Total PrP Research Articles

Fluid Biomarkers in Individuals at Risk for Genetic Prion Disease up to Disease Conversion.

To longitudinally characterize disease-relevant CSF and plasma biomarkers in individuals at risk for genetic prion disease up to disease conversion. This single-center longitudinal cohort study has followed known carriers of PRNP pathogenic variants at risk for prion disease, individuals with a close relative who died of genetic prion disease but who have not undergone predictive genetic testing, and controls. All participants were asymptomatic at first visit and returned roughly annually. We determined PRNP genotypes, measured NfL and GFAP in plasma, and RT-QuIC, total PrP, NfL, T-tau, and beta-synuclein in CSF. Among 41 carriers and 21 controls enrolled, 28 (68%) and 15 (71%) were female, and mean ages were 47.5 and 46.1. At baseline, all individuals were asymptomatic. We observed RT-QuIC seeding activity in the CSF of 3 asymptomatic E200K carriers who subsequently converted to symptomatic and died of prion disease. 1 P102L carrier remained RT-QuIC negative through symptom conversion. No other individuals developed symptoms. The prodromal window from detection of RT-QuIC positivity to disease onset was 1 year long in an E200K individual homozygous (V/V) at PRNP codon 129 and 2.5 and 3.1 years in 2 codon 129 heterozygotes (M/V). Changes in neurodegenerative and neuroinflammatory markers were variably observed prior to onset, with increases observed for plasma NfL in 4/4 converters, and plasma GFAP, CSF NfL, CSF T-tau, and CSF beta-synuclein each in 2/4 converters, although values relative to age and fold changes relative to individual baseline were not remarkable for any of these markers. CSF PrP was longitudinally stable with mean coefficient of variation 9.0% across all individuals over up to 6 years, including data from converting individuals at RT-QuIC-positive timepoints. CSF prion seeding activity may represent the earliest detectable prodromal sign in E200K carriers. Neuronal damage and neuroinflammation markers show limited sensitivity in the prodromal phase. CSF PrP levels remain stable even in the presence of RT-QuIC seeding activity. ClinicalTrials.gov NCT05124392 posted 2017-12-01, updated 2023-01-27.

Multidimensional features of sporadic Creutzfeldt-Jakob disease in the elderly: a case report and systematic review.

As a rare neurodegenerative disease, sporadic Creutzfeldt-Jakob disease (sCJD) is poorly understood in the elderly populace. This study aims to enunciate the multidimensional features of sCJD in this group. A case of probable sCJD was reported in a 90-year-old Chinese man with initial dizziness. Then, available English literature of the elderly sCJD cases (aged 80 years and over) was reviewed and analyzed. Patients (15 cases) were subdivided and compared geographically. In the elderly sCJD cohort, the onset age was 84.9 ± 4.5 years and the median disease duration was 6.8 months, with respiratory infection/failure as the commonest death cause. Various clinical symptoms were identified, with cognitive disorder (86.7%) as the commonest typical symptom and speech impairment (66.7%) as the most atypical one. Restricted hyperintensities were reported in 60.0% cases on DWI, periodic sharp wave complexes in 73.3% cases on electroencephalogram, and cerebral hypoperfusion/hypometabolism in 26.7% cases on molecular imaging. The sensitive cerebrospinal fluid biomarkers were total tau (83.3%), 14-3-3 protein (75.0%), and PrP RT-QuIC (75.0%). Neuropathological profiles in the cerebral cortex revealed vacuolar spongiosis, neuronal loss, gliosis, and aging-related markers, with synaptic deposit as the commonest PrP pattern (60.0%). The polymorphic PRNP analysis at codon 129 was M/M (90.9%), with MM1 and MM2C as the primary molecular phenotypes. Latency to first clinic visit, hyperintense signals on DWI, and disease duration were significantly different between the patient subgroups. The characteristics of sCJD are multidimensional in the elderly, deepening our understanding of the disease and facilitating an earlier recognition and better care for this group.

