Control of NFAT Isoform Activation and NFAT-Dependent Gene Expression through Two Coincident and Spatially Segregated Intracellular Ca2+ Signals.

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.

Disentangling juxtacrine from paracrine signalling in dynamic tissue.

Prolactin is a major hormone product of the pituitary gland, the central endocrine regulator. Despite its physiological importance, the cell-level mechanisms of prolactin production are not well understood. Having significantly improved the resolution of real-time-single-cell-GFP-imaging, the authors recently revealed that prolactin gene transcription is highly dynamic and stochastic yet shows space-time coordination in an intact tissue slice. However, it still remains an open question as to what kind of cellular communication mediates the observed space-time organization. To determine the type of interaction between cells we developed a statistical model. The degree of similarity between two expression time series was studied in terms of two distance measures, Euclidean and geodesic, the latter being a network-theoretic distance defined to be the minimal number of edges between nodes, and this was used to discriminate between juxtacrine from paracrine signalling. The analysis presented here suggests that juxtacrine signalling dominates. To further determine whether the coupling is coordinating transcription or post-transcriptional activities we used stochastic switch modelling to infer the transcriptional profiles of cells and estimated their similarity measures to deduce that their spatial cellular coordination involves coupling of transcription via juxtacrine signalling. We developed a computational model that involves an inter-cell juxtacrine coupling, yielding simulation results that show space-time coordination in the transcription level that is in agreement with the above analysis. The developed model is expected to serve as the prototype for the further study of tissue-level organised gene expression for epigenetically regulated genes, such as prolactin.

Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration.

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Phenotypic dissection of the mouse Ren1d knockout by complementation with human renin.

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding renin-angiotensin system interactions. A 55-kb transgene encompassing the human renin locus was crossed onto the mouse Ren1d-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labeling. After stimulation of the renin-angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum, and electron-lucent granular structures. However, complementation of Ren1d-/- mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren1d-/- non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin-angiotensin system.

Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function.

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Zebrafish mesonephric renin cells are functionally conserved and comprise two distinct morphological populations.

Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury, and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin (ren), smooth muscle actin (acta2), and platelet-derived growth factor receptor-beta (pdgfrb) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren-expressing cells occupied niches at the preglomerular arteries and afferent arterioles, forming intermittent epithelioid-like multicellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates, and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren-expressing cells.

C9orf72 Hexanucleotide Expansions Are Associated with Altered Endoplasmic Reticulum Calcium Homeostasis and Stress Granule Formation in Induced Pluripotent Stem Cell-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons, decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis, reduced levels of the antiapoptotic protein Bcl-2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063-2078.

Oocyte glycoproteins regulate the form and function of the follicle basal lamina and theca cells.

Maintaining follicle integrity during development, whereby each follicle is a functional unit containing a single oocyte, is essential for the generation of healthy oocytes. However, the mechanisms that regulate this critical function have not been determined. In this paper we investigate the role of the oocyte in maintaining follicle development. To investigate this role, we use a mouse model with oocyte-specific deletion of C1galt1 which is required for the generation of core 1-derived O-glycans. The loss of oocyte-generated O-glycans results in the joining of follicles and the generation of Multiple-Oocyte Follicles (MOFs). The aim was to determine how Mutant follicle development is modified thus enabling follicles to join. Extracellular matrix and follicle permeability were studied using histology, immunohistochemistry and electron microscopy (EM). In ovaries containing Mutant Oocytes, the Follicle basal lamina (FBL) is altered both functionally and structurally from the primary stage onwards with Mutant follicles possessing unexpectedly thicker FBL. In Mutant ovaries, the theca cell layer is also modified with intermingling of theca between adjacent follicles. MOF function was analysed but despite increased numbers of preantral MOFs in Mutants, these do not reach the preovulatory stage after gonadotrophin stimulation. We propose a model describing how oocyte initiated changes in FBL and theca cells result in follicles joining. These data reveal new and important roles for the oocyte in follicle development and follicle integrity.

Human kidney pericytes produce renin.

