Dominique Bétemps

A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice

The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.

Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological α-synuclein by enhanced ELISA

BackgroundThe accumulation of misfolded proteins appears as a fundamental pathogenic process in human neurodegenerative diseases. In the case of synucleinopathies such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB), the intraneuronal deposition of aggregated alpha-synuclein (αS) is a major characteristic of the disease, but the molecular basis distinguishing the disease-associated protein (αSD) from its normal counterpart remains poorly understood. However, recent research suggests that a prion-like mechanism could be involved in the inter-cellular and inter-molecular propagation of aggregation of the protein within the nervous system.ResultsOur data confirm our previous observations of disease acceleration in a transgenic mouse line (M83) overexpressing a mutated (A53T) form of human αS, following inoculation of either brain extracts from sick M83 mice or fibrillar recombinant αS. A similar phenomenon is observed following a “second passage” in the M83 mouse model, including after stereotactic inoculations into the hippocampus or cerebellum. For further molecular analyses of αSD, we designed an ELISA test that identifies αSD specifically in sick mice and in the brain regions targeted by the pathological process in this mouse model. αSD distribution, mainly in the caudal brain regions and spinal cord, overall appears remarkably uniform, whatever the conditions of experimental challenge. In addition to specific detection of αSD immunoreactivity using an antibody against Ser129 phosphorylated αS, similar results were observed in ELISA with several other antibodies against the C-terminal part of αS, including an antibody against non phosphorylated αS. This also indicated consistent immunoreactivity of the murine αS protein specifically in the affected brain regions of sick mice.ConclusionsPrion-like behaviour in propagation of the disease-associated αS was confirmed with the M83 transgenic mouse model, that could be followed by an ELISA test. The ELISA data question their possible relationship with the conformational differences between the disease-associated αS and its normal counterpart.

Transmissibility of atypical scrapie in ovine transgenic mice: major effects of host prion protein expression and donor prion genotype.

Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrPres and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain ∼0.25, 1.5 or 6× the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrPc measurements that appeared to reflect the different PrPc expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrPc expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing ∼6× the normal PrPc level died after a survival periods of 400 days, those with ∼1.5× the normal PrPc level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF141RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrPres in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrPres products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein.

Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein.

There is a growing interest in the potential roles of misfolded protein interactions in neurodegeneration. To investigate this issue, we inoculated 3 prion strains intracerebrally into transgenic (TgM83) mice that overexpress human A53T &agr;-synuclein. In comparison to nontransgenic controls, there was a striking decrease in the incubation periods of scrapie, classic and H-type bovine spongiform encephalopathies(C-BSE and H-BSE), with conservation of the histopathologic and biochemical features characterizing these 3 prion strains. TgM83 mice died of scrapie or C-BSE prion diseases before accumulating the insoluble and phosphorylated forms of &agr;-synuclein specific to late stages of synucleinopathy. In contrast, the median incubation time for TgM83 mice inoculated with H-BSE was comparable to that observed when these mice were uninfected, thereby allowing the development of molecular alterations of &agr;-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with &agr;-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T &agr;-synuclein after intracerebral inoculation with an H-BSE prion strain.

Specific Pesticide-Dependent Increases in α-Synuclein Levels in Human Neuroblastoma (SH-SY5Y) and Melanoma (SK-MEL-2) Cell Lines

Epidemiological studies indicate a role of genetic and environmental factors in Parkinsons disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinsons disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinsons disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinsons disease.