The APOE4 Allele Exacerbates Pathology in a Mouse Model of Prion Disease

AbstractBackgroundThe APOE4 allele is the strongest genetic risk factor in sporadic Alzheimer’s disease (AD). In allele‐dose dependent manner APOE4 increases AD risk, lowers age of onset, and accelerates the rate of dementia progression. Like AD, prionoses are conformational neurodegenerative disorders, which neuropathological hallmarks include accumulation of disease specific misfolded protein(s) and presence of reactive astrocytes and microglia. Accumulation of PrPSc, a toxic conformer of the cellular prion protein‐PrPC, is the initial culprit in prion pathogenesis. PrPSc buildup is associated with the presence of A1 neurotoxic astrocytes and neurodegenerative phenotype microglia (MGnD), which proinflammatory character contributes importantly to neurodegeneration in prionoses. Despite similarities between AD and prionoses, and established effects of APOE4 on misfolded protein accumulation and MGnD activation in AD, effects of the APOE genotype on prion pathology remain uninvestigated.MethodAPOE2, APOE3, and APOE4 targeted replacement mice were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate (NBH) and subjected to repetitive behavioral testing. They were sacrificed at 15 or 23 weeks post inoculation (WPI), when they remained presymptomatic or showed overt neurological symptoms. Neuropathological and biochemical analyses were performed at either timepoint, while NanoStringTM analysis of microglia and astrocyte transcriptomic markers was done at 23WPI.ResultAPOE4 mice feature significantly shorter incubation period and more severe burden of neurological symptoms following prion inoculation than APOE2 and APOE3 mice. Neuropathological analysis reveals significantly higher burden of spongiforms changes in the brain cortex and greater load of reactive astrocytes and microglia in APOE4 mice compared to other APOE genotypes. APOE4 mice also show the highest level of total PrP and PrPSc and insoluble/soluble PrP index. Transcriptomic analysis reveals prion inoculation effects marked elevation of PAN and A1 reactive astrocyte markers, MGnD markers, and in pro‐inflammatory chemokines and cytokine receptors transcripts across all APOE genotypes but the NBH‐relative increase is the highest in APOE4 mice.ConclusionThe APOE4 allele promotes prion neurodegeneration through two separate mechanisms. It is associated with greater PrPSc accumulation and toxicity suggesting its role in the PrPSc metabolism and clearance. It also promotes acquisition of A1 and MGnD neurotoxic phenotypes by astrocytes and microglia, respectively.

Absence of Apolipoprotein E Exacerbates Prion Pathology and Promote Microglial Mediated Neurodegeneration

AbstractBackgroundBoth Alzheimer’s disease and prionoses are neurogenerative disorders, which involve misfolded protein deposition and transition of homeostatic microglia to their neurodegenerative phenotype. In prionoses, accumulation of toxic PrPSc protein constitutes the culprit of pathogenesis. Neurodegenerative phenotype microglia (MGnD) appear early in the course of disease and play opposing roles in PrPSc mediated neurodegeneration. Whilst clearance of PrPSc has a disease‐limiting effect, MGnD‐driven neuroinflammation is deleterious to neurons. Apolipoprotein (apo) E is a pleiotropic protein, which controls MGnD phagocytic and inflammatory characteristics. Despite prominent role of microglia in prionoses, involvement of apoE in their pathogenesis has not been established.MethodWild type (WT) and Apoe‐/‐ mice, both on the B6 background, were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate. Neuropathological and biochemical analyses were performed 15 and 23 weeks post inoculation (WPI) on asymptomatic and overly symptomatic animals, respectively. NanoStringTM analysis of microglia and astrocytic transcriptomic markers was performed at 23WPI.ResultPrion infection of WT mice upregulates total brain apoE level and effects cell‐type shift in the apoE expression, downregulating astrocytic apoE while increasing microglial apoE 24‐folds. 22L Apoe‐/‐ mice have significantly shorter disease incubation period, higher load of spongiform changes, and increased total brain PrP and PrPSc levels compared to 22LWT mice. In 22L Apoe ‐/‐ mice astro and micro gliosis appears earlier and is more robust compared to 22LWT mice. Transcriptomic analysis shows significantly higher level of PAN and A1, but not A2 astrocytic markers and MGnD markers in 22L Apoe ‐/‐ mice compared to 22LWT animals. Microglia in 22L Apoe ‐/‐ mice feature impaired neuronal phagocytosis and produce increased level of pro‐inflammatory interleukins and cytokines. 22L Apoe ‐/‐ mice show higher neuronal loss and greater tempo of neuronal degeneration than 22LWT mice.ConclusionOur results indicate the net effect of apoE in prion pathogenesis is protective. ApoE facilitates clearance of PrPSc and dying neurons by microglia, which becomes ineffective in the absence of apoE. Remnants of disintegrating neurons promote MGnD phenotype transition and inflammatory cascade propagating the vicious cycle of neuronal death and neuroinflammation. Our data also indicate homeostatic microglia transit to the MGnD even if they do not express apoE.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

