Alice Accorsi

Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challenge

In human and rodents, the transcriptional response of neurons to stress is related to epigenetic modifications of both DNA and histone proteins. To assess the suitability of simple invertebrate models in studying the basic mechanisms of stress-related epigenetic modifications, we analyzed epigenetic modifications in neurons of the freshwater snail Pomacea canaliculata after the injection of Escherichia coli-derived lipopolysaccharide (LPS). The phospho-acetylation of histone H3, together with the induction of stress-related factors, c-Fos and HSP70, were evaluated in large and small neurons of the pedal ganglia of sham- and LPS-injected snails. Immunocytochemical investigations showed that after LPS injection, the immunopositivity towards phospho (Ser10)-acetyl (Lys14)-histone H3 and c-Fos increases in the nuclei of small gangliar neurons. Western blot analysis confirmed a significant increase of phospho (Ser10)-acetyl (Lys14)-histone H3 in nuclear extracts from 2h LPS-injected animals. c-Fos protein levels were significantly augmented 6h after LPS injection. Immunocytochemistry and western blot indicated that no changes occurred in HSP70 distribution and protein levels. To our knowledge this is the first demonstration of epigenetic changes in molluscan neurons after an immune challenge and indicate the gastropod P. canaliculata as a suitable model for evolutionary and translational studies on stress-related epigenetic modifications.

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium-binding adapter molecule 1 (Iba1/alias aif-1).

The Ionized calcium‐Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF‐1), is a 17 kDa cytokine‐inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF‐1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events.

The evolution of pro-opiomelanocortin: Looking for the invertebrate fingerprints

The presence and role of the pro-opiomelanocortin (POMC) gene and encoded peptides in invertebrates are here summarized and discussed. Some of the POMC-derived peptides show a significant similarity regarding their functions, suggesting their appearance before the split of protostomian-deuterostomian lineages and their maintenance during evolution. The basic mechanisms that govern the exchange of information between cells are usually well conserved, and this could have also been for POMC-derived peptides, that are mainly involved in fundamental functions such as immune and neuroendocrine responses. However, the presence and functions that POMC-derived peptides exhibit in taxonomically distant models, are not always reflected by the expected gene homology, leaving the problem of POMC evolution in invertebrates in need of additional study.

Effects of repeated hemolymph withdrawals on the hemocyte populations and hematopoiesis in Pomacea canaliculata

Pomacea canaliculata is a freshwater gastropod considered an invasive pest by several European, North American and Asiatic countries. This snail presents a considerable resistance to pollutants and may successfully face stressful events. Thanks to the unusual possibility to perform several hemolymph collections without affecting its survival, P. canaliculata is a good model to study the hematopoietic process and the hemocyte turnover in molluscs. Here we have analyzed the effects of repeated hemolymph withdrawals on circulating hemocyte populations and pericardial organs, i.e., the heart, the main vessels entering and leaving the heart and the ampulla, of P. canaliculata. Our experiments revealed that the circulating hemocyte populations were maintained constant after 3 collections performed in 48 h. The tissue organization of the heart and the vessels remained unaltered, whereas the ampulla buffered the effects of hemolymph collections acting as hemocyte reservoir, and its original organization was progressively lost by the repeated hemolymph withdrawals. The hematopoietic tissue of P. canaliculata was evidenced here for the first time. It is positioned within the pericardial cavity, in correspondence of the principle veins. Mitoses within the hematopoietic tissue were not influenced by hemolymph collections, and circulating hemocytes never presented mitotic activity.

Molluscs as models for translational medicine.

This paper describes the advantages of adopting a molluscan model for studying the biological basis of some central nervous system pathologies affecting humans. In particular, we will focus on the freshwater snail Lymnaea stagnalis, which is already the subject of electrophysiological studies related to learning and memory, as well as ecotoxicological studies. The genome of L. stagnalis has been sequenced and annotated but the gene characterization has not yet been performed. We consider the characterization of the gene networks that play crucial roles in development and functioning of the central nervous system in L. stagnalis, an important scientific development that comparative biologists should pursue. This important effort would add a new experimental model to the limited number of invertebrates already used in studies of translational medicine, the discipline that seeks to improve human health by taking advantage of knowledge collected at the molecular and cellular levels in non-human organisms.

A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata.

Abstract The golden apple snail Pomacea canaliculata is an invasive pest originating from South America. It has already been found in Asia, the southern United States and more recently in the EU. Aiming to target the immune system of the snail as a way to control its spreading, we have developed organ‐specific transcriptomes and looked for molecules controlling replication and differentiation of snail hemocytes. The prokineticin domain‐containing protein Astakine 1 is the only cytokine known thus far capable of regulating invertebrate hematopoiesis, and we analyzed the transcriptomes looking for molecules containing a prokineticin domain. We have identified a prokineticin‐like protein (PlP), that we called Pc‐plp and we analyzed by real‐time PCR (qPCR) its expression. In control snails, highest levels of Pc‐plp were detected in the digestive gland, the ampulla (i.e., a hemocyte reservoir) and the pericardial fluid (i.e., the hematopoietic district). We tested Pc‐plp expression after triggering hematopoiesis via multiple hemolymph withdrawals, or during bacterial challenge through LPS injection. In both cases a reduction of Pc‐plp mRNA was observed. The multiple hemolymph withdrawals caused a significant decrease of Pc‐plp mRNA in pericardial fluid and circulating hemocytes, while the LPS injection promoted the Pc‐plp mRNA drop in anterior kidney, mantle and gills, organs that may act as immune barrier in molluscs. Our data indicate an important role for prokineticin domain‐containing proteins as immunomodulators also in gastropods and their dynamic expression may serve as a biosensor to gauge the effectiveness of immunological interventions aimed at curtailing the spreading of the gastropod pest P. canaliculata. HighlightsA prokineticin‐like protein (Pc‐PlP) is expressed in several organs of Pomacea canaliculata.Pc‐plp is maximally expressed in digestive gland, ampulla and hematopoietic tissue.Pc‐plp expression is specifically altered in different organs by diverse immune challenges.Pc‐plp mRNA appears to be post‐transcriptionally regulated as astakines in crustaceans.Pc‐plp may represent a biosensor to evaluate the immune system status after different challenges.

Drosophila Helical factor is an inducible protein acting as an immune-regulated cytokine in S2 cells

The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stimulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial challenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated factor, with functions comparable to those of secreted vertebrate cytokines.

Hands-On Classroom Activities for Exploring Regeneration and Stem Cell Biology with Planarians

Abstract Regeneration has long fascinated humanity, and its documentation has progressed from simple descriptive observations to the intense molecular and cellular investigations of today. The overarching goal of this work is to make the key methods and tools being used in modern regeneration and stem cell biology research accessible to docents and students in the classroom. We have designed a series of experimental activities with accompanying protocols using four inexpensive, commercially available planarian species indigenous to North America: Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracilis. These planarians are fast and robust regenerators, and can easily be maintained in the classroom. The activities presented here can be used to guide students through hypothesis-driven experiments, and range from simple manipulations aimed at high school students (e.g., planarian husbandry, feeding, and cutting experiments) to gene expression and protein function analyses suitable for college students. Regeneration time courses, the more complex whole worm in situ hybridizations, and RNA interference for gene knock-down experiments are described for each of the four species. Cumulatively, the suggested methods and experiments will facilitate the exploration of animal regeneration biology and promote curiosity-driven, hands-on application of the scientific method.

Hematopoiesis and Hemocytes in Pancrustacean and Molluscan Models

The hematopoietic districts and their organization are here described in detail for some of the principal pancrustacean and molluscan models. Pancrustaceans present an overall similar organization of hematopoietic organs, while among mollusks the hematopoietic organs and tissues are more diversified. The morphologies, functions, and main lineages of circulating hemocytes are recapitulated, and the common traits between different taxa are evidenced.

Thymic Maturation and Programmed Cell Death

The thymus plays a crucial role in the development and maintenance of the immune system, being the main site of T cell differentiation and maturation throughout life. Associated to dramatic structural changes, its function seems to markedly diminish with time, never the less, there are several data indicating that, despite organ atrophy, at least part of the thymus remains active throughout one’s lifetime. In the last decades, several studies, aiming to understand the significance of age-dependent changes in thymic structure and function, highlighted the concept that developmental and maturational stages strongly depend on the balanced and coordinated occurrence of life and death options. In particular, programmed cell death represents a fundamental requirement in order to assure a proper functionality of the immune response and to avoid the formation of uncontrolled and potentially self-damaging lymphocytic clones. By contrast, the time-dependent thymic atrophy is due to progressive replacing of lymphoid with adipose tissue. In the light of the increased knowledge on the factors/mechanisms controlling the process of adipogenesis, it could be suggested that fat accumulation in the thymic stroma might not be considered a passive, deleterious consequence of aging, but instead a potential source of molecules with various biological functions. Therefore, thymus represents a very interesting model in terms of energy expenditure and trade off, tissue homeostasis, immune defence and disease escape. The implications of changes in thymic structure, in the ratio of proliferation and programmed cell death as well as the occurrence of fat involution still represent an open question and will be discussed in the present chapter.