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.

Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic?

In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject.Here we come back on our material as it is possible to study and demonstrate the specificity of prion immunodetection using the PET-Blot method (Paraffin Embedded Tissue--Blot). It is admitted that this method allows detecting the Proteinase K (PK) resistant form of the abnormal prion protein (PrPres) without any confusion with unspecific immunoreaction. We re-analysed the kidney tissue versus adrenal gland and brain samples from the same cheetah affected with TSE using this PET-Blot method. The PET-Blot analysis revealed specific PrPres detection within the brain, adrenal gland and some glomeruli of the kidney, with a complete identicalness compared to our previous detection using immunohistochemistry. In conclusion, these new data enable us to confirm with assurance the presence of specific abnormal prion protein in the adrenal gland and in the kidney of the cheetah affected with FSE. It also emphasizes the usefulness for the re-examination of any available tissue blocks with the PET-Blot method as a sensitive complementary tool in case of doubtful PrP IHC results.

Strain-specific proteolytic processing of the prion protein in prion diseases of ruminants transmitted in ovine transgenic mice.

The cerebral prion protein (PrP) isolated in the absence of proteinase K digestion, from ruminants prion sources transmitted to ovine transgenic mice, was studied by Western blot analysis. A C2 PrP fragment, showing strain-specific cleavages, similar to those observed after proteinase K or thermolysin digestion, accumulated in the brain. 'CH1641-like' scrapie was characterized by the unique accumulation of a more C-terminally cleaved PrP fragment (CTF14). A similar, protease-resistant, PrP product was observed after proteinase K or thermolysin digestion. Whereas classical BSE appeared highly resistant to thermolysin digestion, CH1641 and 'CH1641-like' natural isolates did not show any remarkable feature regarding resistance to thermolysin. Thus, the molecular strain-specific features in the brain of transmissible spongiform encephalopathy infected mice essentially reflect the PrP proteolytic processing occurring in vivo.

Atypical bovine spongiform encephalopathies, France, 2001-2007.

In France, through exhaustive active surveillance, approximately 17.1 million adult cattle were tested for bovine spongiform encephalopathy from July 2001 through July 2007; approximately 3.6 million were >8 years of age. Our retrospective Western blot study of all 645 confirmed cases found that 7 were H-type and 6 were L-type.

Bovine spongiform encephalopathy in Sweden: an H-type variant.

Bovine spongiform encephalopathy (BSE) had never been detected in Sweden until 2006, when the active surveillance identified a case in a 12-year-old cow. The case was an unusual form, because several molecular features of the protease-resistant prion protein (PrP(res)) were different from classical BSE. The differences included higher susceptibility for proteinase K, higher molecular weight of the PrP(res) bands, affinity to the N-terminus-specific antibodies 12B2 and P4, and peculiar banding pattern with antibody SAF84 showing an additional band at the 14 kDa position. The molecular characteristics were in accordance to previous descriptions of H-type BSE. This report shows that a range of Western blot techniques and antibodies can be applied to confirm H-type BSE and further describes that the ratio of the amounts of PrPres#1 and PrPres#2, after deglycosylation, depends on the antibody used during processing. Immunohistochemistry on sections of medulla at the level of the obex applying antibodies with epitopes covering a broad range of the PrP sequence showed accumulation of disease-specific PrP (PrP(d)) in the gray matter. Fine punctate deposition in the neuropil was the most predominant type and was more severe in BSE target nuclei. The types of PrP(d) deposition are described in comparison with classical BSE. PrP-gene sequencing showed 6 copy octarepeat alleles and no abnormalities. It is postulated that the disease had a spontaneous origin, rather than having had been acquired in the BSE epidemic.

H-type bovine spongiform encephalopathy: complex molecular features and similarities with human prion diseases.

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant prion protein (PrP(res)) in Western blot, with a 1-2 kDa higher apparent molecular mass of the unglycosylated PrP(res) associated with labelling by antibodies against the 86-107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrP(res), we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrP(res) (PrP(res) #2), which, in unglycosylated form, migrated as a approximately 14 kDa fragment. Furthermore, a PrP(res) fragment of approximately 7 kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrP(res) #2 and approximately 7 kDa PrP(res) were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.

Transmission of new bovine prion to mice.

We previously reported that cattle were affected by a prion disorder that differed from bovine spongiform encephalopathy (BSE) by showing distinct molecular features of disease-associated protease-resistant prion protein (PrP(res). We show that intracerebral injection of such isolates into C57BL/6 mice produces a disease with preservation of PrP(res) molecular features distinct from BSE.

An alternative pretreatment procedure in animal transmissible spongiform encephalopathies diagnosis using PrPsc immunohistochemistry.

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect).

Comparison of abnormal prion protein glycoform patterns from transmissible spongiform encephalopathy agent-infected deer, elk, sheep, and cattle.

Analysis of abnormal prion protein glycoform patterns from chronic wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and cattle, and cattle with bovine spongiform encephalopathy failed to identify patterns capable of reliably distinguishing these transmissible spongiform encephalopathy diseases. However, PrP-res patterns sometimes differed among individual animals, suggesting infection by different or multiple CWD strains in some species.

Transmissible spongiform encephalopathy diagnosis using PrPsc immunohistochemistry on fixed but previously frozen brain samples.

The histological diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE), relies on identification in the brain of spongiosis, gliosis, and neuron loss without inflammatory lesions. Because of its sensitivity, immunohistochemistry of abnormal prion protein (PrPsc) is of great help in this diagnosis and can be used on its own or complementary to the biochemical detection of PrPsc. However, in some cases no formalin-fixed material is available, rendering its use as a complementary method impossible. For that purpose, we studied the possibility of detecting PrPsc immunohistochemically in fixed brain samples that had been previously frozen and used for Western blotting analysis. We compared freshly and fixed-frozen brain samples originating from the same sheep, either affected or unaffected with scrapie. We also studied fixed-frozen brain samples from scrapie-affected goats and from cows showing BSE. We showed that in all the species tested, despite damage to the histological structures, PrPsc was still detectable in the fixed-frozen brain sections without unspecific background staining. Notwithstanding the limited number of cases thus far analyzed, we have already demonstrated the possibility of using PrPsc immunohistochemistry on fixed-frozen brain samples with very good efficacy, thus rendering possible its use for diagnostic purposes in TSEs.