Leptin Targets in the Mouse Brain

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin`s cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRbI-RES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor. J. Comp. Neurol. 514:518-532, 2009. (C) 2009 Wiley-Liss, Inc.

Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

Investigation of the hypothalamic defensive system in the mouse

The hypothalamus plays especially important roles in various endocrine, autonomic, and behavioral responses that guarantee the survival of both the individual and the species. In the rat, a distinct hypothalamic defensive circuit has been defined as critical for integrating predatory threats, raising an important question as to whether this concept could be applied to other prey species. To start addressing this matter, in the present study, we investigated, in another prey species (the mouse), the pattern of hypothalamic Fos immunoreactivity in response to exposure to a predator (a rat, using the Rat Exposure Test). During rat exposure, mice remained concealed in the home chamber for a longer period of time and increased freezing and risk assessment activity. We were able to show that the mouse and the rat present a similar pattern of hypothalamic activation in response to a predator. Of particular note, similar to what has been described for the rat, we observed in the mouse that predator exposure induces a striking activation in the elements of the medial hypothalamic defensive system, namely, the anterior hypothalamic nucleus, the dorsomedial part of the ventromedial hypothalamic nucleus and the dorsal premammillary nucleus. Moreover, as described for the rat, predator-exposed mice also presented increased Fos levels in the autonomic and parvicellular parts of the paraventricular hypothalamic nucleus, lateral preoptic area and subfornical region of the lateral hypothalamic area. In conclusion, the present data give further support to the concept that a specific hypothalamic defensive circuit should be preserved across different prey species. (C) 2008 Elsevier B.V. All rights reserved.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell ( due to the. ring of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K(ATP) channel conductance (G(K,ATP)) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC(50) of approximately 4 mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca(2+)](i) waves to spread with a speed of approximately 80 mu m s(-1), similar to that observed experimentally in confocal [Ca(2+)](i) imaging.

Molecular Mechanisms by Which Saturated Fatty Acids Modulate TNF-alpha Expression in Mouse Macrophage Lineage

Many macrophage functions are modulated by fatty acids (FAs), including cytokine release, such as tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is of great interest due to its role in the inflammation process observed in several diseases such as rheumatoid arthritis, atherosclerosis, and obesity. However, the mechanisms by which FA effects occur have not been completely elucidated yet. In this study, we used a mouse monocyte lineage (J774 cells) to evaluate the effect of 50 and 100 mu M of saturated (palmitic and stearic acids), monounsaturated (oleic acid) and polyunsaturated (linoleic acid) FAs on TNF-alpha production. Alterations in gene expression, poly(A) tail length and activation of transcription factors were evaluated. Oleic and linoleic acids, usually known as neutral or pro-inflammatory FA, inhibited LPS-induced TNF-alpha secretion by the cells. Saturated FAs were potent inducers of TNF-alpha expression and secretion under basal and inflammatory conditions (in the presence of LPS). Although the effect of the saturated FA was similar, the mechanism involved in each case seem to be distinct, as palmitic acid increased EGR-1 and CREB binding activity and stearic acid increased mRNA poly(A) tail. These results may contribute to the understanding of the molecular mechanisms by which saturated FAs modulate the inflammatory response and may lead to design of associations of dietary and pharmacological strategies to counteract the pathological effects of TNF-alpha.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK. gene transcription.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.org

Hemopressins and other hemoglobin-derived peptides in mouse brain: comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice

P>Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpefat/fat mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpefat/fat mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpefat/fat mouse brain regions.

Tissue distribution of quiescin Q6/sulfhydryl oxidase (QSOX) in developing mouse

Quiescin Q6/sulfhydryl oxidases (QSOX) are revisited thiol oxidases considered to be involved in the oxidative protein folding, cell cycle control and extracellular matrix remodeling. They contain thioredoxin domains and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation, likely altering the redox environment. Since it is known that several developmental processes are regulated by the redox state, here we assessed if QSOX could have a role during mouse fetal development. For this purpose, an anti-recombinant mouse QSOX antibody was produced and characterized. In E-13.5, E-16.5 fetal tissues, QSOX immunostaining was confined to mesoderm- and ectoderm-derived tissues, while in P1 neonatal tissues it was slightly extended to some endoderm-derived tissues. QSOX expression, particularly by epithelial tissues, seemed to be developmentally-regulated, increasing with tissue maturation. QSOX was observed in loose connective tissues in all stages analyzed, intra and possibly extracellularly, in agreement with its putative role in oxidative folding and extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from mesoderm and ectoderm, suggesting it could be of relevance during developmental processes.

Metastatic melanoma positively influences pregnancy outcome in a mouse model: could a deadly tumor support embryo life?

The incidence of melanoma is increasing worldwide. It is one of the leading cancers in pregnancy and the most common malignancy to metastasize to placenta and fetus. There are no publications about experimental models of melanoma and pregnancy. We propose a new experimental murine model to study the effects of melanoma on pregnancy and its metastatic process. We tested several doses of melanoma cells until we arrived at the optimal dose, which produced tumor growth and allowed animal survival to the end of pregnancy. Two control groups were used: control (C) and stress control (SC) and three different routes of inoculation: intravenous (IV), intraperitoneal (IP) and subcutaneous (SC). All the fetuses and placentas were examined macroscopically and microscopically. The results suggest that melanoma is a risk factor for intrauterine growth restriction but does not affect placental weight. When inoculated by the SC route, the tumor grew only in the site of implantation. The IP route produced peritoneal tumoral growth and also ovarian and uterine metastases in 60% of the cases. The IV route produced pulmonary tumors. No placental or fetal metastases were obtained, regardless of the inoculation route. The injection of melanoma cells by any route did not increase the rate of fetal resorptions. Surprisingly, animals in the IV groups had no resorptions and a significantly higher number of fetuses. This finding may indicate that tumoral factors released in the host organism to favor tumor survival may also have a pro-gestational action and consequently improve the reproductive performance of these animals.

Localization of Cathepsins D and B at the Maternal-Fetal Interface and the Invasiveness of the Trophoblast during the Postimplantation Period in the Mouse

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface. Copyright (C) 2010 S. Karger AG, Basel

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B(1) receptor gene

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ(10 mu M). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 mu M), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice. (C) 2009 Elsevier Inc. All rights reserved.

Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin

Stings by Polistes wasps can cause life-threatening allergic reactions, pain and inflammation. We examined the changes in microvascular permeability and neutrophil influx caused by the venom of Polistes lanio a paper wasp found in southeastern Brazil. The intradermal injection of wasp venom caused long-lasting paw oedema and dose-dependently increased microvascular permeability in mouse dorsal skin. SR140333, an NK(1) receptor antagonist, markedly inhibited the response, but the NK(2) receptor antagonist SR48968 was ineffective. The oedema was reduced in capsaicin-treated rats, indicating a direct activation of sensory fibres. Dialysis of the venom partially reduced the oedema and the remaining response was further inhibited by SR140333. Mass spectrometric analysis of the venom revealed two peptides (QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM) with sequence similarities to the C-terminal region of tachykinin-like peptides found in Phoneutria nigniventer spider venom and vertebrates. Wasp venom failed to release histamine from mast cells in vitro and spectrofluorometric assay of the venom revealed a negligible content of histamine in the usual dose of P.l. lanio venom (1 nmol of histamine/7 mu g of venom)that was removed by dialysis. The histamine H(1) receptor antagonist pyrilamine, but not bradykinin B(1) or B(2) receptor antagonists, inhibited venom-induced oedema. In conclusion, P. l. lanio venom induces potent oedema and increases vascular permeability in mice, primarily through activation of tachykinin NK(1) receptors by substance P released from sensory C fibres, which in turn releases histamine from dermal mast cells. This is the first description of a neurovascular mechanism for P. l. lanio venom-mediated inflammation. The extent to which the two tachykinin-like peptides identified here contribute to this neurogenic inflammatory response remains to be elucidated. (c) 2008 Elsevier Ltd. All rights reserved.