Perineuronal nets are phagocytosed by MMP-9 expressing microglia and astrocytes in the SOD1G93A ALS mouse model.

AIMS: Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated the spatio-temporal timeline of PNN breakdown and the contributing cellular factors in the SOD1G93A strain, a fast-onset ALS mouse model. METHODS: This was conducted at the presymptomatic (P30), onset (P70), mid-stage (P130), and end-stage disease (P150) using immunofluorescent microscopy, as this characterisation has not been conducted in the SOD1G93A strain. RESULTS: We observed a significant breakdown of PNNs around α-motor neurons in the ventral horn of onset and mid-stage disease SOD1G93A mice compared with wild-type controls. This was observed with increased numbers of microglia expressing matrix metallopeptidase-9 (MMP-9), an endopeptidase that degrades PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Further increases in microglia and astrocyte number, MMP-9 expression, and engulfment of PNN components by glia were observed in mid-stage SOD1G93A mice. This was observed with increased expression of fractalkine, a signal for microglia engulfment, within α-motor neurons of SOD1G93A mice. Following PNN breakdown, α-motor neurons of onset and mid-stage SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death. CONCLUSIONS: Our observations suggest that increased numbers of MMP-9 expressing glia and their subsequent engulfment of PNNs around α-motor neurons render these neurons sensitive to oxidative damage and eventual death in the SOD1G93A ALS model mouse.

Genome-wide transcriptomic analysis of the forebrain of postnatal Slc13a4+/- mice.

OBJECTIVE: Sulfation is an essential physiological process that regulates the function of a wide array of molecules involved in brain development. We have previously shown expression levels for the sulfate transporter Slc13a4 to be elevated during postnatal development, and that sulfate accumulation in the brains of Slc13a4+/- mice is reduced, suggesting a role for this transporter during this critical window of brain development. In order to understand the pathways regulated by cellular sulfation within the brain, we performed a bulk RNA-sequencing analysis of the forebrain of postnatal day 20 (P20) Slc13a4 heterozygous mice and wild-type litter mate controls. DATA DESCRIPTION: We performed an RNA transcriptomic based sequencing screen on the whole forebrain from Slc13a4+/- and Slc13a4+/+mice at P20. Differential expression analysis revealed 90 differentially regulated genes in the forebrain of Slc13a4+/- mice (a p-value of 0.1 was considered as significant). Of these, 55 were upregulated, and 35 were downregulated in the forebrain of heterozygous mice. Moreover, when we stratified further with a ± 1.2 fold-change, we observed 38 upregulated, and 16 downregulated genes in the forebrain of heterozygous mice. This resource provides a useful tool to interrogate which pathways may require elevated sulfate levels to drive normal postnatal development of the brain.

The Placental Ferroxidase Zyklopen Is Not Essential for Iron Transport to the Fetus in Mice.

BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P < 0.01). CONCLUSIONS: Our data indicate that Zp is not essential for the efficient transfer of iron to the fetus in mice and is localized predominantly in the maternal decidua. The increase in placental size observed when Zp is knocked out in the entire placenta suggests that this protein may play a role in placental development.

Cell-extrinsic requirement for sulfate in regulating hippocampal neurogenesis.

Sulfate is a key anion required for a range of physiological functions within the brain. These include sulfonation of extracellular proteoglycans to facilitate local growth factor binding and to regulate the shape of morphogen gradients during development. We have previously shown that mice lacking one allele of the sulfate transporter Slc13a4 exhibit reduced sulfate transport into the brain, deficits in social behaviour, reduced performance in learning and memory tasks, and abnormal neurogenesis within the ventricular/subventricular zone lining the lateral ventricles. However, whether these mice have deficits in hippocampal neurogenesis was not addressed. Here, we demonstrate that adult Slc13a4+/- mice have increased neurogenesis within the subgranular zone (SGZ) of the hippocampal dentate gyrus, with elevated numbers of neural progenitor cells and intermediate progenitors. In contrast, by 12 months of age there were reduced numbers of neural stem cells in the SGZ of heterozygous mice. Importantly, we did not observe any changes in proliferation when we isolated and cultured progenitors in vitro in neurosphere assays, suggestive of a cell-extrinsic requirement for sulfate in regulating hippocampal neurogenesis. Collectively, these data demonstrate a requirement for sulfate transport during postnatal brain development to ensure normal adult hippocampal neurogenesis.

