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.

Towards understanding the role of insulin in the brain: lessons from insulin-related signaling systems in the invertebrate brain.

Insulin is a molecule that has played a key role in several of the most important landmarks in medical and biological research. It is one of the most extensively studied protein hormones, and its structure and function have been elucidated in many vertebrate species, ranging from man to hagfish and turkey. The structure, function as well as tissue of synthesis of vertebrate insulins are strictly conserved. The structural identification of insulin-related peptides from invertebrates has disrupted the picture of an evolutionary stable peptide hormone. Insulin-related peptides in molluscs and insects turned out to be a structurally diverse group encoded by large multi-gene families that are uniquely expressed in the brain and serve functions different from vertebrate insulin. In this review, we discuss invertebrate insulins in detail. We examine how these peptides relate to the model role that vertebrate insulin has played over the years; however, more importantly, we discuss several unique principles that can be learned from them. We show how diversity of these peptides is generated at the genetic level and how the structural diversity of the peptides is linked to the exclusive presence of a single type of neuronal insulin receptor-related receptor. We also discuss the fact that the invertebrate peptides, in addition to a hormonal role, may also act in a synaptic and/or nonsynaptic fashion as transmitters/neuromodulators on neurons in the brain. It can be expected that the use of well-defined neuronal preparations in invertebrates may lead to a further understanding of these novel functions and may act as guide preparations for a possible role of insulin and its relatives in the vertebrate brain.

Functional implications of neurotransmitter expression during axonal regeneration: serotonin, but not peptides, auto-regulate axon growth of an identified central neuron.

We studied the regenerative properties of one of two electrically coupled molluscan neurons, the serotonergic cerebral giant cells (CGCs) of Lymnaea stagnalis, after axotomy. The CGCs play a crucial role in feeding behavior, and when both cells are disconnected from their target neurons, animals no longer feed. When one CGC was permanently disconnected from its targets and the other was reversibly damaged by a nerve crush, the latter one regenerated over a period of 2 weeks to reform functional synapses with specific target neurons. At the same time, recovery of the feeding behavior was observed. After the crush, neuropeptide gene expression in the CGC was downregulated to approximately 50%. Serotonin synthesis, on the other hand, remained unaffected, suggesting that serotonin might have an active role in regeneration. In primary neuron culture, CGCs failed to extend neurites in the presence of serotonin; in cells that extended neurites in the absence of serotonin, focally applied serotonin, but not neuropeptides, induced growth cone collapse. Using serotonin-sensitive sniffer cells, we show that CGC neurites and growth cones release serotonin in culture. Finally, both the spontaneous and stimulation-induced release of serotonin from CGCs in culture resulted in growth cone collapse responses that could be blocked by the serotonin receptor antagonist methysergide. Our data suggest that auto-released serotonin is inhibitory to CGC neurite outgrowth in vitro. During regeneration in vivo, serotonin release might fine-tune axon guidance and branching by inducing local collapse responses in extending neurites.

Synapse formation between central neurons requires postsynaptic expression of the MEN1 tumor suppressor gene.

Synapse formation is a crucial step in the development of neuronal circuits and requires precise coordination of presynaptic and postsynaptic activities. However, molecular mechanisms that control the formation of functionally mature synaptic contacts, in particular between central neurons, remain poorly understood. To identify genes that are involved in the formation of central synapses, we made use of molluscan neurons that in culture form synaptic contacts between their somata (soma-soma synapses) in the absence of neurite outgrowth. Using single-cell mRNA differential display, we have identified a molluscan homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene encoding the transcription factor menin as a gene that is upregulated during synapse formation. In vitro antisense knock-down of MEN1 mRNA blocks the formation of mature synapses between different types of identified central neurons. Moreover, immunocytochemistry and cell-specific knock-down of MEN1 mRNA show that postsynaptic but not presynaptic expression is required for synapses to form. Together, our data demonstrate that menin is a synaptogenic factor that is critically involved in a general postsynaptic mechanism of synapse formation between central neurons.

Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis.

The growth-controlling neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express a family of genes encoding structurally related, yet distinct, molluscan insulin-related peptides (MIPs). In the present study one of these peptides, MIP II, has been isolated and structurally identified. MIP II is a heterodimer of A and B chains connected by disulfide bonds. Both chains are N-terminally blocked with pyroglutamate. After cleaving of the A and B chains and deblocking with pyroglutamate amino-peptidase their sequences have been determined as: A chain: pQRTTNLVCECCFNYCTPDVVRKYCY and B chain: pQSSCSLSSRPHPRGICGSNLAGFRAFICSNQNSPS. In comparison with the MIP II sequence based on complementary DNA studies, it is clear that the two C-terminal amino acid residues of the B chain are posttranslationally removed. In addition, the glutamic acid residue in A chain was recovered in very low yields during Edman degradation, suggesting that the residue may be posttranslationally modified.

Processing and targeting of a molluscan egg-laying peptide prohormone as revealed by mass spectrometric peptide fingerprinting and peptide sequencing.

The neuroendocrine cerebral caudodorsal cells of Lymnaea stagnalis initiate and coordinate ovulation and egg mass production and associated behaviors through the release of a complex set of peptides that are derived from the caudodorsal cell hormone-I (CDCH-I) precursor. We have previously characterized the CDCH-I peptide. In the present study, we isolated and amino acid sequenced by conventional peptide chemistry five additional peptides, epsilon-peptide, calfluxin, alpha-caudodorsal cell peptide, delta-peptide, and carboxyl-terminally located peptide, from the cerebral commissure, the neurohemal area of the caudodorsal cells. Fingerprinting by matrix-assisted laser desorption mass spectrometry of peptides in the commissure demonstrated the presence of all sequenced peptides and, in addition, could identify two other peptides derived from pro-CDCH-1, the beta 1- and beta 3-peptides. These findings together with previous immunocytochemical studies enabled us to define cleavage sites and major processing events of pro-CDCH-1. Pro-CDCH-1 is initially cleaved in the Golgi apparatus into carboxyl- and amino-terminal parts, each of which is sorted into distinct vesicle classes that traffic to different intracellular sites. As a result, in the commissure, peptides derived from the carboxyl-terminal part, including CDCH-1, are present at a many-fold higher concentration than those derived from the amino-terminal part.

Characterization of a putative molluscan insulin-related peptide receptor.

In the pond snail Lymnaea stagnalis (Ls), growth and associated processes are likely to be controlled by a family of molluscan insulin-related peptides (MIP). Here we report on the cloning of a cDNA encoding a putative receptor for these MIP. This cDNA was isolated from Ls via PCR with degenerate oligodeoxynucleotides corresponding to conserved parts of the tyrosine kinase domain of the human insulin receptor and its Drosophila homologue. Many of the typical insulin-receptor features, including a cysteine-rich domain, a single transmembrane domain and a tyrosine-kinase domain are conserved in the predicted, 1607-amino acid (aa) protein. Comparison of the aa sequence of the molluscan receptor to other insulin-receptor sequences revealed strong variations in the percentage of sequence identity for the different domains, ranging from 70% sequence identity in the tyrosine-kinase domain to virtually no sequence identity in the C-terminal sequence. Striking differences are the absence of a clear tetrabasic cleavage site, and the extremely long C-terminus of 308 aa that contains seven Tyr residues. Southern blot analyses at varying stringencies, extensive screening of cDNA- and genomic libraries, and PCR experiments indicate the presence of a single putative MIP receptor. This suggests that the four different MIP may exert their functional role in Ls by binding to the same receptor.

Molluscan putative prohormone convertases: structural diversity in the central nervous system of Lymnaea stagnalis.

In the cerebral ganglia of the central nervous system of the freshwater snail Lymnaea stagnalis many neuropeptides are proteolytically processed from larger prohormones at sites marked by both single and multiple basic amino acids. In the present study we identified cloned cDNA and PCR products corresponding to three putative endoproteases that may be involved in prohormone processing. The cDNA encodes a protein of 653 residues with an overall sequence identity of 60%, 41%, 35%, 40%, and 27% with the recently characterized endoproteases PC2, PC1/3, PC4, furin and Kex2, respectively. The Lymnaea preproconvertase has approximately 80% homology with the catalytic domain, and approximately 40% and approximately 50% with the N- and C-terminal part, respectively, of the vertebrate PC2. Two cloned PCR products, Lfur 1 and Lfur 2, show highest sequence identity to furin. Expression of the LPC2 gene is exclusively in the central nervous system, where two LPC2 transcripts of 3.0 and 4.8 kb were detected.

Structural characterization of a Lymnaea putative endoprotease related to human furin.

