Growth cone MKK7 mRNA targeting regulates MAP1b-dependent microtubule bundling to control neurite elongation.

Local mRNA translation in neurons has been mostly studied during axon guidance and synapse formation but not during initial neurite outgrowth. We performed a genome-wide screen for neurite-enriched mRNAs and identified an mRNA that encodes mitogen-activated protein kinase kinase 7 (MKK7), a MAP kinase kinase (MAPKK) for Jun kinase (JNK). We show that MKK7 mRNA localizes to the growth cone where it has the potential to be translated. MKK7 is then specifically phosphorylated in the neurite shaft, where it is part of a MAP kinase signaling module consisting of dual leucine zipper kinase (DLK), MKK7, and JNK1. This triggers Map1b phosphorylation to regulate microtubule bundling leading to neurite elongation. We propose a model in which MKK7 mRNA localization and translation in the growth cone allows for a mechanism to position JNK signaling in the neurite shaft and to specifically link it to regulation of microtubule bundling. At the same time, this uncouples activated JNK from its functions relevant to nuclear translocation and transcriptional activation.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

Cholinergic regulation of neuropeptide Y synthesis and release in human neuroblastoma cells.

The biosynthesis and release of neuropeptide Y (NPY) is regulated by several factors. Here, the effect of the muscarinic agonist carbachol on NPY biosynthesis and release was analyzed utilizing the SH-SY5Y human neuroblastoma cell line. We observed that: (a) carbachol moderately increased the post-translational cleavage of proNPY to NPY; (b) carbachol treatment stimulated NPY accumulation into the medium in a time- and dose-related manner; (c) protein kinase C activation is involved in carbachol-mediated NPY synthesis/release (>6h). In conclusion, the present observations support the hypothesis that muscarinic receptor activation regulates the biosynthesis and secretion of NPY.

Ghrelin and cannabinoids require the ghrelin receptor to affect cellular energy metabolism.

INTRODUCTION: Ghrelin is a potent orexigenic brain-gut peptide with lipogenic and diabetogenic effects, possibly mediated by growth hormone secretagogue receptor (GHS-R1a). Cannabinoids also have orexigenic and lipogenic effects. AMPK is a regulator of energy homeostasis and we have previously shown that ghrelin and cannabinoids stimulate hypothalamic AMPK activity while inhibiting it in the liver and adipose tissue, suggesting that AMPK mediates both the central appetite-inducing and peripheral effects of ghrelin and cannabinoids. AIMS: Using GHS-R KO mice, we investigated whether the known ghrelin receptor GHS-R1a is required for the tissue-specific effects of ghrelin on AMPK activity, and if an intact ghrelin signalling pathway is necessary for the effects of cannabinoids on AMPK activity. METHODS: Wild-type and GHS-R KO mice were treated intraperitoneally with ghrelin 500 ng/g bodyweight or CB1 agonist HU210 20 ng/g and hypothalamic, hepatic and adipose AMPK activity was studied using a functional kinase assay. RESULTS: Ghrelin and HU210 significantly stimulated hypothalamic AMPK activity in wild-type animals (mean±SEM, 122.5±5.2% and 128±11.6% of control, p<0.05) and inhibited it in liver (55.1±4.8% and 62.2±14.5%, p<0.01) and visceral fat (mesenteric fat (MF): 54.6±16% and 52.0±9.3%, p<0.05; epididymal fat (EF): 47.9±12.1% and 45.6±1.7%, p<0.05). The effects of ghrelin, and interestingly also HU210, on hypothalamic, visceral fat and liver AMPK activity were abolished in the GHS-R KO mice (hypothalamus: 107.9±7.7% and 87.4±13.3%, liver: 100.5±11.6% and 116.7±5.4%, MF: 132.1±29.9% and 107.1±32.7%, EF: 89.8±7.3% and 91.7±18.3%, p>0.05). CONCLUSIONS: Ghrelin requires GHS-R1a for its effect on hypothalamic, liver and adipose tissue AMPK activity. An intact ghrelin signalling pathway is necessary for the effects of cannabinoids on AMPK activity.

Assessment of Rho GTPase signaling during neurite outgrowth.

Rho GTPases are key regulators of the cytoskeleton during the process of neurite outgrowth. Based on overexpression of dominant-positive and negative Rho GTPase constructs, the classic view is that Rac1 and Cdc42 are important for neurite elongation whereas RhoA regulates neurite retraction in response to collapsing agents. However, recent work has suggested a much finer control of spatiotemporal Rho GTPase signaling in this process. Understanding this complexity level necessitates a panel of more sensitive tools than previously used. Here, we discuss a novel assay that enables the biochemical fractionation of the neurite from the soma of differentiating N1E-115 neuronal-like cells. This allows for spatiotemporal characterization of a large number of protein components, interactions, and post-translational modifications using classic biochemical and also proteomics approaches. We also provide protocols for siRNA-mediated knockdown of genes and sensitive assays that allow quantitative analysis of the neurite outgrowth process.

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth.

BACKGROUND: The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. METHODOLOGY/PRINCIPAL FINDINGS: We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. CONCLUSIONS/SIGNIFICANCE: We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Regulation of prostate cancer cell proliferation by somatostatin receptor activation.

Although some evidence supports the antitumoral effects of somatostatin (SRIF) and related agonists, the available data in prostate cancer (PCa) model systems and clinical studies are few, conflicting and not conclusive. This study investigated the effects of lanreotide and new mono- and bi-specific SRIF agonists on proliferation, ligand-driven SRIF receptor (sst) dimerization and secretory pattern of the IGF system in LNCaP cells, a model of androgen-dependent PCa. LNCaP expressed all sst(s), but sst(4). Among them, sst(1) and sst(3) were inversely regulated by serum concentration. sst(1)/sst(2) and sst(2)/sst(5) dimers were constitutively present and further stabilized by treatment with BIM-23704 (sst(1)/sst(2)) and BIM-23244 (sst(2)/sst(5)), respectively. Dose-response studies showed that lanreotide and BIM-23244 were significantly more potent in inhibiting LNCaP cell proliferation than BIM-23120 (sst(2)) and BIM-23206 (sst(5)) alone or in combination. Treatment with BIM-23926 [corrected] (sst(1)) markedly reduced cell proliferation, whereas exposure to BIM-23704 resulted in a lower cell growth inhibition. The antiproliferative effects of BIM-23244, lanreotide and BIM-23704 were unchanged, reduced and abolished by the sst(2) antagonist BIM-23627, respectively. All SRIF analogs caused a significant induction in p27(KipI) and p21 and down-regulation of protein expression of cyclin E, as well as reduced IGF-I and IGF-II secretion. In particular, the administration of exogenous IGF-I, at variance to IGF-II, counteracted the inhibitory effect on cell proliferation of these compounds. Moreover, SRIF agonists reduced endogenous IGFBP-3 proteolysis. These results show that, in LNCaP cells, activation of sst(1) and sst(2)/sst(5) results in relevant antiproliferative/antisecretive actions.