Pericytes, perivascular cells embedded in the microvascular wall, are crucial for vascular homeostasis. These cells also play diverse roles in tissue development and regeneration as multi-lineage progenitors, immunomodulatory cells and as sources of trophic factors. Here, we establish that pericytes are renin producing cells in the human kidney. Renin was localized by immunohistochemistry in CD146 and NG2 expressing pericytes, surrounding juxtaglomerular and afferent arterioles. Similar to pericytes from other organs, CD146+CD34-CD45-CD56- renal fetal pericytes, sorted by flow cytometry, exhibited tri-lineage mesodermal differentiation potential in vitro. Additionally, renin expression was triggered in cultured kidney pericytes by cyclic AMP as confirmed by immuno-electron microscopy, and secretion of enzymatically functional renin, capable of generating angiotensin I. Pericytes derived from second trimester human placenta also expressed renin in an inducible fashion although the renin activity was much lower than in renal pericytes. Thus, our results confirm and extend the recently discovered developmental plasticity of microvascular pericytes, and may open new perspectives to the therapeutic regulation of the renin-angiotensin system.

Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications.

The blood-brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1(-/-) mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function.

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.

Annexin A1: a central player in the anti-inflammatory and neuroprotective role of microglia.

The brain microenvironment is continuously monitored by microglia with the detection of apoptotic cells or pathogens being rapidly followed by their phagocytosis to prevent inflammatory responses. The protein annexin A1 (ANXA1) is key to the phagocytosis of apoptotic leukocytes during peripheral inflammatory resolution, but the pathophysiological significance of its expression in the CNS that is restricted almost exclusively to microglia is unclear. In this study, we test the hypothesis that ANXA1 is important in the microglial clearance of apoptotic neurons in both noninflammatory and inflammatory conditions. We have identified ANXA1 to be sparingly expressed in microglia of normally aged human brains and to be more strongly expressed in Alzheimer's disease. Using an in vitro model comprising microglial and neuronal cell lines, as well as primary microglia from wild-type and ANXA1 null mice, we have identified two distinct roles for microglial ANXA1: 1) controlling the noninflammatory phagocytosis of apoptotic neurons and 2) promoting resolution of inflammatory microglial activation. In particular, we showed that microglial-derived ANXA1 targets apoptotic neurons, serving as both an "eat me" signal and a bridge between phosphatidylserine on the dying cell and formyl peptide receptor 2 on the phagocytosing microglia. Moreover, inflammatory activation of microglia impairs their ability to discriminate between apoptotic and nonapoptotic cells, an ability restored by exogenous ANXA1. We thus show that ANXA1 is fundamental for brain homeostasis, and we suggest that ANXA1 and its peptidomimetics can be novel therapeutic targets in neuroinflammation.

Hypothalamic remodeling of thyroid hormone signaling during hibernation in the arctic ground squirrel.

Hibernation involves prolonged intervals of profound metabolic suppression periodically interrupted by brief arousals to euthermy, the function of which is unknown. Annual cycles in mammals are timed by a photoperiodically-regulated thyroid-hormone-dependent mechanism in hypothalamic tanycytes, driven by thyrotropin (TSH) in the pars tuberalis (PT), which regulates local TH-converting deiodinases and triggers remodeling of neuroendocrine pathways. We demonstrate that over the course of hibernation in continuous darkness, arctic ground squirrels (Urocitellus parryii) up-regulate the retrograde TSH/Deiodinase/TH pathway, remodel hypothalamic tanycytes, and activate the reproductive axis. Forcing the premature termination of hibernation by warming animals induced hypothalamic deiodinase expression and the accumulation of secretory granules in PT thyrotrophs and pituitary gonadotrophs, but did not further activate the reproductive axis. We suggest that periodic arousals may allow for the transient activation of hypothalamic thyroid hormone signaling, cellular remodeling, and re-programming of brain circuits in preparation for the short Arctic summer.

LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model.

