Creutzfeldt-Jakob Disease Patients Research Articles

Analysis of the advantage features of Beijing surveillance network for Creutzfeldt-Jakob disease

ABSTRACT. Since 2006, China has conducted the surveillance program for Creutzfeldt-Jakob disease (CJD) and other subtypes of prion diseases covering 12 provinces. In this study, the characteristics and special role of Beijing CJD surveillance network in the national CJD surveillance system were analyzed. Based on the registered permanent resident places, all reporting suspected CJD cases and diagnosed CJD cases via Beijing CJD surveillance network between 2006 and 2013 were grouped as the cases from Beijing and from outside of Beijing. Both numbers of the suspected and diagnosed CJD cases via Beijing CJD surveillance network constantly increased along with the years, totally 532 reporting cases and 192 diagnosed CJD cases were obtained in the past 8 y. About 75% of suspected and diagnosed CJD cases via Beijing CJD surveillance network came from other provinces, mainly from neighboring provinces. Altogether, 46 different hospitals in the Beijing region have reported suspected CJD cases to the CJD surveillance system during the period from 2006 to 2013. Five hospitals continually reported suspected CJD cases during those 8 y and 5 other hospitals had reported cases except for 1 to 2 y. Additionally, we found that the diagnosed CJD patients from Beijing region had less numbers of hospital transfer and shorter interval from the disease onset to the final diagnosis than those outside of Beijing. It indicates that as the most important component, Beijing CJD surveillance network functions more actively, which supplies the special medical services not only for Beijing residents but also for patients from all of China.

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

The influence of PRNP polymorphisms on human prion disease susceptibility: an update.

Two normally occurring polymorphisms of the human PRNP gene, methionine (M)/valine (V) at codon 129 and glutamic acid (E)/lysine (K) at codon 219, can affect the susceptibility to prion diseases. It has long been recognized that 129M/M homozygotes are overrepresented in sporadic Creutzfeldt-Jakob disease (CJD) patients and variant CJD patients, whereas 219E/K heterozygotes are absent in sporadic CJD patients. In addition to these pioneering findings, recent progress in experimental transmission studies and worldwide surveillance of prion diseases have identified novel relationships between the PRNP polymorphisms and the prion disease susceptibility. For example, although 219E/K heterozygosity confers resistance against the development of sporadic CJD, this genotype is not entirely protective against acquired forms (iatrogenic CJD and variant CJD) or genetic forms (genetic CJD and Gerstmann-Sträussler-Scheinker syndrome) of prion diseases. In addition, 129M/V heterozygotes predispose to genetic CJD caused by a pathogenic PRNP mutation at codon 180. These findings show that the effects of the PRNP polymorphisms may be more complicated than previously thought. This review aims to summarize recent advances in our knowledge about the influence of the PRNP polymorphisms on the prion disease susceptibility.

Subtype and regional regulation of prion biomarkers in sporadic Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is a rapid progressive neurological disease leading to dementia and death. Prion biomarkers are altered in the cerebrospinal fluid (CSF) of CJD patients, but the pathogenic mechanisms underlying these alterations are still unknown. The present study examined prion biomarker levels in the brain and CSF of sporadic CJD (sCJD) cases and their correlation with neuropathological lesion profiles. The expression levels of 14-3-3, Tau, phospho-Tau and α-synuclein were measured in the CSF and brain of sCJD cases in a subtype- and region-specific manner. In addition, the activity of prion biomarker kinases, the expression levels of CJD hallmarks and the most frequent neuropathological sCJD findings were analysed. Prion biomarkers levels were increased in the CSF of sCJD patients; however, correlations between mRNA, total protein and their phosphorylated forms in brain were different. The observed downregulation of the main Tau kinase, GSK3, in sCJD brain samples may help to explain the differential phospho-Tau/Tau ratios between sCJD and other dementias in the CSF. Importantly, CSF biomarkers levels do not necessarily correlate with sCJD neuropathological findings. Present findings indicate that prion biomarkers levels in sCJD tissues and their release into the CSF are differentially regulated following specific modulated responses, and suggest a functional role for these proteins in sCJD pathogenesis.

A genome wide association study links glutamate receptor pathway to sporadic Creutzfeldt-Jakob disease risk.

