Processing of the FMRFamide Precursor Protein in the Snail Lymnaea stagnalis: Characterization and Neuronal Localization of a Novel Peptide, 'SEEPLY'

In the pulmonate snail Lymnaea stagnalis , FMRFamide-like neuropeptides are encoded by a multi-exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post-translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDWLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post-translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor-like peptides.     

Identification, Distribution and Physiological Activity of Three Novel Neuropeptides of Lymnaea: FLRlamide and pQFYRlamide Encoded by the FMRFamide Gene, and a Related Peptide

We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamideand QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif –FXRlamide. In this paper we adopt a novel approach to first identify and characterize –FXRlamide-like peptides in extracts from the central nervous system of Lymnaea. By a combination of high-performance liquid chromatography (HPLC) and continuous-flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post-translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immuno reactive to all three peptides, revealed EFLRlamide-like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post-translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardio excitatory motoneurons, the E_he cells (E heart excitors of the visceral ganglion) and penialmotoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that the serespective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide-like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many centralneurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory response was followed by a long-lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post-translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.     

Unique accumulation of neuropeptides in an insect: FMRFamide-related peptides in the cockroach, Periplaneta americana

FMRFamides belong to the most extensively studied neuropeptides in invertebrates and exhibit diverse physiological effects on different target organs, such as muscles, intestine and the nervous system. This study on the American cockroach confirms for the first time that extended FMRFamides occur in non-dipteran insects. By means of tandem mass spectrometry, these neuropeptides were structurally elucidated, and sequence information was used for subsequent cloning of the cockroach FMRFamide gene. This precursor gene encodes for 24 putative peptides and shows sufficient similarity with the Drosophila FMRFamide gene. Of the 24 peptides, 23 were detected by mass spectrometric methods; it is the highest number of neuropeptide forms shown to be expressed from a single precursor in any insect. The expression was traced back to single neurons in the thoracic ganglia. The unique accumulation of these FMRFamide-related peptides in thoracic perisympathetic organs provides the definite evidence for a tagma-specific distribution of peptidergic neurohormones in neurohaemal release sites of the insect CNS. Excitatory effects of the cockroach FMRFamides were observed on antenna-heart preparations. In addition, the newly described FMRFamides reduce the spike frequency of dorsal-unpaired median neurons and reduce the intracellular calcium concentration, which may affect the peripheral release of the biogenic amine octopamine.     

The Beneficial Effects of Early Short-Term Exercise in the Offspring of Obese Mothers are Accompanied by Alterations in the Hypothalamic Gene Expression of Appetite Regulators and FTO (Fat Mass and Obesity Associated) Gene

Maternal overnutrition is implicated in the development of adult metabolic disease, and has been shown to alter the expression of genes involved in energy homeostasis. In the present study, we aimed to test whether a short period of voluntary exercise, followed by a sedentary period, would regulate hypothalamic markers involved in appetite. Adult female Sprague–Dawley rats were fed either normal chow or high-fat diet (HFD) ad lib. for 5 weeks, mated and continued on their assigned diet during gestation/lactation. At weaning males, were separated into chow or HFD groups; half were exercised (running wheels), whereas the remainder were sedentary. At week 10, wheels were removed and rats remained sedentary for 5 weeks, prior to tissue collection. Maternal obesity increased offspring adiposity at 15 weeks and this was exacerbated by postnatal HFD (P < 0.05). Body weight and fat mass were reduced in offspring of obese mothers if they exercised, and this was maintained even after 5 weeks without exercise. At 15 weeks, fasting plasma insulin, leptin and triglyceride concentrations were significantly reduced by exercise in offspring of lean and obese mothers consuming chow, with little benefit in those consuming HFD. Hypothalamic mRNA expression of pro-opiomelanocortin was increased by exercise but only in offspring of lean mothers. Exercise reduced hypothalamic FTO (fat mass and obesity associated) mRNA in offspring of lean dams regardless of diet. A short period of exercise early in life had lasting beneficial effects on body weight, adiposity and hormone profile of male offspring from obese and lean dams, despite being followed by a period of inactivity. The effects of exercise on hypothalamic appetite regulators were more marked in offspring of lean dams.