Infectivity in Skeletal Muscle of Cattle with Atypical Bovine Spongiform Encephalopathy

The amyloidotic form of bovine spongiform encephalopathy (BSE) termed BASE is caused by a prion strain whose biological properties differ from those of typical BSE, resulting in a clinically and pathologically distinct phenotype. Whether peripheral tissues of BASE-affected cattle contain infectivity is unknown. This is a critical issue since the BASE prion is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible. We carried out bioassays in transgenic mice overexpressing bovine PrP (Tgbov XV) and found infectivity in a variety of skeletal muscles from cattle with natural and experimental BASE. Noteworthy, all BASE muscles used for inoculation transmitted disease, although the attack rate differed between experimental and natural cases (∼70% versus ∼10%, respectively). This difference was likely related to different prion titers, possibly due to different stages of disease in the two conditions, i.e. terminal stage in experimental BASE and pre-symptomatic stage in natural BASE. The neuropathological phenotype and PrPres type were consistent in all affected mice and matched those of Tgbov XV mice infected with brain homogenate from natural BASE. The immunohistochemical analysis of skeletal muscles from cattle with natural and experimental BASE showed the presence of abnormal prion protein deposits within muscle fibers. Conversely, Tgbov XV mice challenged with lymphoid tissue and kidney from natural and experimental BASE did not develop disease. The novel information on the neuromuscular tropism of the BASE strain, efficiently overcoming species barriers, underlines the relevance of maintaining an active surveillance

Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP(Sc). Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP(Sc) were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1+/+), hemizygous (Pin1+/−) or knock-out (Pin1−/−) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease

Synthetic Prion Selection and Adaptation

Prion pathologies are characterized by the conformational conversion of the cellular prion protein (PrPC) into a pathological infectious isoform, known as PrPSc. The latter acquires different abnormal conformations, which are associated with specific pathological phenotypes. Recent evidence suggests that prions adapt their conformation to changes in the context of replication. This phenomenon is known as either prion selection or adaptation, where distinct conformations of PrPSc with higher propensity to propagate in the new environment prevail over the others. Here, we show that a synthetically generated prion isolate, previously subjected to protein misfolding cyclic amplification (PMCA) and then injected in animals, is able to change its biochemical and biophysical properties according to the context of replication. In particular, in second transmission passage in vivo, two different prion isolates were found: one characterized by a predominance of the monoglycosylated band (PrPSc-M) and the other characterized by a predominance of the diglycosylated one (PrPSc-D). Neuropathological, biochemical, and biophysical assays confirmed that these PrPSc possess distinctive characteristics. Finally, PMCA analysis of PrPSc-M and PrPSc-D generated PrPSc (PrPSc-PMCA) whose biophysical properties were different from those of both inocula, suggesting that PMCA selectively amplified a third PrPSc isolate. Taken together, these results indicate that the context of replication plays a pivotal role in either prion selection or adaptation. By exploiting the ability of PMCA to mimic the process of prion replication in vitro, it might be possible to assess how changes in the replication environment influence the phenomenon of prion selection and adaptation

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases

<p>Pin1 is a peptidyl-prolyl isomerase that induces the <i>cis-trans</i> conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1^+/+), hemizygous (Pin1^+/−) or knock-out (Pin1^−/−) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.</p

PK digestion assay of amyloid #4, #19, #28 and #32.

<p>Western blotting of PK digestion assay (amyloids #4, #19, #28, #32) showed partial protease K (PK) resistance of recMoPrP(23–231) (amyloids #4 and #28). RecMoPrP(23–231) amyloids (PK- lanes) were digested with PK at ratio 1:10 (w/w) (PK+ lanes) and 1:1 (w/w) (PK++ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences)</p

PK digestion assay of GT1 and N2a cells collected at different passages after infection with amyloid fibrils.

<p>Western blotting of GT1 and N2a cell lines infected with PrP amyloid #4 was observed throughout, from first passage (P1) to sixth passage (P6) and after treatment with proteinase K at ratio 1:500 (w/w). Western blot was performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences).</p

Seeding assay of N2a and GT1 cell cultures with synthetic amyloid fibrils.

<p>Seeding of recMoPrP(23–231) amyloid preparations induced the conversion of endogenous PrP^C to mildly PK resistant forms (A) and accumulation (B) in mouse neuroblastoma N2a and mouse hypothalamic GT1 amyloid-infected cell lines analyzed six passages after the infection (P6). Western blotting shows the partial protease K (PK) resistance of N2a and GT1 amyloid fibril-infected cell lysates. Fibril-infected cell lysates (PK-lanes) were digested with PK at ratio 1:500 (w/w) (PK+ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL) for GT1 infected cells and Clone-P (1μg/mL) for N2a infected cells. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences) (A). Immunofluorescence imaging shows the accumulations of PrP in N2a and GT1 amyloid fibril-infected cell lines. The deposition and level of PrP (green) in amyloid fibril-infected cell lines after six passages were detected by Fab D18 monoclonal antibody (10 μg/mL final concentration). The nuclei (blue) were stained with DAPI. Scale bar is 20μm (B).</p