The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons.

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads.

The Link of the Prion Protein with Ca2+ Metabolism and ROS Production, and the Possible Implication in Aß Toxicity.

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

Age-dependent neuromuscular impairment in prion protein knockout mice.

INTRODUCTION: The cellular prion protein (PrP(C) ) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP(C) is still ambiguous. Evidence has suggested that PrP(C) is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. METHODS: We analyzed the age-dependent influence of PrP(C) on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP(C) . RESULTS: We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrP-expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. CONCLUSION: PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons.

Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.

Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle.

The fraudulent treatment of cattle with growth promoting agents (GPAs) is a matter of great concern for the European Union (EU) authorities and consumers. It has been estimated that 10% of animals are being illegally treated in the EU. In contrast, only a much lower percentage of animals (<0.5%) are actually found as being noncompliant by conventional analytical methods. Thus, it has been proposed that methods should be developed that can detect the use of the substances via the biological effects of these substances on target organs, such as the alteration of protein expression profiles. Here we present a study aimed at evaluating if a correlation exists between the treatment with GPAs and alterations in the two-dimensional electrophoresis (2DE) protein pattern obtained from the biceps brachii skeletal muscle from mixed-bred cattle. After image analysis and statistical evaluation, protein spots that differentiate between treated and control groups were selected for analysis by mass spectrometry. A set of proteins could be defined that accurately detect the use of glucocorticoids and ß(2)-agonists as growth promoters through the changes caused in muscle differentiation. As a further validation, we repeated the analysis using an independent set of samples from a strain of pure-bred cattle and verified these proteins by Western blot analysis.

Cellular prion protein is implicated in the regulation of local Ca2+ movements in cerebellar granule neurons.

The cellular prion protein (PrP(C)) is a cell-surface glycoprotein mainly expressed in the CNS. The structural conversion of PrP(C) generates the prion, the infectious agent causing transmissible spongiform encephalopathies, which are rare and fatal diseases affecting animals and humans. Despite decades of intensive research, the mechanism of prion-associated neurodegeneration and the physiologic role of PrP(C) are still obscure. Recent evidence, however, supports the hypothesis that PrP(C) may be involved in the control of Ca(2+) homeostasis. Given the universal significance of Ca(2+) as an intracellular messenger for both the life and death of cells, this possibility may help explain the complex, often controversial, dataset accumulated on PrP(C) physiology, and the events leading to prion-associated neuronal demise. In this study, we have compared local Ca(2+) movements in cerebellar granule neurons (CGN) derived from wild-type (WT), or PrP-knockout (KO), mice, by means of the Ca(2+)-sensitive photo-probe, aequorin, genetically targeted to specific intracellular domains and delivered to CGN by lentiviral vectors. The use of an aequorin that localizes to the cytosolic domains proximal to the plasma membrane has allowed us to demonstrate that there was a dramatic increase of store-operated Ca(2+) entry in PrP-KO CGN compared to WT neurons. Notably, this phenotype was rescued upon restoring PrP(C) expression. The Ca(2+)-phenotype of PrP-KO neurons can in part be explained by the lower expression of two major Ca(2+)-extruding proteins, namely the plasma membrane and the sarco-endoplasmic reticulum Ca(2+)-ATPases. The lower sarco-endoplasmic reticulum Ca(2+)-ATPase content may also contribute to explain why PrP-KO CGN accumulated less Ca(2+) in the endoplasmic reticulum than the WT counterpart.

The Prion Protein Regulates Synaptic Transmission by Controlling the Expression of Proteins Key to Synaptic Vesicle Recycling and Exocytosis.

The cellular prion protein (PrPC), whose misfolded conformers are implicated in prion diseases, localizes to both the presynaptic membrane and postsynaptic density. To explore possible molecular contributions of PrPC to synaptic transmission, we utilized a mass spectrometry approach to quantify the release of glutamate from primary cerebellar granule neurons (CGN) expressing, or deprived of (PrP-KO), PrPC, following a depolarizing stimulus. Under the same conditions, we also tracked recycling of synaptic vesicles (SVs) in the two neuronal populations. We found that in PrP-KO CGN these processes decreased by 40 and 60%, respectively, compared to PrPC-expressing neurons. Unbiased quantitative mass spectrometry was then employed to compare the whole proteome of CGN with the two PrP genotypes. This approach allowed us to assess that, relative to the PrPC-expressing counterpart, the absence of PrPC modified the protein expression profile, including diminution of some components of SV recycling and fusion machinery. Subsequent quantitative RT-PCR closely reproduced proteomic data, indicating that PrPC is committed to ensuring optimal synaptic transmission by regulating genes involved in SV dynamics and neurotransmitter release. These novel molecular and cellular aspects of PrPC add insight into the underlying mechanisms for synaptic dysfunctions occurring in neurodegenerative disorders in which a compromised PrPC is likely to intervene.

