Prion Protein Expression and Processing in Human Mononuclear Cells: The Impact of the Codon 129 Prion Gene Polymorphism

BACKGROUND:So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD), thought to result from the Bovine Spongiform Encephalopathy (BSE) prion agent, have shown Methionine-Methionine (M/M) homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion. METHODOLOGY/PRINCIPAL FINDINGS:We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs) from three blood donor populations with Methionine-Methionine (M/M), Valine-Valine (V/V) and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism. CONCLUSIONS/SIGNIFICANCE:These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues

Two Dimensional Gel Electrophoresis Analysis of Mesenchymal Stem Cells

International audienceProteomic 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

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

Peripheral Blood and Salivary Biomarkers of Blood-Brain Barrier Permeability and Neuronal Damage:Clinical and Applied Concepts

International audienceWithin the neurovascular unit (NVU), the blood-brain barrier (BBB) operates as a key cerebrovascular interface, dynamically insulating the brain parenchyma from peripheral blood and compartments. Increased BBB permeability is clinically relevant for at least two reasons: it actively participates to the etiology of central nervous system (CNS) diseases, and it enables the diagnosis of neurological disorders based on the detection of CNS molecules in peripheral body fluids. In pathological conditions, a suite of glial, neuronal, and pericyte biomarkers can exit the brain reaching the peripheral blood and, after a process of filtration, may also appear in saliva or urine according to varying temporal trajectories. Here, we specifically examine the evidence in favor of or against the use of protein biomarkers of NVU damage and BBB permeability in traumatic head injury, including sport (sub)concussive impacts, seizure disorders, and neurodegenerative processes such as Alzheimer's disease. We further extend this analysis by focusing on the correlates of human extreme physiology applied to the NVU and its biomarkers. To this end, we report NVU changes after prolonged exercise, freediving, and gravitational stress, focusing on the presence of peripheral biomarkers in these conditions. The development of a biomarker toolkit will enable minimally invasive routines for the assessment of brain health in a broad spectrum of clinical, emergency, and sport settings

Unravelling landscape variables with multiple approaches to overcome scarce species knowledge: a landscape genetic study of the slow worm

Landscape genetics was developed to detect landscape elements shaping genetic population structure, including the effects of fragmentation. Multifarious environmental variables can influence gene flow in different ways and expert knowledge is frequently used to construct friction maps. However, the extent of the migration and the movement of single individuals are frequently unknown, especially for non-model species, and friction maps only based on expert knowledge can be misleading. In this study, we used three different methods: isolation by distance (IBD), least-cost modelling and a strip-based approach to disentangle the human implication in the fragmentation process in the slow worm (Anguis fragilis), as well as the specific landscape elements shaping the genetic structure in a highly anthropized 16km2 area in Switzerland. Friction maps were constructed using expert opinion, but also based on the combination of all possible weightings for all landscape elements. The IBD indicated a significant effect of geographic distance on genetic differentiation. Further approaches demonstrated that highways and railways were the most important elements impeding the gene flow in this area. Surprisingly, we also found that agricultural areas and dense forests seemed to be used as dispersal corridors. These results confirmed that the slow worm has relatively unspecific habitat requirements. Finally, we showed that our models based on expert knowledge performed poorly compared to cautious analysis of each variable. This study demonstrated that landscape genetic analyses should take expert knowledge with caution and exhaustive analyses of each landscape element without a priori knowledge and different methods can be recommende

Cerebrospinal fluid phospho-tau T217 outperforms T181 as a biomarker for the differential diagnosis of Alzheimer\u27s disease and PET amyloid-positive patient identification

BACKGROUND: Cerebrospinal fluid biomarker profiles characterized by decreased amyloid-beta peptide levels and increased total and phosphorylated tau levels at threonine 181 (pT181) are currently used to discriminate between Alzheimer\u27s disease and other neurodegenerative diseases. However, these changes are not entirely specific to Alzheimer\u27s disease, and it is noteworthy that other phosphorylated isoforms of tau, possibly more specific for the disease process, have been described in the brain parenchyma of patients. The precise detection of these isoforms in biological fluids remains however a challenge. METHODS: In the present study, we used the latest quantitative mass spectrometry approach, which achieves a sensitive detection in cerebrospinal fluid biomarker of two phosphorylated tau isoforms, pT181 and pT217, and first analyzed a cohort of probable Alzheimer\u27s disease patients and patients with other neurological disorders, including tauopathies, and a set of cognitively normal controls. We then checked the validity of our results on a second cohort comprising cognitively normal individuals and patients with mild cognitive impairments and AD stratified in terms of their amyloid status based on PiB-PET imaging methods. RESULTS: In the first cohort, pT217 but not pT181 differentiated between Alzheimer\u27s disease patients and those with other neurodegenerative diseases and control subjects much more specificity and sensitivity than pT181. T217 phosphorylation was increased by 6.0-fold in patients with Alzheimer\u27s disease whereas T181 phosphorylation was only increased by 1.3-fold, when compared with control subjects. These results were confirmed in the case of a second cohort, in which the pT217 cerebrospinal fluid levels marked out amyloid-positive patients with a sensitivity and a specificity of more than 90% (AUC 0.961; CI 0.874 to 0.995). The pT217 concentrations were also highly correlated with the PiB-PET values (correlation coefficient 0.72; P \u3c 0.001). CONCLUSIONS: Increased cerebrospinal fluid pT217 levels, more than those of pT181, are highly specific biomarkers for detecting both the preclinical and advanced forms of Alzheimer\u27s disease. This finding should greatly improve the diagnosis of Alzheimer\u27s disease, along with the correlations found to exist between pT217 levels and PiB-PET data. It also suggests that pT217 is a promising potential target for therapeutic applications and that a link exists between amyloid and tau pathology

Diagnosis of Methionine/Valine Variant Creutzfeldt-Jakob Disease by Protein Misfolding Cyclic Amplification

A patient with a heterozygous variant of Creutzfeldt-Jakob disease (CJD) with a methionine/valine genotype at codon 129 of the prion protein gene was recently reported. Using an ultrasensitive and specific protein misfolding cyclic amplification–based assay for detecting variant CJD prions in cerebrospinal fluid, we discriminated this heterozygous case of variant CJD from cases of sporadic CJD

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in ‘functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP

Blood amyloid and tau biomarkers as predictors of cerebrospinal fluid profiles

Blood biomarkers represent a major advance for improving the management, diagnosis, and monitoring of Alzheimer's disease (AD). However, their context of use in relation to routine cerebrospinal fluid (CSF) analysis for the quantification of amyloid peptides and tau proteins remains to be determined. We studied in two independent cohorts, the performance of blood biomarkers in detecting "nonpathological" (A−/T−/N−), amyloid (A+) or neurodegenerative (T+ /N+) CSF profiles. Plasma Aβ/Aβ ratio and phosphorylated tau (p-tau(181)) were independent and complementary predictors of the different CSF profile and in particular of the nonpathological (A−/T−/N−) profile with a sensitivity and specificity close to 85%. These performances and the corresponding biomarker thresholds were significantly different from those related to AD detection. The use of blood biomarkers to identify patients who may benefit from secondary CSF testing represents an attractive stratification strategy in the clinical management of patients visiting memory clinics. This could reduce the need for lumbar puncture and foreshadow the use of blood testing on larger populations. The online version contains supplementary material available at 10.1007/s00702-022-02474-9