Tiago G. Santos

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain

The prion protein (PrPC) is highly expressed in the nervous system, and its abnormal con‐former is associated with prion diseases. PrPC is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrPC‐mediated intracellular signaling. Binding of laminin (Ln) to PrPC modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrPC‐Ln interaction in order to identify transmembrane proteins involved in the transduction of PrPC‐Ln signals. The Ln γl‐chain peptide, which contains the Ln binding site for PrPC, induced neuritogenesis through activation of phos‐pholipase C (PLC), Ca2+ 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 PrPC‐null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluRl/5) associate with PrPC. Expression of either mGluRl or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrPC‐Ln γl peptide interaction. Specific inhibitors of these receptors impaired PrPC‐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 PrPC‐Ln, and they support the notion that PrPC 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

Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins

The physiological functions of the cellular prion protein, PrPC, as a cell surface pleiotropic receptor are under debate. We report that PrPC interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrPC-vitronectin interaction were mapped to residues 105-119 of PrPC and the residues 307-320 of vitronectin. The two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrPC antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrPC-null mice. Functional assays demonstrated that relative to wild-type cells, PrPC-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater αvβ3 activity. Our findings indicate that PrPC plays an important role in axonal growth, and this function may be rescued in PrPC-knockout animals by integrin compensatory mechanisms.

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrPC) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrPC extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrPC engagement induces an increase in intracellular Ca2+ levels. This effect was not detected in PrPC-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrPC. Using a best candidate approach to test for potential channels involved in Ca2+ influx evoked by STI1-PrPC, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrPC-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrPC and allowed reconstitution of signaling by PrPC-STI1 interaction. These results indicate that STI1 can interact with the PrPC·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Neuropeptide Y Modulation of Interleukin-1β (IL-1β)-induced Nitric Oxide Production in Microglia

Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y1 receptor agonist and selective antagonists for Y1, Y2, and Y5 receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y1 receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y1 receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y1 receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.

Enhanced neural progenitor/stem cells self-renewal via the interaction of stress-inducible protein 1 with the prion protein.

Prion protein (PrPC), when associated with the secreted form of the stress‐inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrPC‐STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild‐type (Prnp+/+) and PrPC‐null (Prnp0/0) mice were maintained for several passages without the loss of self‐renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrPC suggest that they may associate and function as a complex in neurosphere‐derived stem cells. The formation of neurospheres from Prnp0/0 mice was reduced significantly when compared with their wild‐type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrPC, with specific antibodies, impaired Prnp+/+ neurosphere formation without further impairing the formation of Prnp0/0 neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrPC but not in cells from Prnp0/0 mice. The STI1‐PrPC interaction was able to stimulate cell proliferation in the neurosphere‐forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1‐PrPC complex may play a critical role in neural progenitor/stem cells self‐renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126–1136

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein

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

Laminin-γ1 chain and stress inducible protein 1 synergistically mediate PrPC-dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons

Prion protein (PrPC) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrPC interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimers diseases. PrPC co‐opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross‐talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrPC‐mediated axonogenesis in peripheral neurons in response to STI1 and laminin‐γ1 chain‐derived peptide (Ln‐γ1). STI1 and Ln‐γ1 promoted robust axonogenesis in wild‐type neurons, whereas no effect was observed in neurons from PrPC‐null mice. PrPC binding to Ln‐γ1 or STI1 led to an increase in intracellular Ca2+ levels via distinct mechanisms: STI1 promoted extracellular Ca2+ influx, and Ln‐γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln‐γ1, but depends on, C‐type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrPC‐mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrPC. These results suggest a role for PrPC as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.

Prion potency in stem cells biology.

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

Synaptosomal glutamate release and uptake in mice lacking the cellular prion protein

Glutamate plays a central role in the fast excitatory synaptic transmission and is a key neurotransmitter involved in several neurophysiological processes. Glutamate levels on the synaptic cleft are related to neural excitability, neuroplasticity, and neuronal damage associated with excitotoxicity. Mice lacking the cellular prion protein (PrP(c)) gene (Prnp) present a decreased astrocytic glutamate uptake in cultures, higher neuronal excitability in vitro and sensitivity to pro-convulsant drugs in vivo, and age-dependent memory impairment. Here, we investigate if PrP(c) might be involved in neuronal uptake and release of glutamate. For this purpose, we compared synaptosomal preparations from the cerebral cortex, entorhinal cortex, hippocampus, cerebellum, and olfactory bulb of 3- or 9-month-old PrP(c) null mice and with respective wild-type controls. Although we observed differences in synaptosomal glutamate release and uptake regarding the age of mice and the brain structure studied, these differences were similar for PrP(c) null mice and their respective wild-type controls. Therefore, despite a possible correlation between neuronal glutamate transporters, excitability, and neuronal damage, our results suggest that PrP(c) expression is not critical for neuronal glutamate transport.

Targeting prion protein interactions in cancer

ABSTRACT In recent years, prion protein (PrPC) has been considered as a promising target molecule for cancer therapies, due its direct or indirect participation in tumor growth, metastasis, and resistance to cell death induced by chemotherapy. PrPC functions as a scaffold protein, forming multiprotein complexes on the plasma membrane, which elicits distinct signaling pathways involved in diverse biological phenomena and could be modulated depending on the cell type, complex composition, and organization. In addition, PrPC and its partners participate in self-renewal of embryonic, tissue-specific stem cells and cancer stem cells, which are suggested to be responsible for the origin, maintenance, relapse, and dissemination of tumors. Interference with protein–protein interaction has been recognized as an important therapeutic strategy in cancer; indeed, the possible interference in PrPC engagement with specific partners is a novel strategy. Recently, our group successfully used that approach to interfere with the interaction between PrPC and HSP-90/70 organizing protein (HOP, also known as stress-inducible protein 1 - STI1) to control the growth of human glioblastoma in animal models. Thus, PrPC-organized multicomplexes have emerged as feasible candidates for anti-tumor therapy, warranting further exploration.