Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation.

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.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: Potential involvement of proteins from hemocytes revealed by a proteomic approach.

As an approach to investigate the suspected involvement of cellular factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns from hemocytes collected from susceptible and resistant snails. This proteomic approach revealed that twelve hemocytic proteins exhibited significant differences in their apparent abundance. The genes corresponding to five of them were characterized by a combination of mass spectrometry and molecular cloning. They encode an aldolase, an intermediate filament protein, a cytidine deaminase, the ribosomal protein P1 and the histone H4. Furthermore, we investigated their expression in parasite-exposed or -unexposed snails. These last experiments revealed changes in transcript levels corresponding to intermediate filament and histone H4 proteins post-infection.

Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni.

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.

Association of ATP synthase alpha-chain with neurofibrillary degeneration in Alzheimer's disease.

Amyloid deposits and neurofibrillary tangles (NFT) are the two hallmarks that characterize Alzheimer's disease (AD). In order to find the molecular partners of these degenerating processes, we have developed antibodies against insoluble AD brain lesions. One clone, named AD46, detects only NFT. Biochemical and histochemistry analyses demonstrate that the labeled protein accumulating in the cytosol of Alzheimer degenerating neurons is the alpha-chain of the ATP synthase. The cytosolic accumulation of the alpha-chain of ATP synthase is observed even at early stages of neurofibrillary degenerating process. It is specifically observed in degenerating neurons, either alone or tightly associated with aggregates of tau proteins, suggesting that it is a new molecular event related to neurodegeneration. Overall, our results strongly suggest the implication of the alpha-chain of ATP synthase in neurofibrillary degeneration of AD that is illustrated by the cytosolic accumulation of this mitochondrial protein, which belongs to the mitochondrial respiratory system. This regulatory subunit of the respiratory complex V of mitochondria is thus a potential target for therapeutic and diagnostic strategies.

Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor.

A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx. The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield. The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far. It was shown that the protein is monomeric and possesses two conserved cysteines (Cys). The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx). Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif. The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.

Therostasin, a novel clotting factor Xa inhibitor from the rhynchobdellid leech, Theromyzon tessulatum.

Therostasin is a potent naturally occurring tight-binding inhibitor of mammalian Factor Xa (K(i), 34 pm), isolated from the rhynchobdellid leech Theromyzon tessulatum. Therostasin is a cysteine-rich protein (8991 Da) consisting of 82 amino acid residues with 16 cysteine residues. Its amino acid sequence has been determined by a combination of techniques, including Edman degradation, enzymatic cleavage, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-beta-pyridylethylated compound. Sequence analysis reveals that it shares no significant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of the endothelin family, potent vasoconstrictors isolated in mammal and snake venom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid residues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and stored in the cells of the leech salivary glands.

Shift from fetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated with okadaic acid.

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.

Biochemical characterization of Tau proteins during cerebral aging of the lemurian primate Microcebus murinus.

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.

General cortical involvement in a late-onset case of Alzheimer disease. A biochemical approach by quantitation of abnormal tau proteins.

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.

[Antigenic changes of Tau protein induced by glutamate on primary cultures of neurons: immunocytochemistry study]. / Modifications antigéniques de la protéine Tau induites par le glutamate sur des cultures primaires de neurones: études immunocytochemiques.

Degenerating neurons in Alzheimer's disease are characterized by the presence of neurofibrillary tangles constituted by paired helical filaments (PHF). Abnormally phosphorylated Tau protein, a microtubule associated protein is one of the major component of PHF. Abnormal phosphorylation seems to be located in the C-terminal domain but also in the N-terminal region of Tau proteins. Previous studies demonstrated that calcium-mediated glutamate toxicity produces a dose-dependent increase of Tau immunolabellings in neuronal cultures. Biochemical results revealed that these changes could be associated with abnormal Tau migrations on immunoblots. Using three anti-Tau antibodies the present study shows that glutamate toxicity induces in neuronal cultures, Tau modifications localized in the N- and C-terminal domains of the protein. These findings suggest the possibility that glutamate toxicity can induce Tau antigenic changes involving probably the whole molecule.

[Detection of Alzheimer type pathological epitopes on Tau proteins of neuroblastoma cells after treatment with okadaic acid]. / Détection d'épitopes pathologiques de type Alzheimer sur les protéines Tau de cellules de neuroblastome après traitement à l'acide okadaïque.

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.

Tau antigenic changes induced by glutamate in rat primary culture model: a biochemical approach.

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.