High performance anion exchange chromatographic and colorimetric methods for quality assessment of total and free polysaccharide content in Haemophilus influenzae type b conjugate vaccine containing lactose.

The presence of lactose as a stabilizer in Haemophilus influenzae type b (Hib) conjugate vaccine is a challenge for chromatographic resolution of its total and free poly ribosyl ribitol phosphate (PRP) content. Sample pretreatment using ultrafiltration was performed and had removed ≥95% of lactose in shorter time compared to the conventional dialysis process. Separation of free unconjugated PRP was performed using solid-phase extraction C4 cartridges. Hib conjugate vaccine was then analyzed for determination of total and free PRP, using two validated techniques: high performance anion exchange chromatography with pulsed amperometry (HPAEC-PAD) for ribitol determination and a colorimetric assay for phosphorus determination. Lactose removal had enabled a rapid chromatographic assay via fast depolymerization of PRP using high temperature treatment. Modifying the burning process in the colorimetric assay reduced the analysis time significantly compared to the pharmacopoeial method. Linearity was obtained over the range of 0.10-10.0 μg mL-1 for the HPAEC method and in the range of 1.0-8.0 μg mL-1 for the colorimetric one. Stability of Hib conjugate vaccine was investigated. The HPAEC results revealed about a 35% increase in free PRP content after storage under stressed conditions (moisture and temperature). The proposed methods offered a reliable and economic platform for assessing the immunogenicity, efficacy and stability of Hib conjugate vaccine containing lactose for the biopharmaceutical industry.

Analysis of the Electrophoretic Profiles of Prion Protein in Carcinous and Pericarcinous Lysates of Six Different Types of Cancers

ObjectiveTo find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric, colon, liver, lung, thyroid, and laryngeal cancers. MethodsSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to test the amounts and electrophoretic patterns of total PrP and the tolerance of PK (protease K) digestion among six various cancer tissue types. ResultsA mass of PrP signals with a large molecular weight were identified in the homogenates of peripheral tissues. The amounts and electrophoretic patterns of total PrP did not differ significantly between carcinous and pericarcinous tissues. PrPs in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types. ConclusionThe study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar. Unlike PrP-specific immunohistochemical assay, evaluation of PrP electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.

A General Mass Spectrometry-Based Method of Quantitating Prion Polymorphisms from Heterozygous Chronic Wasting Disease-Infected Cervids.

