A systematic investigation of production of synthetic prions from recombinant prion protein

Abstract

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

Prion disease: experimental models and reality

“…The objective of generating synthetic PrP is to study the physiological function of PrP, its role in disease pathogenesis, and conformational properties. Whilst the formation of prions from recombinant PrP in a cell-free system has been demonstrated by some groups [102], a robust approach to reproducibly and systematically generate infectious prions from recombinant PrP in vitro has yet to be developed [161]. The systematic studies of conditions that lead to in vitro aggregation have also aided the development of cell-free assays for the detection of prions.…”

Prokaryotic SPHINX 1.8 REP protein is tissue‐specific and expressed in human germline cells

“…Such features include accumulation of host-encoded amyloids and various fibril aggregates, microglial activation, and molecular markers such as Clusterin (Apo J) and MIP1α. 11 Independent investigators have not been able to generate infectivity from recPrP itself despite many robust attempts, for example, Barron et al, Schmidt et al, and Timmes et al 6,7,12 Nevertheless, amyloid may have other important functions. 2,[6][7][8][9] Large amounts of amyloid injected into the brain will collect as deposits that can be toxic to neurons in AD but are not transmissible in serial passages, 10 and the popular belief that recombinant PrP (recPrP) amyloid is infectious is based on a single brain homogenate from very old transgenic mice that were inoculated with large amounts of "seeded" recPrP amyloid fibrils.…”

The prokaryotic SPHINX 1.8 REP protein is tissue-specific and expressed in human germline cells

“…Such features include accumulation of host-encoded amyloids and various fibril aggregates, microglial activation, and molecular markers such as Clusterin (Apo J) and MIP1a (1)(2)(3)(4). Therapeutic targeting of AD amyloid in humans has repeatedly failed, even with drugs that reduce amyloid plaques (5), because b-amyloid, as prion protein (PrP) amyloid, is not the initiating cause of disease, but rather, a late stage pathological product (1,(6)(7)(8)(9). Large amounts of amyloid injected into the brain will collect as deposits that can be toxic to neurons in AD but are not transmissible in serial passages (10), and the popular belief that recombinant PrP (recPrP) amyloid is infectious is based on a single brain homogenate from very old transgenic mice that were inoculated with large amounts of "seeded" recPrP amyloid fibrils.…”

“…These "seeded" mice transmitted only laboratory RML scrapie on serial passages (11). Independent investigators have not been able to generate infectivity from recPrP itself despite many robust attempts, e.g., (7,8,12). Nevertheless, amyloid may have other important functions.…”