Edit Pollák

Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.

Identical cellular distribution of all abundant neuropeptides in the major abdominal neurohemal system of an insect (Periplaneta americana)

The median neurosecretory cells in abdominal ganglia of insects synthesize a number of putative hormones, which are abundant in the abdominal perisympathetic organs (PSOs). The peptide inventory of these prominent neurohemal release sites is best investigated in the American cockroach and strongly differs from that of head/thoracic neurohemal organs. In this study, we found a complete colocalization of all abundant neuropeptides in this hormonal system, including periviscerokinin‐1 and ‐2, pyrokinin‐5, YLSamide, VEAacid, and SKNacid. The first immunoreactive cells were detected on day 18 of embryonic development and already contained the complete set of peptides. By using antisera against the above‐mentioned peptides, the development of this neurohormonal system could be studied and is described in detail. Subsequent electron microscopic immunogold stainings in PSO preparations revealed the costorage of PSO peptides in a single vesicle species. Surprisingly, all these peptides were found in axons containing clear vesicles, whereas all axons with dense core vesicles were totally devoid of immunoreactivity. Unlike the axons with dense core vesicles, immunostained axons ramify in the center of the PSO but exhibit only rare morphological signs of exocytosis. Instead, putative release sites of the clear vesicle‐containing axons were detected peripherally to the PSOs, namely, on the hyperneural muscle. J. Comp. Neurol. 452:264–275, 2002.

Morphology and metamorphosis of the peptidergic Va neurons and the median nerve system of the fruit fly, Drosophila melanogaster

Metamorphosis is a fundamental developmental process and has been intensively studied for various neuron types of Drosophila melanogaster. However, detailed accounts of the fate of identified peptidergic neurons are rare. We have performed a detailed study of the larval morphology and pupal remodelling of identified peptidergic neurons, the CAPA-expressing Va neurons of D. melanogaster. In the larva, Va neurons innervate abdominal median and transverse nerves that are typically associated with perisympathetic organs (PSOs), major neurohaemal release sites in insects. Since median and transverse nerves are lacking in the adult, Va neurites have to undergo substantial remodelling during metamorphosis. We have examined the hitherto uncharacterised gross morphology of the thoracic PSOs and the abdominal median and transverse nerves by scanning electron microscopy and found that the complete reduction of these structures during metamorphosis starts around pupal stage P7 and is completed at P9. Concomitantly, neurite pruning of the Va neurons begins at P6 and is preceded by the high expression of the ecdysone receptor (EcR) subtype B1 in late L3 larvae and the first pupal stages. New neuritic outgrowth mainly occurs from P7-P9 and coincides with the expression of EcR-A, indicating that the remodelling of the Va neurons is under ecdysteroid control. Immunogold-labelling has located the CAPA peptides to large translucent vesicles, which are released from the transverse nerves, as suggested by fusion profiles. Hence, the transverse nerves may serve a neurohaemal function in D. melanogaster.

Peptidomics of identified neurons demonstrates a highly differentiated expression pattern of FXPRLamides in the neuroendocrine system of an insect

FXPRLamides are insect neuropeptides that mediate such diverse functions as pheromone biosynthesis, visceral muscle contraction, and induction of diapause. Although multiple forms occur in every insect studied so far, little is known about a possible functional differentiation and/or differences in the cellular expression pattern of these messenger molecules. In this study, we performed a mass spectrometric survey of all FXPRLamide‐expressing neurosecretory neurons in the CNS of Periplaneta americana. That species combines a very well characterized peptidergic system with relatively easy accessible neurosecretory cells suitable for dissection. In addition to the extensive mass spectrometric analyses of single cells, the projection of the FXPRLamide‐expressing neurons was studied with three antisera specifically recognizing different FXPRLamides. The following conclusions can be drawn from this first comprehensive peptidomic approach on insect neurons. 1) A high degree of differentiation in the expression of FXPRLamides exists; not fewer then four cell types containing different sets of FXPRLamides were observed. 2) A low level of colocalization with other neuropeptides was found in these neurons. 3) A comparison with FXPRLamide‐expressing neurons of other insects shows a high degree of conservation in the localization and projection of these neurons, which is not corroborated by a similar conservation of the corresponding peptide sequences. 4) Although the methods for cell identification, dissection, and sample preparation for mass spectrometry were kept as simple as possible, it was unambiguously shown that this approach is generally suitable for routine analysis of single identified neurons of insects. J. Comp. Neurol. 500:498–512, 2007.