We performed a genome-wide association (GWA) study in 434 sporadic Creutzfeldt-Jakob disease (sCJD) patients and 1939 controls from the United Kingdom, Germany and The Netherlands. The findings were replicated in an independent sample of 1109 sCJD and 2264 controls provided by a multinational consortium. From the initial GWA analysis we selected 23 SNPs for further genotyping in 1109 sCJD cases from seven different countries. Five SNPs were significantly associated with sCJD after correction for multiple testing. Subsequently these five SNPs were genotyped in 2264 controls. The pooled analysis, including 1543 sCJD cases and 4203 controls, yielded two genome wide significant results: rs6107516 (p-value=7.62x10-9) a variant tagging the prion protein gene (PRNP); and rs6951643 (p-value=1.66x10-8) tagging the Glutamate Receptor Metabotropic 8 gene (GRM8). Next we analysed the data stratifying by country of origin combining samples from the pooled analysis with genotypes from the 1000 Genomes Project and imputed genotypes from the Rotterdam Study (Total n=12967). The meta-analysis of the results showed that rs6107516 (p-value=3.00x10-8) and rs6951643 (p-value=3.91x10-5) remained as the two most significantly associated SNPs. Rs6951643 is located in an intronic region of GRM8, a gene that was additionally tagged by a cluster of 12 SNPs within our top100 ranked results. GRM8 encodes for mGluR8, a protein which belongs to the metabotropic glutamate receptor family, recently shown to be involved in the transduction of cellular signals triggered by the prion protein. Pathway enrichment analyses performed with both Ingenuity Pathway Analysis and ALIGATOR postulates glutamate receptor signalling as one of the main pathways associated with sCJD. In summary, we have detected GRM8 as a novel, non-PRNP, genome-wide significant marker associated with heightened disease risk, providing additional evidence supporting a role of glutamate receptors in sCJD pathogenesis.

Phosphatidylinositol-glycan-phospholipase D is involved in neurodegeneration in prion disease.

PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer’s disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.

Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD.

Creutzfeldt-Jakob disease: clinical and diagnostic aspects

In this article, authors analyzed a modern approach to the diagnosis of Creutzfeldt-Jakob disease (CJD) based on the clinical signs, cerebrospinal fluid markers, electroencephalography and magnetic resonance imaging. It was demonstrated for the first time that patients with late-onset CJD differed from younger CJD patients with respect to MRI profiles and initial clinical presentation. To date, cerebrospinal fluid (CSF) analysis, particularly protein 14-3-3 testing, presents an important approach to the identification of disease cases. A spectrum of differential diagnosis of rapid progressive dementia varied from neurodegenerative dementias to dementia due to acute neurological conditions. Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein. Recent studies applied the RT-QuIC methodology to CSF for the diagnosis of human prion diseases.

Pin1 and neurodegeneration: a new player for prion disorders?

Pin1 is a peptidyl-prolyl isomerase that catalyzes the cis/trans conversion of phosphorylated proteins at serine or threonine residues which precede a proline. The peptidyl-prolyl isomerization induces a conformational change of the proteins involved in cell signaling process. Pin1 dysregulation has been associated with some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. Proline-directed phosphorylation is a common regulator of these pathologies and a recent work showed that it is also involved in prion disorders. In fact, prion protein phosphorylation at the Ser-43-Pro motif induces prion protein conversion into a disease-associated form. Furthermore, phosphorylation at Ser-43-Pro has been observed to increase in the cerebral spinal fluid of sporadic Creutzfeldt-Jakob Disease patients. These findings provide new insights into the pathogenesis of prion disorders, suggesting Pin1 as a potential new player in the disease. In this paper, we review the mechanisms underlying Pin1 involvement in the aforementioned neurodegenerative pathologies focusing on the potential role of Pin1 in prion disorders.

