Diagnostic tests for human and animal prion diseases

The potential existence of clinically silent cases of bovine spongiform encephalopathy (BSE) among cattle, and of humans incubating the new variant Creutzfeldt-Jakob disease (nvCJD) is still a major public health concern. Therefore, the development of screening tests for transmissible subacute spongiform encephalopathies (TSSE) in man and animals remains a priority. In the first part of this paper, we review the main methods used to diagnose generally clinical TSSE, such as brain imaging, electroencephalogram (EEG) analysis, and cerebrospinal fluid (CSF) analysis. In the second part, we present the post-mortem tests used to confirm a TSSE diagnosis, such as inoculation to laboratory animals, histological examination, and identification of abnormal prion protein (PrPres) using biochemical methods. Finally, the third part presents so-called rapid tests (Prionics, Bio-rad, Enfer), validated by the European Commission (EC) for post-slaughter BSE diagnosis in cattle. Now used on a large scale in Europe, these tests have helped assess the extent of the epizooty and eliminate from the food chain animals presenting a risk for human consumption. Since 2002, they have been used for the post-slaughter diagnosis of scrapie in small ruminants. New tests have recently been evaluated by the EC, but it is too soon to predict their role in the field.L'existence potentielle de bovins en phase de latence cliniquement silencieuse d'encéphalopathie spongiforme bovine (ESB) et d'individus en période d'incubation de la nouvelle variante de la maladie de Creutzfeldt-Jakob représente constamment un grand risque pour la santé publique. Par conséquent, le développement de tests de dépistage des encéphalopathies spongiformes subaiguës transmissibles (ESST) humaines et animales constitue toujours une priorité. Dans la première partie de cet article, sont décrites les principales méthodes d'orientation permettant d'aider au diagnostic d'une ESST le plus souvent clinique, comme l'imagerie médicale cérébrale, l'analyse de l'électroencéphalogramme (EEG) et l'examen du liquide céphalo-rachidien. Dans la deuxième partie, sont présentés les tests de confirmation post mortem du diagnostic des ESST, comme l'inoculation à l'animal de laboratoire, l'examen histologique et la recherche de la PrPres par des méthodes biochimiques. La troisième partie est consacrée aux tests dits « rapides » (Prionics, Bio-rad, Enfer), validés en 1999 par la Commission Européenne (CE), pour le diagnostic post mortem de l'ESB à l'abattoir chez les bovins. Utilisés à grande échelle en Europe, ils ont permis de préciser l'étendue réelle de l'épizootie et d'éliminer efficacement de la chaîne alimentaire les animaux présentant un risque pour l'homme. Depuis 2002, ils sont également utilisés pour le diagnostic post mortem des petits ruminants. De nouveaux tests ont été récemment évalués par la CE, mais il est trop tôt pour évaluer la place qu'ils tiendront sur le terrain

HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tau pathology

Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. in Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 +/- 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 +/- 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P-301L mutation hTau P-301L, and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-kappa B p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau P-301L, that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer's disease.Association France Alzheimer & Maladies ApparenteesSATT Idf InnovCONACyT, MexicoFrench Ministry of Higher Education and ResearchInstitute de Recherche ServierUniv Paris Est, CNRS, Lab Cell Growth Tissue Repair & Regenerat CRRET, UPEC,EA 4397,ERL 9215, F-94000 Creteil, FranceUPMC, Univ Paris 04, Inst Cerveau & Moelle Epiniere, CNRS,UMR 7225,INSERM,U1127,UM75, Paris, FranceHop Robert Debre, INSERM, UMR 1141, F-75019 Paris, FranceSorbonne Paris Cite, Univ Paris Diderot, Paris, FranceUniversidade Federal de São Paulo, Aging & Neurodegenerat Dis Brain Bank Invest Lab, BR-04023062 São Paulo, BrazilGrp Hosp Pitie Salpetriere, Biochim Malad Neurometab, F-75013 Paris, FranceRadboud Univ Nijmegen, Med Ctr, Radboud Inst Mol Life Sci, NL-6525 ED Nijmegen, NetherlandsUniv Strasbourg, INSERM, U1119, FMTS, F-67000 Strasbourg, FranceUniversidade Federal de São Paulo, Aging & Neurodegenerat Dis Brain Bank Invest Lab, BR-04023062 São Paulo, BrazilCONACyT, Mexico: 308978Web of Scienc

Nouvelles approches thérapeutiques des maladies à prions (étude des mimétiques des héparanes sulfates)

