In vitro screen of prion disease susceptibility genes using the scrapie cell assay.

Prion diseases (transmissible spongiform encephalopathies) are fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. While genome-wide association studies in human and quantitative trait loci mapping in mice have provided evidence for multiple susceptibility genes, few of these have been confirmed functionally. Phenotyping mouse models is generally the method of choice. However, this is not a feasible option where many novel genes, without pre-existing models, would need to be tested. We have therefore developed and applied an in-vitro screen to triage and prioritize candidate modifier genes for more detailed future studies which is faster, far more cost effective and ethical relative to mouse bioassay models. An in vitro prion bioassay, the scrapie cell assay, uses a neuroblastoma-derived cell line (PK1) that is susceptible to RML prions and able to propagate prions at high levels. In this study, we have generated stable gene silencing and/or overexpressing PK1-derived cell lines to test whether perturbation of 14 candidate genes affects prion susceptibility. While no consistent differences were determined for seven genes, highly significant changes were detected for Zbtb38, Sorcs1, Stmn2, Hspa13, Fkbp9, Actr10 and Plg, suggesting that they play key roles in the fundamental processes of prion propagation or clearance. Many neurodegenerative diseases involve the accumulation of misfolded protein aggregates and 'prion-like' seeding and spread has been implicated in their pathogenesis. It is therefore expected that some of these prion-modifier genes may be of wider relevance in neurodegeneration.

Overexpression of the Hspa13 (Stch) gene reduces prion disease incubation time in mice.

Prion diseases are fatal neurodegenerative disorders that include bovine spongiform encephalopathy (BSE) and scrapie in animals and Creutzfeldt-Jakob disease (CJD) in humans. They are characterized by long incubation periods, variation in which is determined by many factors including genetic background. In some cases it is possible that incubation time may be directly correlated to the level of gene expression. To test this hypothesis, we combined incubation time data from five different inbred lines of mice with quantitative gene expression profiling in normal brains and identified five genes with expression levels that correlate with incubation time. One of these genes, Hspa13 (Stch), is a member of the Hsp70 family of ATPase heat shock proteins, which have been previously implicated in prion propagation. To test whether Hspa13 plays a causal role in determining the incubation period, we tested two overexpressing mouse models. The Tc1 human chromosome 21 (Hsa21) transchromosomic mouse model of Down syndrome is trisomic for many Hsa21 genes including Hspa13 and following Chandler/Rocky Mountain Laboratory (RML) prion inoculation, shows a 4% reduction in incubation time. Furthermore, a transgenic model with eightfold overexpression of mouse Hspa13 exhibited highly significant reductions in incubation time of 16, 15, and 7% following infection with Chandler/RML, ME7, and MRC2 prion strains, respectively. These data further implicate Hsp70-like molecular chaperones in protein misfolding disorders such as prion disease.

Microglial Cx3cr1 knockout reduces prion disease incubation time in mice.

BACKGROUND: Microglia are resident mononuclear phagocytes of the brain that become activated in response to insults including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and prion disease. In the central nervous system the chemokine Cx3cl1 (Fractalkine) is expressed by neurons and its exclusive receptor Cx3cr1 is expressed solely on microglia. Cx3cl1/Cx3cr1 signalling is thought to maintain microglia in their resting state and disrupting this equilibrium may allow microglia to become activated. In prion disease, microglial proliferation has been suggested to contribute to overall disease progression, however, in different mouse models of neurodegeneration, loss of Cx3cr1 has been shown to either worsen or improve the phenotype depending on the paradigm. RESULTS: To investigate the role of Cx3cl1/Cx3cr1 signalling in prion disease we infected Cx3cr1 null mice with three different strains of prions. Following challenge with Chandler/RML, ME7 and MRC2 prion strains, Cx3cr1 knockout mice showed highly significant reductions in incubation time. No differences were seen in the pattern and localisation of activated microglia in the brain or in the mRNA expression levels of chemokines/cytokines (Cxcl10, Il-12b, Il-1b, Arg-1 and Cxc3l1). CONCLUSION: Our data suggest a protective role for Cx3cl1/Cx3cr1 cross-talk in prion disease.