Mutant PrPCJD prevails over wild-type PrPCJD in the brain of V210I and R208H genetic Creutzfeldt–Jakob disease patients

Creutzfeldt–Jakob disease (CJD) is a neurodegenerative disorder characterized by the deposition of the pathological conformer (PrPCJD) of the host encoded cellular prion protein (PrPC). In genetic CJD associated with V210I or R208H PrP substitutions, the pathogenic role of mutant residues is still poorly understood. To understand how V210I or R208H PrP mutations facilitate the development of the disease, we determined by mass spectrometry the quantitative ratio of mutant/wild-type PrPCJD allotypes in brains from affected subjects. We found that the mutant PrPCJD allotypes moderately exceeds of 2- or 3-fold the amount of the wild-type counterpart suggesting that these mutations mainly exert their pathogenic effect on the onset of the pathogenic cascade.Different mechanisms can be hypothesized to explain the pathogenic role of mutant residues: V210I and R208H substitutions can increase the concentration of PrPC and the probability to form insoluble aggregates, or they may facilitate the formation of pathological intermediates, or, alternatively, they may increase the affinity for ligands that are involved in the initial phases of PrPCJD formation and aggregation.Whatever the mechanism, the enrichment found for the mutated PrPCJD species indicates that these altered structures are more prone, with respect to the non-mutated ones, to be captured in the polymerization process either at the onset or during the development of the disease.

Analysis of the compliance and the related influence factors in the follow-up process of surveillance for Creutzfeldt-Jakob disease in China

Creutzfeldt-Jakob disease (CJD) is a kind of rare, rapidly progressive fatal central nervous system disorders. In China, the surveillance for CJD has started since 2006. As one of the major issues in CJD surveillance, the follow-up process via telephone plays important role in CJD diagnosis and surveillance. Although the follow-up process was conducted by the experiential staffs from CJD surveillance center in China CDC, it is frequently encountered that some interviewed family members do not cooperate well during follow-up. To screen the possible factors influence on the compliances of the interviewees during CJD follow-up, 11 independent variables from patient aspect and 4 variables from interviewee aspect were selected and a questionnaire was prepared. Based on 199 suspected sporadic CJD cases reported to CJD surveillance center in 2013, a telephone-inquiring was conducted and the degree of compliances of the interviewees were given as good, fair or poor. After screened with univariate analysis and evaluated ordinal logistic regression analysis, several indictors, such as the patient gender, CJD diagnosis, numbers of clinical symptoms, continual medical treatment after diagnosis, medical treatment mode, as well as the relationship with the patient and CJD knowledge of the interviewees, showed influence on the compliance in CJD follow-up process significantly. The data here provide for the first time the factors related with the compliances of the interviewed family members of the suspected CJD patients during follow-up process, which supplies useful clue for us to improve CJD follow-up process and increase the capacity of CJD surveillance.

A pilot study of a genetic CJD risk factor (E200K) in the general Slovak population.

Creutzfeldt-Jakob disease (CJD) is a fatal, incurable human transmissible spongiform encephalopathy, or prion disease. Classical CJD has been recognised since the 1920s. CJD occurs in three forms : sporadic, of unknown origin, genetic, related to disease specific point mutations, insertions or deletions of the prion protein gene (PRNP) and iatrogenic (iCJD), caused by accidental transmission of the disease in the course of medical or surgical treatment. A new variant of CJD, which is causally related to bovine spongiform encephalopathy (BSE) was first described by Will et al. in 1996. The risk of horizontal transmission of CJD has been confirmed in classical [1] as well as in variant CJD. Precautions and measures to prevent iCJD are focused mainly on clinical CJD patients, but a potential risk from preclinical cases, for example asymptomatic carriers of CJDspecific mutations of the PRNP, cannot be excluded. These carriers represent a ‘‘genetic CJD-risk group’’ in the general population. More than 37 disease-specific mutations have been described in the PRNP coding sequence. The most common point mutation associated with genetic CJD is at codon 200 (E200K) [2]. While in the majority of countries the genetic form represents about 10–15 % of all CJD patients, in Slovakia it has never been \65 % since 1975. [3]. Except for one case with the R208H mutation, all 202 genetic CJD patients in Slovakia carry the E200K mutation (gCJD). Most of these cases are concentrated in two clusters in central Slovakia [4]. Voluntary genetic testing of healthy blood relatives has revealed that 35 % carry the E200K mutation [3]. Besides the risk of gCJD (59 % of carriers will develop the disease) (3), ‘‘healthy’’ carriers could act as a source of iatrogenic infection. To our knowledge, the prevalence of the mutation E200K has not been studied. The aim of this pilot study is to investigate the occurrence and frequency of asymptomatic carries of E200K in the Slovak general population, comparing the areas of focal accumulation of gCJD with extrafocal areas, i.e. comparison of regions with contrasting incidence rates of gCJD. In the present study the occurrence of the mutation E200K and the distribution of the M129 V polymorphism of PRNP were analysed in 2,662 subjects with no known link to patients with gCJD. Tested were DNA from two different sources: 1. anonymised DNA samples of newborns provided by Slovak Newborn Screening Center were from regions with different incidence of gCJD: 1a) 893 samples from two focal accumulations (clusters) of gCJD in central Slovakia (mean incidence of gCJD 3.55/million) and 1b) 757 samples from extrafocal regions in western and E. Mitrova D. Kosorinova M. Gajdos I. Tomeckova Medical Faculty, Slovak Medical University, Bratislava, Slovakia

Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration.

The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour γ-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer's disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases.

Increased α-synuclein levels in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease.

Recent studies have shown that cerebrospinal fluid (CSF) levels of α-synuclein (α-syn) are highly elevated in patients with Creutzfeldt-Jakob disease (CJD) compared to controls. However, the diagnostic value of CSF α-syn in CJD has not been established. To confirm whether CSF α-syn is increased in CJD and is a useful marker for this disease, two independent enzyme-linked immunoabsorbent assays (ELISAs) specific for α-syn were used: ELISA 211-FL140, which is specific for full-length α-syn, and ELISA N19-FL140, which is specific for the full-length and associated C-terminal truncated forms of α-syn. CSF samples from 24 patients with CJD and 24 controls were assessed in this study. We found that samples from the CJD patients showed significantly higher levels of CSF α-syn compared to controls in both ELISA (211-FL140 or N19-FL140) tests (P = 0.0467 and P = 0.0010, respectively). However, there was a considerable overlap in the concentration ranges of the two groups of subjects. We also measured the levels of total tau (t-tau) protein in these samples and found that CSF t-tau levels were 5-10-times higher in the CJD group (P < 0.0001) compared with the controls. When the CSF t-tau and α-syn levels were combined, the area under the ROC curve (AUC) was slightly increased in clinically diagnosed CJD cases (AUC of 0.964) relative to an AUC of 0.943 for increased CSF t-tau alone. The combined use of CSF α-syn and t-tau levels may be a useful biomarker for the diagnosis of CJD.

Diagnostic Performance of Cerebrospinal Fluid Total Tau and Phosphorylated Tau in Creutzfeldt-Jakob Disease

Identifying a clinical distinction between the invariably lethal prion disease Creutzfeldt-Jakob disease (CJD) and nonprion rapidly progressive dementias is important and sometimes difficult; thus, reliable tools for diagnosis are in great demand. To test the diagnostic performance of dementia cerebrospinal fluid (CSF) biomarkers total tau (T-tau), phosphorylated tau (P-tau), and the T-tau to P-tau ratio for CJD by analyzing the results from a large database of routine clinical samples in combination with diagnosis information from the Swedish Mortality Registry. This was a retrospective cohort study. We cross-referenced the Swedish Mortality Registry with a data set of CSF measurements of T-tau and P-tau performed in routine clinical testing at the Clinical Neurochemistry Laboratory of the Sahlgrenska University Hospital, which serves most of Sweden. The data set consisted of 9765 deceased individuals with CSF measures, including 93 with CJD, with 52 autopsy-verified samples (56%). For each patient, T-tau and P-tau levels in CSF were measured and the T-tau to P-tau ratio was calculated. Biomarker levels (adjusted for age and sex) were analyzed in relation to the recorded cause of death and time of death. We specifically tested a previously defined CJD biomarker profile (T-tau >1400 ng/L and T-tau to P-tau-ratio >25). Patients who died of CJD had elevated CSF T-tau levels and T-tau to P-tau ratio, but not elevated CSF P-tau levels, compared with patients who died of Alzheimer disease (AD) and other dementias. The previously defined biomarker profile had a specificity of 99.0%, a sensitivity of 78.5%, and a positive likelihood ratio of 79.9. When tested against common differential diagnoses, the sensitivity, specificity, and positive likelihood ratio of this profile was 78.5%, 99.6%, and 196.6, respectively, in relation to AD and 78.5%, 99.3%, and 109.3, respectively, in relation to other dementias. In CJD individuals (n = 30) with repeated measurements, but not in those with AD (n = 242) or other dementias (n = 258), T-tau levels and T-tau to P-tau ratios increased over time. In this routine clinical setting, the combination of increased T-tau levels and increased T-tau to P-tau ratios in CJD patients has a very high specificity against important differential diagnoses to CJD and may serve as a clinically useful diagnostic test.

