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Zika virus (ZIKV) is a mosquito-transmitted virus that has caused significant public health concerns around the world, partly because of an association with microcephaly in babies born to mothers who were infected with ZIKV during pregnancy. As a recently emerging virus, little is known as to how the virus interacts with the host cell machinery. A yeast-2-hybrid screen for proteins capable of interacting with the ZIKV E protein domain III, the domain responsible for receptor binding, identified 21 proteins, one of which was the predominantly ER resident chaperone protein GRP78. The interaction of GRP78 and ZIKV E was confirmed by co-immunoprecipitation and reciprocal co-immunoprecipitation, and indirect immunofluorescence staining showed intracellular and extracellular co-localization between GRP78 and ZIKV E. Antibodies directed against the N-terminus of GRP78 were able to inhibit ZIKV entry to host cells, resulting in significant reductions in the levels of ZIKV infection and viral production. Consistently, these reductions were also observed after down-regulation of GRP78 by siRNA. These results indicate that GRP78 can play a role mediating ZIKV binding, internalization and replication in cells. GRP78 is a main regulator of the unfolded protein response (UPR), and the study showed that expression of GRP78 was up-regulated, and the UPR was activated. Increases in CHOP expression, and activation of caspases 7 and 9 were also shown in response to ZIKV infection. Overall these results indicate that the interaction between GRP78 and ZIKV E protein plays an important role in ZIKV infection and replication, and may be a potential therapeutic target.

Arginine vasopressin (AVP) is synthesised in magnocellular neurons (MCNs) of supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. In response to the hyperosmotic stressors of dehydration (complete fluid deprivation, DH) or salt loading (drinking 2% salt solution, SL), AVP synthesis increases in MCNs, which over-burdens the protein folding machinery in the endoplasmic reticulum (ER). ER stress and the unfolded protein response (UPR) are signaling pathways that improve ER function in response to the accumulation of misfold/unfold protein. We asked whether an ER stress response was activated in the SON and PVN of DH and SL rats. We observed increased mRNA expression for the immunoglobulin heavy chain binding protein (BiP), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), and cAMP responsive element binding protein 3 like 1 (Creb3l1) in both SON and PVN of DH and SL rats. Although we found no changes in the splicing pattern of X box-binding protein 1 (Xbp1), an increase in the level of the unspliced form of Xbp1 (Xbp1U) was observed in DH and SL rats. CREB3L1, a novel ER stress inducer, has been shown to be activated by ER stress to regulate the expression of target genes. We have previously shown that CREB3L1 is a transcriptional regulator of the AVP gene; however, a role for CREB3L1 in the response to ER stress has yet to be investigated in MCNs. Here, we used lentiviral vectors to introduce a dominant negative form of CREB3L1 (CREB3L1DN) in the rat SON. Expression of CREB3L1DN in the SON decreased Chop and Xbp1U mRNA levels, but not BiP and Atf4 transcript expression. CREB3L1 is thus implicated as a transcriptional mediator of the ER stress response in the osmotically stimulated SON.

Salt appetite, the primordial instinct to favorably ingest salty substances, represents a vital evolutionary important drive to successfully maintain body fluid and electrolyte homeostasis. This innate instinct was shown here in Sprague-Dawley rats by increased ingestion of isotonic saline (IS) over water in fluid intake tests. However, this appetitive stimulus was fundamentally transformed into a powerfully aversive one by increasing the salt content of drinking fluid from IS to hypertonic saline (2% w/v NaCl, HS) in intake tests. Rats ingested HS similar to IS when given no choice in one-bottle tests and previous studies have indicated that this may modify salt appetite. We thus investigated if a single 24 h experience of ingesting IS or HS, dehydration (DH) or 4% high salt food (HSD) altered salt preference. Here we show that 24 h of ingesting IS and HS solutions, but not DH or HSD, robustly transformed salt appetite in rats when tested 7 days and 35 days later. Using two-bottle tests rats previously exposed to IS preferred neither IS or water, whereas rats exposed to HS showed aversion to IS. Responses to sweet solutions (1% sucrose) were not different in two-bottle tests with water, suggesting that salt was the primary aversive taste pathway recruited in this model. Inducing thirst by subcutaneous administration of angiotensin II did not overcome this salt aversion. We hypothesised that this behavior results from altered gene expression in brain structures important in thirst and salt appetite. Thus we also report here lasting changes in mRNAs for markers of neuronal activity, peptide hormones and neuronal plasticity in supraoptic and paraventricular nuclei of the hypothalamus following rehydration after both DH and HS. These results indicate that a single experience of drinking HS is a memorable one, with long-term changes in gene expression accompanying this aversion to salty solutions.

Cyclic AMP (cAMP) inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1) is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH). We have recently shown that Creb3l1 expression is upregulated by cAMP pathways , however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway is dictated by the level of methylation of a CpG island within the proximal promoter/5'UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3′ poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress. Cells are only able to work properly if they maintain a more or less constant balance of water and salts. In mammals, a hormone called arginine vasopressin regulates water and salt levels in the whole body. This hormone is made by cells in a region of the brain called the hypothalamus, and is then transported to the pituitary gland. When the level of water relative to the level of salts in the blood starts to drop (i.e., during dehydration), arginine vasopressin is released into the blood and travels to the kidneys where it acts as a signal to retain more water in the body. However, if water levels continue to remain low, the stores of arginine vasopressin in the pituitary gland may run out and so more protein needs to be made in the hypothalamus. Like all proteins, arginine vasopressin is made by first copying a template encoded in a particular gene into a molecule called messenger ribonucleic acid (mRNA). During dehydration, the cells in the hypothalamus produce more of these corresponding mRNA molecules. Also, the mRNAs are slightly larger than normal because they have longer ‘polyA tails’ (structures added to the ends of all newly-made mRNAs). However, it was not clear how or why this happens. Here, Konopacka et al. studied the production of arginine vasopressin in rats. The experiments show that a protein called Caprin-2 accumulates in hypothalamic neurons when rats are dehydrated. Furthermore, Caprin-2 is able to directly bind to the mRNA that encodes arginine vasopressin and is responsible for increasing the length of the polyA tail. To test whether this interaction is important for regulating the balance of water and salts, Konopacka et al. decreased the levels of Caprin-2 protein in the hypothalamus of live rats. When these rats became dehydrated, they had lower levels of the arginine vasopressin mRNA and these mRNAs had shorter polyA tails. Furthermore, the rats drank less water and urinated less than normal rats. Further experiments show that Caprin-2 helps to stabilize the structure of these mRNAs so that they accumulate in cells. Together, Konopacka et al.'s findings show that Caprin-2 regulates the production of arginine vasopressin by interacting with and modifying its corresponding mRNA in the rat hypothalamus. The next challenge is to find out which other mRNAs in the hypothalamus are regulated by Caprin-2, and to determine their roles in the body.

Background Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression. Results The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1 . The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress. Conclusion Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.

Background Rasd1 is a member of the Ras family of monomeric G proteins that was first identified as a dexamethasone inducible gene in the pituitary corticotroph cell line AtT20. Using microarrays we previously identified increased Rasd1 mRNA expression in the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in response to increased plasma osmolality provoked by fluid deprivation and salt loading. RASD1 has been shown to inhibit adenylyl cyclase activity in vitro resulting in the inhibition of the cAMP-PKA-CREB signaling pathway. Therefore, we tested the hypothesis that RASD1 may inhibit cAMP stimulated gene expression in the brain. Results We show that Rasd1 is expressed in vasopressin neurons of the PVN and SON, within which mRNA levels are induced by hyperosmotic cues. Dexamethasone treatment of AtT20 cells decreased forskolin stimulation of c-Fos , Nr4a1 and phosphorylated CREB expression, effects that were mimicked by overexpression of Rasd1 , and inhibited by knockdown of Rasd1 . These effects were dependent upon isoprenylation, as both farnesyltransferase inhibitor FTI-277 and CAAX box deletion prevented Rasd1 inhibition of cAMP-induced gene expression. Injection of lentiviral vector into rat SON expressing Rasd1 diminished, whereas CAAX mutant increased, cAMP inducible genes in response to osmotic stress. Conclusions We have identified two mechanisms of Rasd1 induction in the hypothalamus, one by elevated glucocorticoids in response to stress, and one in response to increased plasma osmolality resulting from osmotic stress. We propose that the abundance of RASD1 in vasopressin expressing neurons, based on its inhibitory actions on CREB phosphorylation, is an important mechanism for controlling the transcriptional responses to stressors in both the PVN and SON. These effects likely occur through modulation of cAMP-PKA-CREB signaling pathway in the brain.

Ageing is associated with altered neuroendocrine function. In the context of the hypothalamic supraoptic nucleus, which makes the antidiuretic hormone vasopressin, ageing alters acute responses to hyperosmotic cues, rendering the elderly more susceptible to dehydration. Chronically, vasopressin has been associated with numerous diseases of old age, including type 2 diabetes and metabolic syndrome. Bulk RNAseq transcriptome analysis has been used to catalogue the polyadenylated supraoptic nucleus transcriptomes of adult (3 months) and aged (18 months) rats in basal euhydrated and stimulated dehydrated conditions. Gene ontology and Weighted Correlation Network Analysis revealed that ageing is associated with alterations in the expression of extracellular matrix genes. Interestingly, whilst the transcriptomic response to dehydration is overall blunted in aged animals compared to adults, there is a specific enrichment of differentially expressed genes related to neurodegenerative processes in the aged cohort, suggesting that dehydration itself may provoke degenerative consequences in aged rats.