Leucine rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD) although LRRK2 function remains unclear. We report a new role for LRRK2 in regulating autophagy and describe the recruitment of LRRK2 to the endosomal-autophagic pathway and specific membrane subdomains. Using a novel human genomic reporter cellular model, we found LRRK2 to locate to membrane microdomains such as the neck of caveolae, microvilli/filopodia and intraluminal vesicles of multivesicular bodies (MVBs). In human brain and in cultured human cells LRRK2 was present in cytoplasmic puncta corresponding to MVBs and autophagic vacuoles (AVs). Expression of the common R1441C mutation from a genomic DNA construct caused impaired autophagic balance evident by the accumulation of MVBs and large AVs containing incompletely degraded material and increased levels of p62. Furthermore, the R1441C mutation induced the formation of skein-like abnormal MVBs. Conversely, LRRK2 siRNA knockdown increased autophagic activity and prevented cell death caused by inhibition of autophagy in starvation conditions. The work necessitated developing a new, more efficient recombineering strategy, which we termed Sequential insertion of Target with ovErlapping Primers (STEP) to seamlessly fuse the green fluorescent protein-derivative YPet to the human LRRK2 protein in the LRRK2 genomic locus carried by a bacterial artificial chromosome. Taken together our data demonstrate the functional involvement of LRRK2 in the endosomal-autophagic pathway and the recruitment to specific membrane microdomains in a physiological human gene expression model suggesting a novel function for this important PD-related protein.

Hyperprolactinemia in a male pituitary androgen receptor knockout mouse is associated with female-like lactotroph development.

BACKGROUND: Circulating prolactin concentration in rodents and humans is sexually dimorphic. Oestrogens are a well-characterised stimulator of prolactin release. Circulating prolactin fluctuates throughout the menstrual/oestrous cycle of females in response to oestrogen levels, but remains continually low in males. We have previously identified androgens as an inhibitor of prolactin release through characterisation of males of a mouse line with a conditional pituitary androgen receptor knockout (PARKO) which have an increase in circulating prolactin, but unchanged lactotroph number. OBJECTIVES: In the present study, we aimed to specify the cell type that androgens act on to repress prolactin release. MATERIALS AND METHODS: PARKO, lactotroph-specific, Pit1 lineage-specific and neural-specific conditional androgen receptor knockout male mice were investigated using prolactin ELISA, pituitary electron microscopy, immunohistochemistry and qRT-PCR. RESULTS: Lactotroph-specific, Pit1 lineage-specific and neural-specific conditional AR knockouts did not duplicate the high circulating prolactin seen in the PARKO line. Using electron microscopy to examine ultrastructure, we showed that pituitary androgen receptor knockout male mice develop lactotrophs that resemble those seen in female mice. Castrated PARKO males have significantly reduced circulating prolactin compared to intact males. When expression of selected oestrogen-regulated anterior pituitary genes was examined, there were no differences in expression level between controls and knockouts. DISCUSSION: The cell type that androgens act on to repress prolactin release is not the lactotroph, cells in the Pit1-lineage, or the dopaminergic neurons in the hypothalamus. PARKO males develop a female-specific lactotroph ultrastructure that this is likely to contribute to the increase in circulating prolactin. Castrated PARKO males have significantly reduced circulating prolactin compared to intact males, which suggests that removal of both circulating oestrogens and androgens reduces the stimulation of pituitary prolactin release. CONCLUSION: Further investigation is needed into prolactin regulation by changes in androgen-oestrogen balance, which is involved sexual dimorphism of development and diseases including hyperprolactinemia.

Role of Annexin A1 in mouse myoblast cell differentiation.

Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein involved in a broad range of cellular events. This study used molecular and microscopy approaches to explore the role of ANXA1 in mouse myoblast C2C12 cell differentiation. We report that ANXA1 expression increases during differentiation and that the down-regulation of ANXA1 significantly inhibits the differentiation process. ANXA1 is expressed in vivo in both quiescent and activated satellite cells and is highly localized in the cells that migrate in the lumen of regenerating fibers after an acute injury. Endogenous ANXA1 co-localizes with actin fibers at the protruding ends of undifferentiated but not differentiated cells suggesting a role of the protein in cell migration. Furthermore, ANXA1 neutralizing antibody reduces MyHC expression, decreases myotube formation and significantly inhibits cell migration. The data reported here suggest for the first time that ANXA1 plays a role in myogenic differentiation.

The apical (hPepT1) and basolateral peptide transport systems of Caco-2 cells are regulated by AMP-activated protein kinase.

