Early life exposure to air pollution particulate matter (PM) as risk factor for attention deficit/hyperactivity disorder (ADHD): Need for novel strategies for mechanisms and causalities.

Epidemiological studies have demonstrated that air pollution particulate matter (PM) and adsorbed toxicants (organic compounds and trace metals) may affect child development already in utero. Recent studies have also indicated that PM may be a risk factor for neurodevelopmental disorders (NDDs). A pattern of increasing prevalence of attention deficit/hyperactivity disorder (ADHD) has been suggested to partly be linked to environmental pollutants exposure, including PM. Epidemiological studies suggest associations between pre- or postnatal exposure to air pollution components and ADHD symptoms. However, many studies are cross-sectional without possibility to reveal causality. Cohort studies are often small with poor exposure characterization, and confounded by traffic noise and socioeconomic factors, possibly overestimating the study associations. Furthermore, the mechanistic knowledge how exposure to PM during early brain development may contribute to increased risk of ADHD symptoms or cognitive deficits is limited. The closure of this knowledge gap requires the combined use of well-designed longitudinal cohort studies, supported by mechanistic in vitro studies. As ADHD has profound consequences for the children affected and their families, the identification of preventable risk factors such as air pollution exposure should be of high priority.

High and low concentration of 17α-estradiol protect cerebellar granule neurons in different time windows.

17α-estradiol is a hormonally inactive isomer of 17ß-estradiol, but with similar potency as neuroprotector. However, we have previously reported that pretreatment with high concentration (10 µM) of both estrogens abolishes their neuroprotection in rat cerebellar granule neurons. Here, we have examined neuroprotective properties of 17α-estradiol against glutamate-induced excitotoxicity in chicken cerebellar granule neurons using low (1 nM) and high concentration.17α-Estradiol, 1 nM, was neuroprotective when glutamate was administered after a pretreatment period of 24 h, but not when coadministered with glutamate. In contrast, 10 µM was protective when coadministered with glutamate, but was not efficient when glutamate was administered after a pretreatment period. The difference in protection was linked to a stronger calcium response during glutamate exposure in the non-protective treatments. In addition, the pretreatment period with 10 µM was accompanied by increased protein level of the N-methyl-d-aspartate receptor subunit NR2B and reduced glutathione level. Thus, 17α-estradiol has a concentration and time dependent protective effect against glutamate-induced cell death.

Synthesis and initial in vitro biological evaluation of two new zinc-chelating compounds: comparison with TPEN and PAC-1.

The lipophilic, cell-penetrating zinc chelator N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 1) and the zinc chelating procaspase-activating compound PAC-1 (2) both have been reported to induce apoptosis in various cell types. The relationship between apoptosis-inducing ability and zinc affinity (Kd), have been investigated with two new model compounds, ZnA-DPA (3) and ZnA-Pyr (4), and compared to that of TPEN and PAC-1. The zinc-chelating o-hydroxybenzylidene moiety in PAC-1 was replaced with a 2,2'-dipicoylamine (DPA) unit (ZnA-DPA, 3) and a 4-pyridoxyl unit (ZnA-Pyr, 4), rendering an order of zinc affinity TPEN>ZnA-Pyr>ZnA-DPA>PAC-1. The compounds were incubated with the rat pheochromocytoma cell line PC12 and cell death was measured in combination with ZnSO4, a caspase-3 inhibitor, or a ROS scavenger. The model compounds ZnA-DPA (3) and ZnA-Pyr (4) induced cell death at higher concentrations as compared to PAC-1 and TPEN, reflecting differences in lipophilicity and thereby cell-penetrating ability. Addition of ZnSO4 reduced cell death induced by ZnA-Pyr (4) more than for ZnA-DPA (3). The ability to induce cell death could be reversed for all compounds using a caspase-3-inhibitor, and most so for TPEN (1) and ZnA-Pyr (4). Reactive oxygen species (ROS), as monitored using dihydro-rhodamine (DHR), were involved in cell death induced by all compounds. These results indicate that the Zn-chelators ZnA-DPA (3) and ZnA-Pyr (4) exercise their apoptosis-inducing effect by mechanisms similar to TPEN (1) and PAC-1 (2), by chelation of zinc, caspase-3 activation, and ROS production.

Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF.

NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

Cell death induced by novel procaspase-3 activators can be reduced by growth factors.

