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 K d 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.

Understanding the physiological function of the cellular prion (PrP^c) depends on the investigation of PrP^c-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrP^cligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrP^cinteraction with STI1 in hippocampal neurons. Both PrP^cand STI1 are abundantly expressed and highly colocalized in the hippocampusin situ, indicating that they can interactin vivo. Recombinant STI1 (His₆-STI1) added to hippocampal cultures interacts with PrP^cat the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrP^c-null cells. This effect was abolished by antibodies against either PrP^cor STI1 and was dependent on the STI1 domain that binds PrP^c. 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 PrP^cbinding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrP^c–STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.

The prion protein (PrP ^C ) is highly expressed in the nervous system, and its abnormal con‐former is associated with prion diseases. PrP ^C is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP ^C ‐mediated intracellular signaling. Binding of laminin (Ln) to PrP ^C modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP ^C ‐Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP ^C ‐Ln signals. The Ln γl‐chain peptide, which contains the Ln binding site for PrP ^C , induced neuritogenesis through activation of phos‐pholipase C (PLC), Ca ²⁺ mobilization from intracellular stores, and protein kinase C and extracellular signalregulated kinase (ERK1/2) activation in primary cultures of neurons from wild‐type, but not PrP ^C ‐null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluRl/5) associate with PrP ^C . Expression of either mGluRl or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP ^C ‐Ln γl peptide interaction. Specific inhibitors of these receptors impaired PrP ^C ‐Ln γl peptide‐induced signaling and neuri‐togenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP ^C ‐Ln, and they support the notion that PrP ^C participates in the assembly of multiprotein complexes with physiological functions on neurons.—Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F. _, Roffe, M., Hajj, G. N., Lee, K. S., Magalhães, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Amãrico, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γl chain. FASEB J. 25, 265–279 (20ll). www.fasebj.org

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrP(C) interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrP(C)-STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrP(C)-STI1 binding is corrupted in neuronal cell lines persistently infected with PrP(Sc), as well as in primary cultured hippocampal neurons acutely exposed to PrP(Sc). Consistent with this, high levels of eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation were found in PrP(Sc)-infected cells and in neurons acutely exposed to PrP(Sc). These data indicate that modulation of protein synthesis is critical for PrP(C)-STI1 neurotrophic functions, and point to the impairment of this process during PrP(Sc) infection as a possible contributor to neurodegeneration.

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP^C) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP^C trafficking and tested whether this process controls PrP^C-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP^C, induced PrP^C endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP^C; however, heterologous expression of PrP^C reconstituted both PKA and ERK1/2 activation. In contrast, a PrP^C mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP^C endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

Stress‐inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase‐3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1‐mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild‐type astrocytes by 3‐fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia‐mediated neuronal death in a prion protein‐dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience.—Beraldo, F. H., Soares, I. N., Goncalves, D. F., Fan, J., Thomas, A. A., Santos, T. G., Mohammad, A. H., Roffe, M., Calder, M. D., Nikolova, S., Hajj, G. N., Guimaraes, A. N., Massensini, A. R., Welch, I., Betts, D. H., Gros, R., Drangova, M., Watson, A. J., Bartha, R., Prado, V. F., Martins, V. R., and Prado, M. A. M., Stress‐inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein. FASEB J. 27, 3594–3607 (2013). www.fasebj.org