contributed equally to this work 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 twodimensional protein gels followed by sequencing the speci®c spot, we have now identi®ed 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 speci®c and high af®nity 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.

Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.

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 cellto-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.

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.

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