Caspase-3 is known as the key executioner caspase, activated in both the intrinsic and extrinsic apoptotic pathway, and an effector far downstream in the apoptotic cascade. Procaspase-activating compound-1 (PAC-1) and 1541 were launched as direct activators of procaspase-3 to caspase-3, and anticipated to be promising therapeutic agents for the treatment of cancer. PAC-1 has recently been evaluated in a phase I preclinical trial. However, little is known about the effect of these substances in cells. Activation of caspase-3 in whole cells may be more complicated than thought, as it is likely that this key protease is tightly regulated both in development and apoptosis. In this study, we investigated the effect of epidermal growth factor (EGF) on PAC-1-induced caspase-3 activity and cell death. We show that EGF can block caspase-3 activity generated by PAC-1, and protect both PC12 cells and primary cerebellar granule neurons against PAC-1-induced death. Similar results were obtained with 1541. Both substances reduced cellular p-ERK levels. Crosstalk between caspase-3 and growth factor signaling pathways may present a challenge for the use of such caspase-3-activating substances in cancer therapy, since aberrant growth factor signaling is frequently seen in malignant cells. This study adds important knowledge about cellular effects of procaspase-3 activators like PAC-1 and 1541. Effects mediated by these substances may also contribute to the understanding of caspase signaling in cells.

Delayed translocation of NGFI-B/RXR in glutamate stimulated neurons allows late protection by 9-cis retinoic acid.

Nuclear receptor and apoptosis inducer NGFI-B translocates out of the nucleus as a heterodimer with RXR in response to different apoptosis stimuli, and therefore represents a potential pharmacological target. We found that the cytosolic levels of NGFI-B and RXRα were increased in cultures of cerebellar granule neurons 2h after treatment with glutamate (excitatory neurotransmitter in the brain, involved in stroke). To find a time-window for potential intervention the neurons were transfected with gfp-tagged expressor plasmids for NGFI-B and RXR. The default localization of NGFI-Bgfp and RXRgfp was nuclear, however, translocation out of the nucleus was observed 2-3h after glutamate treatment. We therefore hypothesized that the time-window between treatment and translocation would allow late protection against neuronal death. The RXR ligand 9-cis retinoic acid was used to arrest NGFI-B and RXR in the nucleus. Addition of 9-cis retinoic acid 1h after treatment with glutamate reduced the cytosolic translocation of NGFI-B and RXRα, the cytosolic translocation of NGFI-Bgfp observed in live neurons, as well as the neuronal death. However, the reduced translocation and the reduced cell death were not observed when 9-cis retinoic acid was added after 3h. Thus, late protection from glutamate induced death by addition of 9-cis retinoic acid is possible in a time-window after apoptosis induction.

Exposure to human relevant mixtures of halogenated persistent organic pollutants (POPs) alters neurodevelopmental processes in human neural stem cells undergoing differentiation.

Halogenated persistent organic pollutants (POPs) like perfluorinated alkylated substances (PFASs), brominated flame retardants (BFRs), organochlorine pesticides and polychlorinated biphenyls (PCBs) are known to cause cancer, immunotoxicity, neurotoxicity and interfere with reproduction and development. Concerns have been raised about the impact of POPs upon brain development and possibly neurodevelopmental disorders. The developing brain is a particularly vulnerable organ due to dynamic and complex neurodevelopmental processes occurring early in life. However, very few studies have reported on the effects of POP mixtures at human relevant exposures, and their impact on key neurodevelopmental processes using human in vitro test systems. Aiming to reduce this knowledge gap, we exposed mixed neuronal/glial cultures differentiated from neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) to reconstructed mixtures of 29 different POPs using concentrations comparable to Scandinavian human blood levels. Effects of the POP mixtures on neuronal proliferation, differentiation and synaptogenesis were evaluated using in vitro assays anchored to common key events identified in the existing developmental neurotoxicity (DNT) adverse outcome pathways (AOPs). The present study showed that mixtures of POPs (in particular brominated and chlorinated compounds) at human relevant concentrations increased proliferation of NSCs and decreased synapse number. Based on a mathematical modelling, synaptogenesis and neurite outgrowth seem to be the most sensitive DNT in vitro endpoints. Our results indicate that prenatal exposure to POPs may affect human brain development, potentially contributing to recently observed learning and memory deficits in children.

Does the food processing contaminant acrylamide cause developmental neurotoxicity? A review and identification of knowledge gaps.