Molecular analysis of the human placental cysteine dioxygenase type 1 gene.

Sulfate is essential for healthy fetal growth and development. Cysteine dioxygenase type 1 (CDO1) plays an important role in the catabolism of cysteine to sulfate. Cdo1 knockout mice exhibit severe and lethal fetal phenotypes but the involvement of CDO1 gene variants in human development is unknown. We searched the NCBI and Ensembl gene databases and identified four alternatively spliced CDO1 coding mRNA transcripts, as well as 148 validated CDO1 gene variants, including 138 missense, 6 nonsense, 1 frameshift, 1 in-frame deletion, and 2 splice site variants. In silico analyses predicted 68 of the missense variants to be deleterious to CDO1 protein structure and function. We examined the relative abundance of the four CDO1 coding mRNA transcripts in human term placentas using qRT-PCR. CDO1 mRNA variant 2 was the most abundant transcript, with intermediate levels of variant 4 and lower levels of variants 1 and 3. Using in situ hybridization, we localised CDO1 mRNA expression to the syncytiotrophoblast layer of human term placenta. To investigate the regulation of CDO1 gene expression, we analysed the transcriptional activity of the human CDO1 5'-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Transcriptional activities were identified in the regions -5 to -269 and - 269 to -1200 nucleotides upstream of the CDO1 transcription initiation site. Mutational analyses of a single nucleotide polymorphism -289C > G that is common in the general population (allele frequency = 0.37) and a putative transcription factor binding motif (CCAAT enhancer binding protein beta) did not alter transcriptional activity of the CDO1 5'-flanking region. Collectively, this study provides an overview and analysis of human CDO1 for future investigations of this gene in human health.

Complex patterns of cell growth in the placenta in normal pregnancy and as adaptations to maternal diet restriction.

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

Secondary Placental Defects in Cxadr Mutant Mice.

The Coxsackie virus and adenovirus receptor (CXADR) is an adhesion molecule known for its role in virus-cell interactions, epithelial integrity, and organogenesis. Loss of Cxadr causes numerous embryonic defects in mice, notably abnormal development of the cardiovascular system, and embryonic lethality. While CXADR expression has been reported in the placenta, the precise cellular localization and function within this tissue are unknown. Since impairments in placental development and function can cause secondary cardiovascular abnormalities, a phenomenon referred to as the placenta-heart axis, it is possible placental phenotypes in Cxadr mutant embryos may underlie the reported cardiovascular defects and embryonic lethality. In the current study, we determine the cellular localization of placental Cxadr expression and whether there are placental abnormalities in the absence of Cxadr. In the placenta, CXADR is expressed specifically by trophoblast labyrinth progenitors as well as cells of the visceral yolk sac (YS). In the absence of Cxadr, we observed altered expression of angiogenic factors coupled with poor expansion of trophoblast and fetal endothelial cell subpopulations, plus diminished placental transport. Unexpectedly, preserving endogenous trophoblast Cxadr expression revealed the placental defects to be secondary to primary embryonic and/or YS phenotypes. Moreover, further tissue-restricted deletions of Cxadr suggest that the secondary placental defects are likely influenced by embryonic lineages such as the fetal endothelium or those within the extraembryonic YS vascular plexus.

Periconceptional alcohol exposure causes female-specific perturbations to trophoblast differentiation and placental formation in the rat.