Les encéphalopathies subaiguës spongiformes transmissibles (ESST), plus communément appelées maladies à prions, sont des maladies lentes neurodégénératives transmissibles strictement confinées au système nerveux central (SNC). Elles touchent à la fois l homme et l animal. Les ESST sont caractérisées par l accumulation dans le cerveau de la forme anormale (PrPres) d une protéine de l hôte (PrPc). La PrPres constitue le seul marqueur spécifique des maladies à prions. L inhibition de son accumulation est largement utilisée pour évaluer l efficacité des différentes molécules étudiées à des fins thérapeutiques. À l heure actuelle, il n existe aucune thérapie pour les ESST. Donc, le développement d une nouvelle thérapie pour les ESST humaine est d une importance cruciale. L étude comprend trois volets : 1) un travail impliquant la synthèse chimique d une bibliothèque de molécules de type héparane mimétique (HMs) incluant des composés différents (poids moléculaires, degrés de sulfatation et carboxyméthylation, ainsi que la présence ou pas de groupements hydrophobes), 2) l étude de la capacité des molécules synthétisées à lier la PrP de prion recombinante bovine et 3) l étude de leurs activités inhibitrices dans l accumulation de la PrPres in vitro (modèle ScGT1-7). Ces études ont permis d étudier l effet des modifications structurales de ces molécules sur l activité anti-prion et de sélectionner les composés les plus actifs in vitro. Nous avons montré que l activité anti-prion peut être optimisée notamment avec des molécules moyennement sulfatées et que ces activités peuvent être améliorées par l introduction d un noyau hydrophobe. Cette étude nous a permis également de montrer que des molécules de très faible poids moléculaire appelées des HM-oligosaccharides sont aussi efficaces pour inhiber l accumulation de la protéine du prion pathologique (PrPres) que les polyanions de haut poids moléculaire. De plus, nous avons démontré in vitro la capacité de ces molécules oligomériques à traverser la barrière hémato-encéphalique (BHE). Ces molécules constituent à l heure actuelle des outils thérapeutiques potentiels pour le traitement des maladies à prions voire même d autres maladies neurodégénératives où les héparanes sulfates semblent avoir des rôles régulateurs comme la maladie de Parkinson ou encore la maladie d Alzheimer.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Nouvelles approches thérapeutiques des maladies à prions (étude des mimétiques des héparanes sulfates)

Les encéphalopathies subaiguës spongiformes transmissibles (ESST), plus communément appelées maladies à prions, sont des maladies lentes neurodégénératives transmissibles strictement confinées au système nerveux central (SNC). Elles touchent à la fois l homme et l animal. Les ESST sont caractérisées par l accumulation dans le cerveau de la forme anormale (PrPres) d une protéine de l hôte (PrPc). La PrPres constitue le seul marqueur spécifique des maladies à prions. L inhibition de son accumulation est largement utilisée pour évaluer l efficacité des différentes molécules étudiées à des fins thérapeutiques. À l heure actuelle, il n existe aucune thérapie pour les ESST. Donc, le développement d une nouvelle thérapie pour les ESST humaine est d une importance cruciale. L étude comprend trois volets : 1) un travail impliquant la synthèse chimique d une bibliothèque de molécules de type héparane mimétique (HMs) incluant des composés différents (poids moléculaires, degrés de sulfatation et carboxyméthylation, ainsi que la présence ou pas de groupements hydrophobes), 2) l étude de la capacité des molécules synthétisées à lier la PrP de prion recombinante bovine et 3) l étude de leurs activités inhibitrices dans l accumulation de la PrPres in vitro (modèle ScGT1-7). Ces études ont permis d étudier l effet des modifications structurales de ces molécules sur l activité anti-prion et de sélectionner les composés les plus actifs in vitro. Nous avons montré que l activité anti-prion peut être optimisée notamment avec des molécules moyennement sulfatées et que ces activités peuvent être améliorées par l introduction d un noyau hydrophobe. Cette étude nous a permis également de montrer que des molécules de très faible poids moléculaire appelées des HM-oligosaccharides sont aussi efficaces pour inhiber l accumulation de la protéine du prion pathologique (PrPres) que les polyanions de haut poids moléculaire. De plus, nous avons démontré in vitro la capacité de ces molécules oligomériques à traverser la barrière hémato-encéphalique (BHE). Ces molécules constituent à l heure actuelle des outils thérapeutiques potentiels pour le traitement des maladies à prions voire même d autres maladies neurodégénératives où les héparanes sulfates semblent avoir des rôles régulateurs comme la maladie de Parkinson ou encore la maladie d Alzheimer.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

The role of heparan sulfates in protein aggregation and their potential impact on neurodegeneration

Neurodegenerative disorders, such as Alzheimer´s, Parkinson´s, and prion diseases, are directly linked to the formation and accumulation of protein aggregates in the brain. These aggregates, principally made of proteins or peptides that clamp together after acquisition of β-folded structures, also contain heparan sulfates. Several lines of evidence suggest that heparan sulfates centrally participate in the protein aggregation process. In vitro, they trigger misfolding, oligomerization, and fibrillation of amyloidogenic proteins, such as Aβ, tau, α-synuclein, prion protein, etc. They participate in the stabilization of protein aggregates, protect them from proteolysis, and act as cell-surface receptors for the cellular uptake of proteopathic seeds during their spreading. This review focuses attention on the importance of heparan sulfates in protein aggregation in brain disorders including Alzheimer´s, Parkinson´s, and prion diseases. The presence of these sulfated polysaccharides in protein inclusions in vivo and their capacity to trigger protein aggregation in vitro strongly suggest that they might play critical roles in the neurodegenerative process. Further advances in glyco-neurobiology will improve our understanding of the molecular and cellular mechanisms leading to protein aggregation and neurodegeneration.Fil: Maïza, Auriane. Universite de Paris; FranciaFil: Chantepie, Sandrine. Universite de Paris; FranciaFil: Vera, Claudia Cecilia. Universite de Paris; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Fifre, Alexandre. Universite de Paris; FranciaFil: Huynh, Minh Bao. Universite de Paris; FranciaFil: Stettler, Olivier. Universite de Paris; FranciaFil: Ouidja, Mohand Ouidir. Universite de Paris; FranciaFil: Papy Garcia, Dulce. Universite de Paris; Franci

Glycosaminoglycans from Alzheimer’s disease hippocampus have altered capacities to bind and regulate growth factors activities and to bind tau

International audienceGlycosaminoglycans (GAGs), including heparan sulfates and chondroitin sulfates, are major components of the extracellular matrix. Upon interacting with heparin binding growth factors (HBGF), GAGs participate to the maintaintenance of tissue homeostasis and contribute to self-healing. Although several processes regulated by HBGF are altered in Alzheimer’s disease, it is unknown whether the brain GAG capacities to bind and regulate the function of HBGF or of other heparin binding proteins, as tau, are modified in this disease. Here, we show that total sulfated GAGs from hippocampus of Alzheimer’s disease have altered capacities to bind and potentiate the activities of growth factors including FGF-2, VEGF, and BDNF while their capacity to bind to tau is remarkable increased. Alterations of GAG structures and capacities to interact with and regulate the activity of heparin binding proteins might contribute to impaired tissue homeostasis in the Alzheimer’s disease brain

Self-evolving oxidative stress with identifiable pre- and postmitochondrial phases in PC12 cells

International audienc

New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease

International audienceThe causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neu-roblastoma cell model of PD induced by MPP + , the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway , regulating cathD activity and affecting two main biological processes, α-synuclein ag-gregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology

Structure-activity studies of heparan mimetic polyanions for anti-prion therapies.

Polysulfated molecules, as the family of heparan mimetics (HMs) and pentosan polysulfate, are considered among the more promising drugs used in experimental models of prion diseases. Regardless of their therapeutic potential, structure-function studies on these polyanions are still missing. Here, we report the syntheses of a library of HMs of different molecular sizes, containing various sulfation and carboxylation levels, and substituted or not by different hydrophobic cores. The HMs capacities to inhibit the accumulation of PrPres in chronically infected cells (ScGT1-7) and their PrPc binding abilities were examined. Our results showed that an optimal size and sulfation degree are needed for optimum activity, that incorporation of hydrophobic moieties increases compounds efficacy and that the presence of carboxymethyl moieties decreases it. These structural features should be considered on the modelling of polyanionic compounds for optimum anti-prion activities and for advancing in the understanding the mechanisms involved in their biological actions

Characterization of MPP⁺-induced apoptosis.

<p>Total, mitochondrial and cytosolic extracts were obtained from normal and MPP⁺-stressed SHSY5Y cells (0.5 mM, 6 h).a) CathD activity in cytosolic extracts. CathD activity was normalized by cell number in each sample and expressed as percentage of the activity in normal cells. Results are expressed in percentage of normal cell extract levels and presented as mean ± S.E.M. of four independent experiments in triplicate. *** p<0.01 compared to control cells. b) Immunofluorescence detection of total cathD in cells. Left: unstressed control cells (Ctrl), right: MPP⁺-stressed cells (6 h). Cells were observed with a confocal microscope Zeiss Axio Observer Z.1. Scale bar represents 10 μm. Negative control omitting the first antibody but in the presence of the second one (anti-goat Fluo 546) did not show any signal. c) Analysis by western blot of Bax relocation in mitochondrial membranes at 6 h, just after the end of stress. The amount of protein was normalized by the number of cells to avoid taking into account the increase of protein amount induced by the MPP⁺ stress. Succinate dehydrogenase-A (SDHA) was used as the loading control. This immunoblot is representative of three independent experiments in duplicate. d) Assessment of respiratory chain function 24 h after MPP⁺ treatment. The respiratory control index (RCI) i.e. ratio [state 3 rate] / [state 4 rate] was calculated. Results are expressed as percentage of unstressed control group and represent three independent experiments in triplicate. Results are presented as the mean ± S.E.M. *** p<0.01 compared to control cells.</p