Single-nucleus chromatin accessibility profiling highlights distinct astrocyte signatures in progressive supranuclear palsy and corticobasal degeneration.

Tauopathies such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) exhibit characteristic neuronal and glial inclusions of hyperphosphorylated Tau (pTau). Although the astrocytic pTau phenotype upon neuropathological examination is the most guiding feature in distinguishing both diseases, regulatory mechanisms controlling their transitions into disease-specific states are poorly understood to date. Here, we provide accessible chromatin data of more than 45,000 single nuclei isolated from the frontal cortex of PSP, CBD, and control individuals. We found a strong association of disease-relevant molecular changes with astrocytes and demonstrate that tauopathy-relevant genetic risk variants are tightly linked to astrocytic chromatin accessibility profiles in the brains of PSP and CBD patients. Unlike the established pathogenesis in the secondary tauopathy Alzheimer disease, microglial alterations were relatively sparse. Transcription factor (TF) motif enrichments in pseudotime as well as modeling of the astrocytic TF interplay suggested a common pTau signature for CBD and PSP that is reminiscent of an inflammatory immediate-early response. Nonetheless, machine learning models also predicted discriminatory features, and we observed marked differences in molecular entities related to protein homeostasis between both diseases. Predicted TF involvement was supported by immunofluorescence analyses in postmortem brain tissue for their highly correlated target genes. Collectively, our data expand the current knowledge on risk gene involvement (e.g., MAPT, MAPK8, and NFE2L2) and molecular pathways leading to the phenotypic changes associated with CBD and PSP.

Seizure prevalence in neurodegenerative diseases-a study of autopsy proven cases.

BACKGROUND AND PURPOSE: Knowledge about the seizure prevalence in the whole symptomatic course, from disease onset to death, in neurodegenerative diseases (ND) is lacking. Therefore, the aim was to investigate seizure prevalence and associated clinical implications in neuropathologically diagnosed ND. METHODS: Clinical records of cases from the Neurobiobank Munich, Germany, were analyzed. Neuropathological diagnoses of the assessed cases included Alzheimer disease (AD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). Seizure prevalence during the whole symptomatic disease phase was assessed and compared amongst ND. Associations between first clinical symptom and seizure prevalence and between seizures and disease duration were examined. RESULTS: In all, 454 patients with neuropathologically diagnosed ND and with available and meaningful clinical records were investigated (AD, n = 144; LBD, n = 103; PSP, n = 93; FTLD, n = 53; MSA, n = 36; CBD, n = 25). Seizure prevalence was 31.3% for AD, 20.0% for CBD, 12.6% for LBD, 11.3% for FTLD, 8.3% for MSA and 7.5% for PSP. Seizure prevalence was significantly higher in AD compared to FTLD (p = 0.005), LBD (p = 0.001), MSA (p = 0.005) and PSP (p < 0.001). No other significant differences regarding seizure prevalence were found between the studied ND. Cognitive first symptoms in ND were associated with an increased seizure prevalence (21.1% vs. 11.0% in patients without cognitive first symptoms) and motor first symptoms with a decreased seizure prevalence (10.3% vs. 20.5% in patients without motor first symptoms). Seizures were associated with a longer disease duration in MSA (12.3 vs. 7.0 years in patients without seizures; p = 0.017). CONCLUSIONS: Seizures are a clinically relevant comorbidity in ND, particularly in AD. Knowledge of the first clinical symptom in ND may allow for estimation of seizure risk.

Phenotypic diversity of genetic Creutzfeldt-Jakob disease: a histo-molecular-based classification.

The current classification of sporadic Creutzfeldt-Jakob disease (sCJD) includes six major clinicopathological subtypes defined by the physicochemical properties of the protease-resistant core of the pathologic prion protein (PrPSc), defining two major PrPSc types (i.e., 1 and 2), and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein gene (PRNP). How these sCJD subtypes relate to the well-documented phenotypic heterogeneity of genetic CJD (gCJD) is not fully understood. We analyzed molecular and phenotypic features in 208 individuals affected by gCJD, carrying 17 different mutations, and compared them with those of a large series of sCJD cases. We identified six major groups of gCJD based on the combination PrPSc type and codon 129 genotype on PRNP mutated allele, each showing distinctive histopathological characteristics, irrespectively of the PRNP associated mutation. Five gCJD groups, named M1, M2C, M2T, V1, and V2, largely reproduced those previously described in sCJD subtypes. The sixth group shared phenotypic traits with the V2 group and was only detected in patients carrying the E200K-129M haplotype in association with a PrPSc type of intermediate size ("i") between type 1 and type 2. Additional mutation-specific effects involved the pattern of PrP deposition (e.g., a "thickened" synaptic pattern in E200K carriers, cerebellar "stripe-like linear granular deposits" in those with insertion mutations, and intraneuronal globular dots in E200K-V2 or -M"i"). A few isolated cases linked to rare PRNP haplotypes (e.g., T183A-129M), showed atypical phenotypic features, which prevented their classification into the six major groups. The phenotypic variability of gCJD is mostly consistent with that previously found in sCJD. As in sCJD, the codon 129 genotype and physicochemical properties of PrPSc significantly correlated with the phenotypic variability of gCJD. The most common mutations linked to CJD appear to have a variable and overall less significant effect on the disease phenotype, but they significantly influence disease susceptibility often in a strain-specific manner. The criteria currently used for sCJD subtypes can be expanded and adapted to gCJD to provide an updated classification of the disease with a molecular basis.

First symptom guides diagnosis and prognosis in neurodegenerative diseases-a retrospective study of autopsy proven cases.

BACKGROUND AND PURPOSE: Clinical diagnostic criteria for neurodegenerative diseases have been framed based on clinical phenomenology. However, systematic knowledge about the first reported clinical symptoms in neurodegenerative diseases is lacking. Therefore, the aim was to determine the prevalence and clinical implications of the first clinical symptom (FS) as assessed by medical history in neuropathologically proven neurodegenerative diseases. METHODS: Neuropathological diagnoses from the Neurobiobank Munich, Germany, were matched with clinical records for analyses of the diagnostic and prognostic values of FSs. RESULTS: In all, 301 patients with the neuropathological diagnoses Alzheimer disease (AD), progressive supranuclear palsy (PSP), frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD) including the neuropathologically indistinguishable clinical phenotypes Parkinson disease and dementia with Lewy bodies, multiple system atrophy (MSA) and corticobasal degeneration (CBD) were studied. Memory disturbance was the most common FS in AD (34%), FTLD (19%) and LBD (26%), gait disturbance in PSP (35%) and MSA (27%) and aphasia and personality changes in CBD (20%, respectively). In a model adjusting for prevalence in the general population, AD was predicted by memory disturbance in 79.0%, aphasia in 97.2%, personality changes in 96.0% and by cognitive disturbance in 99.0%. Gait disturbance and tremor predicted LBD in 54.6% and 97.3%, coordination disturbance MSA in 59.4% and dysarthria FTLD in 73.0%. Cognitive FSs were associated with longer survival in AD (12.0 vs. 5.3 years; p < 0.001) and FTLD (8.2 vs. 4.1 years; p = 0.005) and motor FSs with shorter survival in PSP (7.2 vs. 9.7; p = 0.048). CONCLUSIONS: Assessing FSs in neurodegenerative diseases may be beneficial for accuracy of diagnosis and prognosis and thereby may improve clinical care and precision of study recruitment.

Sporadic Fatal Insomnia in Europe: Phenotypic Features and Diagnostic Challenges.

