Prion strains are differentially released through the exosomal pathway.

Cell-to-cell transfer of prions is a crucial step in the spreading of prion infection through infected tissue. At the cellular level, several distinct pathways including direct cell-cell contacts and release of various types of infectious extracellular vesicles have been described that may potentially lead to infection of naïve cells. The relative contribution of these pathways and whether they may vary depending on the prion strain and/or on the infected cell type are not yet known. In this study we used a single cell type (RK13) infected with three different prion strains. We showed that in each case, most of the extracellular prions resulted from active cell secretion through the exosomal pathway. Further, quantitative analysis of secreted infectivity indicated that the proportion of prions eventually secreted was dramatically dependent on the prion strain. Our data also highlight that infectious exosomes secreted from cultured cells might represent a biologically pertinent material for spiking experiments. Also discussed is the appealing possibility that abnormal PrP from different prion strains may differentially interact with the cellular machinery to promote secretion.

Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway.

Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation.

Autoantibody profiling on high-density protein microarrays for biomarker discovery in the cerebrospinal fluid.

Detection of autoantibodies, which are involved in tissue injury and/or the reporters from the immune system of various pathologic events, has an important potential for diagnosis, prognosis, disease staging and treatment selection. This explains the interest for new proteomics technologies, such as the high-density protein microarray used here, that allow a high-throughput, multiplexed and sensitive detection of specific autoantibodies. So far, most of the research has been performed on blood. In this note, we focus on the cerebrospinal fluid in an attempt to address autoimmune events associated with neurological disorders. Importantly, the cerebrospinal fluid is quite different from the blood in terms of protein composition and concentration. We had therefore to adapt the available blood protocols. We present here the result of our optimization that will be useful to carry out full scale immunological studies of the cerebrospinal fluid using high-density protein microarrays.

Isolation of Exosomes and Microvesicles from Cell Culture Systems to Study Prion Transmission.

Extracellular vesicles (EVs) are composed of microvesicles and exosomes. Exosomes are small membrane vesicles (40-120 nm sized) of endosomal origin released in the extracellular medium from cells when multivesicular bodies fuse with the plasma membrane, whereas microvesicles (i.e., shedding vesicles, 100 nm to 1 µm sized) bud from the plasma membrane. Exosomes and microvesicles carry functional proteins and nucleic acids (especially mRNAs and microRNAs) that can be transferred to surrounding cells and tissues and can impact multiple dimensions of the cellular life. Most of the cells, if not all, from neuronal to immune cells, release exosomes and microvesicles in the extracellular medium, and all biological fluids including blood (serum/plasma), urine, cerebrospinal fluid, and saliva contain EVs.Prion-infected cultured cells are known to secrete infectivity into their environment. We characterized this cell-free form of prions and showed that infectivity was associated with exosomes. Since exosomes are produced by a variety of cells, including cells that actively accumulate prions, they could be a vehicle for infectivity in body fluids and could participate to the dissemination of prions in the organism. In addition, such infectious exosomes also represent a natural, simple, biological material to get key information on the abnormal PrP forms associated with infectivity.In this chapter, we describe first a method that allows exosomes and microvesicles isolation from prion-infected cell cultures and in a second time the strategies to characterize the prions containing exosomes and their ability to disseminate the prion agent.

Interest of major serum protein removal for Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) proteomic blood profiling.

BACKGROUND: Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) has been proposed as new approach for blood biomarker discovery. However, results obtained so far have been often disappointing as this technique still has difficulties to detect low-abundant plasma and serum proteins. RESULTS: We used a serum depletion scheme using chicken antibodies against various abundant proteins to realized a pre-fractionation of serum prior to SELDI-TOF profiling. Depletion of major serum proteins by immunocapture was confirmed by 1D and 2D gel electrophoresis. SELDI-TOF analysis of bound and unbound (depleted) serum fractions revealed that this approach allows the detection of new low abundant protein peaks with satisfactory reproducibility. CONCLUSION: The combination of immunocapture and SELDI-TOF analysis opens new avenues into proteomic profiling for the discovery of blood biomarkers.

Two dimensional gel electrophoresis analysis of mesenchymal stem cells.

Proteomic analysis is a powerful tool to follow physiological modifications and phenotypes of mesenchymal stem cells (MSC). This approach generates informative data on expression and post-translational modifications of proteins which are of interest to assess the true potential of MSC in regenerative medicine. No matter the technologies used, proteomic analysis is always a challenge as the proteome is extremely diverse (in terms of constituents and concentrations), is changing with time, and is highly sensitive to pre-analytical conditions. In the framework of a European project (GENOSTEM http://www.genostem.org/), we have set up a multisite two dimensional gel electrophoresis (2DE) proteomic comparison of MSC. The goal is to compare cells from different origins, to follow their differentiation and to ultimately define a specific MSC proteomic signature. One important initial task is the optimization of 2DE protocols such that they are robust enough to be used in a multisite project. In this chapter, we detail these protocols which can be used not only for MSC but also for other cells in culture.

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells.

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP(C)) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP(C) is converted into an abnormally folded isoform, called PrP(Sc), that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP(C) and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrP(Sc) production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.

Depletion of one, six, twelve or twenty major blood proteins before proteomic analysis: the more the better?

Depletion of major blood proteins is one of the most promising approaches to access low abundant biomarkers using proteomics. Immunocapture columns often used for this purpose exist in different formats depending on the number of major proteins removed. In this article, we compared the relative interest of depleting either one (albumin), six (albumin, IgG, IgA, transferrin, alpha1-antitrypsin, and haptoglobin), twelve (the previous six and apo A-I and -II, orosomucoid, alpha2-macroglobulin, fibrinogen, IgM) or twenty blood proteins (the previous twelve and IgD, ceruloplasmin, apo B, complement C1q, C3, C4, plasminogen, and prealbumin). Such study raises interesting issues related to the reproducibility, practicability, specificity of the immunocapture, and to the impact of removing not only the selected molecules, but also associated peptides and proteins. Depleted sera were here analysed using different proteomic approaches, including two dimensional electrophoresis and SELDI-TOF. Altogether, our results clearly confirmed the interest of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins. This conclusion is however related to the technologies that we have used, and we believe that it is necessary to adapt the immunocapture to the analytical method employed, and to the ratio between wanted and unwanted proteins removed.

Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines.

The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.