There is a worldwide concern on adverse health effects of dietary exposure to acrylamide (AA) due to its presence in commonly consumed foods. AA is formed when carbohydrate rich foods containing asparagine and reducing sugars are prepared at high temperatures and low moisture conditions. Upon oral intake, AA is rapidly absorbed and distributed to all organs. AA is a known human neurotoxicant that can reach the developing foetus via placental transfer and breast milk. Although adverse neurodevelopmental effects have been observed after prenatal AA exposure in rodents, adverse effects of AA on the developing brain has so far not been studied in humans. However, epidemiological studies indicate that gestational exposure to AA impair foetal growth and AA exposure has been associated with reduced head circumference of the neonate. Thus, there is an urgent need for further research to elucidate whether pre- and perinatal AA exposure in humans might impair neurodevelopment and adversely affect neuronal function postnatally. Here, we review the literature with emphasis on the identification of critical knowledge gaps in relation to neurodevelopmental toxicity of AA and its mode of action and we suggest research strategies to close these gaps to better protect the unborn child.

Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons.

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

Mono(2-ethylhexyl) phthalate induces both pro- and anti-inflammatory responses in rat alveolar macrophages through crosstalk between p38, the lipoxygenase pathway and PPARalpha.

Airway inflammation is important in asthma pathogenesis. Recent epidemiological data have indicated an association between asthma symptoms in children and exposure to di(2-ethylhexyl) phthalate (DEHP). Thus, we have studied inflammatory responses in primary rat alveolar macrophages (AMs) after exposure to mono(2-ethylhexyl) phthalate (MEHP), the major primary metabolite of DEHP. First, we show that MEHP induces a dose-dependent release of the pro-inflammatory tumour necrosis factor-alpha (TNF-alpha) in AMs, giving a maximal (5-fold) increase at 0.7 mM. This concentration also induced some cell death. MEHP also induced phosphorylation of MAPK p38, while the p38 inhibitor SB 202190 reduced MEHP-induced TNF-alpha, suggesting a p38-dependent cytokine production. Next, we elucidated possible effects of MEHP on the 5-lipoxygenase (5-LO) pathway and found that MEHP caused increased leukotriene (LTB(4)) release. Further, we found that the 5-LO inhibitor nordihydrogualaretic acid (NDGA) significantly reduced both MEHP-induced TNF-alpha release and MEHP-induced formation of reactive oxygen species (ROS), supporting an involvement of the 5-LO pathway in MEHP induced inflammatory reactions. Last, we found that MK-886, a known inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), increased the MEHP-induced TNF-alpha response. This indicates that MEPH-PPARalpha binding mediates an anti-inflammatory signal.

Exploring the overlapping binding sites of ifenprodil and EVT-101 in GluN2B-containing NMDA receptors using novel chicken embryo forebrain cultures and molecular modeling.

N-methyl-d-aspartate receptors (NMDAR) are widely expressed in the brain. GluN2B subunit-containing NMDARs has recently attracted significant attention as potential pharmacological targets, with emphasis on the functional properties of allosteric antagonists. We used primary cultures from chicken embryo forebrain (E10), expressing native GluN2B-containing NMDA receptors as a novel model system. Comparing the inhibition of calcium influx by well-known GluN2B subunit-specific allosteric antagonists, the following rank order of potency was found: EVT-101 (EC 50 22 ± 8 nmol/L) > Ro 25-6981 (EC 50 60 ± 30 nmol/L) > ifenprodil (EC 50 100 ± 40 nmol/L) > eliprodil (EC 50 1300 ± 700 nmol/L), similar to previous observations in rat cortical cultures and cell lines overexpressing chimeric receptors. The less explored Ro 04-5595 had an EC 50 of 186 ± 32 nmol/L. Venturing to explain the differences in potency, binding properties were further studied by in silico docking and molecular dynamics simulations using x-ray crystal structures of GluN1/GluN2B amino terminal domain. We found that Ro 04-5595 was predicted to bind the recently discovered EVT-101 binding site, not the ifenprodil-binding site. The EVT-101 binding pocket appears to accommodate more structurally different ligands than the ifenprodil-binding site, and contains residues essential in ligand interactions necessary for calcium influx inhibition. For the ifenprodil site, the less effective antagonist (eliprodil) fails to interact with key residues, while in the EVT-101 pocket, difference in potency might be explained by differences in ligand-receptor interaction patterns.