RARB and STMN2 polymorphisms are not associated with sporadic Creutzfeldt–Jakob disease (CJD) in the Korean population

Polymorphisms in the prion protein gene (PRNP) can affect the susceptibility of humans to prion diseases. Recently, aside from PRNP, single nucleotide polymorphisms (SNPs) of two candidate genes for susceptibility to human prion diseases have been identified by human genome-wide association studies (GWAS) in the British population. One SNP of retinoic acid receptor beta (RARB), which is correlated with prion disease incubation time in mice, was associated with human prion diseases such as variant and iatrogenic CJD in the British population. The other SNP of the gene that encodes SCG10 (STMN2), which is related to clinical onset of sporadic CJD, was also associated with variant CJD and kuru. In order to investigate whether two polymorphisms located in upstream of RARB and STMN2 are associated with sporadic CJD in the Korean population, we compared genotype and allele frequencies of these polymorphisms in 217 sporadic CJD patients and 216 healthy Koreans. The genotype distribution and allele frequencies in upstream of the RARB and STMN2 polymorphisms were not significantly different between healthy controls and Korean sporadic CJD patients. This finding indicates that the two SNPs are not correlated with genetic susceptibility to sporadic CJD in the Korean population. This is the first genetic association study of RARB and STMN2 with sporadic CJD in an Asian population.

A proposal of new diagnostic pathway for fatal familial insomnia

BackgroundIn absence of a positive family history, the diagnosis of fatal familial insomnia (FFI) might be difficult because of atypical clinical features and low sensitivity of diagnostic tests. FFI patients...

Global Protein Differential Expression Profiling of Cerebrospinal Fluid Samples Pooled from Chinese Sporadic CJD and non-CJD Patients

The shotgun proteomic based on the approach of tandem mass tag (TMT) labeling has received increasing attention for neuroproteomics analysis and becomes an effective tool for the identification and quantification of a large number of proteins for the purpose of revealing key proteins involved in the neuronal dysfunction and an inflammatory response associated with neurodegenerative disorders. To assess the potential expression difference of proteins in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, the pooled CSF samples from 39 Chinese probable sporadic CJD (sCJD) patients and from 52 non-CJD cases were comparably analyzed with the methodology of TMT labeling and RP-RP-UPLC-MS/MS. Totally, 437 possible proteins were identified in the tested CSF specimen, among them, 49 proteins with 95% confidence interval. Differential assays showed among those 49 CSF proteins, 12 were upregulated and 13 were downregulated significantly in the sCJD compared to non-CJD. The most affected pathway of the differential expression proteins in CSF of sCJD was complement and coagulation cascade. Western blots for six selected changed proteins in the pooled CSF samples revealed the similar altering profiles in the groups of sCJD and non-CJD as proteomics. Furthermore, CSF samples from 24 CJD patients and 24 non-CJD patients were randomly selected and subjected individually into the Western blots of an increased protein (phosphoglycerate mutase 1) and a decreased one (alpha-1-antichymotrysin), which also confirmed the altering tendency of these identified proteins. Those data indicate that proteomic assay of CSF is a powerful technique not only for selection of the potential biomarkers for the development of diagnostic tool of CJD but also for supplement of useful scientific clues for understanding the CSF homeostasis during the pathogenesis of prion diseases.

Characterization of Variant Creutzfeldt-Jakob Disease Prions in Prion Protein-humanized Mice Carrying Distinct Codon 129 Genotypes

To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.

Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP-FRET-enabled high throughput assay (PrP-FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood-brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.