Antiserum to an eye-specific protein identifies photoreceptor and circadian pacemaker neuron projections in Aplysia

The marine gastropod Aplysia has a circadian clock in each eye that generates a circadian rhythm of optic nerve activity. The axons of pacemaker neurons carry the rhythmic activity to the brain where it can be recorded from various ganglionic connectives as it is distributed throughout the CNS. We had previously identified an eye-specific 48-kD protein using an antiserum, anti-S, that recognizes the period gene product of Drosophila. We have now obtained two partial amino acid sequences of the 48-kD protein and raised a polyclonal antiserum using a synthetic peptide with the amino acid sequence of one of them. The antiserum recognizes a family of spots of M_r 47–48 kD and P_i 5.9–6.0 on 2D immunoblots of eye proteins. The immunoblot staining intensity does not exhibit a circadian rhythm. Used in immunocytochemistry, the antiserum recognizes fibers in the optic nerve and retinal neuropil, pacemaker neurons, certain photoreceptors, and the photoreceptor rhabdom layer. It stains the optic nerve fibers and optic fiber terminals in the cerebral optic ganglion and recognizes the cerebral optic tracts, putative synaptic exchange areas, and optic tract projections from the cerebral ganglion into various head nerves and interganglionic connectives. The function of the 48-kD protein is not known but it could be involved in the maintenance or regulation of the retinal afferent pathways, including the pacemaker neuron axons, known from previous axonal transport and electrical recording studies to be the circadian output pathway. © 1993 John Wiley & Sons, Inc.     

Identification of a 70 kD protein with sequence homology to squid neurofilament protein in glial cells of the leech CNS

A monoclonal antibody G39, generated against a protein extract of leech central nervous system, labels specific cell types in adult, embryonic, and regenerating preparations. The antibody stained glial cells, microglial cells, and connective tissue cells, but nor neurons or muscle on cryosections. The staining pattern resembled that of an intracellular network. Affinity purification of the antigen revealed a 70 kD protein. Peptide sequencing showed significant homology of a stretch of 15 amino acids to squid neural filament protein. The same mAb G39 delineated glial cells as they formed during development of the CNS and showed that the giant neuropil glial cells appear before those in the packets. The antigen recognized by mAb G39 represents a nonneuronal intermediate filament of the leech Hirudo medicinalis found in various cell-types such as glia, microglia, and some cells of the connective tissue. 1994 John Wiley & Sons, Inc.     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

Sensorin-A immunocytochemistry reveals putative mechanosensory neurons inLymnaea CNS

The pond snailLymnaea stagnalis is a useful model system for studying the neural basis of behaviour but the mechanosensory inputs that impact on behaviours such as respiration, locomotion, reproduction and feeding are not known. InAplysia, the peptide sensorin-A appears to be specific to a class of central mechanosensory neurons. We show that in theLymnaea central nervous system sensorin-A immunocytochemistry reveals a discrete pattern of staining involving well over 100 neurons. Identifiable sensorin positive clusters of neurons are located in the buccal and cerebral ganglia, and a single large neuron is immunopositive in each pedal ganglion. These putative mechanosensory neurons are not in the same locations as previously identified motoneurons, interneurons or neurosecretory cells. As would be expected for a mechanoafferent, sensorin positive fibres were found in nerve tracts innervating the body wall. This study lays the foundation for future electrophysiological and behavioural analysis of these putative mechanosensory neurons.     

Deer mice: "The Drosophila of North American mammalogy"

Summary: Oocyte‐somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte‐secreted proteins remain unknown. A novel cDNA encoding mouse oo cyte‐s ecreted p rotein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202‐amino acid protein that contains a 21‐amino acid signal peptide sequence, 5 putative N‐linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental‐specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12–11q13. The identification of OOSP1 as a new oocyte‐secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis. genesis 31:105–110, 2001. © 2001 Wiley‐Liss, Inc.     

Urotensin II in Invertebrates: From Structure to Function in Aplysia californica

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.     

Modulation of growth of Aplysia neurons by an endogenous lectin

We have purified and characterized a galactose-binding lectin from the gonads of the mollusk Aplysia californica that modulates neurite outgrowth from cultured Aplysia neurons. Agglutination of sheep red blood cells (RBC) by this lectin, termed Aplysia gonad lectin (AGL), is inhibited strongly by galactose and to a lesser extent by fucose. On SDS-PAGE, AGL appears as a single species with a molecular weight of 34 kD under reducing conditions, and 65 kD under nonreducing conditions. This suggests that AGL is a disulfide-linked dimer in its native state. Amino terminal sequence analysis of purified AGL indicates a similarity to another galactose-binding lectin, phytohemagglutinin-E (E-PHA), found in red kidney beans. By using polyclonal antibodies prepared against AGL, we have found that the lectin is present in the gonads and eggs but not in other tissues of adult Aplysia californica. We have examined biological actions of AGL on Aplysia neurons growing in primary cell culture. AGL affects several properties of these neurons. The addition of 100 nM AGL to cultured neurons enhances neurite outgrowth from the cell soma, resulting in a greater number of primary processes. In addition, AGL acts as a neurotrophic agent, increasing neurite viability in vitro. This trophic effect is not seen with concanavalin A (con A), another lectin known to affect several properties of cultured Aplysia neurons. The results are consistent with the suggestion that AGL may play a role in neuronal differentiation and/or maintenance of viability. © 1992 John Wiley & Sons, Inc.     

The CFTR-derived peptides as a model of sequence-specific protein aggregation

Protein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.     

Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis.

Summary The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic -galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.     

Steroid induction of nerve growth factor synthesis in cell culture

Nerve growth factor (NGF) is a peptide hormone which is necessary for the development of sympathetic neurons. Exposing a rat central nervous system glioma cell line (C-6) to the steroid hormone 17β-estradiol increases the amount of NGF secreted by these cells into the surrounding medium. This induction is highly specific to 17β-estradiol in that similar steroids do not increase NGF levels. Both NGF activity and protein levels increase upon estradiol stimulation and there is a parallel increase in NGF $\text{de}$$\text{novo}$ synthesis. The estradiol effect can be blocked with actinomycin D but not with puromycin or cycloheximide. This is the first report demonstrating regulation of NGF synthesis by a steroid hormone in a clonal cell line of glial origin. We propose this system as a model system for the study of the regulation of NGF synthesis and the isolation and analysis of putative precursors to the NGF molecule.     

Branch architecture of the fly larval abdominal serotonergic neurons

In the metazoan central nervous system (CNS), serotonergic neurons send projections throughout the synaptic neuropil. Little is known about the rules that govern these widespread neuromodulatory branching patterns. In this study, we utilize the Drosophila as a model to examine serotonergic branching. Using single cell GFP labeling we show that within each segment of the Drosophila ventral nerve cord (VNC), each of two serotonergic neurons tiles distinct innervation patterns in the contralateral neuropil. In addition, branches extend only a short distance from the target segment. Through ablation-mediated isolation of serotonergic cells, we demonstrate that the distinct areas of innervation are not maintained through competition between neighboring like-serotonergic neurites. Furthermore, the basic branching pattern of serotonergic neurons within the neuropil remains unchanged despite alterations of initial axonal trajectories.     

Rhipicephalus appendiculatus: characterization of a testis-associated protein

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.     

A CUB‐serine protease in the olfactory organ of the spiny lobster Panulirus argus

csp, a gene encoding a protein with high sequence identity to trypsinlike serine protease and CUB domains, was identified from a cDNA library from the olfactory organ (antennular lateral flagellum) of the spiny lobster Panulirus argus. The full‐length cDNA sequence of csp is 1801 bp, encoding a protein of 50.25 kD, with three domains: signal peptide, trypsinlike serine protease, and CUB (named for a class of compounds including C omplement subcomponents Clr/Cls, U egf, and B one morphogenic protein‐1). RT‐PCR, Northern blots, and immunoblots showed that csp is predominantly expressed in the lateral flagellum and eyestalk. Immunocytochemistry showed that Csp is present in olfactory (aesthetasc) sensilla around auxiliary cells (glia that surround the inner dendrites of olfactory receptor neurons, ORNs) and ORN outer dendrites. We propose that Csp is expressed and secreted by auxiliary cells, associates with ORN cell membranes or extracellular matrix via the CUB domain, and has trypsinlike activity. In the eyestalk, Csp is associated with cells surrounding axons between neuropils of the eyestalk ganglia. Possible functions in the olfactory organ and eyestalk are discussed. To our knowledge, this is the first report from any olfactory system of a gene encoding a protein with serine protease and CUB domains. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 277–302, 2001     

Autoradiographic localization of 3H-histamine accumulation by the visual system of the locust

Summary The uptake of [³H]-histamine into the retina and optic lobe of the locust, Schistocerca americana gregaria was studied by means of autoradiography at the light- and electron-microscopic levels. Light-microscopic autoradiography showed a significant accumulation of [³H]-histamine in several regions of the optic lobe. Dense accumulations of silver grains were concentrated along the medial border of the medullary neuropil and around the entire periphery of the lobula. No significant accumulations of grains were present within the retina or the neuropil zones of the lamina, medulla or lobula.Electron-microscopic autoradiography showed histamine-accumulating cells along the border of the medulla to exhibit electron density and morphology typical of glial cells. Labelled histamine was present within both glial cell bodies and their processes. In the region surrounding the neuropil of the lobula, [³H]-histamine was concentrated within fine glial processes wrapped around neuronal cell bodies and their axons. No neuronal cell bodies or axons showed accumulation of silver grains above background.These results are consistent with previous studies showing the glial uptake of amino acid and biogenic amine putative neurotransmitters. However, the lack of a demonstration of a specific uptake of histamine in neuropil zones makes it difficult to assess the role of histamine uptake in the inactivation of neurally released histamine in the locust visual system.     

Aminergic neurons: State control and plasticity in three model systems

Aminergic neurons have particular functions in many systems, and in this review their role is discussed and compared in three systems: those parts of the central nervous system controlling sleep and waking in the cat; the superior cervical ganglion; and the isolated nervous system of Aplysia.In the cat the aminergic neurons are most important in a waking state during which time external information is received, processed, and can be retrieved, and during which time habituation and sensitization occur. Aminergic neurons appear to have similar roles in state control in plasticity in both the Aplysianervous system and the superior cervical ganglion. The striking similarities in the role of aminergic neurons in these three systems support the speculation that aminergic neurons have uniquely important roles in regulation of the plastic properties of neurons.     

Stress protein synthesis by crayfish CNS tissue in vitro

Some crustacean axons remain functional for months after injury. This unusual property may require stress proteins synthesized by those neurons or provided to them by glial cells. To begin to explore this hypothesis, we examined the conditions that stimulated stress protein synthesis by crayfish CNS tissue in vitro. Incubation for 1–15 h with arsenite or at temperatures about 15°C higher than the acclimation temperature of 20°C induced transient expression of several stress proteins. The heat stress response was blocked by Actinomycin D, suggesting that synthesis of new mRNA was required. In addition, the major crayfish 66 kD stress protein and its mRNA had sequence identities with the 70 kD stress proteins of mammals. Since the crayfish stress response has much in common with that of other organisms, the unique advantages of the crayfish nervous system can be used to study the impact of stress proteins on glial and neuronal function.