Low-potency glucocorticoid hydrocortisone has similar neurotoxic effects as high-potency glucocorticoid dexamethasone on neurons in the immature chicken cerebellum.

High-potency glucocorticoids (GC) are used in the prophylaxis and treatment of neonatal bronchopulmonal dysplasia, but there is concern about side effects on the developing brain. Recently, the low-potency GC hydrocortisone (HC) has been suggested as an alternative to high-potency GC. We compared the neurotoxic effects of HC with the high-potency GC dexamethasone (DEX) in chicken cerebellum. A single dose of GC was injected into the egg at embryonic day 16 and the death of granule neurons in histologic sections of the cerebellar cortex was examined 24 h later. DEX and HC showed a similar dose-dependent induction of morphological apoptosis and caspase-3 activation in the internal granular layer. A doubling of the apoptosis rate compared to the basal rate was seen for the highest dose of DEX (5 mg/kg) and medium dose of HC (1 mg/kg). In cultures of embryonic chicken cerebellar granule cells, DEX and HC increased cell death and induced rapid caspase-3 activation in a similar dose-dependent manner. Transfection of granule cells with a luciferase reporter gene revealed that the dose needed for the activation of gene transcription (classical signalling pathway) with DEX was much lower than for HC. In conclusion, HC does not present itself as a safer drug than DEX in this model. In addition, it appears that DEX and HC induce apoptosis in immature granule neurons via a non-genomic (non-classical) mechanism.

Differential effects of some novel synthetic oestrogen analogs on oxidative PC12 cell death caused by serum deprivation.

Oestrogens with no or reduced oestrogen receptor (ER) binding properties are reported to have neuroprotective functions. However, we have previously shown that the hormonally inactive isomer of 17ß-estradiol (17ß-E), 17α-estradiol (17α-E), down-regulates glutathione (GSH) synthesis, and fails to rescue serum deprivation-induced cell death in the rat pheochromocytoma cell line PC12 in micromolar concentration. The present study examined cellular protective effects of new 17ß-E analogs and 2-methoxyestradiol (2-ME) analogs with no or little oestrogen activity. 17ß-E, 17α-E, 2-ME, and an antagonist of the G protein-coupled oestrogen receptor (GPER), G36, were also included. Both 17α-E and 2-ME protected against deprivation-induced cell death in PC12 cells at 1 nM, but they enhanced the deprivation-induced cell death accompanied by caspase 3 activity and decreased intracellular GSH levels during deprivation at 10 µM. In addition, 10 µM 17α-E activated the p38 mitogen activated protein kinase pathway, which was linked to the enhanced death and reduced GSH levels. Analogs of 2-ME modified with a 6-isoquinoline moiety (6iq) protected against deprivation-induced cell death at 1 nM and did not interfere with the GSH levels nor increase p38 protein levels at 10 µM. The promoter activity of the catalytic subunit of the rate-limiting enzyme, glutamate cysteine ligase (GCLC) in GSH synthesis as well as protein levels of GCLC and Nrf2, increased with the 2-ME analogs at 10 µM. In conclusion, the steroids have differential protective effects, and modifying 2-ME may give the steroid more favourable properties than 17α-E, 2-ME, and G36 in regard to GSH regulation.

Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson's disease.

More than 90% of the cases of Parkinson's disease have unknown etiology. Gradual loss of dopaminergic neurons of substantia nigra is the main cause of morbidity in this disease. External factors such as environmental toxins are believed to play a role in the cell loss, although the cause of the selective vulnerability of dopaminergic neurons remains unknown. We have previously shown that aquaglyceroporin AQP9 is expressed in dopaminergic neurons and astrocytes of rodent brain. AQP9 is permeable to a broad spectrum of substrates including purines, pyrimidines, and lactate, in addition to water and glycerol. Here we test our hypothesis that AQP9 serves as an influx route for exogenous toxins and, hence, may contribute to the selective vulnerability of nigral dopaminergic (tyrosine hydroxylase-positive) neurons. Using Xenopus oocytes injected with Aqp9 cRNA, we show that AQP9 is permeable to the parkinsonogenic toxin 1-methyl-4-phenylpyridinium (MPP+). Stable expression of AQP9 in HEK cells increases their vulnerability to MPP+ and to arsenite-another parkinsonogenic toxin. Conversely, targeted deletion of Aqp9 in mice protects nigral dopaminergic neurons against MPP+ toxicity. A protective effect of Aqp9 deletion was demonstrated in organotypic slice cultures of mouse midbrain exposed to MPP+ in vitro and in mice subjected to intrastriatal injections of MPP+ in vivo. Seven days after intrastriatal MPP+ injections, the population of tyrosine hydroxylase-positive cells in substantia nigra is reduced by 48% in Aqp9 knockout mice compared with 67% in WT littermates. Our results show that AQP9 -selectively expressed in catecholaminergic neurons-is permeable to MPP+ and suggest that this aquaglyceroporin contributes to the selective vulnerability of nigral dopaminergic neurons by providing an entry route for parkinsonogenic toxins. To our knowledge this is the first evidence implicating a toxin permeable membrane channel in the pathophysiology of Parkinson's disease.

Secretory PLA2-IIA and ROS generation in peripheral mitochondria are critical for neuronal death.

In this study the role of mitochondrial secretory PLA2-IIA in glutamate-induced cell death in cultured cerebellar granule neurons has been investigated. Inhibition of secretory PLA2-IIA blocked glutamate-induced cell death. Since PLA2 may generate reactive oxygen species (ROS), we have investigated ROS production, detected as dihydrorhodamine 123 oxidation and nitrotyrosine modifications of proteins, following glutamate treatment in the absence or presence of an inhibitor of secretory PLA2-IIA. There was an increased generation of ROS in both glutamate- and buffer-treated neurons compared to untreated neurons. Scavenging with dihydrorhodamine 123 reduced glutamate-induced death (60%), showing that ROS detected in glutamate-treated neurons were associated with cell death. However, ROS detected in buffer-treated neurons were not associated with toxicity. Glutamate treatment led to ROS production predominantly in peripheral mitochondria, whereas buffer treatment led to ROS production in somal mitochondria. Inhibition of secretory PLA2-IIA (i) reduced the generation of ROS after glutamate treatment, (ii) reduced the ROS production in peripheral mitochondria in glutamate-treated neurons, consistent with the fact that calcium entry through glutamate (NMDA) receptors has a privileged access to peripheral mitochondria, and (iii) did not reduce the generation of ROS after buffer treatment. In conclusion, activation of NMDA receptors induces ROS, which is critical for neuronal death, due to secretory PLA2-IIA associated with peripheral mitochondria.

Chicken cerebellar granule neurons rapidly develop excitotoxicity in culture.

Rat cerebellar granule cell culture is widely used as a model to study factors that control neuronal differentiation and death (e.g. excitotoxicity). However, a main drawback of this model is its dependence on depolarizing culture condition (25 mM potassium). In addition, it is quite expensive to maintain and requires animal facilities. Here we report that cerebellar granule neuron cultures from chicken may be used as an alternative model to study excitotoxicity. Surprisingly, fetal chicken cells may be grown in a physiological potassium concentration (5 mM potassium). They develop excitotoxicity rapidly in culture (fully developed at 3 days in vitro), and respond to glutamate excitotoxicity similar to rat cultures (ROS production and activation of caspase-3).

Dexamethasone induces cell death which may be blocked by NMDA receptor antagonists but is insensitive to Mg2+ in cerebellar granule neurons.

Since dexamethasone may elevate the Ca2+ influx through NMDA receptors, we have investigated mechanisms of dexamethasone toxicity in rat cerebellar granule neurons. Dexamethasone concentrations over 0.1 microM induced cell death that reached about 20% of the death induced by glutamate. Dexamethasone-induced cell death was reduced by more than 80% by the mineralocorticoid antagonist RU 28318 or the NMDA receptor antagonists MK 801 and CGP 39551, whereas RU 28318 rescued only approximately 30% of cells treated with glutamate, indicating that dexamethasone requires NMDA receptors to induce acute neuronal toxicity and that a fraction of the neurons showed this toxicity. Mg2+ reduced the cell death induced by glutamate at potassium concentrations of 1 mM and 5 mM, but not at 25 mM. In contrast, cell death induced by dexamethasone was not significantly reduced by Mg2+ in any of the potassium concentrations. Both glutamate and dexamethasone induced toxicity with translocation of the apoptosis inducer NGFI-B to the mitochondria seen after 30 min-2 h concomitant with activation of apoptosis inducing factor (AIF) and caspase-3. In conclusion, dexamethasone induces a rapid toxicity which is blocked by NMDA receptor antagonists other than Mg2+, and involves mitochondrial apoptosis inducer NGFI-B.

