Vilma R. Martins

Physiology of the Prion Protein

Prion diseases are transmissible spongiform encephalopathies (TSEs), attributed to conformational conversion of the cellular prion protein (PrP(C)) into an abnormal conformer that accumulates in the brain. Understanding the pathogenesis of TSEs requires the identification of functional properties of PrP(C). Here we examine the physiological functions of PrP(C) at the systemic, cellular, and molecular level. Current data show that both the expression and the engagement of PrP(C) with a variety of ligands modulate the following: 1) functions of the nervous and immune systems, including memory and inflammatory reactions; 2) cell proliferation, differentiation, and sensitivity to programmed cell death both in the nervous and immune systems, as well as in various cell lines; 3) the activity of numerous signal transduction pathways, including cAMP/protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt pathways, as well as soluble non-receptor tyrosine kinases; and 4) trafficking of PrP(C) both laterally among distinct plasma membrane domains, and along endocytic pathways, on top of continuous, rapid recycling. A unified view of these functional properties indicates that the prion protein is a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior.

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 PrPc, whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrPc‐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 Kd of 10−7 M. The interaction sites were mapped to amino acids 113–128 from PrPc and 230–245 from STI1. Cell surface binding and pull‐down experiments showed that recombinant PrPc binds to cellular STI1, and co‐immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrPc interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuropro tective signals that rescue cells from apoptosis.

Cellular prion protein transduces neuroprotective signals

To test for a role for the cellular prion protein (PrPc) in cell death, we used a PrPc‐binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild‐type rodents, but not from PrPc‐null mice. Neuroprotection was abolished by treatment with phosphatidylinositol‐specific phospholipase C, with human peptide 106–126, with certain antibodies to PrPc or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrPc that increased cAMP also induced neuroprotection. Thus, engagement of PrPc transduces neuroprotective signals through a cAMP/PKA‐dependent pathway. PrPc may function as a trophic receptor, the activation of which leads to a neuroprotective state.

Cellular prion protein binds laminin and mediates neuritogenesis

Laminin (LN) plays a major role in neuronal differentiation, migration and survival. Here, we show that the cellular prion protein (PrPc) is a saturable, specific, high-affinity receptor for LN. The PrPc-LN interaction is involved in the neuritogenesis induced by NGF plus LN in the PC-12 cell line and the binding site resides in a carboxy-terminal decapeptide from the gamma-1 LN chain. Neuritogenesis induced by LN or its gamma-1-derived peptide in primary cultures from rat or either wild type or PrP null mice hippocampal neurons, indicated that PrPc is the main cellular receptor for that particular LN domain. These results point out to the importance of the PrPc-LN interaction for the neuronal plasticity mechanism.

Interaction of Cellular Prion and Stress-Inducible Protein 1 Promotes Neuritogenesis and Neuroprotection by Distinct Signaling Pathways

Understanding the physiological function of the cellular prion (PrPc) depends on the investigation of PrPc-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrPc ligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrPc interaction with STI1 in hippocampal neurons. Both PrPc and STI1 are abundantly expressed and highly colocalized in the hippocampus in situ, indicating that they can interact in vivo. Recombinant STI1 (His6-STI1) added to hippocampal cultures interacts with PrPc at the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrPc-null cells. This effect was abolished by antibodies against either PrPc or STI1 and was dependent on the STI1 domain that binds PrPc. Binding of these proteins induced the phosphorylation/activation of the mitogen-activated protein kinase, which was essential for STI1-promoted neuritogenesis. His6-STI1, but not its counterpart lacking the PrPc binding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrPc–STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.

Increased Sensitivity to Seizures in Mice Lacking Cellular Prion Protein

Summary: Purpose: The physiologic role of the cellular prion protein (PrPc) is unknown. Mice devoid of PrPc develop normally and show only minor deficits. However, electrophysiologic and histologic alterations found in these mice suggest a possible role for PrPc in seizure threshold and/or epilepsy.

Cellular prion protein: on the road for functions

Cellular prion (PrPc) is a plasma membrane glycosyphosphatidylinositol‐anchored protein present in neurons but also in other cell types. Protein conservation among species suggests that PrPc may have important physiological roles. Cellular and molecular approaches have established several novel features of the regulation of PrPc expression, cellular trafficking as well as its participation in copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling and cell survival. It is therefore likely that PrPc plays pleiotropic roles in neuronal and non‐neuronal cells, and as such the loss of function of PrPc may be an important component of various diseases.

Tumor-cell-derived microvesicles as carriers of molecular information in cancer

Purpose of review Exosomes and microvesicles are secreted particles of 30–200 nm in diameter, delimited by a lipid bilayer and containing a wide range of membrane-bound or free proteins and nucleic acids (in particular mRNA and miRNA). Here, we review the properties of tumor-cell-derived microvesicles as carriers of molecular information in relation to cancer progression and promotion of metastasis. Recent findings Microvesicles from tumor cells operate as signaling platforms that diffuse in the extracellular space to target cells in the microenvironment, modulating the interactions of tumor cells with stromal, inflammatory, dendritic, immune or vascular cells and priming the formation of the metastatic niche. Summary Because of their stability, exosomes and microvesicles can be retrieved in bodily fluids as biomarkers for cancer detection and monitoring. They offer a range of molecular targets for controlling cell–cell interactions during invasion and metastasis.

Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells

The cellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI)‐anchored plasma membrane protein whose conformational altered forms (PrPsc) are known to cause neurodegenerative diseases in mammals. In order to investigate the intracellular traffic of mammalian PrPc in living cells, we have generated a green fluorescent protein (GFP) tagged version of PrPc. The recombinant protein was properly anchored at the cell surface and its distribution pattern was similar to that of the endogenous PrPc, with labeling at the plasma membrane and in an intracellular perinuclear compartment. Comparison of the steady‐state distribution of GFP‐PrPc and two N‐terminal deletion mutants (Δ32‐121 and Δ32‐134), that cause neurological symptoms when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP‐PrPc. In addition, GFP‐PrPc, but not the two mutants, internalized from the plasma membrane in response to Cu2+ treatment and accumulated at a perinuclear region in SN56 cells. Our data suggest that GFP‐PrPc can be used to follow constitutive and induced PrPc traffic in living cells.

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC‐12 cell model to explore the biological role of LN–PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN‐coated culture plaques. Furthermore, chromophore‐assisted laser inactivation of cell surface PrPc perturbs LN‐induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.