Chronic wasting disease (CWD) is the only prion disease naturally transmitted among farmed and free-ranging cervids (deer, elk, moose, etc.). These diseases are always fatal and have long asymptomatic incubation periods. By 2019, CWD-infected cervids had been detected in 26 states, three Canadian provinces, South Korea, Norway, Finland, and Sweden. Prions (PrPSc) replicate by inducing a normal cellular prion protein (PrPC) to adopt the prion conformation. This prion templated conformational conversion is influenced by PrPC polymorphisms. Cervid PrPC contains at least 20 different polymorphic sites. By using chymotrypsin, trypsin, or trypsin followed by chymotrypsin to digest denatured cervid PrP, 19 peptides suitable for multiple reaction monitoring (MRM)-based analysis and spanning positions 30-51, 61-112, and 114-231 of cervid PrP were identified. Ten of these peptides span polymorphism-containing regions of cervid PrP. The other nine contain no polymorphisms, so they can be used as internal standards. Calibration curves relating the area ratios of MRM signals from polymorphism-containing peptides to appropriate internal standard peptides were linear and had excellent correlation coefficients. Samples from heterozygous (G96/S96) white-tailed deer orally dosed with CWD from homozygous (G96/G96) deer were analyzed. The G96 polymorphism comprised 75 ± 5% of the total PrP from the G96/S96 heterozygotes. Heterozygous animals facilitate conversion of different PrPC polymorphisms into PrPSc. This approach can be used to quantitate the relative amounts of the polymorphisms present in other animal species and even humans.

Early existence and biochemical evolution characterise acutely synaptotoxic PrPSc.

Although considerable evidence supports that misfolded prion protein (PrPSc) is the principal component of “prions”, underpinning both transmissibility and neurotoxicity, clear consensus around a number of fundamental aspects of pathogenesis has not been achieved, including the time of appearance of neurotoxic species during disease evolution. Utilizing a recently reported electrophysiology paradigm, we assessed the acute synaptotoxicity of ex vivo PrPSc prepared as crude homogenates from brains of M1000 infected wild-type mice (cM1000) harvested at time-points representing 30%, 50%, 70% and 100% of the terminal stage of disease (TSD). Acute synaptotoxicity was assessed by measuring the capacity of cM1000 to impair hippocampal CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in explant slices. Of particular note, cM1000 from 30% of the TSD was able to cause significant impairment of LTP and PTP, with the induced failure of LTP increasing over subsequent time-points while the capacity of cM1000 to induce PTP failure appeared maximal even at this early stage of disease progression. Evidence that the synaptotoxicity directly related to PrP species was demonstrated by the significant rescue of LTP dysfunction at each time-point through immuno-depletion of >50% of total PrP species from cM1000 preparations. Moreover, similar to our previous observations at the terminal stage of M1000 prion disease, size fractionation chromatography revealed that capacity for acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, with the profile appearing maximised by 50% of the TSD. Using enhanced sensitivity western blotting, modestly proteinase K (PK)-resistant PrPSc was detectable at very low levels in cM1000 at 30% of the TSD, becoming robustly detectable by 70% of the TSD at which time substantial levels of highly PK-resistant PrPSc was also evident. Further illustrating the biochemical evolution of acutely synaptotoxic species the synaptotoxicity of cM1000 from 30%, 50% and 70% of the TSD, but not at 100% TSD, was abolished by digestion of immuno-captured PrP species with mild PK treatment (5μg/ml for an hour at 37°C), demonstrating that the predominant synaptotoxic PrPSc species up to and including 70% of the TSD were proteinase-sensitive. Overall, these findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrPSc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, albeit with eventual plateauing of transmitting conformers.

Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species.

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.

Determining the Relative Susceptibility of Four Prion Protein Genotypes to Atypical Scrapie.

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). To determine the relative amounts of PrP polymorphisms present in atypical scrapie, total PrP was digested with chymotrypsin to generate characteristic peptides spanning relevant polymorphisms at positions 136, 141, 154, 171, and 172 of sheep PrPC. A multiple reaction monitoring method (MRM), employing 15N-labeled internal standards, was used to detect and quantify these polymorphisms present in both the PrPSc and PrPC from heterozygous (ALRRY and ALHQY or ALRQD or AFRQY) atypical scrapie-infected or uninfected control sheep. Both polymorphisms of the full length and truncated (C1) natively expressed PrPC are produced in equal amounts. The overall amount of PrPC present in the infected or uninfected animals was similar. PrPSc isolated from heterozygotes was composed of significant amounts of both PrP polymorphisms, including the ALRRY polymorphism which is highly resistant to classical scrapie. Thus, an atypical scrapie infection does not result from an overexpression of sheep PrPC. The replication of all atypical scrapie prions occurs at comparable rates, despite polymorphisms at positions 141, 154, 171, or 172.

