Amyloidogenesis and Responses to Stress

Amyloidogenesis is a primitive, physiological response that seems to be an ancient process widely distributed in different cell types of evolutionary distant organisms. The amyloid fibril synthesis is part of a more general inflammatory response to stressful conditions all entailing overproduction of reactive oxygen species (ROS). Interestingly, this event has been integrated into additional physiological functions: (i) the formation of a scaffold promoting the activation and packaging of melanin; (ii) the formation of a scaffold to compartmentalize hormones in the cytoplasm; (iii) the ability to reversibly link different types of molecules to drive close to the nonself; (iv) the construction of a framework to close body lesions. Amyloid fibril formation is a cellular response harmonically integrated with the stress response but for a deregulation in assembling/dismantling, dangerous depots, as in a lot of pathologies, can occur

Dopaminergic inhibition of human neutrophils is exerted through D1-like receptors and affected by bacterial infection

Dopamine (DA) affects immune functions in healthy subjects (HS) and during disease by acting on D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopaminergic receptors (DR); however, its effects on human polymorphonuclear leukocytes (PMN) are still poorly defined. We investigated DR expression in human PMN and the ability of DA to affect cell migration and reactive oxygen species (ROS) production. Experiments were performed on cells from HS and from patients (Pts) with bacterial infections as well, during the acute phase and after recovery. Some experiments were also performed in mice knockout (KO) for the DRD5 gene. PMN from HS express both D1-like and D2-like DR, and exposure to DA results in inhibition of activation-induced morphological changes, migration and ROS production which depend on the activation of D1-like DR. In agreement with these findings, DA inhibited migration of PMN obtained from wild-type mice, but not from DRD5KO mice. In Pts with bacterial infections, during the febrile phase D1-like DRD5 on PMN were downregulated and DA failed to affect PMN migration. Both D1-like DRD5 expression and DA-induced inhibition of PMN migration were however restored after recovery. Dopaminergic inhibition of human PMN is a novel mechanism which is likely to play a key role in the regulation of innate immunity. Evidence obtained in Pts with bacterial infections provides novel clues for the therapeutic modulation of PMN during infectious disease

Spatio-Temporal Changes of Extracellular Matrix (ECM) Stiffness in the Development of the Leech <i>Hirudo verbana</i>

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation

USE OF THE ANIMAL MODEL HIRUDO VERBANA (LEECH) TO EVALUATE THE POSSIBLE CORRELATION BETWEEN IMMUNE RESPONSE, AMYOLOIDOGENESIS AND CHRONIC INFLAMMATION

Toll-like receptors, essential pattern-recognition receptors (PRRs) of the innate immune system, recognize a range of conserved molecules of invading pathogens. Among them, TLR4 is expressed on the cell surface on both hematopoietic and non-hematopoietic cells, including cells of the central nervous system, playing a crucial role in both innate and neuroimmune responses. It is the receptor of LPS (Lipopolysaccharide) endotoxins, the major outer membrane components of Gram- bacteria and a potent activator of innate immunity and inflammatory response, inducing the expression of the proinflammatory molecules IL-18 and TNF-\u3b1. Recently, amyloidogenesis has been identified as new player in innate immune responses and has been proposed as a detoxifying event to fight ROS (reactive oxygen species) increase, given that an excessive oxidative stress becomes harmful to cells when their antioxidant capacities result insufficient to retain the appropriate redox state. Thus, an uncontrolled activation of the innate immune system can lead to amyloid fibrils accumulation and chronic inflammation. In addition, it has been shown that LPS and TLR4 are associated with Alzheimer disease (AD), characterized by the accumulation of amyloid fibrils and neuroinflammation. Within this context, microglia in the brain and monocytes/neutrophils in the periphery have a prominent role in initiating and regulating inflammation processes. Here we propose the use of the medicinal leech, Hirudo verbana, as a powerful model system to understand the role of TLR4 in innate immune response and neuroimmune activation. The advantages of using this model is found in its innate immune system that is very similar to Vertebrate\u2019s one, but lacking the complex cross-talk typical of adaptive immunity. Our in vivo and in vitro approaches, by means of histological, ultrastructural, immunohistochemical and Western Blot techniques aim to correlate amyloid fibrils and ROS production with LPS treatment, clarifying the relationship between peripheral and central nervous system immune responses. Furthermore, by blocking TLR4 intracellular cascade we demonstrate that both macrophages and microglia cells undergo to a rescue process that implicate amyloid fibrils degradation and restoration of physiological conditions. In conclusion, our study is promising to gain novel insight about the correlation between peripheral/neuro inflammation and amyloid accumulation

Amyloid fil rouge from invertebrate up to human ageing: A focus on Alzheimer&nbsp;disease

Amyloid fibrils and fibril-like structures are currently estimated to represent many different products of several genes in humans and play a key role in many types of proteinopathies, commonly associated with ageing process. They share the mutual feature of aggregation-prone proteins and the building up of molecular-supramolecular structure, such as inter-neuronal plaques in the brain of Alzheimer's Disease (AD) patients, characterized by an extraordinary strength. Noteworthy, this type of structure has been reported in different organisms, in particular in invertebrates. The aim of the current review is to focus on alpha and beta amyloids i.e., SAAs, SAP and APP, elucidating the structure and function of amyloid proteins in invertebrates (such as nematods, annelids, molluscs, insects, ascidians) and highlighting their striking pattern of functional conservation when compared to human amyloid-like fibrils, thus focusing on possible new studies and applications for innovative therapies, particularly for AD, the most common and worldwide type of dementia

Hirudo verbana as a freshwater invertebrate model to assess the effects of polypropylene micro and nanoplastics dispersion in freshwater

Plastics are a heterogeneous class of synthetic compounds that, due to their unique characteristics find numerous applications both in industrial and civil fields. However, despite the great advantages that these materials brought in everyday life, the plastic wastes resulting from their massive use represent one of the main envi-ronmental problems at the global level. Once released, plastics persist for a long time and are subjected both to biotic and abiotic processes leading to the formation of small particles, known as micro and to nanoplastics, that interact with organisms, accumulating inside tissues and risking to enter in the trophic chain. Among the different types of plastic, polypropylene (PP) is one of the diffused, widely exploited in food and textile industries for disposable packaging and to produce surgical masks. Owing to the huge distribution and the resultant abundant presence of PP waste products, it results necessary investigate the possible toxicity on living organisms. For these reasons, here we analyzed the effects of PP micro and nanoplastics dispersed in freshwater, using the medicinal leech Hirudo verbana as invertebrate model. To better follow the plastics fate, fluorescent particles, labeled with a fluorophore, have been used. Animals were examined at various timings after plastics exposure and results were analyzed by means of microscopy, immunofluorescent and molecular biology analyses. After assessing the entrance of PP fragments into leech tissues, the activation of the innate immune response was evaluated. The results show that the presence of micro and nanoplastics induces an initial physical protection that consists in the secretion of mucus, followed by an increase of blood vessels and the recruitment of immune cells, in particular macrophages. Moreover, macrophages were directly involved in both phagocytic and encapsulation processes, as demonstrated by acid phosphatase (ACP) histoenzymatic and Thioflavin-T assays, expressing specific pro-inflammatory factors, such as HvRNASET2 and HmAIF-1, as demonstrated by immuno-localization and qPCR experiments. Finally, the expression levels of genes related to oxidative stress-induced enzymes have been investigated, in order to evaluate the possible increase in reactive oxygen species (ROS), due to the entry into the leech tissues of PP micro and nanoplastics. This work allows deepening the current knowledge of the possible harmful effects on human health deriving from micro and nanoplastics dispersion, leading new insight about freshwater ecosystems that often represent the first environments interested in plastic pollution

3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech&rsquo;s innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health

A new cellular type in invertebrates: first evidence of telocytes in leech Hirudo medicinalis

Abstract Telocytes, a peculiar cell type, were recently found in vertebrates. Hence this cell system has been reported as ubiquitous in the bodies of mammals and interpreted as an important player in innate immunity and tissue regeneration, it is reasonable to look for it also in invertebrates, that rely their integrity solely by innate immunity. Here we describe, at morphological and functional level, invertebrate telocytes from the body of leech Hirudo medicinalis (Annelida), suggesting how these cells, forming a resident stromal 3D network, can influence or participate in different events. These findings support the concepts that leech telocytes: i) are organized in a cellular dynamic and versatile 3D network likewise the vertebrate counterpart; ii) are an evolutionarily conserved immune-neuroendocrine system; iii) form an immuno-surveillance system of resident cells responding faster than migrating immunocytes recruited in stimulated area; iv) communicate with neighbouring cells directly and indirectly, via cell-cell contacts and soluble molecules secreted by multivesicular bodies; v) present within neo-vessels, share with immunocytes the mesodermal lineage; vi) are involved in regenerative processes. In conclusion, we propose that HmTCs, integrating so different functions, might explain the innate immune memory and can be associated with several aged related diseases

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme