BAG3 localizes in axonal structures during neuronal differentiation and is expressed in cellular processes of migrating cells in mouse cerebral cortex

BAG3 protein belongs to the family of co-chaperones involved in protein quality control and in the clearance of misfolded proteins [1]. Few studies have addressed BAG3 distribution and function in the central nervous system (CNS) and little is known about the cellular localization of BAG3 during neuronal differentiation in vitro and migration in vivo. Therefore we analysed by immunofluorescence microscopy the cellular distribution of BAG3 in the PC12 cell model treated or not with NGF and in developing and adult cortex of mice brain. Our results shows that BAG3 localizes mainly in vesicle structures of the neuritic domain during cell differentiation, while in undifferentiated cells it appears confined to the cytoplasm near the nuclear membrane. These observations were corroborated by transmission electron microscopy (TEM) which revealed that in NGF-differentiated PC12 cells, BAG3 localizes into electron-dense vesicles clustered along the axon and showing the typical aspect of the large dense core vesicles (LDCVs). Interestingly, the change of BAG3 localization during neuronal differentiation was associated only to a slight increase in the total BAG3 immunoreactivity as shown by western blot analysis. In order to provide further insights on the role of BAG3 in neuronal differentiation and migration, we also analysed BAG3 localization in mice developing and adult cerebral cortex. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, while in adult brain a low expression was detected in neuronal cell bodies and glial cells. In conclusion, our findings suggest that the presence and differential expression of BAG3 might be required for the correct development of the nervous system as well as for the maintenance of protein homeostasis

The endocannabinoid anandamide inhibits colon cancer cell growth by modulating different survival and proliferating pathways

The Endocannabinoid System (ECS) comprising the CB1 and CB2 receptors and their endogenous ligands is a central signalling system regulating food intake and energy balance. It is also present in peripheral tissues where it is involved in cell proliferation and survival. It has been shown that in colon cancer cells, the CB1 receptor antagonist SR171416 reduces colon cancer cell growth by acting as an inverse agonist rather than an antagonist [1]. Starting from this observation and from evidence indicating that some biological responses to cannabinoids depend on estrogen levels and some selective estrogen receptor modulators can bind the CB1 receptor [2], we aimed to study the effects of the CB1 receptor ligand anandamide (AEA) on colon cancer cell proliferation and its ability to modulate some survival and proliferating pathways including Akt, MAPK/ERK and estrogen receptor (ER) b signalling which is the predominant ER pathway in colonic epithelium. We used an AEA-analogue and a selective inhibitor of fatty acid amide hydrolase (FAAH) that enhances intracellular levels of AEA and studied proliferation and cell cycle progression on human adenocarcinoma cells DLD1 and SW620. Our results showed that increased levels of AEA significantly reduced cell proliferation in both cell lines at 24 and 48 h also inducing an S phase cell cycle accumulation. The AEA-induced inhibition of cell growth was mediated by a reduced expression of phoshoAkt and phosphoERK and, at the same time, by an induction of ERβ expression. These data suggest that AEA can reduces colon cancer cell proliferation by interfering with different signalling pathways

The isoprenoid end product N6-isopentenyladenosine reduces inflammatory response through the inhibition of the NF\uce\ubaB and STAT3 pathways in cystic fibrosis cells

N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation. TNF alpha-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NF kappa B activity, luciferase assay was performed on TNF alpha-stimulated HEK293/T cells transfected with a NF kappa B reporter plasmid. We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNF alpha-stimulated CF cells and this effect is mediated by increasing the expression of the direct NF kappa B inhibitor I kappa B alpha and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNF alpha-mediated NF kappa B activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins. Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF

Protease-activated receptor-1 regulates cytokine productionand induces the suppressor of cytokine signaling-3 in microglia

Protease-activated receptors (PARs) are cleaved and activated by thrombin and other extracellular proteases which are released during tissue trauma and inflammation. PAR-1 is the prototypic member of the PAR family and has been shown to be upregulated in several brain pathologies being expressed by neurons and glial cells. The present experiments show that the administration of the PAR-1 activating peptides (TRAP6 and TFLLR) inhibits the production of the pro-inflammatory cytokines TNF-alpha and IL-6 in microglial cells treated with lipopolysaccharide (LPS) while promoting the release of the anti-inflammatory cytokine IL-10. Conversely, the addition of the specific PAR-2 agonist SLIGRL had no effect on the amount of cytokines released following LPS treatment. Consistent with these data PAR-1, but not PAR-2, stimulation upregulates the expression of the suppressor of cytokine signaling-3 (SOCS-3). The present data support the hypothesis that in microglia PAR-1 may be involved in the regulation of inflammatory reactions modulating the balance between pro- and anti-inflammatory cytokines possibly through SOCS induction

Regulation of neurotrophic functions: proteinase inhibitors.

[No abstract available

Bovine pancreatic trypsin inhibitor and homologous polypeptide inhibitors in nephron cells

Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules)

BAG3 is involved in neuronal differentiation and migration

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels

The ultrastructure of peripheral neurofibroma: the role of mast cells and their interaction with perineurial cells.

The authors analyze the ultrastructure of mast cells and perineurial cells when both are present in neurofibroma of the nerve sheath. Samples of pathologic tissue taken from three patients with neurofibroma of a peripheral nerve sheath were analyzed by light and transmission electron microscopy. The observations document the characteristics of the tumor cells (Schwann cells and perineurial cells) as well as the presence of numerous mast cells, typically in close contact with the perineurial cells and never with the Schwann cells. Many electron-dense vesicles were found between the cells; these vesicles are created when the cell membrane of the mast cell buds, and then they come into contact with the adjacent perineurial cell. Endocytosis vesicles are often present in the cytoplasm of perineurial cells. Analysis of these observations led the authors to assume the existence of a metabolic interaction between the two cell type in contact with each other and an active role of the mast cells in the evolution of the tumor. The following two theories are plausible: either the mast cells actively stimulate tumor growth, or they alter the phenotype of the tumor cell. These findings could have interesting clinical applications. The use of treatment protocols which inhibit mast cell activity could, in theory, stop either the proliferation of the neurofibroma or its malignant transformation