Abnormalities in Brainstem Auditory Evoked Potentials in Sheep with Transmissible Spongiform Encephalopathies and Lack of a Clear Pathological Relationship.

Scrapie is transmissible spongiform encephalopathy (TSE), which causes neurological signs in sheep, but confirmatory diagnosis is usually made postmortem on examination of the brain for TSE-associated markers like vacuolar changes and disease-associated prion protein (PrPSc). The objective of this study was to evaluate whether testing of brainstem auditory evoked potentials (BAEPs) at two different sound levels could aid in the clinical diagnosis of TSEs in sheep naturally or experimentally infected with different TSE strains [classical and atypical scrapie and bovine spongiform encephalopathy (BSE)] and whether any BAEP abnormalities were associated with TSE-associated markers in the auditory pathways. BAEPs were recorded from 141 clinically healthy sheep of different breeds and ages that tested negative for TSEs on postmortem tests to establish a reference range and to allow comparison with 30 sheep clinically affected or exposed to classical scrapie (CS) without disease confirmation (test group 1) and 182 clinically affected sheep with disease confirmation (test group 2). Abnormal BAEPs were found in 7 sheep (23%) of group 1 and 42 sheep (23%) of group 2. The proportion of sheep with abnormalities did not appear to be influenced by TSE strain or PrPSc gene polymorphisms. When the magnitude of TSE-associated markers in the auditory pathways was compared between a subset of 12 sheep with and 12 sheep without BAEP abnormalities in group 2, no significant differences in the total PrPSc or vacuolation scores in the auditory pathways could be found. However, the data suggested that there was a difference in the PrPSc scores depending on the TSE strain because PrPSc scores were significantly higher in sheep with BAEP abnormalities infected with classical and L-type BSE, but not with CS. The results indicated that BAEPs may be abnormal in sheep infected with TSEs but the test is not specific for TSEs and that neither vacuolation nor PrPSc accumulation appears to be responsible for the clinical abnormalities.

Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked.

Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent.

A novel Enzyme-Linked Immuno-Sorbent Assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b–Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib–TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive.

Effects of metal binding on solubility and resistance of physiological prions depend on tissues and glycotypes

Prion diseases entail the conversion of a normal host-encoded prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). Various PrP(C) types differing in banding profiles and detergent solubility are present in different tissues, but only few PrP(Sc) types have been generated although PrP(C) acts as substrate. We hypothesize that distinct PrP(C) subtypes may be converted more efficiently to PrP(Sc) than others. One prerequisite for the analysis is the identification of the PrP(C) subtypes present in the protein complexes. Metal binding to PrP(C) is one of the most prominent features of the protein which induces increased proteolysis resistance and structural changes which might play an important role in the conversion process. Here we analyzed the metal-induced structural PrP(C) transformation of two different Triton X-100 soluble PrP(C) types derived from human platelets and brains by changes in protein solubility. We found that zinc and copper rendered approximately half of total PrP(C) and mainly un- and low-glycosylated PrP(C) to the Triton insoluble fraction. Our results indicate the presence of at least two distinct PrP(C) subtypes by metal interactions. The differentiation of high and low soluble metal bound PrP(C) offers precious information about PrP(C) protein composition and provides approaches for analyzing the transformation efficiency to PrP(Sc).

