy, suggesting that TH signaling could affect early brain development. Studying TH signaling in early embryogenesis in mammals is experimentally challenging. In contrast, free-living embryos, such as Xenopus laevis, permit physiological experimentation independent of maternal factors. We detailed key elements of TH signaling: ligands, receptors (TR), and deiodinases during early X. laevis development, before embryonic thyroid gland formation. Dynamic profiles for all components were found. Between developmental stages 37 and 41 (~48 h after hatching, coincident with a phase of continuing neurogenesis) significant increases in T(3) levels as well as in mRNA encoding deiodinases and TR occurred. Exposure of embryos at this developmental stage for 24 h to either a TH antagonist, NH-3, or to tetrabromobisphenol A, a flame retardant and known TH disruptor, differentially modulated the expression of a number of TH target genes implicated in neural stem cell function or neural differentiation. Moreover, 24-h exposure to either NH-3 or tetrabromobisphenol A diminished cell proliferation in the brain. Thus, these data show first, that TH signaling exerts regulatory roles in early X. laevis neurogenesis and second, that this period represents a potential window for endocrine disruption.

Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor.

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Thyroid status co-regulates thyroid hormone receptor and co-modulator genes specifically in the hypothalamus.

Regulation of Thyrotropin Releasing Hormone (TRH) transcription in the hypothalamus represents the central control point of thyroid function. To examine the expression of potential TRH regulatory components, we simultaneously amplified, by semi-quantitative multiplex PCR system, nine key genes from < or = 100 ng total RNA from two brain areas (hypothalamus and cortex) under different thyroid states. Expression of TR1 and TR2 isoforms, key elements in TRH regulation, was modified by thyroid status in the hypothalamus but not in the cortex. Similarly, hypothyroidism increased specifically hypothalamic levels of three co-modulator genes. This study provides the first demonstration of tissue specific co-regulation of a set of genes by thyroid status within a defined brain area.

Nuclear corepressor and silencing mediator of retinoic and thyroid hormone receptors corepressor expression is incompatible with T(3)-dependent TRH regulation.

Ligand-independent repression by thyroid hormone (T(3)) receptors on positive T(3)-responsive genes requires corepressor proteins. However, the role of corepressors in regulating genes such as hypothalamic TRH, which are under negative control by T(3), is largely unknown. We examined the expression of mRNAs encoding the corepressors NCoR (nuclear corepressor) and SMRT (silencing mediator of retinoic and thyroid hormone receptors) in the TRH-producing paraventricular nucleus of the mouse hypothalamus. Further, we carried out in vivo functional studies by overexpression of both corepressors. Three lines of evidence show that NCoR and SMRT expression is incompatible with physiological regulation of TRH. First, Northern blotting revealed TRH and NCoR mRNA expressions to be inversely correlated during postnatal development and as a function of thyroid status. Second, in situ hybridization showed that NCoR and SMRT mRNA expression profiles in the paraventricular nucleus were distinct from that of TRH mRNA. Third, over-expression of full length NCoR and SMRT in the hypothalamus abolished T(3)-dependent repression of TRH-luciferase. However, over-expression of NCoR or SMRT did not affect either T(3)-independent activation of TRH-luciferase transcription, or transcription from a positively regulated T(3)-response element. We conclude that T(3) -dependent feedback on TRH expression is unlikely to involve the corepressors NCoR or SMRT.

Xenopus Bcl-X(L) selectively protects Rohon-Beard neurons from metamorphic degeneration.

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-X(L) plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-X(L) homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal beta-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

Polyethylenimines for in vivo gene delivery.

Branched and linear polyethylenimines (PEIs) are proving to be efficient, non-toxic and versatile agents for in vivo gene delivery by a number of routes. A major factor in the successful use of PEIs seems to be the small size of PEI/DNA complexes which can be achieved under controlled conditions of formulation, mainly by using PEIs of low molecular weights. This review considers the in vivo use of PEI, from formulation to delivery and analysis of gene expression. PEI delivery is already used for the analysis of numerous physiological processes. It is hoped that scrutiny of the mechanisms involved with PEI-based gene delivery at different levels of the transfection process and in different in vivo contexts will aid the transition towards its use in therapeutic situations.

