Almost a century of prion protein(s): From pathology to physiology, and back to pathology.

Prions are one of the few pathogens whose name is renowned at all population levels, after the dramatic years pervaded by the fear of eating prion-infected food. If now this, somehow irrational, scare of bovine meat inexorably transmitting devastating brain disorders is largely subdued, several prion-related issues are still unsolved, precluding the design of therapeutic approaches that could slow, if not halt, prion diseases. One unsolved issue is, for example, the role of the prion protein (PrPC), whole conformational misfolding originates the prion but whose physiologic reason d'etre in neurons, and in cells at large, remains enigmatic. Preceded by a historical outline, the present review will discuss the functional pleiotropicity ascribed to PrPC, and whether this aspect could fall, at least in part, into a more concise framework. It will also be devoted to radically different perspectives for PrPC, which have been recently brought to the attention of the scientific world with unexpected force. Finally, it will discuss the possible reasons allowing an evolutionary conserved and benign protein, as PrPC is, to turn into a high affinity receptor for pathologic misfolded oligomers, and to transmit their toxic message into neurons.

The cellular prion protein is expressed in olfactory sensory neurons of adult mice but does not affect the early events of the olfactory transduction pathway.

A conformational conversion of the cellular prion protein (PrP(C)) is now recognized as the causal event of fatal neurodegenerative disorders, known as prion diseases. In spite of long-lasting efforts, however, the physiological role of PrP(C) remains unclear. It has been reported that PrP(C) is expressed in various areas of the olfactory system, including the olfactory epithelium, but its precise localization in olfactory sensory neurons (OSNs) is still debated. Here, using immunohistochemistry tools, we have reinvestigated the expression and localization of PrP(C) in the olfactory epithelium of adult congenic mice expressing different PrP(C) amounts, that is, wild-type, PrP-knockout, and transgenic PrP(C)-overexpressing animals. We found that PrP(C) was expressed in OSNs, in which, however, it was unevenly distributed, being detectable at low levels in cell bodies, dendrites and apical layer, and more abundantly in axons. We also studied the involvement of PrP(C) in the response of the olfactory epithelium to odorants, by comparing the electro-olfactograms of the 3 mouse lines subjected to different stimulation protocols. We found no significant difference between the 3 PrP genotypes, supporting previous reports that exclude a direct action of PrP(C) in the early signal transduction activity of the olfactory epithelium.

Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains of epithelial FRT cells.

Dpl (doppel) is a paralogue of the PrPC (cellular prion protein), whose misfolded conformer (the scrapie prion protein, PrPSc) is responsible for the onset of TSEs (transmissible spongiform encephalopathies) or prion diseases. It has been shown that the ectopic expression of Dpl in the brains of some lines of PrP-knockout mice provokes cerebellar ataxia, which can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where, and under which conditions, this event may occur. In the present study we addressed this issue by analysing the intracellular localization and the interaction between Dpl and PrPC in FRT (Fischer rat thyroid) cells stably expressing the two proteins separately or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both singly and doubly transfected clones. Interestingly we found that they associate with DRMs (detergent-resistant membranes) or lipid rafts, from where they can be co-immunoprecipitated in a cholesterol-dependent fashion. Although the interaction between Dpl and PrPC has been suggested before, our results provide the first clear evidence that this interaction occurs in rafts and is dependent on the integrity of these membrane microdomains. Furthermore, both Dpl and PrPC could be immunoprecipitated with flotillin-2, a raft protein involved in endocytosis and cell signalling events, suggesting that they share the same lipid environment.

From cell protection to death: may Ca2+ signals explain the chameleonic attributes of the mammalian prion protein?

It is now accepted that a conformational change of the cellular prion protein (PrP(C)) generates the prion, the infectious agent responsible for lethal neurodegenerative disorders, named transmissible spongiform encephalopathies, or prion diseases. The mechanisms of prion-associated neurodegeneration are still obscure, as is the cell role of PrP(C), although increasing evidence attributes to PrP(C) important functions in cell survival. Such a behavioral dichotomy thus enables the prion protein to switch from a benign role under normal conditions, to the execution of neurons during disease. By reviewing data from models of prion disease and PrP(C)-null paradigms, which suggest a relation between the prion protein and Ca(2+) homeostasis, here we discuss the possibility that Ca(2+) is the factor behind the enigma of the pathophysiology of PrP(C). Ca(2+) features in almost all processes of cell signaling, and may thus tell us much about a protein that pivots between health and disease.