Hampered Lung Maturation in Methimazole-Induced Hypothyroidism in Fetal Chicken: Morphological and Molecular Correlates to Human Fetal Development.

BACKGROUND: Molecular understanding of lung development is crucial for developing therapies and diagnostic tools. Animal models with altered thyroid hormone signaling provide mechanistic insight into thyroid-dependent neonatal lung disease. Repression of Klf2 (Krüppel-like factor 2), a suggested T3 target gene, is associated with disrupted lung development in mice. Klf2 is proposed to be specifically involved in type I pneumocyte differentiation. OBJECTIVES: To explore mechanisms of thyroid-dependent lung disease, we studied developing chicken fetuses with experimentally induced hypothyroidism. METHODS: Morphology and the expression of a panel of molecules linked to Klf2 were assessed using histology, immunohistochemistry, Western blot and qPCR. RESULTS: Methimazole injections at E14 hampered lung maturation. The effects of methimazole were evident in several tissue compartments, and impacted on both pneumocyte and vascular differentiation, suggesting cellular and molecular pleiotropy. CONCLUSIONS: Concomitant expression changes in a panel of selected microRNAs regulated by Klf2 suggest importance in lung development. These microRNAs may thus represent potential clinical targets and diagnostic and prognostic tools in thyroid-dependent lung disease.

Calcium-induced apoptosis of developing cerebellar granule neurons depends causally on NGFI-B.

Immediate early gene nerve growth factor-induced clone B (NGFI-B), a nuclear receptor important for differentiation and apoptosis, is expressed in mice and rat cerebellum from an early stage of postnatal development. Following apoptotic stimuli NGFI-B translocates to mitochondria to initiate cell death processes. Controlled cell death is critical for correct cerebellar development. Immunohistochemical analysis of NGFI-B in sections of mice cerebella showed NGFI-B to be expressed in granule neurons in vivo at a time (P8-11) when apoptosis is known to occur. The importance of NGFI-B for apoptosis of cultured rat cerebellar granule neurons was investigated by inducing apoptosis with calcium ionophore A23187 (CaI, 0.1µM). Imaging studies of gfp-tagged NGFI-B confirmed that mitochondrial translocation of NGFI-B occurred following treatment with CaI and was reduced by addition of 9-cis-retinoic acid (1µM), a retinoid X receptor (RXR) agonist that prevents dimerization of RXR and NGFI-B that is known to occur before translocation. Consequently, 9-cis-retinoic acid partly reduced cell death. To address the causality of NGFI-B in apoptosis further, knock-down by siRNA was performed and it removed 85% of the NGFI-B protein. This resulted in a complete inhibition of apoptosis after CaI exposure. Together these findings suggest that NGFI-B plays a role in controlling correct cerebellar development.

Type I and II pneumocyte differentiation in the developing fetal chicken lung: conservation of pivotal proteins from birds to human in the struggle for life at birth.

BACKGROUND: Antenatal corticosteroids and surfactant replacement therapy have dramatically reduced mortality caused by lung disease in premature babies. Knowledge about mechanisms regulating epithelial differentiation of the respiratory membrane is limited, as are effects of pharmacological interventions. The chicken fetus is a valuable model for exploring pharmacological actions on developing organs. However, more precise information about the timing of developmental events in the chicken lung is needed for human correlation. OBJECTIVES: Characterization of morphological development and protein expression in the respiratory membrane of the developing chicken lung to create a platform for pharmacological testing in a human context. METHODS: Fetal chicken lungs, embryonic days (E) 7-20, were characterized by morphology and protein expression of epithelial differentiation markers. This was compared with publications on the same processes during human lung development. RESULTS: The respiratory membranes of developing chicken and human lungs show basic similarities. In chicken, surfactant protein B is expressed in cuboidal type II epithelial cells from E17. Aquaporin 5 is expressed in the epithelium from E7 and selectively in type I pneumocytes from E17. The type I pneumocyte and endothelial marker, caveolin 1, is expressed in the endothelium from E7 to E20. CONCLUSION: Despite phylogenetic distance, central aspects of cellular development in the chicken and human lung are similar. The fetal chicken model has important additional advantages to mammalian models, including fetal independence and short incubation, and is thus well suited for in vivo studies of lung maturation relevant to human development.