Protease-Sensitive and -Resistant Forms of Human and Murine Alpha-Synucleins in Distinct Brain Regions of Transgenic Mice (M83) Expressing the Human Mutated A53T Protein.

Human neurodegenerative diseases associated with the misfolding of the alpha-synuclein (aS) protein (synucleinopathies) are similar to prion diseases to the extent that lesions are spread by similar molecular mechanisms. In a transgenic mouse model (M83) overexpressing a mutated (A53T) form of human aS, we had previously found that Protein Misfolding Cyclic Amplification (PMCA) triggered the aggregation of aS, which is associated with a high resistance to the proteinase K (PK) digestion of both human and murine aS, a major hallmark of the disease-associated prion protein. In addition, PMCA was also able to trigger the aggregation of murine aS in C57Bl/6 mouse brains after seeding with sick M83 mouse brains. Here, we show that intracerebral inoculations of M83 mice with C57Bl/6-PMCA samples strikingly shortens the incubation period before the typical paralysis that develops in this transgenic model, demonstrating the pathogenicity of PMCA-aggregated murine aS. In the hind brain regions of these sick M83 mice containing lesions with an accumulation of aS phosphorylated at serine 129, aS also showed a high PK resistance in the N-terminal part of the protein. In contrast to M83 mice, old APPxM83 mice co-expressing human mutated amyloid precursor and presenilin 1 proteins were seen to have an aggregation of aS, especially in the cerebral cortex, hippocampus and striatum, which also contained the highest load of aS phosphorylated at serine 129. This was proven by three techniques: a Western blot analysis of PK-resistant aS; an ELISA detection of aS aggregates; or the identification of aggregates of aS using immunohistochemical analyses of cytoplasmic/neuritic aS deposits. The results obtained with the D37A6 antibody suggest a higher involvement of murine aS in APPxM83 mice than in M83 mice. Our study used novel tools for the molecular study of synucleinopathies, which highlight similarities with the molecular mechanisms involved in prion diseases.

Seeded propagation of α-synuclein aggregation in mouse brain using protein misfolding cyclic amplification.

α-Synuclein (α-syn) protein aggregation is associated with several neurodegenerative disorders collectively referred to as synucleinopathies, including Parkinson's disease. We used protein misfolding cyclic amplification (PMCA) to study α-syn aggregation in brain homogenates of wild-type or transgenic mice expressing normal (D line) or A53T mutant (M83 line) human α-syn. We found that sonication-incubation cycles of M83 mouse brain gradually produce large quantities of SDS-resistant α-syn aggregates, involving both human and mouse proteins. These PMCA products, containing partially proteinase K-resistant α-syn species, are competent to accelerate the onset of neurologic symptoms after intracerebral inoculation to young M83 mice and to seed aggregate formation of α-syn following PMCA, including in D and wild-type mouse brain substrates. PMCA seeding activity in the M83 diseased brain correlates positively with regions mostly targeted by the α-syn pathology in this model. Our data indicate that similar to prions, PMCA can reproduce some characteristics of α-syn aggregation and seeded propagation in vitro in a complex milieu. This opens new opportunities for the molecular study of synucleinopathies.-Nicot, S., Verchère, J., Bélondrade, M., Mayran, C., Bétemps, D., Bougard, D., Baron, T. Seeded propagation of α-synuclein aggregation in mouse brain using protein misfolding cyclic amplification.

Investigating the neuroprotective effect of AAV-mediated ß-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 ß-synuclein (ß-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 ß-syn gene (AAVß-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was - or not - accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics. AAV mediated ß-syn overexpression did not delay the disease onset or reduce the α-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant ß-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing ß-syn after inoculation of AAVß-syn. This study indicated for the first time that viral vector-mediated ß-syn overexpression could form aggregates in a model of synucleinopathy.

'Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum.

The M83 transgenic mouse is a model of human synucleinopathies that develops severe motor impairment correlated with accumulation of the pathological Ser129-phosphorylated α-synuclein (α-synP ) in the brain and spinal cord. M83 disease can be accelerated by intracerebral inoculation of brain extracts from sick M83 mice. This has also recently been described using peripheral routes, injecting recombinant preformed α-syn fibrils into the muscle or the peritoneum. Here, we inoculated homozygous and/or hemizygous M83 neonates via the intraperitoneal and/or intracerebral routes with two different brain extracts: one from sick M83 mice inoculated with brain extract from other sick M83 mice, and the other derived from a human multiple system atrophy source passaged in M83 mice. Detection of α-synP using ELISA and western blot confirmed the disease in mice. The distribution of α-synP in the central nervous system was similar, independently of the inoculum or inoculation route, consistent with previous studies describing M83 disease. ELISA tests revealed higher levels of α-synP in homozygous than in hemizygous sick M83 mice, at least after IC inoculation. Interestingly, the immunoreactivity of α-synP detected by ELISA was significantly lower in M83 mice inoculated with the multiple system atrophy inoculum than in M83 mice inoculated with the M83 inoculum, at the first two passages. 'Prion-like' propagation of the synucleinopathy up to the clinical disease was accelerated by both intracerebral and intraperitoneal inoculations of brain extracts from sick mice. This acceleration, however, depends on the levels of α-syn expression by the mouse and the type of inoculum.

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein.

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.

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

BACKGROUND: 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 (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. RESULTS: Our 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. CONCLUSIONS: Prion-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.

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 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-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 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.

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 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.

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 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.

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 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.

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 (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.