Expression of PACAP-Like Compounds During the Caudal Regeneration of the Earthworm Eisenia fetida

The regeneration of the ventral nerve cord ganglion and peripheral tissues was investigated by radioimmunoassay and immunohistochemistry in the model animal, Eisenia fetida (Annelida, Oligochaeta). It is now well-established that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic factor, playing important roles in the development of the nervous system in vertebrate animals. Based on the apparent evolutionary conservation of PACAP and on the several common mechanisms of vertebrate and invertebrate nervous regeneration, the question was raised whether PACAP has any role in the regeneration of the earthworm nervous system. As a first step, we studied the distribution, concentration, and time-course of PACAP-like immunoreactivity during caudal regeneration of both lost segments and the ventral nerve cord ganglia in E. fetida. A strong upregulation of PACAP-like immunoreactivity was observed in most tissues following injury as determined by radioimmunoassay and immunohistochemistry. Significant increases in the concentration of PACAP-like compounds were found in the body wall, alimentary canal, and in coelomocytes. The most characteristic morphological feature was the accumulation of immunolabeled neoblasts in the injured tissues, especially in the ventral nerve cord ganglion that initiates and mediates regeneration processes. Our present results show that PACAP/PACAP-like peptides accumulate in the regenerating tissues of the earthworm, suggesting trophic functions of these compounds in earthworm tissues similarly to vertebrate species.

Identification of Novel Neuropeptides in the Ventral Nerve Cord Ganglia and Their Targets in an Annelid Worm, Eisenia fetida

Periviscerokinins (PVKs) and pyrokinins (PKs) are neuropeptides known in several arthropod species. Sequence homology of these peptides with the molluscan small cardioactive peptides reveals that the occurrence of PVKs and PKs is not restricted to arthropods. Our study focuses on the biochemical and immunocytochemical identification of neuropeptides with sequence homology to PVKs and PKs in the central and peripheral nervous system of the earthworm Eisenia fetida. By means of affinity chromatography, nanoflow liquid chromatography, and high accuracy mass spectrometry, six peptides, SPFPR(L/I)amide, APFPR(L/I)amide, SPLPR(L/I)amide, SFVR(L/I)amide, AFVR(L/I)amide, and SPAFVR(L/I)amide, were identified in the central nervous system with the common −XR(L/I)amide C‐terminal sequence. The exact anatomical position of 13 labeled XR(I/L)amide expressing neuron groups and numerous peptide‐containing fibers were determined by means of immunocytochemistry and confocal laser scanning microscopy in whole‐mount preparations of ventral nerve cord ganglia. The majority of the stained neurons were interneurons with processes joining the distinct fine‐fibered polysegmental tracts in the central neuropil. Some stained fibers were seen running in each segmental nerve that innervated metanephridia and body wall. Distinct groups of neurosecretory cells characterized by small round soma and short processes were also identified. Based on immunoelectron microscopy six different types of labeled cells were described showing morphological heterogeneity of earthworm peptides containing elements. Our findings confirm that the sequence of the identified earthworm neuropeptides homologous to the insect PVKs and PKs suggesting that these peptides are phylogenetically conservative molecules and are expressed in sister‐groups of animals such as annelids, mollusks, and insects. J. Comp. Neurol. 514:415–432, 2009.

Differential sorting and packaging of capa-gene related products in an insect.

