Medicinal Leech CNS as a Model for Exosome Studies in the Crosstalk between Microglia and Neurons.

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-ß) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

Calreticulin contributes to C1q-dependent recruitment of microglia in the leech Hirudo medicinalis following a CNS injury.

BACKGROUND: The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech's CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR). MATERIAL AND METHODS: Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS. RESULTS: A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process. CONCLUSIONS: These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.

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.

Proteome modifications of the medicinal leech nervous system under bacterial challenge.

Once considered as lacking intrinsic immune mechanisms, the CNS of vertebrates is now known to be capable of mounting its own innate immune response. Interestingly, while invertebrates have been very useful in the interpretation of general vertebrate innate immunity mechanisms, only scarce data are available on the immune response of nervous tissue within this group. This study provides new data on the innate immune response of medicinal leech Hirudo medicinalis CNS. We identified several spots in 2-D gels of leech CNS proteins that showed specific changes following bacterial challenge, thus demonstrating the ability of the leech nervous system to mount a response to an immune stress. Protein identifications were based on comparison of sequence data with publicly available databases and a recently established leech ESTs database. The broad nature of the identified proteins suggests a clear involvement of cytoskeletal rearrangements, endoplasmic reticulum stress, modulation of synaptic activity and calcium mobilization, all during the first 24 hours of this response. Moreover, several of these proteins are specifically expressed in glial cells, suggesting an important role for glial cells in the immune response of the leech nervous system, similar to what has been observed in vertebrates.

Up-regulation of neurohemerythrin expression in the central nervous system of the medicinal leech, Hirudo medicinalis, following septic injury.

We report here some results of a proteomic analysis of changes in protein expression in the leech Hirudo medicinalis in response to septic injury. Comparison of two-dimensional protein gels revealed several significant differences between normal and experimental tissues. One protein found to be up-regulated after septic shock was identified, through a combination of Edman degradation, mass spectrometry, and molecular cloning, as a novel member of the hemerythrin family, a group of non-heme-iron oxygen transport proteins found in four invertebrate phyla: sipunculids, priapulids, brachiopods, and annelids. We found by in situ hybridization and immunocytochemistry that the new leech protein, which we have called neurohemerythrin, is indeed expressed in the leech central nervous system. Both message and protein were detected in the pair of large glia within the ganglionic neuropile, in the six packet glia that surround neuronal somata in each central ganglion, and in the bilateral pair of glia that separate axonal fascicles in the interganglionic connective nerves. No expression was detected in central neurons or in central nervous system microglia. Expression was also observed in many other, non-neuronal tissues in the body wall. Real-time PCR experiments suggest that neurohemerythrin is up-regulated posttranscriptionaly. We consider potential roles of neurohemerythrin, associated with its ability to bind oxygen and iron, in the innate immune response of the leech nervous system to bacterial invasion.