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrPc). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrPc, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

Discovery of a Novel, Monocationic, Small-Molecule Inhibitor of Scrapie Prion Accumulation in Cultured Sheep Microglia and Rov Cells

Prion diseases, including sheep scrapie, are neurodegenerative diseases with the fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC) to disease-associated prion protein (PrPSc). Chemical inhibition of prion accumulation is widely investigated, often using rodent-adapted prion cell culture models. Using a PrPSc-specific ELISA we discovered a monocationic phenyl-furan-benzimidazole (DB772), which has previously demonstrated anti-pestiviral activity and represents a chemical category previously untested for anti-prion activity, that inhibited PrPSc accumulation and prion infectivity in primary sheep microglial cell cultures (PRNP 136VV/154RR/171QQ) and Rov9 cultures (VRQ-ovinized RK13 cells). We investigated potential mechanisms of this anti-prion activity by evaluating PrPC expression with quantitative RT-PCR and PrP ELISA, comparing the concentration-dependent anti-prion and anti-pestiviral effects of DB772, and determining the selectivity index. Results demonstrate at least an approximate two-log inhibition of PrPSc accumulation in the two cell systems and confirmed that the inhibition of PrPSc accumulation correlates with inhibition of prion infectivity. PRNP transcripts and total PrP protein concentrations within cell lysates were not decreased; thus, decreased PrPC expression is not the mechanism of PrPSc inhibition. PrPSc accumulation was multiple logs more resistant than pestivirus to DB772, suggesting that the anti-PrPSc activity was independent of anti-pestivirus activity. The anti-PrPSc selectivity index in cell culture was approximately 4.6 in microglia and 5.5 in Rov9 cells. The results describe a new chemical category that inhibits ovine PrPSc accumulation in primary sheep microglia and Rov9 cells, and can be used for future studies into the treatment and mechanism of prion diseases.

Evidence for Co-infection of Ovine Prion Strains in Classical Scrapie Isolates

The diversity of strains of ovine prions within classical scrapie isolates was investigated by transmission studies in wild type mice. To determine the maximum diversity of prion strains present in each ovine scrapie isolate examined, isolates from mice having the shortest and longest incubation times for terminal disease after primary inoculation were passaged serially. Serial passage of ARQ/ARQ scrapie isolates in RIII mice revealed the ME7 prion strain in mice with short incubation times for terminal prion disease and the 87A strain in those mice with long incubation times. Serial passage of VRQ/VRQ scrapie isolates in RIII mice led to emergence of the 221C prion strain in mice with short incubation times and a variant of the 221C strain in those mice with long incubation times. RIII mice with short incubation times had higher levels of total and proteinase K-resistant PrP(Sc) compared with those RIII mice with long incubation times, while mice with long incubation times had large aggregates and plaques of PrP(Sc). ME7 PrP(Sc) differed in stability compared with the 87A prion strain, while PrP(Sc) associated with 221C had similar stability to that of the 221C variant. Serial passage in VM mice led to identification of ME7 and 87V in the same scrapie isolate. The data show that different prion strains can emerge from the same ovine scrapie isolate following serial passage in wild type mice and that the transmission properties of these strains correlate with distinct patterns of PrP(Sc) deposition.

A Retrospective Study on the Quality of Haemophilus Influenzae Type B Vaccines Used in the U.K. Between 1996 and 2004

Following the reduction in efficacy of Hib-TT vaccines in the primary immunization schedule observed in the U.K. between 1999 and 2003, batches of vaccine manufactured by two different companies were retrospectively examined by the National Institute for Biological Standards and Control. The study evaluated 41 batches of the Hib-TT vaccines manufactured between 1994 and 2003, assaying potency (total PRP saccharide content), integrity (% free saccharide), consistency (molecular sizing), and immunogenicity, as well as reviewing data previously obtained at the time of release. The study indicated the stability of the lyophilized final fill vaccines to extend well past their assigned shelf-lives, and found no trends in the endotoxin content, total saccharide or % free saccharide content. A trend towards slightly larger conjugates was observed over time in Hib-TT A, evidenced in both the manufacturer’s data obtained at the time that samples were submitted for testing and in data obtained from the retrospective analysis. The study confirmed that that there had been no significant change in the quality of the Hib vaccines that could possibly account for the change reported in their protective efficacy in the U.K. The study also demonstrated the value of independent testing of vaccines from the time of licensure and in the ongoing monitoring and re-examination of selected batches, as necessary, to assure their continuing quality, safety and consistency.

Insoluble Aggregates and Protease-resistant Conformers of Prion Protein in Uninfected Human Brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.