A number of peptides have been identified in the central nervous system of the freshwater snail, Lymnaea stagnalis, that function as hormones and neurotransmitters/neuromodulators. These peptides are typically proteolytically processed from larger prohormones mostly at sites composed of single or multiple basic amino acid residues. Previously we demonstrated a diversity of putative prohormone convertases that may be involved in prohormone processing in the Lymnaea brain. In the present report, we have characterized a cDNA clone encoding a putative endoprotease of 837 amino acids. The primary structure of endoprotease (Lfur2) was comparable to that of human furin and contained a putative catalytic domain, a Cys-rich domain, and a transmembrane region. The catalytic domain of Lfur2 demonstrated about 70% residue identity when compared with human furin, PACE4 and Drosophila Dfur1 and dKLIP-1. The Lfur2 gene was expressed in the central nervous system as well as various peripheral tissues of Lymnaea.

Evolutionary conservation of the insulin gene structure in invertebrates: cloning of the gene encoding molluscan insulin-related peptide III from Lymnaea stagnalis.

Although insulins and structurally related peptides are found in vertebrates as well as in invertebrates, it is not clear whether the genes encoding these hormones have emerged from a single ancestral (insulin)-type of gene or, alternatively, have arisen independently through convergent evolution from different types of gene. To investigate this issue, we cloned the gene encoding the molluscan insulin-related peptide III (MIP III) from the freshwater snail, Lymnaea stagnalis. The predicted MIP III preprohormone had the overall organization of preproinsulin, with a signal peptide and A and B chains, connected by two putative C peptides. Although MIP III was found to share key features with vertebrate insulins, it also had unique structural characteristics in common with the previously identified MIPs I and II, thus forming a distinct class of MIP peptides within the insulin superfamily. MIP III is synthesized in neurones in the brain. It is encoded by a gene with the overall organization of the vertebrate insulin genes, with three exons and two introns, of which the second intron interrupts the coding region of the C peptides. Our data therefore demonstrate that in the Archaemetazoa, the common ancestor of the vertebrates and invertebrates, a primordial peptide with a two-chain insulin configuration encoded by a primordial insulin-type gene must have been present.

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

De novo protein synthesis in isolated axons of identified neurons.

Neurons are highly polarized cells that contain a wealth of cytoplasmic and membrane proteins required for neurotransmission, synapse formation and various forms of neuronal plasticity. Typically, these proteins are differentially distributed over somatic, dendritic and axonal compartments. Until recently, it was believed that all proteins destined for various neuronal sites were synthesized exclusively in the somata and were subsequently targeted to appropriate extrasomal compartments. The discovery of various messenger RNA molecules in both dendrites and axons is suggestive of de novo protein synthesis in extrasomatic regions. The latter process has been demonstrated in few neuronal svrstems, but direct proof for the axonal transcription of a specific protein from a given messenger RNA is still lacking. This lack of fundamental knowledge in the field of cellular and molecular neurobiology is due primarily to both anatomical and experimental difficulties encountered in most animal preparations studied thus far. In this study we developed a neuronal experimental system comprising of individually identified neurons and their isolated axons from the mollusc Lymnaea stagnalis. We injected a foreign messenger RNA encoding a peptide precursor into the isolated axons of cultured neurons; and utilizing cellular, molecular and immunocytochemical techniques, we provide direct evidence for specific protein synthesis in isolated axons. The Lymnaea model provides us with an opportunity to examine the role and specificity of de novo protein synthesis in the extrasomal regions.

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.

Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis.

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C alpha peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNIVCECCMKPCTLSELRQYCP; B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C alpha peptide has also been isolated and its sequence was determined as NAETDLDDPLRNIKLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.

Characterization and evolutionary aspects of a transcript encoding a neuropeptide precursor of Lymnaea neurons, VD1 and RPD2.

We isolated and characterized a cDNA clone encoding the major prohormone of VD1 and RPD2, two electrotonically coupled identified neurons in the central nervous system of the freshwater snail, Lymnaea stagnalis. The VD1/RPD2 prohormone may be cleaved to generate a set of 4 different neuropeptides, called epsilon, delta, alpha 1 and beta peptides, as well as a single aspartate. Since VD1 and RPD2 probably are involved in O2 perception and modulation of cardio-respiratory functions, it is thought that the neuropeptides synthesized and released by these neurons coordinate the adaptive physiological and behavioural processes that occur in response to changes in O2 availability. Comparison of the Lymnaea VD1/RPD2 precursor with two related precursors, prohormones R15-1 and R15-2, identified from neuron R15 in the marine mollusc Aplysia californica revealed a similar pattern of organization of the preprohormones. The overall homology is rather low, however, detailed comparisons show a highly differential pattern of conservation of peptide regions on the precursors.