OBJECTIVE: Comprehensively describe the phenotypic spectrum of sporadic fatal insomnia (sFI) to facilitate diagnosis and management of this rare and peculiar prion disorder. METHODS: A survey among major prion disease reference centers in Europe identified 13 patients diagnosed with sFI in the past 20 years. We undertook a detailed analysis of clinical and histopathological features and the results of diagnostic investigations. RESULTS: Mean age at onset was 43 years, and mean disease duration 30 months. Early clinical findings included psychiatric, sleep, and oculomotor disturbances, followed by cognitive decline and postural instability. In all tested patients, video-polysomnography demonstrated a severe reduction of total sleep time and/or a disorganized sleep. Cerebrospinal fluid (CSF) levels of proteins 14-3-3 and t-tau were unrevealing, the concentration of neurofilament light protein (NfL) was more consistently increased, and the real-time quaking-induced conversion assay (RT-QuIC) revealed a positive prion seeding activity in 60% of cases. Electroencephalography and magnetic resonance imaging showed nonspecific findings, whereas fluorodeoxyglucose positron emission tomography (FDG-PET) demonstrated a profound bilateral thalamic hypometabolism in 71% of cases. Molecular analyses revealed PrPSc type 2 and methionine homozygosity at PRNP codon 129 in all cases. INTERPRETATION: sFI is a disease of young or middle-aged adults, which is difficult to reconcile with the hypothesis of a spontaneous etiology related to stochastic, age-related PrP misfolding. The combination of psychiatric and/or sleep-related symptoms with oculomotor abnormalities represents an early peculiar clinical feature of sFI to be valued in the differential diagnosis. Video-polysomnography, FDG-PET, and especially CSF prion RT-QuIC and NfL constitute the most promising supportive diagnostic tests in vivo. Ann Neurol 2018;84:347-360.

Effect of Polymorphisms at Codon 146 of the Goat PRNP Gene on Susceptibility to Challenge with Classical Scrapie by Different Routes.

This report presents the results of experimental challenges of goats with scrapie by both the intracerebral (i.c.) and oral routes, exploring the effects of polymorphisms at codon 146 of the goat PRNP gene on resistance to disease. The results of these studies illustrate that while goats of all genotypes can be infected by i.c. challenge, the survival distribution of the animals homozygous for asparagine at codon 146 was significantly shorter than those of animals of all other genotypes (chi-square value, 10.8; P = 0.001). In contrast, only those animals homozygous for asparagine at codon 146 (NN animals) succumbed to oral challenge. The results also indicate that any cases of infection in non-NN animals can be detected by the current confirmatory test (immunohistochemistry), although successful detection with the rapid enzyme-linked immunosorbent assay (ELISA) was more variable and dependent on the polymorphism. Together with data from previous studies of goats exposed to infection in the field, these data support the previously reported observations that polymorphisms at this codon have a profound effect on susceptibility to disease. It is concluded that only animals homozygous for asparagine at codon 146 succumb to scrapie under natural conditions.IMPORTANCE In goats, like in sheep, there are PRNP polymorphisms that are associated with susceptibility or resistance to scrapie. However, in contrast to the polymorphisms in sheep, they are more numerous in goats and may be restricted to certain breeds or geographical regions. Therefore, eradication programs must be specifically designed depending on the identification of suitable polymorphisms. An initial analysis of surveillance data suggested that such a polymorphism in Cypriot goats may lie in codon 146. In this study, we demonstrate experimentally that NN animals are highly susceptible after i.c. inoculation. The presence of a D or S residue prolonged incubation periods significantly, and prions were detected in peripheral tissues only in NN animals. In oral challenges, prions were detected only in NN animals, and the presence of a D or S residue at this position conferred resistance to the disease. This study provides an experimental transmission model for assessing the genetic susceptibility of goats to scrapie.

Overexpression of chimaeric murine/ovine PrP (A136H154Q171) in transgenic mice facilitates transmission and differentiation of ruminant prions.

Development of transgenic mouse models expressing heterologous prion protein (PrP) has facilitated and advanced in vivo studies of prion diseases affecting humans and animals. Here, novel transgenic mouse lines expressing a chimaeric murine/ovine (Mu/Ov) PrP transgene, including amino acid residues alanine, histidine and glutamine at ovine polymorphic codons 136, 154 and 171 (A136H154Q171), were generated to provide a means of assessing the susceptibility of the ovine AHQ allele to ruminant prion diseases in an in vivo model. Transmission studies showed that the highest level of transgene overexpression, in Tg(Mu/OvPrP(AHQ))EM16 (EM16) mice, conferred high susceptibility to ruminant prions. Highly efficient primary transmission of atypical scrapie from sheep was shown, irrespective of donor sheep PrP genotype, with mean incubation periods (IPs) of 154­178 days post-inoculation (p.i.), 100% disease penetrance and early Western blot detection of protease-resistant fragments (PrP(res)) of the disease-associated isoform, PrP(Sc), in EM16 brain from 110 days p.i. onwards. EM16 mice were also highly susceptible to classical scrapie and bovine spongiform encephalopathy (BSE), with mean IPs 320 and 246 days faster, respectively, than WT mice. Primary passage of atypical scrapie, classical scrapie and BSE showed that the PrP(res) profiles associated with disease in the natural host were faithfully maintained in EM16 mice, and were distinguishable based on molecular masses, antibody reactivities and glycoform percentages. Immunohistochemistry was used to confirm PrP(Sc) deposition in brain sections from terminal phase transmissible spongiform encephalopathy-challenged EM16 mice. The findings indicate that EM16 mice represent a suitable bioassay model for detection of atypical scrapie infectivity and offer the prospect of differentiation of ruminant prions.