The development of pathologies during pregnancy, including pre-eclampsia, hypertension and fetal growth restriction (FGR), often originates from poor functioning of the placenta. In vivo models of maternal stressors, such as nutrient deficiency, and placental insufficiency often focus on inadequate growth of the fetus and placenta in late gestation. These studies rarely investigate the origins of poor placental formation in early gestation, including those affecting the pre-implantation embryo and/or the uterine environment. The current study characterises the impact on blastocyst, uterine and placental outcomes in a rat model of periconceptional alcohol exposure, in which 12.5% ethanol is administered in a liquid diet from 4 days before until 4 days after conception. We show female-specific effects on trophoblast differentiation, embryo-uterine communication, and formation of the placental vasculature, resulting in markedly reduced placental volume at embryonic day 15. Both sexes exhibited reduced trophectoderm pluripotency and global hypermethylation, suggestive of inappropriate epigenetic reprogramming. Furthermore, evidence of reduced placental nutrient exchange and reduced pre-implantation maternal plasma choline levels offers significant mechanistic insight into the origins of FGR in this model.

TRPM7 is the central gatekeeper of intestinal mineral absorption essential for postnatal survival.

Zn2+, Mg2+, and Ca2+ are essential minerals required for a plethora of metabolic processes and signaling pathways. Different categories of cation-selective channels and transporters are therefore required to tightly control the cellular levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these essential minerals are poorly understood. Herein, we identify a central and indispensable role of the channel-kinase TRPM7 for organismal mineral homeostasis. The function of TRPM7 was assessed by single-channel analysis of TRPM7, phenotyping of TRPM7-deficient cells in conjunction with metabolic profiling of mice carrying kidney- and intestine-restricted null mutations in Trpm7 and animals with a global "kinase-dead" point mutation in the gene. The TRPM7 channel reconstituted in lipid bilayers displayed a similar permeability to Zn2+ and Mg2+ Consistently, we found that endogenous TRPM7 regulates the total content of Zn2+ and Mg2+ in cultured cells. Unexpectedly, genetic inactivation of intestinal rather than kidney TRPM7 caused profound deficiencies specifically of Zn2+, Mg2+, and Ca2+ at the organismal level, a scenario incompatible with early postnatal growth and survival. In contrast, global ablation of TRPM7 kinase activity did not affect mineral homeostasis, reinforcing the importance of the channel activity of TRPM7. Finally, dietary Zn2+ and Mg2+ fortifications significantly extended the survival of offspring lacking intestinal TRPM7. Hence, the organismal balance of divalent cations critically relies on one common gatekeeper, the intestinal TRPM7 channel.

Deletion of the Syncytin A receptor Ly6e impairs syncytiotrophoblast fusion and placental morphogenesis causing embryonic lethality in mice.

Fetal growth and survival is dependent on the elaboration and propinquity of the fetal and maternal circulations within the placenta. Central to this is the formation of the interhaemal membrane, a multi-cellular lamina facilitating exchange of oxygen, nutrients and metabolic waste products between the mother and fetus. In rodents, this cellular barrier contains two transporting layers of syncytiotrophoblast, which are multinucleated cells that form by cell-cell fusion. Previously, we reported the expression of the GPI-linked cell surface protein LY6E by the syncytial layer closest to the maternal sinusoids of the mouse placenta (syncytiotrophoblast layer I). LY6E has since been shown to be a putative receptor for the fusogenic protein responsible for fusion of syncytiotrophoblast layer I, Syncytin A. In this report, we demonstrate that LY6E is essential for the normal fusion of syncytiotrophoblast layer I, and for the proper morphogenesis of both fetal and maternal vasculatures within the placenta. Furthermore, specific inactivation of Ly6e in the epiblast, but not in placenta, is compatible with embryonic development, indicating the embryonic lethality reported for Ly6e-/- embryos is most likely placental in origin.

Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction.