HECTD2 is associated with susceptibility to mouse and human prion disease.

Prion diseases are fatal transmissible neurodegenerative disorders, which include Scrapie, Bovine Spongiform Encephalopathy (BSE), Creutzfeldt-Jakob Disease (CJD), and kuru. They are characterised by a prolonged clinically silent incubation period, variation in which is determined by many factors, including genetic background. We have used a heterogeneous stock of mice to identify Hectd2, an E3 ubiquitin ligase, as a quantitative trait gene for prion disease incubation time in mice. Further, we report an association between HECTD2 haplotypes and susceptibility to the acquired human prion diseases, vCJD and kuru. We report a genotype-associated differential expression of Hectd2 mRNA in mouse brains and human lymphocytes and a significant up-regulation of transcript in mice at the terminal stage of prion disease. Although the substrate of HECTD2 is unknown, these data highlight the importance of proteosome-directed protein degradation in neurodegeneration. This is the first demonstration of a mouse quantitative trait gene that also influences susceptibility to human prion diseases. Characterisation of such genes is key to understanding human risk and the molecular basis of incubation periods.

A Copine family member, Cpne8, is a candidate quantitative trait gene for prion disease incubation time in mouse.

Prion disease incubation time in mice is determined by many factors including genetic background. The prion gene itself plays a major role in incubation time; however, other genes are also known to be important. Whilst quantitative trait loci (QTL) studies have identified multiple loci across the genome, these regions are often large, and with the exception of Hectd2 on Mmu19, no quantitative trait genes or nucleotides for prion disease incubation time have been demonstrated. In this study, we use the Northport heterogeneous stock of mice to reduce the size of a previously identified QTL on Mmu15 from approximately 25 to 1.2 cM. We further characterised the genes in this region and identify Cpne8, a member of the copine family, as the most promising candidate gene. We also show that Cpne8 mRNA is upregulated at the terminal stage of disease, supporting a role in prion disease. Applying these techniques to other loci will facilitate the identification of key pathways in prion disease pathogenesis.

Shadoo (Sprn) and prion disease incubation time in mice.

Prion diseases are transmissible neurodegenerative disorders of mammalian species and include scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease (vCJD). The prion protein (PrP) plays a key role in the disease, with coding polymorphism in both human and mouse influencing disease susceptibility and incubation time, respectively. Other genes are also thought to be important and a plausible candidate is Sprn, which encodes the PrP-like protein Shadoo (Sho). Sho is expressed in the adult central nervous system and exhibits neuroprotective activity reminiscent of PrP in an in vitro assay. To investigate the role of Sprn in prion disease incubation time we sequenced the open reading frame (ORF) in a diverse panel of mice and saw little variation except in strains derived from wild-trapped mice. Sequencing the untranslated regions revealed polymorphisms that allowed us to carry out an association study of incubation period in the Northport heterogeneous stock of mice inoculated with Chandler/RML prions. We also examined the expression level of Sprn mRNA in the brains of normal and prion-infected mice and saw no correlation with either genotype or incubation time. We therefore conclude that Sprn does not play a major role in prion disease incubation time in these strains of mice.

Investigation of mcp1 as a quantitative trait gene for prion disease incubation time in mouse.

The genetic basis of prion disease incubation time is principally determined by polymorphisms in the prion protein gene, Prnp. However, it is now known that other genetic factors are important. Several quantitative trait loci (QTL) have been identified across the genome including a broad region of linkage on Mmu11. Monocyte chemoattractant protein 1 (MCP-1) maps to this region and has been associated with microglial activation and reduced survival in the ME7 mouse scrapie model of prion disease. We have identified 10 polymorphisms, 3 of which are nonsynonomous, in Mcp1 between "long" (CAST) and "short" (SJL or NZW) incubation-time mouse strains. Crosses between these strains and Mcp1(-/-) mice inoculated with the Chandler/RML mouse scrapie prion strain formed the basis of a quantitative complementation test. In these models loss of Mcp1 did not show an increase in incubation time suggesting that the effects of Mcp1 may be specific to the ME7 prion strain and that Mcp1 does not contribute to the QTL described on Mmu11.

