Giuseppe Legname

Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice

There is increasing concern over the extent to which bovine spongiform encephalopathy (BSE) prions have been transmitted to humans, as a result of the rising number of variant Creutzfeldt–Jakob disease (vCJD) cases. Toward preventing new transmissions, diagnostic tests for prions in livestock have been developed using the conformation-dependent immunoassay (CDI), which simultaneously measures specific antibody binding to denatured and native forms of the prion protein (PrP). We employed high-affinity recombinant antibody fragments (recFab) reacting with residues 95–105 of bovine (Bo) PrP for detection and another recFab that recognizes residues 132–156 for capture in the CDI. We report that the CDI is capable of measuring the disease-causing PrP isoform (PrPSc) in bovine brainstems with a sensitivity similar to that of end-point titrations in transgenic (Tg) mice expressing BoPrP. Prion titers were ∼107 ID50 units per gram of bovine brainstem when measured in Tg(BoPrP) mice, a figure ∼10 times greater than that determined by bioassay in cattle and ∼10,000× greater than in wild-type mice. We also report substantial differences in BoPrPSc levels in different areas of the obex region, where neuropathology has been consistently observed in cattle with BSE. The CDI was able to discriminate between PrPSc from BSE-infected cattle and Tg(BoPrP) mice as well as from chronic wasting disease (CWD)-infected deer and elk. Our findings argue that applying the CDI to livestock should considerably reduce human exposure to animal prions.

Copper Coordination in the Full-Length, Recombinant Prion Protein

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991-4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760-13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.

Folding of Prion Protein to Its Native α-Helical Conformation Is under Kinetic Control

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric α-helical or oligomeric β-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the β-rich isoform of MoPrP is thermodynamically more stable than the native α-helical isoform. The conformational transition from the α-helical to β-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the α-helical isoform and folds directly to the thermodynamically more stable β-rich isoform. Our data demonstrate that the folding of the prion protein to its native α-helical monomeric conformation is under kinetic control.

A change in the conformation of prions accompanies the emergence of a new prion strain.

To investigate the role of the pathogenic prion protein (PrP(Sc)) in controlling susceptibility to foreign prions, two Syrian hamster (SHa) prion strains, Sc237 and DY, were transmitted to transgenic mice expressing chimeric SHa/mouse PrP genes, Tg(MH2M). First passage of SHa(Sc237) prions exhibited prolonged incubation times, diagnostic of a species barrier. PrP(Sc) of the new MH2M(Sc237) strain possessed different structural properties from those of SHa(Sc237), as demonstrated by relative conformational stability measurements. This change was accompanied by a disease phenotype different from the SHa(Sc237) strain. Conversely, transmission of SHa(DY) prions to Tg(MH2M) mice showed no species barrier, and the MH2M(DY) strain retained the conformational and disease-specific properties of SHa(DY). These results suggest a causal relationship between species barriers, changes in PrP(Sc) conformation, and the emergence of new prion strains.

Design and construction of diverse mammalian prion strains

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.

Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes

On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn·HCl) concentration required to denature 50% of disease-causing prion protein (PrPSc) molecules, denoted as the [Gdn·HCl]1/2 value. For the two prion isolates enciphering shorter and longer incubation times, [Gdn·HCl]1/2 values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the [Gdn·HCl]1/2 values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of PrPSc structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different PrPSc conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of PrPSc remains to be determined.

Prion detection by an amyloid seeding assay

Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrPSc, the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt–Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was ≈1 fg. Some prion strains consist largely of protease-sensitive PrPSc (sPrPSc), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrPSc and protease-resistant PrPSc that does not rely on protease digestion or immunodetection.

Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro

While a β‐sheet‐rich form of the prion protein (PrPSc) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrPC), has been elusive. We have studied the effect of purified recombinant prion protein (recPrP) on rat fetal hippocampal neurons in culture. Overnight exposure to Syrian hamster or mouse recPrP, folded into an α‐helical‐rich conformation similar to that of PrPC, resulted in a 1.9‐fold increase in neurons with a differentiated axon, a 13.5‐fold increase in neurons with differentiated dendrites, a fivefold increase in axon length, and the formation of extensive neuronal circuitry. Formation of synaptic‐like contacts was increased by a factor of 4.6 after exposure to recPrP for 7 days. Neither the N‐terminal nor C‐terminal domains of recPrP nor the PrP paralogue doppel (Dpl) enhanced the polarization of neurons. Inhibitors of protein kinase C (PKC) and of Src kinases, including p59Fyn, blocked the effect of recPrP on axon elongation, while inhibitors of phosphatidylinositol 3‐kinase showed a partial inhibition, suggesting that signaling cascades involving these kinases are candidates for transduction of recPrP‐mediated signals. The results predict that full‐length PrPC functions as a growth factor involved in development of neuronal polarity.

Prions in skeletal muscle

Considerable evidence argues that consumption of beef products from cattle infected with bovine spongiform encephalopathy (BSE) prions causes new variant Creutzfeldt–Jakob disease. In an effort to prevent new variant Creutzfeldt–Jakob disease, certain “specified offals,” including neural and lymphatic tissues, thought to contain high titers of prions have been excluded from foods destined for human consumption [Phillips, N. A., Bridgeman, J. & Ferguson-Smith, M. (2000) in The BSE Inquiry (Stationery Office, London), Vol. 6, pp. 413–451]. Here we report that mouse skeletal muscle can propagate prions and accumulate substantial titers of these pathogens. We found both high prion titers and the disease-causing isoform of the prion protein (PrPSc) in the skeletal muscle of wild-type mice inoculated with either the Me7 or Rocky Mountain Laboratory strain of murine prions. Particular muscles accumulated distinct levels of PrPSc, with the highest levels observed in muscle from the hind limb. To determine whether prions are produced or merely accumulate intramuscularly, we established transgenic mice expressing either mouse or Syrian hamster PrP exclusively in muscle. Inoculating these mice intramuscularly with prions resulted in the formation of high titers of nascent prions in muscle. In contrast, inoculating mice in which PrP expression was targeted to hepatocytes resulted in low prion titers. Our data demonstrate that factors in addition to the amount of PrP expressed determine the tropism of prions for certain tissues. That some muscles are intrinsically capable of accumulating substantial titers of prions is of particular concern. Because significant dietary exposure to prions might occur through the consumption of meat, even if it is largely free of neural and lymphatic tissue, a comprehensive effort to map the distribution of prions in the muscle of infected livestock is needed. Furthermore, muscle may provide a readily biopsied tissue from which to diagnose prion disease in asymptomatic animals and even humans.

Doppel-induced cerebellar degeneration in transgenic mice

Doppel (Dpl) is a paralog of the mammalian prion protein (PrP); it is abundant in testes but expressed at low levels in the adult central nervous system. In two Prnp-deficient (Prnp0/0) mouse lines (Ngsk and Rcm0), Dpl overexpression correlated with ataxia and death of cerebellar neurons. To determine whether Dpl overexpression, rather than the dysregulation of genes neighboring the Prn gene complex, was responsible for the ataxic syndrome, we placed the mouse Dpl coding sequence under the control of the Prnp promoter and produced transgenic (Tg) mice on the Prnp0/0-ZrchI background (hereafter referred to as ZrchI). ZrchI mice exhibit neither Dpl overexpression nor cerebellar degeneration. In contrast, Tg(Dpl)ZrchI mice showed cerebellar granule and Purkinje cell loss; the age of onset of ataxia was inversely proportional to the levels of Dpl protein. Crosses of Tg mice overexpressing wild-type PrP with two lines of Tg(Dpl)ZrchI mice resulted in a phenotypic rescue of the ataxic syndrome, while Dpl overexpression was unchanged. Restoration of PrP expression also rendered the Tg(Dpl) mice susceptible to prion infection, with incubation times indistinguishable from non-Tg controls. Whereas the rescue of Dpl-induced neurotoxicity by coexpression of PrP argues for an interaction between the PrP and Dpl proteins in vivo, the unaltered incubation times in Tg mice overexpressing Dpl in the central nervous system suggest that Dpl is unlikely to be involved in prion formation.