Cross-talk between signal transducer and activator of transcription (Stat5) and thyroid hormone receptor-beta 1 (TRbeta1) signaling pathways.

PRL and T3 are involved in antagonistic regulations during various developmental processes in vertebrate species. We have studied cross-talk between transcription factors activated by these signaling pathways, i.e. signal transducer and activator of transcription 5 (Stat5) and thyroid hormone receptor beta1 (TRbeta1). Liganded TRbeta1 in the presence of its heterodimeric partner, retinoid X receptor gamma (RXRgamma), inhibited the PRL-induced Stat5a- and Stat5b-dependent reporter gene expression by up to 60%. This T3-inhibitory effect studied on Stat5 activity was partly reversed by overexpression of a TRbeta1 dominant negative variant mutated within its nuclear localization signal (TR2A). We next showed that TRbeta1 and TR2A in the presence of RXRgamma increased and decreased, respectively, Stat5 localization into the nucleus regardless of hormonal stimulation. Thus, our data suggest that TRbeta1 can be associated with Stat5 in the cytoplasm and may be involved in Stat5 nuclear translocation. In PRL-treated cells overexpressing TRbeta1/RXRgamma, both Stat5 and TRbeta1 were coimmunoprecipitated, indicating physical association of the two transcription factors. In these cells, addition of T3 with ovine (o)PRL decreased the amounts of total and tyrosine-phosphorylated Stat5 in the cytoplasm compared with oPRL-treated cells. In the nucleus, no clear difference was observed on Stat5 DNA-binding after treatment with PRL and T3 vs. PRL alone in TRbeta1/RXRgamma transfected cells. However, antibodies directed against TRbeta1 lowered Stat5-DNA binding and addition of the deacetylase inhibitor trichostatin A (TSA) relieved T3 inhibition on Stat5 transcriptional activity. Thus, we postulated that the negative cross-talk between TR and Stat5 on target genes could involve histone deacetylase recruitment by liganded TRbeta1.

Transcriptional repression of TRH promoter function by T3: analysis by in vivo gene transfer.

We consider how an integrated in vivo model can be used to study the specific transcriptional effects of specific receptors in neuroendocrine systems. Our example is the role of thyroid receptor (TR) isoforms in mediating negative feedback effects of T3 on TRH (thyrotropin releasing hormone) expression. The in vivo transfection method employed polyethylenimine (PEI) to introduce genes directly into specific regions of the brains of mice, rats, and Xenopus tadpoles. In the mouse model, the technique has served to examine TR effects on TRH transcription and on the pituitary-thyroid axis end point: thyroid hormone secretion. When a TRH-luciferase construct is introduced into the hypothalami of newborn mice TRH-luciferase transcription is regulated physiologically, being significantly increased in hypothyroidism and decreased in T3-treated animals. When various T3-binding forms of TRbeta or TRalpha are expressed in the hypothalamus, all TRbeta isoforms give T3-dependent regulation of TRH transcription, whereas TRalpha isoforms block T3-dependent transcription. Moreover, TR transcriptional effects are correlated with physiological consequences on circulating T4. Thus, somatic gene transfer shows TR subtypes to have distinct, physiologically relevant effects on TRH transcription. The approach is an appealing alternative to germinal transgenesis for studying specific neuroendocrine regulations at defined developmental stages in different species.

Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes.

Intravenous administration could become a delivery route of choice for prophylactic and curative gene therapies on condition that genes cross the capillary barrier and reach target tissues without being degraded. We investigated the kinetics and process of transgene delivery through mouse lung capillaries following DNA complexation with linear polyethylenimine (L-PEI) and intravenous injection. Using digoxin-labeled DNA we followed the cellular localization of DNA at different times after injection and correlated these findings with cell markers and transgene expression. At 2 h after injection some DNA was still localized on the interior of the capillary lumen, but other complexes had already crossed the barrier and resulted in gene expression. At 24 h after injection most labeled DNA was localised in pulmonary cells, as was transgene expression. Only rarely was transgene expression found in endothelial cells, suggesting that the complexes cross the capillary barrier rapidly. Levels of caspase-1-like activity did not increase following transfection implying that L-PEI/DNA complexes are transported across cellular barriers by a non-damaging, physiological process, without causing inflammation. The high levels of expression of different transgenes in pneumocytes indicates that transport of L-PEI/DNA complexes through the endothelial barrier does not affect their transfection capacity. These findings open up new possibilities for gene delivery and its application to the lung.