The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) activation of the AMP-activated protein kinase (AMPK) on the transport of the model radiolabeled dipeptide [(3)H]-D-Phe-L-Gln was investigated in the human epithelial colon cancer cell line Caco-2. Uptake and transepithelial fluxes of [(3)H]-D-Phe-L-Gln were carried out in differentiated Caco-2 cell monolayers, and hPepT1 and glucose transporter 2 (GLUT2) protein levels were quantified by immunogold electron microscopy. AICAR treatment of Caco-2 cells significantly inhibited apical [(3)H]-D-Phe-L-Gln uptake, matched by a decrease in brush-border membrane hPepT1 protein but with a concomitant increase in the facilitated glucose transporter GLUT2. A restructuring of the apical brush-border membrane was seen by electron microscopy. The hPepT1-mediated transepithelial (A-to-B) peptide flux across the Caco-2 monolayers showed no significant alteration in AICAR-treated cells. The electrical resistance in the AICAR-treated monolayers was significantly higher compared with control cells. Inhibition of the sodium/hydrogen exchanger 3 (NHE3) had an additive effect to AICAR, suggesting that the AMPK effect is not via NHE3. Fluorescence measurement of intracellular pH showed no reduction in the proton gradient driving PepT1-mediated apical uptake. The reduction in apical hPepT1 protein and dipeptide uptake after AICAR treatment in Caco-2 cells demonstrates a regulatory effect of AMPK on hPepT1, along with an influence on both the microvilli and tight junction structures. The absence of an associated reduction in transepithelial peptide movement implies an additional stimulatory effect of AICAR on the basolateral peptide transport system in these cells. These results provide a link between the hPepT1 transporter and the metabolic state of this model enterocyte.

Annexin A1 regulates hormone exocytosis through a mechanism involving actin reorganization.

The glucocorticoid-regulated protein annexin A1 is a potent inhibitor of hormone exocytosis in the neuroendocrine system, acting in a paracrine/juxtacrine manner. The signaling mechanism employed by annexin A1 in this process is uncertain, although we have recently presented evidence for a role of the formyl peptide receptor in vivo. We sought to characterize the mechanism of action of annexin A1 on exocytosis using the release of adrenocorticotrophin from the corticotroph-like cell line AtT20 as an in vitro model system. Through the comparison of adrenocorticotrophin release from cells expressing either wild-type annexin A1 or mutant forms, we show a critical involvement of phosphorylation on serine 27 and 45 in the translocation of the protein to the membrane and its inhibitory action on exocytosis. Moreover, we show, for the first time, that annexin A1-dependent inhibition of adrenocorticotrophin release involves the enhancement of actin polymerization to prevent exocytosis via formyl peptide receptor and Rho kinase signaling pathways. This finding has significant implications for the inhibitory actions of annexin A1 on exocytosis in other endocrine and immune contexts.

Characterisation of a mural cell network in the murine pituitary gland.

The anterior and intermediate lobes of the pituitary are composed of endocrine cells, as well as vasculature and supporting cells, such as folliculostellate cells. Folliculostellate cells form a network with several postulated roles in the pituitary, including production of paracrine signalling molecules and cytokines, coordination of endocrine cell hormone release, phagocytosis, and structural support. Folliculostellate cells in rats are characterised by expression of S100B protein, and in humans by glial fibrillary acid protein. However, there is evidence for another network of supporting cells in the anterior pituitary that has properties of mural cells, such as vascular smooth muscle cells and pericytes. The present study aims to characterise the distribution of cells that express the mural cell marker platelet derived growth factor receptor beta (PDGFRß) in the mouse pituitary and establish whether these cells are folliculostellate. By immunohistochemical localisation, we determine that approximately 80% of PDGFRß+ cells in the mouse pituitary have a non-perivascular location and 20% are pericytes. Investigation of gene expression in a magnetic cell sorted population of PDGFRß+ cells shows that, despite a mostly non-perivascular location, this population is enriched for mural cell markers but not enriched for rat or human folliculostellate cell markers. This is confirmed by immunohistochemistry. The present study concludes that a mural cell network is present throughout the anterior pituitary of the mouse and that this population does not express well-characterised human or rat folliculostellate cell markers.

Annexin A1 in the brain--undiscovered roles?

Annexin A1 (ANXA1) is an endogenous protein known to have potent anti-inflammatory properties in the peripheral system. It has also been detected in the brain, but its function there is still ambiguous. In this review, we have, for the first time, collated the evidence currently available on the function of ANXA1 in the brain and have proposed several possible mechanisms by which it exerts a neuroprotective or anti-neuroinflammatory function. We suggest that ANXA1, its small peptide mimetics and its receptors might be exciting new therapeutic targets in the management of a wide range of neuroinflammatory diseases, including stroke and neurodegenerative conditions.