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In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep ™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.

Tau proteins extracted from the brain of 12 adult microcebes ranging from 2 to 9 years old were characterized by Western blots, using immunological probes against normal and pathological human Tau proteins. In microcebes, the molecular weight of Tau proteins increases during aging, with variants of 52-54, 64, 67 kDa in the young adult and variants of 60 and 70 kDa in the oldest animal studied. The increase of the apparent molecular weight is due to a change of conformation and a stabilization in the "hyperphosphorylated" state, as revealed with phosphorylation-dependent monoclonal antibodies Tau-1 and AD2. Furthermore, AD1 specifically detected Alzheimer-type epitopes on the 60 kDa Tau isoform from a very old microcebe. These results suggest that Microcebus murinus is an interesting model for the study of the biochemical dysfunctions that occur in the human brain during aging and Alzheimer disease.

We have performed a biochemical mapping of the neurofibrillary degeneration in all cortical areas of Alzheimer patients, using the immunological quantification of pathological tau 55, 64, and 69. These abnormally phosphorylated proteins, which are the basic components of PHF, are reliable markers of the degenerating process in Alzheimer disease. Here, we report our biochemical findings on a brain from a 90-yr-old woman with an 8-yr history of Alzheimer disease who exhibited dramatic and general cortical involvement. The detection of these markers was very high in all Brodmann areas, even in primary motor, somatosensory, or visual cortex. This case report contrasts with other studies, which suggested that a more virulent disease process is generally associated with an early onset and argues for the heterogeneity of the disease. Moreover, we show here that the immunodetection of abnormal tau proteins using the western blot method is a precise, reliable, and reproducible way to quantify the degenerating process in AD.

In Alzheimer's disease, Tau proteins are abnormally phosphorylated. In this paper, we describe a cellular model producing such pathological Tau proteins. After differentiation by NGF and treatment with okadaic acid (an inhibitor of phosphatases 1 and 2 A), neuroblastoma SKNSH-SY 5Y cells produced Tau proteins with an increased apparent molecular weight and a more acidic isoelectric point when compared to Tau proteins from control cells. These modified tau proteins bore Alzheimer-type epitopes detectable by antibodies specific to phosphorylated Alzheimer epitopes. This model is the first step toward a pharmacological approach of neuroprotection.

Primary neuronal cultures were treated with glutamate to induce an increase of Tau immunoreactivity similar to that observed in Alzheimer's disease. The Tau profile of neurones in culture before and after exposure to glutamate was analyzed on immunoblots with anti-Tau, anti-paired helical filaments (PHF) and antibody specific for modified Tau. Differences were observed between treated and control cultures: glutamate induced a shift of immunodetection from the lowest to the highest molecular weight Tau isoform and an acidification of Tau proteins. However, these modifications are not exactly those observed in Alzheimer's disease since we were not able to detect 'Alzheimer-type' epitopes on Tau proteins after the glutamate exposure.