Subtle shifts in the ratio between pro- and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate.

Glucocorticoid receptor (GR) activation induces apoptosis of granule cells in the hippocampus. In contrast, neuroprotection is seen after mineralocorticoid receptor (MR) activation. To date there is no in vivo evidence for direct interactions between corticosteroids and any of the key regulatory molecules of programmed cell death. In this report, we show that the opposing actions of MR and GR on neuronal survival result from their ability to differentially influence the expression of members of the bcl-2 gene family; specifically, in the rat hippocampus, activation of GR induces cell death by increasing the ratio of the proapoptotic molecule Bax relative to the antiapoptotic molecules Bcl-2 or Bcl-x(L); the opposite effect is observed after stimulation of MR. The same results were obtained in both young and aged animals; however, older subjects (which were more susceptible to GR-mediated apoptosis) tended to express the antiapoptotic genes more robustly. Using a loss-of-function mouse model, we corroborated the observations made in the rat, demonstrating Bax to be essential in the GR-mediated cell death-signaling cascade. In addition, we show that GR activation increases and MR activation decreases levels of the tumor suppressor protein p53 (a direct transcriptional regulator of bax and bcl-2 genes), thus providing new information on the early genetic events linking corticosteroid receptors with apoptosis in the nervous system.

Optimisation of polyethylenimine-based gene delivery to mouse brain.

Polyethylenimine (PEI) is proving to be an efficient and versatile vector for gene delivery in vivo. However, a limiting factor is the relatively short duration of gene expression in some sites. Given the particularly high levels of expression seen in the short term we postulated that loss of expression could result from overloading the nucleus with foreign DNA. To address this problem we first followed DNA delivery and localisation with digoxin-labelled plasmid DNA complexed with 22 kD linear PEI and used these complexes for intraventricular injection into brains of anaesthetised newborn mice. At 24 h post-injection, labelled DNA was found exclusively in the nuclear and perinuclear regions. We next carried out a dose response curve using decreasing amounts of DNA, either in a constant volume (2 microl) or at a constant concentration (500 ng/microl). In both conditions, transgene expression yield was maximum at 100 ng DNA per injection. Using this optimal amount of DNA increased yield of transgene expression significantly at 24 h and one week post-injection as compared to 1 microg DNA. A final point addressed was whether co-expressing an anti-apoptotic gene could enhance gene expression in the longer term. Co-expressing bcl-X(L) with luciferase or LacZ significantly increased expression of both these genes at one week post-injection.

A somatic gene transfer approach using recombinant fusion proteins to map muscle-motoneuron projections in Xenopus spinal cord.

A combination of somatic gene transfer with fusion protein technology has been developed, thus providing an innovative means of mapping muscle-motoneuronal connections in Xenopus tadpole spinal cord. We analyzed whether a neuronal tracer created by the fusion of the LacZ gene to the tetanus toxin C fragment (LacZ-TTC) could be produced from plasmid DNA injected into muscle, and whether it could be released and undergo retrograde transport into motoneurons. Plasmids encoding various fusion protein constructions, with or without a signal peptide, were injected into dorsal or caudal muscles of premetamorphic tadpoles. The marker was produced in the muscle at constantly high levels. At one month post-injection, the fusion protein passed the neuromuscular junction and underwent retrograde transport into motoneurons. Transfer into motoneurons was seen for every animal injected, emphasizing the high reproducibility and efficiency of the process. No uptake of beta-gal protein into motoneurons was observed in the absence of the TTC fragment. Furthermore, no enhancement was obtained by adding a signal peptide. These results provide the first demonstration of the synthesis and transport of a TTC fusion protein produced directly from exogenous DNA in a vertebrate system.