A unique costorage of neuropeptides was recently found in the abdominal perisympathetic organs (PSOs) of the American cockroach, Periplaneta americana. Having specific antisera directed against all peptides belonging to this neurosecretory system, we examined the sorting of PSO‐peptides in the soma of the median neurosecretory cells of abdominal ganglia by using immunoelectron microscopic double stainings. The data indicate that all six abundant neuropeptides of this neurohormonal system, which includes three capa‐gene related products, are primarily incorporated into separate vesicles. These vesicles fuse with each other in the cytoplasm and become translucent on their way to the axon hillock. By means of light microscopy and MALDI‐TOF mass spectrometry, an identical population of neuropeptides was found in interneurons of the brain. As revealed by subsequent immunoelectron microscopic analysis, the peptides of these cells are separately packed into dense core vesicles but do not fuse with each other. Thus, hitherto unknown cell‐type–specific sorting mechanisms occur in neurosecretory cells and interneurons, respectively. J. Comp. Neurol. 481:84–95, 2005.

PAC1 receptor localization in a model nervous system: Light and electron microscopic immunocytochemistry on the earthworm ventral nerve cord ganglia

The presence and pattern of pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptors were identified by means of pre- and post-embedding immunocytochemical methods in the ventral nerve cord ganglia (VNC) of the earthworm Eisenia fetida. Light and electron microscopic observations revealed the exact anatomical positions of labeled structures suggesting that PACAP mediates the activity of some interneurons, a few small motoneurons and certain sensory fibers that are located in ventrolateral, ventromedial and intermediomedial sensory longitudinal axon bundles of the VNC ganglia. No labeling was located on large interneuronal systems such as dorsal medial and lateral giant axon systems and ventral giant axons. At the ultrastructural level labeling was mainly restricted to endo- and plasma membranes showing characteristic unequal distribution in various neuron parts. An increasing abundance of PAC1 receptors located on both rough endoplasmic reticulum and plasma membranes was seen from perikarya to neural processes, indicating that intracellular membrane traffic might play a crucial role in the transportation of PAC1 receptors. High number of PAC1 receptors was found in both pre- and postsynaptic membranes in addition to extrasynaptic sites suggesting that PACAP acts as neurotransmitter and neuromodulator in the earthworm nervous system.

Comparative Anatomy of PACAP-Immunoreactive Structures in the Ventral Nerve Cord Ganglia of Lumbricid Oligochaetes

Abstract:  By means of a whole mount immunocytochemical approach, the distribution patterns of pituitary adenylate cyclase‐activating polypeptide (PACAP)‐27 and PACAP‐38 were identified in the ventral nerve cord (VNC) ganglia of the earthworms Eisenia fetida and Lumbricus terrestris. Each PACAP form appears to occur in a distinct neuron population. Positions of these populations, as well as numbers and sizes of the constituting neurons do not essentially differ between the two species. The data suggest that in Lumbricid Oligochaetes, PACAP‐27 and PACAP‐38 neuron populations may mediate distinct physiological processes.

Pituitary Adenylate Cyclase-activating Polypeptide-like Compounds Could Modulate the Activity of Coelomocytes in the Earthworm

By means of radioimmunoassay, we studied the concentration of pituitary adenylate cyclase‐activating polypeptide (PACAP)‐like proteins in intact and regenerating earthworms. Transection of animals increased the concentration of PACAP‐like compounds in coelomocytes, and a decreasing rostrocaudal gradient was detected in the regenerating animals. Western blot analysis revealed a range of PAC1‐receptor proteins with molecular weights from 40 to 80 kDa. Electron microscopic immunocytochemistry showed that PAC1 receptors were located on distinct sets of coelomocytes (mainly on amebocytes and on some granulocytes). Based on our results we hypothesize a link between PACAP and coelomocytes, suggesting that PACAP modulates the function of amebocytes and certain granulocytes that play a role in tissue remodeling of regenerating earthworms.