Neuronal pathophysiology featuring PrPC and its control over Ca2+ metabolism.

Calcium (Ca2+) is an intracellular second messenger that ubiquitously masters remarkably diverse biological processes, including cell death. Growing evidence substantiates an involvement of the prion protein (PrPC) in regulating neuronal Ca2+ homeostasis, which could rationalize most of the wide range of functions ascribed to the protein. We have recently demonstrated that PrPC controls extracellular Ca2+ fluxes, and mitochondrial Ca2+ uptake, in neurons stimulated with glutamate (De Mario et al., J Cell Sci 2017; 130:2736-46), suggesting that PrPC protects neurons from threatening Ca2+ overloads and excitotoxicity. In light of these results and of recent reports in the literature, here we review the connection of PrPC with Ca2+ metabolism and also provide some speculative hints on the physiologic outcomes of this link. In addition, because PrPC is implicated in neurodegenerative diseases, including prion disorders and Alzheimer's disease, we will also discuss possible ways by which disruption of PrPC-Ca2+ association could be mechanistically connected with these pathologies.

Physiopathologic implications of the structural and functional domains of the prion protein.

Prion diseases are invariably fatal neurodegenerative disorders affecting man and various animal species. A large body of evidence supports the notion that the causative agent of these diseases is the prion, which, devoid of nucleic acids, is composed largely, if not entirely, of a conformationally abnormal isoform (PrP(Sc) of the cellular prion protein (PrPc). PrPc is a highly conserved and ubiquitously expressed sialoglycoprotein, the normal function of which is, however, still ill defined. Several modules have been recognised in PrPc structure. Their extensive analysis by different experimental approaches, including transgenic animal models, has allowed to assigning to several modules a putative role in PrPc physiology. Concurrently, it has underscored the possibility that alteration of specific domains may determine the switching from a beneficial role of PrPc into one that becomes detrimental to neurons, and/or promote the conversion of PrPc into the pathogenic PrP(Sc) conformer.

Generation and usage of aequorin lentiviral vectors for Ca(2+) measurement in sub-cellular compartments of hard-to-transfect cells.

Targeted aequorin-based Ca(2+) probes represent an unprecedented tool for the reliable measurement of Ca(2+) concentration and dynamics in different sub-cellular compartments. The main advantages of aequorin are its proteinaceous nature, which allows attachment of a signal peptide for targeting aequorin to virtually any sub-cellular compartment; its low Ca(2+)-binding capacity; the wide range of Ca(2+) concentrations that can be measured, ranging from sub-micromolar to millimolar; its robust performance in aggressive environments, e.g., the strong acidic pH of the lysosomal lumen. Lentiviral vectors represent a popular tool to transduce post-mitotic or hard-to-transfect cells both in vitro and in vivo. Furthermore, it has great potential for gene therapy. Last generation lentiviral vectors represent a perfect compromise for combining large insert size, ease of production and handling, and high degree of biosafety. Here, we describe strategies for cloning aequorin probes - targeted to the cytosol, sub-plasma membrane cytosolic domains, the mitochondrial matrix, and the endoplasmic reticulum lumen - into lentiviral vectors. We describe methods for the production of lentiviral particles, and provide examples of measuring Ca(2+) dynamics by such aequorin-encoding lentiviral vectors in sub-cellular compartments of hard-to-transfect cells, including immortalized striatal neurons, primary cerebellar granule neurons and endothelial progenitor cells, which provide suitable in vitro models for the study of different human diseases.

The prion protein constitutively controls neuronal store-operated Ca(2+) entry through Fyn kinase.

