Alex J. Raeber

Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie

The ‘protein only’ hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N‐terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild‐type PrP or PrP lacking 26 or 49 amino‐proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild‐type and truncated PrPs. Within the framework of the ‘protein only’ hypothesis, this means that the amino‐proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).

Prion (PrPSc)-specific epitope defined by a monoclonal antibody

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPCand PrPScare evident from increased β-sheet content and protease resistance in PrPSc(refs 1,2,3). Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt–Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models forthe PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.

A crucial role for B cells in neuroinvasive scrapie

Although prion proteins are most efficiently propagated through intracerebral inoculation, peripheral administration has caused the diseases kuru, iatrogenic Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and new-variant CJD,. The development of neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system,. Here we investigate the identity of these cells by using a panel of immune-deficient mice inoculated with prions intraperitoneally: we found that defects affecting only T lymphocytes had no apparent effect, but that all mutations that disrupted the differentiation and response of B lymphocytes prevented the development of clinical scrapie. As an absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells, a lack of any of these three components may prevent the development of clinical scrapie. However, we found that scrapie developed after peripheral inoculation in mice expressing immunoglobulins that were exclusively of the M subclass and without detectable specificity for the normal form of the prion PrPc, and in mice which had differentiated B cells but no functional follicular dendritic cells. We conclude that differentiated B cells are crucial for neuroinvasion by scrapie, regardless of the specificity of their receptors.

PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain

Much available evidence points to a pathological isoform of the prion protein PrP being the infectious agent that causes transmissible spongiform encephalopathies, but the mechanisms controlling the neurotropism of prions are still unclear. We have previously shown that mice that do not express PrP (Prnp[o/o] mice) are resistant to infection by prions, and that if a Prnp(+/+) neurograft is introduced into such animals and these are infected intracerebrally with scrapie, the graft but not the surrounding tissue shows scrapie pathology. Here we show that PrP-expressing neurografts in Prnp(o/o) mice do not develop scrapie histopathology after intraperitoneal or intravenous inoculation with scrapie prions. Prion titres were undetectable in spleens of inoculated Prnp(o/o) mice, but were restored to wild-type levels upon reconstitution of the host lymphohaemopoietic system with PrP-expressing cells. Surprisingly, however, i.p. or i.v. inoculation failed to produce scrapie pathology in the neurografts of 27 out of 28 reconstituted animals, in contrast to intracerebral inoculation. We conclude that transfer of infectivity from the spleen to the central nervous system is crucially dependent on the expression of PrP in a tissue compartment that cannot be reconstituted by bone marrow transfer. Thus the requirement for the normal isoform of PrP in peripheral tissues represents a bottleneck for the spread of prions from peripheral sites to the central nervous system.

PrP expression in B lymphocytes is not required for prion neuroinvasion

Prion diseases are typically initiated by infection of peripheral sites, as in the case of bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, kuru and most cases of iatrogenic Creutzfeldt-Jakob disease. In mouse scrapie, prion infectivity accumulates in lymphoid organs, and the absence of mature B lymphocytes prevents peripherally administered prions from inducing central nervous system disease. We have now assessed whether expression of the cellular prion protein, PrPC, is required for B lymphocytes to mediate neuroinvasion. We found that repopulation of SCID and Rag-1-/- mice with fetal liver cells from either PrP-expressing or PrP-deficient mice and from T-cell deficient mice, but not from B-cell deficient mice, is equally efficient in restoring neuroinvasion after intraperitoneal inoculation of scrapie prions. These results indicate that cells whose maturation depends on B cells or their products, such as follicular dendritic cells, may enhance neuroinvasion. Alternatively, B cells may transport prions to the nervous system by a PrP-independent mechanism.

Prion Protein Devoid of the Octapeptide Repeat Region Restores Susceptibility to Scrapie in PrP Knockout Mice

Mice devoid of PrP are resistant to scrapie and fail to replicate the agent. Introduction of transgenes expressing PrP into such mice restores susceptibility to scrapie. We find that truncated PrP devoid of the five copper binding octarepeats still sustains scrapie infection; however, incubation times are longer and prion titers and protease-resistant PrP are about 30-fold lower than in wild-type mice. Surprisingly, brains of terminally ill animals show no histopathology typical for scrapie. However, in the spinal cord, infectivity, gliosis, and motor neuron loss are as in scrapie-infected wild-type controls. Thus, while the region comprising the octarepeats is not essential for mediating pathogenesis and prion replication, it modulates the extent of these events and of disease presentation.

Astrocyte-specific expression of hamster prion protein (PrP) renders PrP knockout mice susceptible to hamster scrapie

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte‐specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 ± 5 days and died 7 ± 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.

Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

ABSTRACT A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 1014-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. IMPORTANCE Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals. Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eQuIC) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

Immunodetection of disease‐associated mutant PrP, which accelerates disease in GSS transgenic mice

The absence of infectivity‐associated, protease‐resistant prion protein (PrPSc) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann–Sträussler Scheinker syndrome, and the failure of gene‐targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease‐sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrPSc‐specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease‐associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre‐existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrPSc characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.

Aggravation of ischemic brain injury by prion protein deficiency : Role of ERK-1/-2 and STAT-1

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp(0/0)), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp(0/0) mice, detectable in wild-type controls and approximately 4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by approximately 200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp(0/0)-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp(0/0)mice. Our data suggest a role of cytosolic signaling pathways in Prnp(0/0)-induced cell death.