Chemical characterization of a novel peptide from the neuroendocrine light yellow cells of Lymnaea stagnalis.

The neuroendocrine light yellow cells of Lymnaea stagnalis form two clusters of cells in the visceral and right parietal ganglion, respectively. These cells are endogenously bursting neurons whose activities are modified during egg-laying and feeding. Using gel permeation chromatography and reverse phase HPLC we have purified two highly related novel peptides from the light yellow cells. These peptides differ only in length, due to truncation of the amino-terminal aspartic acid residue, which causes a major shift in the charge of the molecule. We conclude that the longer peptide is the immediate precursor of the shorter form. Using whole mount immunocytochemistry, it was confirmed that the light yellow cells produce these peptides.

Characterization of a cDNA clone encoding molluscan insulin-related peptide V of Lymnaea stagnalis.

A cDNA clone encoding molluscan insulin-related peptide V (MIP V) was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP II cDNA. The MIP V cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, however, in this case connected by two distinct C peptide, C alpha and C beta, instead of one single C peptide. This phenomenon, which is shared by the MIP II precursor, represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIPs V, I and II, differ remarkably in primary structure; in contrast, the C alpha peptide domains are almost identical. MIP V has only limited sequence similarity with insulins and related peptides. Both MIP V and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I, II and V genes are expressed in a single type of neuron: the growth controlling neuroendocrine light green cells of the Lymnaea CNS.

Structural identification, neuronal synthesis, and role in male copulation of myomodulin-A of Lymnaea: a study involving direct peptide profiling of nervous tissue by mass spectrometry.

We used a strategy combining immunodetection, peptide chemistry, and a novel method, direct peptide fingerprinting of neurons and small pieces of nerve by using matrix-assisted laser desorption ionization mass spectrometry, to structurally identify and localize the neuropeptide myomodulin-A in the mollusc, Lymnaea stagnalis. Lymnaea myomodulin appeared to be identical to Aplysia myomodulin-A and is produced by many central neurons, including neurons located in the ventral lobe of the right cerebral ganglion that innervate the penis complex via the penis nerve. Myomodulin-A could also be characterized from the penis complex, and physiological concentrations of the peptide enhanced the relaxation rate of electrically induced contractions of the penis retractor muscle in vitro in a dose-dependent fashion.

Structure, localization and action of a novel inhibitory neuropeptide involved in the feeding of Lymnaea.

A neuropeptide that strongly inhibits the spontaneous contractions of the oesophagus in Lymnaea has been characterized as GAPRFVamide. Direct mass spectrometry of nervous tissues and immunocytochemical studies show that the peptide is synthesized by neurones in the buccal ganglia and transported to the oesophagus via the dorso-buccal nerve. In accordance with the function of the peptide, immunoreactive fibres are detected within the muscle layer of the oesophagus. Finally, mass spectrometry reveals the presence of a number of unidentified peptides in the nerves that innervate the oesophagus, which suggests that oesophageal activities may be modified by multiple peptides.

Processing, axonal transport and cardioregulatory functions of peptides derived from two related prohormones generated by alternative splicing of a single gene in identified neurons VD1 and RPD2 of Lymnaea.

The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea stagnalis is alternatively spliced to yield two related variants encoding two distinct yet related preprohormones, named the VD1/RPD2-A and -B preprohormones. Here, we report the isolation and structural characterization of alpha 1, alpha 2 and beta peptides from dissected neurons VD1 and RPD2. The alpha 1 and alpha 2 peptides are derived from VD1/RPD2-A and B prohormones, respectively, whereas beta peptide is identical for both prohormones. In addition, we report the isolation and structural characterization of the alpha 2 peptide from the heart, demonstrating that the mature peptides are transported and released in the heart. The pharmacological actions of synthetic alpha 1 and alpha 2 peptides on isolated auricle preparations of the Lymnaea heart were examined. The two alpha peptides have similar excitatory effects on beat rate and beat amplitude, while their potencies differed considerably, indicating that alternative splicing results in structurally and functionally overlapping, through non-identical, sets of peptides.