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

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

Abstract 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. 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 β-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 β-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing β-syn after inoculation of AAVβ-syn. This study indicated for the first time that viral vector-mediated β-syn overexpression could form aggregates in a model of synucleinopathy

Natural scrapie isolates transmission results to TgOvPrP4 ovine transgenic mice.

(1)<p>Animals were inoculated intracerebrally with 2 mg brain tissue.</p>(2)<p>Genotype of sheep or goat (amino acids 136, 141, 154, and 171).</p>(Goat)<p>Goat isolate.</p>(α)<p>Isolates detected in the flock α.</p>(β)<p>Isolates detected in the flock β.</p><p>Natural inoculums were divided into 4 different groups, all originating from active surveillance of TSEs in small ruminants. Group 1a: atypical scrapie cases with <i>prnp</i> alleles associated with increased susceptibility to atypical scrapie (AF₁₄₁RQ or AHQ). Group 1b: atypical scrapie cases without <i>prnp</i> alleles associated with increased susceptibility to atypical scrapie. Group 2: classical usual scrapie cases. Group 3: CH1641-like scrapie cases. Detection of PrP^res/PrP^sc was performed by Western blot or IHC analyses.</p

Effects of variable PrP^c expression levels in TgOvPrP4 mice on TSEs transmission.

<p>Survival periods of PrP^sc positive TgOvPrP4 mice are shown according the level of PrP^c expression (∼0.25, 1.5 or 6× the PrP^c level expressed in a sheep brain control) and to the inoculums; Group 1a: atypical scrapie isolates carrying the AF₁₄₁RQ or AHQ alleles, Group 1b: atypical scrapie isolates without the AF₁₄₁RQ or AHQ alleles, Group 2: usual classical scrapie isolates, Group 3: CH1641-like scrapie isolates, Group 4: experimental isolates (SSBP1, CH1641, BSE^OVINE), Group 5: mouse-adapted strains (BSE, C506M3, 87V). (A) Transmission results. (B) Kaplan-Meier survival curves of PrP^res/PrP^sc positive mice expressing ∼1.5 (blue line) or ∼6× (red line) the PrP^c level expressed in a sheep brain control for each of the 6 different groups. p-values represent log-rank differences in cumulative survival between mice expressing ∼1.5 and ∼6× the PrP^c level expressed in a sheep brain control.</p

Statistical analysis of PrP^c levels influencing survival period of TgOvPrP4 mice inoculated with atypical scrapie.

*<p>PrP^c level set as the baseline category.</p><p>Hazard ratios calculated from model used to identify predictors of survival for mice inoculated with atypical scrapie isolates and illustrating the effect associated with PrP^c sera levels (the mean of each subpopulation were chosen to illustrate the effect of the PrP^c sera level).</p

Levels of transgene expression in transgenic (TgOvPrP4) mice expressing ovine cellular prion protein (PrP^c).

<p>(A) Quantification of seric and cerebral PrP^c levels by ELISA in 47 TgOvPrP4 mice. (B) Repeated measures of PrP^c in the serum for 4 weeks (day D0, D7, D14, D21, D28) in 47 TgOvPrP4 mice representing the 3 subpopulations with distinct PrP^c levels in their brain. Each line represents the average and the standard deviation of 17, 16 and 14 individual mice expressing, respectively, ∼0.25, 1.5 or 6× the PrP^c level expressed in a sheep brain control.</p

Western blot profiles of PrP^res in TgOvPrP4 mice infected with atypical scrapie.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007300#s2" target="_blank">Results</a> are shown before (A) and after (B) PNGase deglycosylation using monoclonal antibodies 4F2 (lanes 1, 5), P4 (lanes 2, 6) (N-terminal), Sha 31 (lanes 4, 8)(core) and 99/97.6.1 (lanes 3, 7)(C-terminal). Western blot profiles with Sha 31 antibody are shown with the curve of chemiluminescence measured along the lanes. Apparent molecular weights were means from 30 mice before PNGase deglycosylation and from 7 mice after PNGase deglycosylation.</p

Schematic representation of PrP^res fragments identified in atypical scrapie.

<p>(A) Diagram illustrating PrP^res A, B and C sequences in atypical scrapie, with location of epitopes recognised by monoclonal antibodies used during the study and estimated cleavages of PrP^res fragments. (B) Diagram of Western blot profiles of PrP^res detected using Sha 31 monoclonal antibody before (1) and after (2) PNGase deglycosylation.</p

Experimental TSEs transmission results to TgOvPrP4 ovine transgenic mice.

(1)<p>Animals were inoculated intracerebrally with 2 mg brain tissue.</p>(2)<p>Genotype of sheep or goat (amino acids 136, 141, 154, and 171).</p><p>Experimental inoculums were divided into 2 different groups. Group 3: small ruminants intracerebrally inoculated with various TSEs. Group 4: experimental strains initially derived from wild-type mice. Detection of PrP^res/PrP^sc was performed by Western blot or IHC analyses.</p