Animal prion diseases.

Prion diseases occur in many animal species, most notably in ruminants. While scrapie in sheep has been recognised for three centuries and goat scrapie has been recognised for decades, BSE in cattle is a relatively novel disease which was first diagnosed in the UK in the mid 1980s. BSE was most likely caused through dietary exposure to animal feed contaminated with prions and disease was subsequently transmitted to people. The BSE epidemic is almost at an end, but the recent identification of so called atypical forms of BSE and scrapie pose many questions about the possible spectrum of prion diseases in animals and their transmissibility to other species, including humans.The pathogenesis of animal prion diseases has been studied both in natural infections and in experimental animal models. Detection of infectivity is greatly helped by suitable rodent models, in particular transgenic mice. Clinically infected animals show characteristic neuropathology in the brain and spinal cord which is accompanied by the accumulation of a conformationally altered, protease-resistant host protein. The post-mortem diagnosis is based on the detection of this protein, PrP(Sc), but despite recent impressive developments a routine ante-mortem diagnostic test has proved elusive.There is no treatment for prion diseases in animals, but disease outbreaks are controlled through a mixture of movement restrictions on holdings, culling of affected animals and herds and, for classical scrapie in sheep, selective breeding for genetic resistance. Prions are very stable and can remain in the environment for prolonged periods. This poses serious practical questions with regard to the decontamination of infected premises. The control of BSE specifically through restrictions in animal feeding practises has been successful, but the changing spectrum of these diseases plus the economic pressures to relax feed bans and reduce levels of surveillance will require constant vigilance to safeguard animal and public health.

Different MAPT haplotypes influence expression of total MAPT in postmortem brain tissue.

The MAPT gene, encoding the microtubule-associated protein tau on chromosome 17q21.31, is result of an inversion polymorphism, leading to two allelic variants (H1 and H2). Homozygosity for the more common haplotype H1 is associated with an increased risk for several tauopathies, but also for the synucleinopathy Parkinson's disease (PD). In the present study, we aimed to clarify whether the MAPT haplotype influences expression of MAPT and SNCA, encoding the protein α-synuclein (α-syn), on mRNA and protein levels in postmortem brains of PD patients and controls. We also investigated mRNA expression of several other MAPT haplotype-encoded genes. Postmortem tissues from cortex of fusiform gyrus (ctx-fg) and of the cerebellar hemisphere (ctx-cbl) of neuropathologically confirmed PD patients (n = 95) and age- and sex-matched controls (n = 81) were MAPT haplotype genotyped to identify cases homozygous for either H1 or H2. Relative expression of genes was quantified using real-time qPCR; soluble and insoluble protein levels of tau and α-syn were determined by Western blotting. Homozygosity for H1 versus H2 was associated with increased total MAPT mRNA expression in ctx-fg regardless of disease state. Inversely, H2 homozygosity was associated with markedly increased expression of the corresponding antisense MAPT-AS1 in ctx-cbl. PD patients had higher levels of insoluble 0N3R and 1N4R tau isoforms regardless of the MAPT genotype. The increased presence of insoluble α-syn in PD patients in ctx-fg validated the selected postmortem brain tissue. Our findings in this small, but well controlled cohort of PD and controls support a putative biological relevance of tau in PD. However, we did not identify any link between the disease-predisposing H1/H1 associated overexpression of MAPT with PD status. Further studies are required to gain a deeper understanding of the potential regulatory role of MAPT-AS1 and its association to the disease-protective H2/H2 condition in the context of PD.

In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation.

BACKGROUND: Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE. RESULTS: PrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification. CONCLUSIONS: PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.

A multiplex pedigree with pathologically confirmed multiple system atrophy and Parkinson's disease with dementia.