Production of a monoclonal antibody, against human α-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the α-synuclein locus

Analyses using antibodies directed against α-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). However, the generation of antibodies against immunogens with significant sequence similarity to host proteins such as α-synuclein is often hindered by host immunotolerance. In contrast to wild-type C57BL/6J and BALB/c mice immunized with recombinant human α-synuclein, C57BL/6S Δsnca mice presenting a natural deletion of the α-synuclein locus, bypassed the immunotolerance process which resulted in a much higher polyclonal antibody response. The native or fibrillized conformation of α-synuclein used as the immunogen did not have an impact on the amounts of specific antibodies in sera of the host. The immunization protocols resulted in the generation of the IgG AS11, raised against fibrillized recombinant human α-synuclein in C57BL/6S Δsnca mice. This monoclonal antibody, recognizing an N-terminal α-synuclein epitope, was selected for its specificity and significant reactivity in Western-blot, immunofluorescence and immunohistochemistry assays. The ability of AS11 to detect both soluble and aggregated forms of α-synuclein present in pathological cytoplasmic inclusions was further assessed using analysis of human brains with PD or MSA, transgenic mouse lines expressing A53T human α-synuclein, and cellular models expressing human α-synuclein. Taken together, our study indicates that novel antibodies helpful to characterize alterations of α-synuclein leading to neurodegeneration in PD and related disorders could be efficiently developed using this original immunization strategy.

RETRACTED ARTICLE: A variety of pesticides trigger in vitro α-synuclein accumulation, a key event in Parkinson’s disease

apologize to the readers of Archives of Toxicology for any inconvenience caused by this retraction. The online version of this article contains the full text of the retracted article as electronic supplementary material. This article has been retracted at the request of the authors and by agreement of the editor. Re-analyses of the raw data did not confirm the published data and they did not support the major conclusions of this publication. The authors

Investigating the neuroprotective effect of AAV-mediated β-synuclein overexpression in a transgenic model of synucleinopathy

Parkinson’s disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of α-synuclein (α-syn) aggregates. The objective of this study was to investigate if β-synuclein (β-syn) overexpression could have beneficial effects by inhibiting the aggregation of α-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of α-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human β-syn gene (AAVβ-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. One or two months later, M83 disease was accelerated or not using brain M83 extracts from mouse (M83) or human (MSA) origins. AAV mediated β-syn overexpression detected by ELISA did not delay the disease onset, regardless of the AAV injection route and of the inoculation of brain extracts. Accordingly, phosphorylated α-syn levels detected by ELISA in sick mice were similar after injecting AAVβ-syn or AAVGFP. Instead, immunohistochemistry analysis of β-syn indicated the presence of proteinase-K resistant β-syn staining specifically in sick M83 mice inoculated with AAVβ-syn. This study indicated for the first time that β-syn could form aggregates in a model of synucleinopathy when it is expressed by a viral vector.

Characterization of a mAb against human α-synuclein

Background: Learning digital instruments is to democratize access to daily technology, include elder people in the information society and preserve independence, autonomy and citizenship. Methods: Forty workshops (130 hours) with 46 communities elder people above 60 years using digital instruments in Local Area Network Houses, between 2004 and 2006, in São Paulo city and Florianópolis city, Brazil. Sociodemographic, health, cognitive and functional variables were measured and compared to learning time of: mouse, electronic games, browser and e-mail. Kaplan-Meier curves were plotted to analyze learning time. Statistical differences between the covariates were evaluated by log-rank test. The impact of covariates on learning time was analyzed by Cox proportional risk model, in SPSS software. Results: The average age was 73 years with 4,2 pathologies per person, 9,1 years of scholarship, 26% presented falls in the last 6 months. The mean learning time (in hours) for ‘‘basic use of the mouse’’ was 8,5 (CI95%:6,610,5), for ‘‘game use’’ was 10,0 (CI95%:6,8-13,2), for ‘‘browser use’’ was 16,0 (CI95%:11,8-20,2) and for ‘‘e-mail use’’ was 27,0 (CI95%:24,629,4). Hypertension, diabetes and falls in the last 6 months were not significant variables even in the bivariate analysis. More than 3 pathologies, more than 3 continuous medications, dislipidemia, hypotiroidism, activities of the daily living losses and low scholarship had no independent significance for the outcomes studied. The cognitive status was an independent factor related to learning time of mouse, games and browser. Age above 75 years and cognitive status were independent factors related to e-mail use. Conclusions: Modifiable factors that influence learning time of elder people must be screened and treated to promote access to the information society by elder people. The ability to conquer new proficiencies is a characteristic of healthy elder people.