The contribution of environmental factors to pancreatic islet damage in type 1 diabetes remains poorly understood. In this study, we crossed mice susceptible to type 1 diabetes, where parental male (CD8+ T cells specific for IGRP206-214; NOD8.3) and female (NOD/ShiLt) mice were randomized to a diet either low or high in AGE content and maintained on this diet throughout pregnancy and lactation. After weaning, NOD8.3+ female offspring were identified and maintained on the same parental feeding regimen for until day 28 of life. A low AGE diet, from conception to early postnatal life, decreased circulating AGE concentrations in the female offspring when compared to a high AGE diet. Insulin, proinsulin and glucagon secretion were greater in islets isolated from offspring in the low AGE diet group, which was akin to age matched non-diabetic C57BL/6 mice. Pancreatic islet expression of Ins2 gene was also higher in offspring from the low AGE diet group. Islet expression of glucagon, AGEs and the AGE receptor RAGE, were each reduced in low AGE fed offspring. Islet immune cell infiltration was also decreased in offspring exposed to a low AGE diet. Within pancreatic lymph nodes and spleen, the proportions of CD4+ and CD8+ T cells did not differ between groups. There were no significant changes in body weight, fasting glucose or glycemic hormones. This study demonstrates that reducing exposure to dietary AGEs throughout gestation, lactation and early postnatal life may benefit pancreatic islet secretion and immune infiltration in the type 1 diabetic susceptible mouse strain, NOD8.3.

Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ.

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a-C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

Molecular analysis of sequence and splice variants of the human SLC13A4 sulfate transporter.

The solute linked carrier 13A4 gene (SLC13A4) is abundantly expressed in the human and mouse placenta where it is proposed to transport nutrient sulfate to the fetus. In mice, targeted disruption of placental Slc13a4 leads to severe and lethal fetal phenotypes, however the involvement of SLC13A4 in human development is unknown. A search of the NCBI and Ensembl gene databases identified two alternatively spliced SLC13A4 mRNA transcripts and 98 SLC13A4 gene variants, including 85 missense, 4 splice site, 5 frameshift and 2 nonsense variants, as well as 2 in-frame deletions. We examined the relative abundance of the two SLC13A4 mRNA transcripts and then compared the sulfate transport function and plasma membrane expression of both isoforms as well as 6 sequence variants that predict disrupted SLC13A4 protein structure and function. SLC13A4 mRNA variant 1 has three additional nucleotides CAG compared to SLC13A4 mRNA variant 2 as a result of alternative splicing at the 5'-end of exon 6. Using qRT-PCR, we show a 4-fold higher abundance of SLC13A4 mRNA variant 1 compared to variant 2 in term human placentas and cultured BeWo and JEG-3 cell lines. The corresponding SLC13A4 protein isoforms 1 and 2 were found to have similar sulfate uptake activity and apical membrane expression in cultured MDCK cells. In addition, sulfate uptake into MDCK cells was similar between SLC13A4 isoform 1 and four missense variants N300S, F310C, E360Q and I570V, whereas V513M and frameshift variant L72Sfs led to partial (≈75% decrease) and complete loss-of-function, respectively. Localisation of these variants in MDCK cells showed N300S, E360Q, V513M and I570V expression on the apical plasma membrane, L72Sfs intracellularly and F310C on both apical and basolateral membranes. Our finding of partial and complete loss-of-function variants warrants further studies of the potential involvement of SLC13A4 in fetal pathophysiology.

Review: Alterations in placental glycogen deposition in complicated pregnancies: Current preclinical and clinical evidence.

Normal placental function is essential for optimal fetal growth. Transport of glucose from mother to fetus is critical for fetal nutrient demands and can be stored in the placenta as glycogen. However, the function of this glycogen deposition remains a matter of debate: It could be a source of fuel for the placenta itself or a storage reservoir for later use by the fetus in times of need. While the significance of placental glycogen remains elusive, mounting evidence indicates that altered glycogen metabolism and/or deposition accompanies many pregnancy complications that adversely affect fetal development. This review will summarize histological, biochemical and molecular evidence that glycogen accumulates in a) placentas from a variety of experimental rodent models of perturbed pregnancy, including maternal alcohol exposure, glucocorticoid exposure, dietary deficiencies and hypoxia and b) placentas from human pregnancies with complications including preeclampsia, gestational diabetes mellitus and intrauterine growth restriction (IUGR). These pregnancies typically result in altered fetal growth, developmental abnormalities and/or disease outcomes in offspring. Collectively, this evidence suggests that changes in placental glycogen deposition is a common feature of pregnancy complications, particularly those associated with altered fetal growth.

Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival.

Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood.

Distinct brainstem and forebrain circuits receiving tracheal sensory neuron inputs revealed using a novel conditional anterograde transsynaptic viral tracing system.

Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.

Evidence for multiple sensory circuits in the brain arising from the respiratory system: an anterograde viral tract tracing study in rodents.

Complex sensations accompany the activation of sensory neurons within the respiratory system, yet little is known about the organization of sensory pathways in the brain that mediate these sensations. In the present study, we employ anterograde viral neuroanatomical tract tracing with isogenic self-reporting recombinants of HSV-1 strain H129 to map the higher brain regions in receipt of vagal sensory neurons arising from the trachea versus the lungs, and single-cell PCR to characterize the phenotype of sensory neurons arising from these two divisions of the respiratory tree. The results suggest that the upper and lower airways are predominantly innervated by sensory neurons derived from the somatic jugular and visceral nodose cranial ganglia, respectively. This coincides with central circuitry that is predominately somatic-like, arising from the trachea, and visceral-like, arising from the lungs. Although some convergence of sensory pathways was noted in preautonomic cell groups, this was notably absent in thalamic and cortical regions. These data support the notion that distinct afferent subtypes, via distinct central circuits, subserve sensations arising from the upper versus lower airways. The findings may explain why sensations arising from different levels of the respiratory tree are qualitatively and quantitatively unique.

The effect of hyperpolarization-activated cyclic nucleotide-gated ion channel inhibitors on the vagal control of guinea pig airway smooth muscle tone.

BACKGROUND AND PURPOSE: Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. EXPERIMENTAL APPROACH: The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs(+) were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. KEY RESULTS: HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs(+) , but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. CONCLUSIONS AND IMPLICATIONS: HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

The basic helix-loop-helix transcription factor Hand1 regulates mouse development as a homodimer.

Hand1 is a basic helix-loop-helix transcription factor that is essential for development of the placenta, yolk sac and heart during mouse development. While Hand1 is essential for trophoblast giant cell (TGC) differentiation, its potential heterodimer partners are not co-expressed in TGCs. To test the hypothesis that Hand1 functions as homodimer, we generated knock-in mice in which the Hand1 gene was altered to encode a tethered homodimer (TH). Some Hand1(TH/-) conceptuses in which the only form of Hand1 is Hand1(TH) are viable and fertile, indicating that homodimer Hand1 is sufficient for mouse survival. ~2/3 of Hand1(TH/-) and all Hand1(TH/TH) mice died in utero and displayed severe placental defects and variable cardial and cranial-facial abnormalities, indicating a dosage-dependent effect of Hand1(TH). Meanwhile, expression of the Hand1(TH) protein did not have negative effects on viability or fertility in all Hand1(TH/+) mice. These data imply that Hand1 homodimer plays a dominant role during development and its expression dosage is critical for survival, whereas Hand1 heterodimers can be either dispensable or play a regulatory role to modulate the activity of Hand1 homodimer in vivo.

Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart.

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and by in situ hybridization can be visualized across the myocardium in isolated cardiac cells. Tas1r1 gene-targeted mice (Tas1r1(Cre)/Rosa26(tdRFP)) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart.