ALK4/5-dependent TGF-ß signaling contributes to the crosstalk between neurons and microglia following axonal lesion.

Neuronal activity is closely influenced by glia, especially microglia which are the resident immune cells in the central nervous system (CNS). Microglia in medicinal leech are the only cells able to migrate to the injury site within the 24 hours post-lesion. The microglia-neuron interactions constitute an important mechanism as there is neither astrocyte nor oligodendrocyte in the leech CNS. Given that axonal sprouting is impaired when microglia recruitment is inhibited, the crosstalk between microglia and neurons plays a crucial role in neuroprotection. The present results show that neurons and microglia both use ALK4/5 (a type of TGF-ß receptor) signaling in order to maintain mutual exchanges in an adult brain following an axonal injury. Indeed, a TGF-ß family member (nGDF) is immediately released by injured axons contributing to the early recruitment of ALK4/5+ microglia to the lesion site. Surprisingly, within the following hours, nGDF from microglia activates ALK4/5+ neurons to maintain a later microglia accumulation in lesion. Taken together, the results demonstrate that ALK4/5 signaling is essential throughout the response to the lesion in the leech CNS and gives a new insight in the understanding of this pathway. This latter is an important signal contributing to a correct sequential mobilization over time of microglia recruitment leading to axon regeneration.

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.

Modulation properties of factors released by bone marrow stromal cells on activated microglia: an in vitro study.

In the present paper we develop a new non-cell based (cell-free) therapeutic approach applied to BV2 microglial cells and spinal cord derived primary microglia (PM) using conditioned media from rat bone marrow stromal cells (BMSCs-CM). First we collected conditioned media (CM) from either naive or injured rat spinal cord tissue (SCI-CM, inflammatory stimulation agent) and from rat bone marrow stromal cells (BMSCs-CM, therapeutic immunomodulation agent). They were both subsequently checked for the presence of chemokines and growth, neurotrophic and neural migration factors using proteomics analysis. The data clearly showed that rat BMSCs-CM contain in vitro growth factors, neural migration factors, osteogenic factors, differentiating factors and immunomodulators, whereas SCI-CM contain chemokines, chemoattractant factors and neurotrophic factors. Afterwards we determined whether the BMSCs-CM affect chemotactic activity, NO production, morphological and pro-apoptotic changes of either BV2 or PM cells once activated with SCI-CM. Our results confirm the anti-migratory and NO-inhibitory effects of BMSCs-CM on SCI-CM-activated microglia with higher impact on primary microglia. The cytotoxic effect of BMSCs-CM occurred only on SCI-CM-stimulated BV2 cells and PM, not on naive BV2 cells, nor on PM. Taken together, the molecular cocktail found in BMSCs-CM is favorable for immunomodulatory properties.

Alterations of protein composition along the rostro-caudal axis after spinal cord injury: proteomic, in vitro and in vivo analyses.

Based on proteomic analyses we investigated the differences of released molecules in the conditioned media (CM) from the spinal cord central lesion and adjacent rostral and caudal segments at 3, 7, and 10 days after spinal cord injury (SCI), in order to specify the molecular environment within greater extent of tissue damage. Proteins found in CM were analyzed by shot-gun MS using nanoLC coupled to an orbitrap. The results showed some specific proteins at each site of the lesion at 3days. Among the proteins from rostral and lesion segments, some are related to chemokines, cytokines or to neurogenesis factors. In contrast, proteins from caudal segments are more related to necrosis factors. The CM from each spinal segment were used in vitro, on microglial BV2 cell lines and DRGs explants, showing a lesion site-dependent impact on microglia activation and DRGs neurite outgrowth. In addition, while naive BV2 cells exhibited insignificant staining for CX3CR1 receptor, the level of CX3CR1 was strongly enhanced in some BV2 cells after their stimulation by CM collected from SCI. The molecular data might correlate with different polarization of activated microglia and macrophages along the rostro-caudal axis following acute injury. This was partially confirmed in vivo with CX3CR1 receptor, revealing higher expression in the rostral segment, with potential neuroprotective action. In addition, the neurotrophic factors released from rostral and lesion segments enhanced outgrowth of DRGs explants. Taken together these data suggest that regionalization in terms of inflammatory and neurotrophic responses may occur between rostral and caudal segments in acute SCI.

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.

The leech nervous system: a valuable model to study the microglia involvement in regenerative processes.

Microglia are intrinsic components of the central nervous system (CNS). During pathologies in mammals, inflammatory processes implicate the resident microglia and the infiltration of blood cells including macrophages. Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects during neuropathological conditions in vivo and in vitro. The medicinal leech Hirudo medicinalis is a well-known model in neurobiology due to its ability to naturally repair its CNS following injury. Considering the low infiltration of blood cells in this process, the leech CNS is studied to specify the activation mechanisms of only resident microglial cells. The microglia recruitment is known to be essential for the usual sprouting of injured axons and does not require any other glial cells. The present review will describe the questions which are addressed to understand the nerve repair. They will discuss the implication of leech factors in the microglial accumulation, the identification of nerve cells producing these molecules, and the study of different microglial subsets. Those questions aim to better understand the mechanisms of microglial cell recruitment and their crosstalk with damaged neurons. The study of this dialog is necessary to elucidate the balance of the inflammation leading to the leech CNS repair.

Hedistin: A novel antimicrobial peptide containing bromotryptophan constitutively expressed in the NK cells-like of the marine annelid, Nereis diversicolor.

A novel antimicrobial peptide, named hedistin was identified from the coelomocytes of Nereis diversicolor. Hedistin shows no obvious similarities with other known peptides and constitutes the first antimicrobial peptide containing bromotryptophans demonstrated in annelids. cDNA and mass spectrometry analysis revealed that, upon bacteria challenge, this peptide is secreted following processing of a precursor containing a signal peptide and prosequences. Hedistin was shown to possess an activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The gene was demonstrated to be constitutively and exclusively expressed in circulating NK cells like known to play an important role in the immunity of the sand worm. These data contrast with those observed in another annelid, the leech, in which genes coding for antimicrobial peptides are upregulated in a specific tissue and peptides are rapidly released into the hemolymph after septic injury.