Prion protein and its ligand stress inducible protein 1 regulate astrocyte development.

Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.

Prion protein ablation increases cellular aggregation and embolization contributing to mechanisms of metastasis.

Cellular Prion Protein (PrP(C)) is a cell surface protein highly expressed in the nervous system, and to a lesser extent in other tissues. PrP(C) binds to the extracellular matrix laminin and vitronectin, to mediate cell adhesion and differentiation. Herein, we investigate how PrP(C) expression modulates the aggressiveness of transformed cells. Mesenchymal embryonic cells (MEC) from wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were immortalized and transformed by co-expression of ras and myc. These cells presented similar growth rates and tumor formation in vivo. When injected in the tail vein, Prnp(0/0)ras/myc cells exhibited increased lung colonization compared with Prnp(+/+)ras/myc cells. Additionally, Prnp(0/0)ras/myc cells form more aggregates with blood components than Prnp(+/+)ras/myc cells, facilitating the arrest of Prnp(0/0)ras/myc cells in the lung vasculature. Integrin alpha(v)beta(3) is more expressed and activated in MEC and in transformed Prnp(0/0) cells than in the respective Prnp(+/+) cells. The blocking of integrin alpha(v)beta(3) by RGD peptide reduces lung colonization in transformed Prnp(0/0) cells to similar levels of those presented by transformed Prnp(+/+) cells. Our data indicate that PrP(C) negatively modulates the expression and activation of integrin alpha(v)beta(3) resulting in a more aggressive phenotype. These results indicate that PrP(C) may have main implications in modulating metastasis formation.

Cellular prion protein expression in astrocytes modulates neuronal survival and differentiation.

The functions of cellular prion protein (PrP(C)) are under intense debate and PrP(C) loss of function has been implicated in the pathology of prion diseases. Neuronal PrP(C) engagement with stress-inducible protein-1 and laminin (LN) plays a key role in cell survival and differentiation. The present study evaluated whether PrP(C) expression in astrocytes modulates neuron-glia cross-talk that underlies neuronal survival and differentiation. Astrocytes from wild-type mice promoted a higher level neuritogenesis than astrocytes obtained from PrP(C)-null animals. Remarkably, neuritogenesis was greatly diminished in co-cultures combining PrP(C)-null astrocytes and neurons. LN secreted and deposited at the extracellular matrix by wild-type astrocytes presented a fibrillary pattern and was permissive for neuritogenesis. Conversely, LN coming from PrP(C)-null astrocytes displayed a punctate distribution, and did not support neuronal differentiation. Additionally, secreted soluble factors from PrP(C)-null astrocytes promoted lower levels of neuronal survival than those secreted by wild-type astrocytes. PrP(C) and stress-inducible protein-1 were characterized as soluble molecules secreted by astrocytes which participate in neuronal survival. Taken together, these data indicate that PrP(C) expression in astrocytes is critical for sustaining cell-to-cell interactions, the organization of the extracellular matrix, and the secretion of soluble factors, all of which are essential events for neuronal differentiation and survival.

Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus.

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.

Is there a role for cellular prion protein in intrathymic T cell differentiation and migration?

The cellular prion protein (PrP(C)) is expressed in the nervous and immune systems. Functionally, PrP(C) has been suggested to participate in neuron survival, neuritogenesis and T lymphocyte activation. Moreover, PrP(C) interaction with laminin influences neuronal adhesion and neurite extension. Nevertheless, so far the physiological role of PrP(C) has not been completely elucidated, particularly in the immune system. The aim of the study was to evaluate the possible participation of PrP(C) in intrathymic T cell development. We evaluated T cell differentiation markers in thymocytes and peripheral lymphocytes, as well as thymocyte death in PrP(C)-null or PrP(C)-overexpressing (Tga20) mice, compared to wild-type controls. In these same animals, we ascertained laminin-driven thymocyte migration. Compared to controls, only marginal differences were found in PrP(C)-null animals. However, Tga20 mice exhibited a severe thymic hypoplasia, with 10-20% lymphocytes compared to wild-type counterparts. In particular, the frequency of CD4+CD8+ cells was largely reduced, and this was accompanied by a dramatic increase in the frequency of CD4-CD8- thymocytes, which could be as high as 60-65% of the whole-cell suspensions. Moreover, Tga20 mice exhibited an increase in thymocyte death, comprising the CD4+CD8+, as well as CD4+ and CD8+ single-positive cells. Additionally, laminin-driven migration was largely impaired in Tga20 mice, in which we also found a significant decrease in total T lymphocytes in the spleen and lymph nodes. Our results show that PrP(C) overexpression alters intrathymic T cell development, a defect that likely has a negative impact in the formation of the T cell peripheral pool.

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.