Multiple system atrophy is considered a sporadic disease, but neuropathologically confirmed cases with a family history of parkinsonism have been occasionally described. Here we report a North-Bavarian (colloquially, Lion's tail region) six-generation pedigree, including neuropathologically confirmed multiple system atrophy and Parkinson's disease with dementia. Between 2012 and 2020, we examined all living and consenting family members of age and calculated the risk of prodromal Parkinson's disease in those without overt parkinsonism. The index case and one paternal cousin with Parkinson's disease with dementia died at follow-up and underwent neuropathological examination. Genetic analysis was performed in both and another family member with Parkinson's disease. The index case was a female patient with cerebellar variant multiple system atrophy and a positive maternal and paternal family history for Parkinson's disease and dementia in multiple generations. The families of the index case and her spouse were genealogically related, and one of the spouse's siblings met the criteria for possible prodromal Parkinson's disease. Neuropathological examination confirmed multiple system atrophy in the index case and advanced Lewy body disease, as well as tau pathology in her cousin. A comprehensive analysis of genes known to cause hereditary forms of parkinsonism or multiple system atrophy lookalikes was unremarkable in the index case and the other two affected family members. Here, we report an extensive European pedigree with multiple system atrophy and Parkinson`s disease suggesting a complex underlying α-synucleinopathy as confirmed on neuropathological examination. The exclusion of known genetic causes of parkinsonism or multiple system atrophy lookalikes suggests that variants in additional, still unknown genes, linked to α-synucleinopathy lesions underlie such neurodegenerative clustering.

Infection of cell lines with experimental and natural ovine scrapie agents.

Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom.

The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection.

Transcriptional changes in the brains of cattle orally infected with the bovine spongiform encephalopathy agent precede detection of infectivity.

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

Prion protein amino acid determinants of differential susceptibility and molecular feature of prion strains in mice and voles.

The bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases.

Real-time PCR detection and identification of prohibited mammalian and avian material in animal feeds.

A method for the detection and identification of "prohibited" mammalian or avian material in animal feed was developed and assessed through the analysis of DNA. A generic real-time PCR assay was designed to detect the presence of mammalian and avian mitochondrial DNA 16S rRNA genes in animal feed samples. Samples positive with this screening method were further investigated using identification assays to detect the 16S rRNA gene from bovine, ovine, porcine, and avian species and to determine whether the DNA originated from species whose material is prohibited from inclusion in farmed animal feed. An internal positive control was coamplified in the 16S real-time PCR assays to monitor PCR amplification efficiency and avoid potential false-negative results. Using vegetable-based feed standards spiked with meat and bone meal generated with a commercial rendering process, 0.1% meat and bone meal could be detected using the general and species-specific 16S assays. The species-specific assays had 100% specificity for the homologous target species. The 16S real-time PCR assays were evaluated alongside existing tests based on protein evaluation or microscopic examination for a wide range of commercial animal feed samples. In total, 111 (0.76%) of 14,678 samples examined contained prohibited material based on the results from at least one of these tests. However, most positive results did not represent noncompliance because they were associated with samples of pet food, which can legitimately contain material prohibited for use in food for farmed animals. The species-specific 16S assays confirmed the presence of prohibited material in 75% of the 111 samples, whereas the existing protein and microscope tests confirmed the presence of this material in 25 and 54% of the samples, respectively.

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE).

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.

Latrogenic Creutzfeldt-Jakob disease with florid plaques.

Florid plaques indistinguishable from those found in vCJD were identified at a postmortem examination in the brain of a 58-year-old clinical suspect case of Creutzfeldt-Jakob disease (CJD). Western blotting of brain tissue revealed an unusual prion protein type. Since the patient had received a dura mater graft 20 years prior to death and florid plaques are not only found in new variant CJD, the findings argue in favor of an iatrogenic origin of the disease with the longest incubation time following a dura mater graft reported to date even though he may have been exposed to BSE. The peculiar pathological, clinical and biochemical features may define a new type of human prion disease.

Enzymatic formulation capable of degrading scrapie prion under mild digestion conditions.

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrP(Sc) signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrP(Sc) at 65 °C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50 °C over 2 h revealed the progressive attenuation of PrP(Sc) intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrP(Sc) infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions.