Cachexia in MAC16 adenocarcinoma:Suppression of hunger despite normal regulation of leptin, insulin and hypothalamic neuropeptide Y

Weight loss normally stimulates hunger, through mechanisms that include falls in circulating leptin and insulin, leading to stimulation of hypothalamic neuropeptide Y (NPY). Here, we investigated the leptin, insulin and NPY to clarify why hunger is suppressed in mice with severe cachexia due to the MAC16 adenocarcinoma. MAC16-bearing mice progressively lost weight (19% below controls) and fat (-61%) over 16 days after tumour transplantation, while total food intake fell by 10%. Pair-fed mice showed less wasting, with final weight being 9% and fat mass 25% below controls. Plasma leptin fell by 85% in MAC16 and 51% in pair-fed mice, in proportion to loss of fat. Plasma insulin was also reduced by 49% in MAC16 and 53% in pair-fed groups. Hypothalamic leptin receptor (OB-Rb) mRNA was significantly increased in both MAC16 (+223%) and pair-fed (+192%) mice. Hypothalamic NPY mRNA was also significantly raised in MAC16 (+152%) and pair-fed (+99%) groups, showing negative correlations with plasma leptin and insulin, and a positive association with OB-Rb mRNA. In MAC16-induced cachexia, leptin production and hypothalamic OB-Rb and NPY expression are regulated appropriately in response to fat depletion. Therefore, suppression of hunger is probably due to tumour products that inhibit NPY transport or release, or that interfere with neuronal targets downstream of NPY.

Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding

Background Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms. Methods A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies. Conclusions AT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion. Significance Immunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion.

Galanin interacts with Neuropeptide Y Y1 receptor in the dentate gyrus of the hippocampus through GALR2/NPYY1R heterodimers

We have shown Galanin(GAL) and Neuropeptide Y Y1(NPYY1) interactions at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers in the amygdala. The aim of this work was to analyze GAL/NPYY1R interactions in the Dentate Gyrus(DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA). Rats(n=6) were sacrificed 15 minutes or 5 hours after icv injections of GAL(3nmol) and DG sections were incubated with NPYY1R agonist [I125]-[Leu31,Pro34]PYY(25 pM) or NPYY1R-33PdATP specific probe, for autoradiography and in situ hybridization respectively. Autoradiograms were analyzed using NIH image analysis system and Student’s unpaired t-test was used. For PLA, DG sections were incubated with anti-GALR2 Rabbit(1:100) and anti-NPYY1R Goat(1:200). PLA signals were detected with PLA PLUS or MINUS probes for rabbit or goat/mouse antibodies. PLA signals were visualized by using a confocal microscope Leica TCS-SL confocal microscope(Leica). We observed that GAL significant increased the NPYY1R agonist [I125]-[Leu31,Pro34]PYY binding in the DG by 20% (p<0,05) and the NPYY1R mRNA expression in the granular layer of DG by 31% (p<0,001). Moreover, PLA-positive red clusters were found specifically in the polymorphic layer and subgranular zone of the DG. No PLA clusters were observed neither in the molecular layer of the DG nor in the corpus callosum, an area that seems to lack of GALR2 receptor. These results demonstrate a novel mechanism of interaction between GAL and NPY1R in the DG at receptor level, probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. Study supported by Junta de Andalucia CVI6476.

Association study of the calcitonin gene-related polypeptide-alpha (CALCA) and the receptor activity modifying 1 (RAMP1) genes with migraine

Migraine is a common neurovascular brain disorder characterised by recurrent attacks of severe headache that may be accompanied by various neurological symptoms. Migraine is thought to result from activation of the trigeminovascular system followed by vasodilation of pain-producing intracranial blood vessels and activation of second-order sensory neurons in the trigeminal nucleus caudalis. Calcitonin gene-related peptide (CGRP) is a mediator of neurogenic inflammation and the most powerful vasodilating neuropeptide, and has been implicated in migraine pathophysiology. Consequently, genes involved in CGRP synthesis or CGRP receptor genes may play a role in migraine and/or increase susceptibility. This study investigates whether variants in the gene that encodes CGRP, calcitonin-related polypeptide alpha (CALCA) or in the gene that encodes a component of its receptor, receptor activity modifying protein 1 (RAMP1), are associated with migraine pathogenesis and susceptibility. The single nucleotide polymorphisms (SNPs) rs3781719 and rs145837941 in the CALCA gene, and rs3754701 and rs7590387 at the RAMP1 locus, were analysed in an Australian Caucasian population of migraineurs and matched controls. Although we find no significant association of any of the SNPs tested with migraine overall, we detected a nominally significant association (p = 0.031) of the RAMP1 rs3754701 variant in male migraine subjects, although this is non-significant after Bonferroni correction for multiple testing.

The role of the glucocorticoids in developing resilience to stress and addiction

There is emerging evidence that individuals have the capacity to learn to be resilient by developing protective mechanisms that prevent them from the maladaptive effects of stress that can contribute to addiction.The emerging field of the neuroscience of resilience is beginning to uncover the circuits and molecules that protect against stress-related neuropsychiatric diseases, such as addiction. Glucocorticoids (GCs) are important regulators of basal and stress-related homeostasis in all higher organisms and influence a wide array of genes in almost every organ and tissue. GCs, therefore, are ideally situated to either promote or prevent adaptation to stress. In this review, we will focus on the role of GCs in the hypothalamic-pituitary adrenocortical axis and extra-hypothalamic regions in regulating basal and chronic stress responses. GCs interact with a large number of neurotransmitter and neuropeptide systems that are associated with the development of addiction. Additionally, the review will focus on the orexinergic and cholinergic pathways and highlight their role in stress and addiction. GCs play a key role in promoting the development of resilience or susceptibility and represent important pharmacotherapeutic targets that can reduce the impact of a maladapted stress system for the treatment of stress-induced addiction.

A Mammalian Circadian Clock Model Incorporating Daytime Expression Elements

Models of the mammalian clock have traditionally been based around two feedback loops-the self-repression of Per/Cry by interfering with activation by BMAL/CLOCK, and the repression of Bmal/Clock by the REV-ERB proteins. Recent experimental evidence suggests that the D-box, a transcription factor binding site associated with daytime expression, plays a larger role in clock function than has previously been understood. We present a simplified clock model that highlights the role of the D-box and illustrate an approach for finding maximum-entropy ensembles of model parameters, given experimentally imposed constraints. Parameter variability can be mitigated using prior probability distributions derived from genome-wide studies of cellular kinetics. Our model reproduces predictions concerning the dual regulation of Cry1 by the D-box and Rev-ErbA/ROR response element (RRE) promoter elements and allows for ensemble-based predictions of phase response curves (PRCs). Nonphotic signals such as Neuropeptide Y (NPY) may act by promoting Cry1 expression, whereas photic signals likely act by stimulating expression from the E/E' box. Ensemble generation with parameter probability restraints reveals more about a model's behavior than a single optimal parameter set.

Gene-based pleiotropy across migraine with aura and migraine without aura patient groups

Introduction: It is unclear whether patients diagnosed according to International Classification of Headache Disorders criteria for migraine with aura (MA) and migraine without aura (MO) experience distinct disorders or whether their migraine subtypes are genetically related. Aim: Using a novel gene-based (statistical) approach, we aimed to identify individual genes and pathways associated both with MA and MO. Methods: Gene-based tests were performed using genome-wide association summary statistic results from the most recent International Headache Genetics Consortium study comparing 4505 MA cases with 34,813 controls and 4038 MO cases with 40,294 controls. After accounting for non-independence of gene-based test results, we examined the significance of the proportion of shared genes associated with MA and MO. Results: We found a significant overlap in genes associated with MA and MO. Of the total 1514 genes with a nominally significant gene-based p value (pgene-based ≤ 0.05) in the MA subgroup, 107 also produced pgene-based ≤ 0.05 in the MO subgroup. The proportion of overlapping genes is almost double the empirically derived null expectation, producing significant evidence of gene-based overlap (pleiotropy) (pbinomial-test = 1.5 × 10–4). Combining results across MA and MO, six genes produced genome-wide significant gene-based p values. Four of these genes (TRPM8, UFL1, FHL5 and LRP1) were located in close proximity to previously reported genome-wide significant SNPs for migraine, while two genes, TARBP2 and NPFF separated by just 259 bp on chromosome 12q13.13, represent a novel risk locus. The genes overlapping in both migraine types were enriched for functions related to inflammation, the cardiovascular system and connective tissue. Conclusions: Our results provide novel insight into the likely genes and biological mechanisms that underlie both MA and MO, and when combined with previous data, highlight the neuropeptide FF-amide peptide encoding gene (NPFF) as a novel candidate risk gene for both types of migraine.

Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation.

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-beta-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Positional cloning and pathway analysis of the asthma susceptibility gene, NPSR1

In the present study, we identified a novel asthma susceptibility gene, NPSR1 (neuropeptide S receptor 1) on chromosome 7p14.3 by the positional cloning strategy. An earlier significant linkage mapping result among Finnish Kainuu asthma families was confirmed in two independent cohorts: in asthma families from Quebec, Canada and in allergy families from North Karelia, Finland. The linkage region was narrowed down to a 133-kb segment by a hierarchial genotyping method. The observed 77-kb haplotype block showed 7 haplotypes and a similar risk and nonrisk pattern in all three populations studied. All seven haplotypes occur in all three populations at frequences > 2%. Significant elevated relative risks were detected for elevated total IgE (immunoglobulin E) or asthma. Risk effects of the gene variants varied from 1.4 to 2.5. NPSR1 belongs to the G protein-coupled receptor (GPCR) family with a topology of seven transmembrane domains. NPSR1 has 9 exons, with the two main transcripts, A and B, encoding proteins of 371 and 377 amino acids, respectively. We detected a low but ubiquitous expression level of NPSR1-B in various tissues and endogenous cell lines while NPSR1-A has a more restricted expression pattern. Both isoforms were expressed in the lung epithelium. We observed aberrant expression levels of NPSR1-B in smooth muscle in asthmatic bronchi as compared to healthy. In an experimental mouse model, the induced lung inflammation resulted in elevated Npsr1 levels. Furthermore, we demonstrated that the activation of NPSR1 with its endogenous agonist, neuropeptide S (NPS), resulted in a significant inhibition of the growth of NPSR1-A overexpressing stable cell lines (NPSR1-A cells). To determine which target genes were regulated by the NPS-NPSR1 pathway, NPSR1-A cells were stimulated with NPS, and differentially expressed genes were identified using the Affymetrix HGU133Plus2 GeneChip. A total of 104 genes were found significantly up-regulated and 42 down-regulated 6 h after NPS administration. The up-regulated genes included many neuronal genes and some putative susceptibility genes for respiratory disorders. By Gene Ontology enrichment analysis, the biological process terms, cell proliferation, morphogenesis and immune response were among the most altered. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), were verified and confirmed by quantitative reverse-transcriptase-PCR. In conclusion, we identified a novel asthma susceptibility gene, NPSR1, on chromosome 7p14.3. NPS-NPSR1 represents a novel pathway that regulates cell proliferation and immune responses, and thus may have functional relevance in the pathogenesis of asthma.

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

Neuronal mechanism of state control in Drosophila melanogaster

The changes in internal states, such as fear, hunger and sleep affect behavioral responses in animals. In most of the cases, these state-dependent influences are “pleiotropic”: one state affects multiple sensory modalities and behaviors; “scalable”: the strengths and choices of such modulations differ depending on the imminence of demands; and “persistent”: once the state is switched on the effects last even after the internal demands are off. These prominent features of state-control enable animals to adjust their behavioral responses depending on their internal demands. Here, we studied the neuronal mechanisms of state-controls by investigating energy-deprived state (hunger state) and social-deprived state of fruit flies, Drosophila melanogaster, as prototypic models. To approach these questions, we developed two novel methods: a genetically based method to map sites of neuromodulation in the brain and optogenetic tools in Drosophila.

