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.

Retina as a Model to Study In Vivo Transmission of α-Synuclein in the A53T Mouse Model of Parkinson's Disease.

Parkinson's disease is a neurodegenerative disorder characterized by accumulation of misfolded α-synuclein within the central nervous system (CNS). Retinal manifestations have been widely described as a prodromal symptom; however, we have a limited understanding of the retinal pathology associated with Parkinson's disease. The strong similarities between the retina and the brain and the accessibility of the retina has potentiated studies to investigate retinal pathology in an effort to identify biomarkers for early detection, as well as for monitoring the progression of disease and efficacy of therapies as they become available. Here, we discuss a study conducted using a transgenic mouse model of Parkinson's disease (TgM83, expressing human α-synuclein containing the familial PD-associated A53T mutation) to demonstrate the effect of the A53T α-synuclein mutation on the retina. Additionally, we show that "seeding" with brain homogenates from clinically ill TgM83 mice accelerates the accumulation of retinal α-synuclein. The work described in this chapter provides insight into retinal changes associated with Parkinson's disease and identifies retinal indicators of Parkinson's disease pathogenesis that could serve as potential biomarkers for early detection.

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.

Binding of the PET radiotracer [¹8F]BF227 does not reflect the presence of alpha-synuclein aggregates in transgenic mice.

Alpha-synuclein (α-syn) aggregation is a neuropathological hallmark of many neurodegenerative diseases, collectively termed synucleinopathies. There is currently no pre-mortem diagnosis tool for these diseases. Although some compounds have been described as potential ligands for α-syn aggregates, no specific PET radiotracer of aggregated α-syn is currently available. Recently, [(18)F]BF227 has been proposed as an α-syn PET radiotracer in the absence of other specific candidates. We proposed here, for the first time, to use this radiotracer in an accelerated mouse model of synucleinopathy presenting α-syn depositions in brainstem and thalamus. Our in vivo and in vitro studies showed that [(18)F]BF227 does not bind to α-syn aggregates. These results highlight the fact that [(18)F]BF227 PET has no suitable characteristics for monitoring this experimental synucleinopathy, justifying the need to develop alternative α-syn PET radiotracers.

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.

An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology.

α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.

Prion-like acceleration of a synucleinopathy in a transgenic mouse model.

Our aim in this study was to investigate experimentally the possible in vivo transmission of a synucleinopathy, using a transgenic mouse model (TgM83) expressing the human A53T mutated α-synuclein. Brain homogenates from old TgM83 mice showing motor clinical signs due to the synucleinopathy and containing insoluble and phosphorylated (pSer129) α-synuclein were intracerebrally inoculated in young TgM83 mice. This triggered an early onset of characteristic motor clinical signs, compared with uninoculated TgM83 mice or to mice inoculated with a brain homogenate from a young, healthy TgM83 mouse. This early disease was associated with insoluble α-synuclein phosphorylated on Ser129, as already identified in old and sick uninoculated TgM83 transgenic mice. Although the molecular mechanisms remain to be determined, acceleration of the pathology following inoculation of mice expressing human mutated α-synuclein with tissues from mice affected by the synucleinopathy, could be consistent with "prion-like" propagation of the 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.

Emergence of classical BSE strain properties during serial passages of H-BSE in wild-type mice.

BACKGROUND: Two distinct forms of atypical spongiform encephalopathies (H-BSE and L-BSE) have recently been identified in cattle. Transmission studies in several wild-type or transgenic mouse models showed that these forms were associated with two distinct major strains of infectious agents, which also differed from the unique strain that had been isolated from cases of classical BSE during the food-borne epizootic disease. METHODOLOGY/PRINCIPAL FINDINGS: H-BSE was monitored during three serial passages in C57BL/6 mice. On second passage, most of the inoculated mice showed molecular features of the abnormal prion protein (PrP(d)) and brain lesions similar to those observed at first passage, but clearly distinct from those of classical BSE in this mouse model. These features were similarly maintained during a third passage. However, on second passage, some of the mice exhibited distinctly different molecular and lesion characteristics, reminiscent of classical BSE in C57Bl/6 mice. These similarities were confirmed on third passage from such mice, for which the same survival time was also observed as with classical BSE adapted to C57Bl/6 mice. Lymphotropism was rarely detected in mice with H-BSE features. In contrast, PrP(d) was detectable, on third passage, in the spleens of most mice exhibiting classical BSE features, the pattern being indistinguishable from that found in C57Bl/6 mice infected with classical BSE. CONCLUSION/SIGNIFICANCE: Our data demonstrate the emergence of a prion strain with features similar to classical BSE during serial passages of H-BSE in wild-type mice. Such findings might help to explain the origin of the classical BSE epizootic disease, which could have originated from a putatively sporadic form of BSE.

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.