The prion protein and its paralogue Doppel affect calcium signaling in Chinese hamster ovary cells.

The function of the prion protein (PrP(c)), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrP(c) on Ca(2+) homeostasis, by analyzing local Ca(2+) fluctuations in cells transfected with PrP(c) and Ca(2+)-sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrP(c) sharply increases the Ca(2+) concentration of subplasma membrane Ca(2+) domains, a feature that may explain the impairment of Ca(2+)-dependent neuronal excitability observed in TSEs. PrP(c) also limits Ca(2+) release from the endoplasmic reticulum and Ca(2+) uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrP(c) paralogue, display opposite effects, which, however, are abolished by the coexpression of PrP(c). These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrP(c) protects, and Doppel sensitizes, cells toward stress conditions.

Generation and validation of novel adeno-associated viral vectors for the analysis of Ca2+ homeostasis in motor neurons.

A finely tuned Ca2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca2+ oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca2+ indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca2+ measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca2+ responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.

Heterogeneous PrPC metabolism in skeletal muscle cells.

Recent reports have shown that prions, the causative agent of transmissible spongiform encephalopathies, accumulate in the skeletal muscle of diseased animals and man. In an attempt to characterise in this tissue the prion protein (PrP(C)), whose conformational rearrangement governs the generation of prions, we have analysed the protein in primary cultured murine myocytes and in different skeletal muscle types. Our results indicate that the expression and cellular processing of PrP(C) change during myogenesis, and in muscle fibres with different contractile properties. These findings imply a potential role for PrP(C) in the skeletal muscle physiology, but may also explain the different capability of muscles to sustain prion replication.

Prions and prion-like pathogens in neurodegenerative disorders.

Prions are unique elements in biology, being able to transmit biological information from one organism to another in the absence of nucleic acids. They have been identified as self-replicating proteinaceous agents responsible for the onset of rare and fatal neurodegenerative disorders-known as transmissible spongiform encephalopathies, or prion diseases-which affect humans and other animal species. More recently, it has been proposed that other proteins associated with common neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, can self-replicate like prions, thus sustaining the spread of neurotoxic entities throughout the nervous system. Here, we review findings that have contributed to expand the prion concept, and discuss if the involved toxic species can be considered bona fide prions, including the capacity to infect other organisms, or whether these pathogenic aggregates share with prions only the capability to self-replicate.

The cellular prion protein counteracts cardiac oxidative stress.

AIMS: The cellular prion protein, PrP(C), whose aberrant isoforms are related to prion diseases of humans and animals, has a still obscure physiological function. Having observed an increased expression of PrP(C) in two in vivo paradigms of heart remodelling, we focused on isolated mouse hearts to ascertain the capacity of PrP(C) to antagonize oxidative damage induced by ischaemic and non-ischaemic protocols. METHODS AND RESULTS: Hearts isolated from mice expressing PrP(C) in variable amounts were subjected to different and complementary oxidative perfusion protocols. Accumulation of reactive oxygen species, oxidation of myofibrillar proteins, and cell death were evaluated. We found that overexpressed PrP(C) reduced oxidative stress and cell death caused by post-ischaemic reperfusion. Conversely, deletion of PrP(C) increased oxidative stress during both ischaemic preconditioning and perfusion (15 min) with H2O2. Supporting its relation with intracellular systems involved in oxidative stress, PrP(C) was found to influence the activity of catalase and, for the first time, the expression of p66(Shc), a protein implicated in oxidative stress-mediated cell death. CONCLUSIONS: Our data demonstrate that PrP(C) contributes to the cardiac mechanisms antagonizing oxidative insults.

Altered behavioral aspects of aged mice lacking the cellular prion protein.

The biological function of the prion protein, which is intimately involved in the onset of prion diseases, remains unclear. To understand whether the prion protein could play a role in animal behavior, a battery of tests was applied to young and aged mice that express, or not, the prion protein. In contrast to the similar results obtained in all young animals, we found that aged mice lacking the prion protein reacted to new and stressful environments differently than their wild-type counterparts. This may suggest that, upon aging, the absence of the prion protein results in altered neural processing at the basis of adaptation to new situations.

Selective and efficient immunoprecipitation of the disease-associated form of the prion protein can be mediated by nonspecific interactions between monoclonal antibodies and scrapie-associated fibrils.

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads.