[Show abstract][Hide abstract] ABSTRACT: In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated α-synuclein (αS(D)) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T αS and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of αS(D) (disease-associated αS) in these mice, an ELISA was designed to specifically quantify αS(D) in sick mice. Analysis of the central nervous system in this mouse model showed the presence of αS(D) mainly in the caudal brain regions and the spinal cord. There were no differences in αS(D) distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of αS(D) immunoreactivity using an antibody against Ser129 phosphorylated αS by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of αS. The propagation of αS(D), suggesting the involvement of a "prion-like" mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.
[Show abstract][Hide abstract] ABSTRACT: The etiology of most human neurodegenerative disorders is believed to be multifactorial and consists of an interaction between environmental factors and genetic predisposition. Regarding Parkinson's disease (PD), the second most frequent neurodegenerative disease characterized by the aggregation of the alpha-synuclein (αS) protein, numerous epidemiological and experimental studies suggested a possible role of exposure to some pesticides. Whereas epidemiological studies largely failed to identify pesticides specifically involved in PD, it is of critical importance to set up in vitro toxicity studies of pesticides. We measured changes of αS levels following pesticide exposures of human cell lines in vitro, using either ELISA detection of endogenous αS or flow cytometry after overexpression using recombinant adenoviruses. We showed that three pesticides (paraquat, rotenone and maneb), which have frequently been associated with PD, produced a dose-dependent increase in cellular αS levels, but also of αS released into the culture medium. Examining an additional series of 20 pesticides from different families and chemical structures, we found that beside some insecticides, including an organophosphate and three pyrethroids, a majority of the 12 studied fungicides were also producing an αS accumulation, three of them (thiophanate-methyl, fenhexamid and cyprodinil) having similar or more pronounced effects than paraquat. A variety of pesticides can disrupt αS homeostasis in vitro; our data illustrate an experimental strategy that could help in the identification of chemicals that could be specifically involved in PD etiology.
Archive für Toxikologie 10/2014; DOI:10.1007/s00204-014-1388-2 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 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 (alphaS) is a major characteristic of the disease, but the molecular basis distinguishing the disease-associated protein (alphaSD) 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.
Our data confirm our previous observations of disease acceleration in a transgenic mouse line (M83) overexpressing a mutated (A53T) form of human alphaS, following inoculation of either brain extracts from sick M83 mice or fibrillar recombinant alphaS. 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 alphaSD, we designed an ELISA test that identifies alphaSD specifically in sick mice and in the brain regions targeted by the pathological process in this mouse model. alphaSD 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 alphaSD immunoreactivity using an antibody against Ser129 phosphorylated alphaS, similar results were observed in ELISA with several other antibodies against the C-terminal part of alphaS, including an antibody against non phosphorylated alphaS. This also indicated consistent immunoreactivity of the murine alphaS protein specifically in the affected brain regions of sick mice.
Prion-like behaviour in propagation of the disease-associated alphaS 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 alphaS and its normal counterpart.
[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease, involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's 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-synuclein adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-synuclein 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 which indicate a potential role of exposure to certain pesticides in the development of Parkinson's 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 Parkinson's disease.
[Show abstract][Hide abstract] ABSTRACT: 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 α-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 α-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 α-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with α-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T α-synuclein after intracerebral inoculation with an H-BSE prion strain.
Journal of Neuropathology and Experimental Neurology 05/2011; 70(5):377-85. DOI:10.1097/NEN.0b013e318217d95f · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Analyses using antibodies directed against α-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinson's 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.
[Show abstract][Hide abstract] ABSTRACT: 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 PrP(res) 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 approximately 0.25, 1.5 or 6x the levels of the PrP(ARQ) ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrP(c) measurements that appeared to reflect the different PrP(c) 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 PrP(c) expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing approximately 6x the normal PrP(c) level died after a survival periods of 400 days, those with approximately 1.5x the normal PrP(c) 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 AF(141)RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrP(res) in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrP(res) 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.
PLoS ONE 10/2009; 4(10):e7300. DOI:10.1371/journal.pone.0007300 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protease-resistant prion protein (PrP(res)) 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 PrP(res) 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 PrP(res) than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrP(res) form (PrP(res) #1) to L-type BSE. However, the "CH1641-like" isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrP(res) product (PrP(res) #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrP(res) #1 and PrP(res) #2 resulted in enrichment in PrP(res) #2, and demonstrated the presence of mono- and diglycosylated PrP(res) products. PrP(res) #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. PrP(res) #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.
[Show abstract][Hide abstract] ABSTRACT: Transgenic mice expressing the prion protein (PrP) of species affected by transmissible spongiform encephalopathies (TSEs) have recently been produced to facilitate experimental transmission of these diseases by comparison with wild-type mice. However, whilst wild-type mice have largely been described for the discrimination of different TSE strains, including differentiation of agents involved in bovine spongiform encephalopathy (BSE) and scrapie, this has been only poorly described in transgenic mice. Here, two ovine transgenic mouse lines (TgOvPrP4 and TgOvPrP59), expressing the ovine PrP (A136 R154 Q171) under control of the neuron-specific enolase promoter, were studied; they were challenged with brainstem or spinal cord from experimentally BSE-infected sheep (AA136 RR154 QQ171 and AA136 RR154 RR171 genotypes) or brainstem from cattle BSE and natural sheep scrapie. The disease was transmitted successfully from all of these sources, with a mean of approximately 300 days survival following challenge with material from two ARQ-homozygous BSE-infected sheep in TgOvPrP4 mice, whereas the survival period in mice challenged with material from the ARR-homozygous BSE-infected sheep was 423 days on average. It was shown that, in the two ovine transgenic mouse lines, the Western blot characteristics of protease-resistant PrP (PrP(res)) were similar, whatever the BSE source, with a low apparent molecular mass of the unglycosylated glycoform, a poor labelling by P4 monoclonal antibody and high proportions of the diglycosylated form. With all BSE sources, but not with scrapie, florid plaques were observed in the brains of mice from both transgenic lines. These data reinforce the potential of this recently developed experimental model for the discrimination of BSE from scrapie agents.
Journal of General Virology 01/2007; 87(Pt 12):3763-71. DOI:10.1099/vir.0.82062-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prion proteins (PrP) from sheep and mouse were produced in large quantities of full-length protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. Both recombinant proteins were recognized, at variable levels, in ELISA using a panel of antibodies recognizing different parts of the PrP molecules, from the octo-repeat region (79-92 human sequence), to the C terminal end of the protein. We show that these recombinant proteins enable polyclonal antisera to be produced in PrP0/0 mice, the sheep prion protein being strongly immunogenic, using either native or guanidium hydrochloride-treated recombinant protein. Sera produced against the sheep protein also reacted in Western blot with bovine, ovine, and murine PrP res, but showed higher reactivity with sheep PrP res. Interestingly, when compared to an antiserum produced against bovine 106-121 peptidic sequence (RB1), we found strikingly different ratios of the PrP res glycoforms, in both cattle with BSE and sheep with natural scrapie, but not in scrapie infected mice. Such results further demonstrate that the assessment of PrP res glycoform ratios, using different antibodies, may depend on antibodies species-specificities.
Biochemical and Biophysical Research Communications 03/2001; 281(1):101-8. DOI:10.1006/bbrc.2001.4326 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot.
[Show abstract][Hide abstract] ABSTRACT: The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)6 p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-beta-d-galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein in Escherichia coli, allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)6 p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.
Protein Expression and Purification 05/1999; 15(3):258-64. DOI:10.1006/prep.1998.1004 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Detection of BIV virus infection by serological means, PCR and virus isolation in experimentally infected calves is described. Viral sequences were specifically detected by PCR in peripheral blood mononuclear cells (PBMCs), with primer systems located in the gag, pol and tat regions of the viral genome. An enzyme-linked oligosorbent assay (ELOSA) in microtiter plates is described, for the detection of PCR products, the sensitivity of which was shown to be comparable to that of membrane hybridization detection. Serological response of the animals against the BIV p26 protein was shown, using a recombinant fusion protein ((His)6p26) expressed in E. coli and purified by metal affinity chromatography, in ELISA and Western blot studies. The presence of infectious virus was demonstrated by its rescue, by virus isolation in cell cultures, from PBMCs during a one year follow-up.
Archives of Virology 01/1998; 143(1):181-9. DOI:10.1007/s007050050278 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the infection by the bovine immunodeficiency-like virus (BIV) in three experimentally infected calves, by polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR), from the peripheral blood mononuclear cells (PBMC). Two primer pairs located in the gag and pol regions of the viral genome allowed to detect the viral genomic DNA by PCR, as well as the unspliced genomic viral RNA transcript, by RT-PCR. We also present the evidence of the presence in peripheral blood mononuclear cells (PBMCs) of a mRNA transcript of the regulatory trans-activator tat gene, according to the splicing pattern of the viral genome, by use of reverse transcription followed by nested PCR. The active expression of the virus in these animals was further assessed by the sequential rescue of the virus from unstimulated PBMCs in cell culture, from 4 weeks until 15 months following the infection.
Archives of Virology 02/1995; 140(8):1461-7. DOI:10.1007/BF01322672 · 2.39 Impact Factor