R.C. van der Schors

CRNF, a Molluscan Neurotrophic Factor That Interacts with the p75 Neurotrophin Receptor

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Protein synthesis in synaptosomes: a proteomics analysis

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre‐synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [35S]methionine and applied two‐dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two‐dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver‐stained gels that were run in parallel. In‐gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear‐encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre‐synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

Co-localized neuropeptides conopressin and Ala-Pro-Gly-Trp-NH2 have antagonistic effects on the vas deferens of Lymnaea

We examined functional aspects of co-localization of neuropeptides involved in the regulation of male copulation behaviour in the simultaneous hermaphrodite snail Lymnaea stagnalis. The copulation behaviour is controlled by several types of peptidergic neurons that include a cluster of neurons in the anterior lobe of the right cerebral ganglion. All anterior lobe neurons express the gene encoding Ala-Pro-Gly-Trp-NH2 (APGWamide), and a subset of neurons also express the vasopressin-related conopressin gene. Immunocytochemical and peptide chemical experiments show that both APGWamide and conopressin are transported to the penis complex and the vas deferens via the penis nerve. Co-localization of the two peptides was also observed in some, but not all, axon bundles that run along the vas deferens. APGWamide and conopressin were structurally identified from the penis complex with vas deferens. Conopressin excites the vas deferens in vitro, whereas APGWamide inhibits the excitatory effects of conopressin, both in a dose-dependent fashion. We propose that the antagonistic effects of these peptides on the vas deferens underlie its peristalsis. Thus, these peptides play an important role in the control of ejaculation of semen during copulation.

Chapter 5 A vasopressin-related peptide in the mollusc Lymnaea stagnalis: peptide structure, prohormone organization, evolutionary and functional aspects of Lymnaea conopressin

Publisher Summary The members of the vasopressin/oxytocin hormone superfamily are widely distributed throughout the animal kingdom, and all hormones belonging to this superfamily are structurally related nonapeptides, having five invariant amino acid residues, including two cysteines, that form a disulphide bridge and an amidated carboxyterminal glycine. This chapter discusses the presence of vasopressin- and oxytocin-related peptides in invertebrates as well as their functions and explores the structural characteristics of the Lymnaea conopressin precursor in an evolutionary perspective. Only four different vasopressin- and oxytocin-related peptides have been structurally identified in invertebrate species, including Lysconopressin from the gastropod molluscs Conus geographus and Lymnaea stagnalis , Arg-conopressin from the gastropod mollusc Conus striatus , the vasopressin-like diuretic hormone from the insect Locusta migratoria , and cephalotocin from the cephalopod mollusc Octopus vufgaris . The results presented in the chapter show for the first time that in invertebrates a vasopressin-related prohormone is present, which has the same organization as its vertebrate counterparts.

Two metal binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage.

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.

Progression and recovery of Parkinsonism in a chronic progressive MPTP-induction model in the marmoset without persistent molecular and cellular damage.

Chronic exposure to low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in marmoset monkeys was used to model the prodromal stage of Parkinsons disease (PD), and to investigate mechanisms underlying disease progression and recovery. Marmosets were subcutaneously injected with MPTP for a period of 12weeks, 0.5mg/kg once per week, and clinical signs of Parkinsonism, motor- and non-motor behaviors were recorded before, during and after exposure. In addition, postmortem immunohistochemistry and proteomics analysis were performed. MPTP-induced parkinsonian clinical symptoms increased in severity during exposure, and recovered after MPTP administration was ended. Postmortem analyses, after the recovery period, revealed no alteration of the number and sizes of tyrosine hydroxylase (TH)-positive dopamine (DA) neurons in the substantia nigra. Also levels of TH in putamen and caudate nucleus were unaltered, no differences were observed in DA, serotonin or nor-adrenalin levels in the caudate nucleus, and proteomics analysis revealed no global changes in protein expression in these brain areas between treatment groups. Our findings indicate that parkinsonian symptoms can occur without detectable damage at the cellular or molecular level. Moreover, we show that parkinsonian symptoms may be reversible when diagnosed and treated early.

Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences

Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.

Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Primary Structure of Insulin of the Black Sea Rockfish Scorpaena porcus

Insulin of the Black Sea rockfish Scorpaena porcus was isolated, purified, and the primary sequence has been determined. The hormone amino acid sequence has been established: the A chain—GIVEQCCNRPCNIFDLQNYCN, and the B chain—AAGPQHLCGSHLVDALYLVCGDRGFFYNPK. The rockfish insulin, in comparison with the human one, has 14 amino acid substitutions; an additional alanine is present at the N-terminal of the B-chain, whereas the 30th amino acid at the C-terminal is absent. In in vitro experiment, the 50% inhibition of the pork 125I-insulin binding to the rat liver plasma membrane was 4 nM, i.e., 50% of the standard pork insulin affinity (2 nM) to the insulin receptors. The pork rockfish insulin biological activity as determined in the mouse convulsion test in vivo was 18 ± 2.2 ME/mg or 75% of the pork hormone activity. It is suggested that the relatively low rockfish insulin biological activity is due to the presence of A8 asparagine position in the hormone structure

ACTIVITY PATTERNS OF LYMNAEA STAGNALIS

Publisher Summary This chapter presents a study on the activity patterns of Lymnaea stagnalis. In the study, on shells of Lymnaea stagnalis with shell heights of 25–30 mm 2 mm Φ capsules containing 137Cs were mounted. Three NaI-detectors each were positioned at 90° different angles from another at ≈ 10 cm from the animals to be observed. The distance between animals and the NaI-detectors was related to the inverse of the radiation intensity on the detectors. The radiation intensities on the three detectors were registered continuously for up to 4 weeks per animal. The study showed that resting was found most from ≈ 1 h after onset of the dark period. Regular movements were most frequent in periods of search for food. Grazing was most active around the onset of light and gradually diminished toward 12 h a.m. In the grazing and in the resting a 3–4 h, rhythm of intensity was observed. It was found that starvation influences the activity pattern, and hunger perception can occur in Lynmaea stagnalis. It was also observed that Lynmaea can decide to feed or not to feed and that food is taken in majority during the early part of the day.