The prion protein (PrP(C)) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrP(C) involvement in prion propagation is well established, PrP(C) physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca(2+) homeostasis. Because PrP(C) binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrP(C) acts as receptor for amyloid-ß (Aß) oligomers associated with Alzheimer's disease (AD), and that PrP(C)-Aß binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca(2+) probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrP(C), allowed us to investigate whether PrP(C) regulates store-operated Ca(2+) entry (SOCE) and the implication of Fyn in this control. Our findings show that PrP(C) attenuates SOCE, and Ca(2+) accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrP(C)-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aß(1-42) oligomers abrogates the control of PrP(C) over Fyn and SOCE, suggesting a PrP(C)-dependent mechanizm for Aß-induced neuronal Ca(2+) dyshomeostasis.

Human Doppel and prion protein share common membrane microdomains and internalization pathways.

Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease. Doppel is considered an N-truncated form of PrPc, and shares with PrPc several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrPc have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrPc were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrPc co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrPc redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrPc in our neuronal cell model.

Possible role for Ca2+ in the pathophysiology of the prion protein?

Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimer's disease.

Cellular prion protein promotes regeneration of adult muscle tissue.

It is now well established that the conversion of the cellular prion protein, PrP(C), into its anomalous conformer, PrP(Sc), is central to the onset of prion disease. However, both the mechanism of prion-related neurodegeneration and the physiologic role of PrP(C) are still unknown. The use of animal and cell models has suggested a number of putative functions for the protein, including cell signaling, adhesion, proliferation, and differentiation. Given that skeletal muscles express significant amounts of PrP(C) and have been related to PrP(C) pathophysiology, in the present study, we used skeletal muscles to analyze whether the protein plays a role in adult morphogenesis. We employed an in vivo paradigm that allowed us to compare the regeneration of acutely damaged hind-limb tibialis anterior muscles of mice expressing, or not expressing, PrP(C). Using morphometric and biochemical parameters, we provide compelling evidence that the absence of PrP(C) significantly slows the regeneration process compared to wild-type muscles by attenuating the stress-activated p38 pathway, and the consequent exit from the cell cycle, of myogenic precursor cells. Demonstrating the specificity of this finding, restoring PrP(C) expression completely rescued the muscle phenotype evidenced in the absence of PrP(C).

Prion and TNFα: TAC(E)it agreement between the prion protein and cell signaling.

Prion diseases are rare and fatal neurodegenerative disorders that occur when the cellular prion protein (PrPC) is converted into a conformationally modified isoform that originates the novel infectious agent, called prion. Although much information is now available on the different routes of prion infection, both the mechanisms underlying prion neurotoxicity and the physiologic role of PrPC remain unclear. By use of a novel paradigm, we have shown in a recent paper that--following a myotoxin-induced degenerative challenge--PrPC is implicated in the morphogenesis of the skeletal muscle of adult mice. PrPC accomplished this task by modulating signaling pathways central to the myogenic process, in particular the p38 kinase pathway. The possibility that PrPC acts in cell signaling has already been suggested after in vitro studies. Using our in vivo approach, we have instead provided proof of the physiologic relevance of PrPC commitment in signaling events, and that PrPC likely performed the task by controlling the activity of the enzyme (TACE) secreting the signaling TNFα molecule. After a brief summary of our data, here we will discuss the suggestion, arising from our and other recent findings, implying that regulation of TACE, and of other members of the protease family TACE belongs to, may be exploited by PrPC in different cell contexts. Notably, this advancement of knowledge on PrPC physiology could also shed light on the defense mechanisms against the onset of a more common neurodegenerative disorder than prion disease, such as Alzheimer disease.

Is, indeed, the prion protein a Harlequin servant of "many" masters?

Tens of putative interacting partners of the cellular prion protein (PrP(C)) have been identified, yet the physiologic role of PrP(C) remains unclear. For the first time, however, a recent paper has demonstrated that the absence of PrP(C) produces a lethal phenotype. Starting from this evidence, here we discuss the validity of past and more recent literature supporting that, as part of protein platforms at the cell surface, PrP(C) may bridge extracellular matrix molecules and/or membrane proteins to intracellular signaling pathways.

Direct detection of soil-bound prions.

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrP(Sc)) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challenged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures.