The overall aim of the work proposed herein (study part 2) is to gain a better understanding of PrP misfolding and aggregation in cattle under physiological and pathological condidtions. To this end, we will analyse the biochemical and biophysical properties of PrPc and PrPd and its truncated metabolites in BSE affected cattle (C-BSE, H-BSE and L-BSE), healthy cattle (of different ages) and in cattle inoculated with the Swiss 2011 BSE isolates in brain and muscle tissues. The biophysical and biochemical features of PrP under investigation are (i) PrP aggregation (ii) PrP insolubility, (iii) protease resistance and (iv) PrP conformation.
Specific objectives of study part 2 are…
- …to investigate the aggregation and solubility of PrPres/2011 in comparison to PrPd in H-BSE, L-BSE and C-BSE and healthy animals.
Little is known on the PrPd aggregation/solubility in cattle affected by the different BSE types. Representative structures of brains and muscle samples of BSE affected cattle and healthy control animals of different age categories will be analysed for the density (size) of PrP aggregates by sucrose gradient centrifugation and for solubility by high-speed ultracentrifugation and NaPTA precipitation. All these techniques have been established in our laboaratory in the framework of the antecedent FSVO funded project 1.13.16. Results will provide further evidence to which extend PrP aggregates in the CNS and in muscle samples are indeed TSE associated or may also occur under physiologiocal conditions, i.e. in aging. Using PrP detection by Western Blot as a read out will allow us to identify which of the truncated PrP species are involved in the aggregation process.
- …to gain insights into structural determinants of PrP in the different BSE types by PrP specifc antibodies and conformation stability assay.
We hypothesize that differences in the biophysical and -chemical properties observed in objective 1 are correlating with distinct secondary and tertiary structures, i.e. with the folding, of PrPd molecules. There are currently no means to directly measure the structure of PrPd within aggregates. Therefore, we will take advantage of a conformation stability immunoassays, to assess structural differences between PrPd types. This assay relies on differential antibody binding capacities to defined epitopes using native and denaturing conditions.
- …to assess protease resistance of PrPd in the different types of BSE, the Swiss 2011 inoculated cattle and in healthy cattle.
PrP misfolding, aggregation insolubility has been associated with increased resistance to proteases such as proteinase K. We will compare the protease resistance of PrP using different types (Proteinase K, Trypsin, Chymotrypsin) and increasing concentrations of protease, followed by Western Blot detection.
Taken, together the results will deliver valuable information to assess the disease status of the Swiss 2011 cases, the cattle of Passage 1 and later also of those of Passage 2. In addition, we will gain knowledge on PrP aggregation states in healthy animals and whether they change with age.
- …to support junior staff development
This project is specially designed for a veterinary doctoral student. Our aim is to promote young postgraduate veterinarians towards a career in research and academia and to train the candidate for a PhD or postdoc position. To this end, the project is further supported by additional financial resources from our institutional budget.