These methods, and genetic perturbations, reveal that the effect of hunger to alter behavioral sensitivity to gustatory cues is mediate by two distinct neuromodulatory pathways. The neuropeptide F (NPF) – dopamine (DA) pathway increases sugar sensitivity under mild starvation, while the adipokinetic hormone (AKH)- short neuropeptide F (sNPF) pathway decreases bitter sensitivity under severe starvation. These two pathways are recruited under different levels of energy demands without any cross interaction. Effects of both of the pathways are mediated by modulation of the gustatory sensory neurons, which reinforce the concept that sensory neurons constitute an important locus for state-dependent control of behaviors. Our data suggests that multiple independent neuromodulatory pathways are underlying pleiotropic and scalable effects of the hunger state.

In addition, using optogenetic tool, we show that the neural control of male courtship song can be separated into probabilistic/biasing, and deterministic/command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, supporting the idea that they constitute a locus of state-dependent influence. Interestingly, moreover, brief activation of the former, but not the latter, neurons trigger persistent behavioral response for more than 10 min. Altogether, these findings and new tools described in this dissertation offer new entry points for future researchers to understand the neuronal mechanism of state control.

The C. elegans ALA neuron : its transcriptome and role in inducing sleep

A long-standing yet to be accomplished task in understanding behavior is to dissect the function of each gene involved in the development and function of a neuron. The C. elegans ALA neuron was chosen in this study for its known function in sleep, an ancient but less understood animal behavior. Single-cell transcriptome profiling identified 8,133 protein-coding genes in the ALA neuron, of which 57 are neuropeptide-coding genes. The most enriched genes are also neuropeptides. In combination with gain-of-function and loss-of-function assays, here I showed that the ALA-enriched FMRFamide neuropeptides, FLP-7, FLP-13, and FLP-24, are sufficient and necessary for inducing C. elegans sleep. These neuropeptides act as neuromodulators through GPCRs, NPR-7, and NPR-22. Further investigation in zebrafish indicates that FMRFamide neuropeptides are sleep-promoting molecules in animals. To correlate the behavioral outputs with genomic context, I constructed a gene regulatory network of the relevant genes controlling C. elegans sleep behavior through EGFR signaling in the ALA neuron. First, I identified an ALA cell-specific motif to conduct a genome-wide search for possible ALA-expressed genes. I then filtered out non ALA-expressed genes by comparing the motif-search genes with ALA transcriptomes from single-cell profiling. In corroborating with ChIP-seq data from modENCODE, I sorted out direct interaction of ALA-expressed transcription factors and differentiation genes in the EGFR sleep regulation pathway. This approach provides a network reference for the molecular regulation of C. elegans sleep behavior, and serves as an entry point for the understanding of functional genomics in animal behaviors.

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel Deuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.

Pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) which belongs to the secretin/glucagon/ VIP family has been originally isolated from the sheep hypothalamus on the basis of its ability to stimulate cAMP formation in culture rat anterior pituitary cells. Post-translational processing of the PACAP precursor generates two biologically active molecular forms, PACAP-38 and PACAP-27. The primary structure of PACAP has been remarkably conserved during evolution. The sequence of PACAP-27 exhibits substantial similarities with those of vasoactive intestinal polypeptide (VIP), glucagon and secretin. The gene encoding the PACAP precursor is widely expressed in brain and various peripheral organs, notably in endocrine glands, gastro-intestinal, urogenital tracts and respiratory system. In vivo, and in vitro studies have shown that PACAP exhibits multiple activities especially a trophic activity during ontogenesis, notably in the adrenal medulla and the central nervous system. The biological effects of PACAP are mediated through three distinct receptor subtypes which exhibit differential affinities for PACAP and VIP. The PAC1 receptor, which shows high selectivity for PACAP, is coupled to several transduction systems. In contrast, VPAC1 and VPAC2, which bind with the same affinity for PACAP and VIP, are mainly coupled to the adenylyl cyclase pathway. In conclusion, PACAP is neuropeptide, and it functions as a hypothalamic hormone, neurohormone, neuromodulator, vasodilator, neurotransmitter or trophic factor in the brain and the various organs.