HECTD2, a candidate susceptibility gene for Alzheimer's disease on 10q.

BACKGROUND: Late onset Alzheimer's disease (LOAD) is a neurodegenerative disorder characterised by the deposition of amyloid plaques and neurofibrillary tangles in the brain and is the major cause of dementia. Multiple genetic loci, including 10q, have been implicated in LOAD but to date, with the exception of APOE, the underlying genes have not been identified. HECTD2 maps to 10q and has been implicated in susceptibility to human prion diseases which are also neurodegenerative conditions associated with accumulation of misfolded host proteins. In this study we test whether the HECTD2 susceptibility allele seen in prion disease is also implicated in LOAD. METHODS: DNA from 320 individuals with Alzheimer's disease and 601 controls were genotyped for a HECTD2 intronic tagging SNP, rs12249854 (A/T). Groups were further analysed following stratification by APOE genotype. RESULTS: The rs12249854 minor allele (A) frequency was higher (5.8%) in the Alzheimer's disease group as compared to the controls (3.9%), however, this was not statistically significant (P = 0.0668). No significant difference was seen in minor allele frequency in the presence or absence of the APOE epsilon4 allele. CONCLUSION: The common haplotypes of HECTD2, tagged by rs12249854, are not associated with susceptibility to LOAD.

Rare structural genetic variation in human prion diseases.

Prion diseases are a diverse group of neurodegenerative conditions, caused by the templated misfolding of prion protein. Aside from the strong genetic risk conferred by multiple variants of the prion protein gene (PRNP), several other variants have been suggested to confer risk in the most common type, sporadic Creutzfeldt-Jakob disease (sCJD) or in the acquired prion diseases. Large and rare copy number variants (CNVs) are known to confer risk in several related disorders including Alzheimer's disease (at APP), schizophrenia, epilepsy, mental retardation, and autism. Here, we report the first genome-wide analysis for CNV-associated risk using data derived from a recent international collaborative association study in sCJD (n = 1147 after quality control) and publicly available controls (n = 5427). We also investigated UK patients with variant Creutzfeldt-Jakob disease (n = 114) and elderly women from the Eastern Highlands of Papua New Guinea who proved highly resistant to the epidemic prion disease kuru, who were compared with healthy young Fore population controls (n = 395). There were no statistically significant alterations in the burden of CNVs >100, >500, or >1000 kb, duplications, or deletions in any disease group or geographic region. After correction for multiple testing, no statistically significant associations were found. A UK blood service control sample showed a duplication CNV that overlapped PRNP, but these were not found in prion disease. Heterozygous deletions of a 3' region of the PARK2 gene were found in 3 sCJD patients and no controls (p = 0.001, uncorrected). A cell-based prion infection assay did not provide supportive evidence for a role for PARK2 in prion disease susceptibility. These data are consistent with a modest impact of CNVs on risk of late-onset neurologic conditions and suggest that, unlike APP, PRNP duplication is not a causal high-risk mutation.

Genetics of prion diseases.

Prion diseases are transmissible, fatal neurodegenerative diseases that include scrapie and bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease (CJD) in human. The prion protein gene (PRNP) is the major genetic determinant of susceptibility, however, several studies now suggest that other genes are also important. Two recent genome wide association studies in human have identified four new loci of interest: ZBTB38-RASA2 in UK CJD cases and MTMR7 and NPAS2 in variant CJD. Complementary studies in mouse have used complex crosses to identify new modifiers such as Cpne8 and provided supporting evidence for previously implicated genes (Rarb and Stmn2). Expression profiling has identified new candidates, including Hspa13, which reduces incubation time in a transgenic model.

Sod1 deficiency reduces incubation time in mouse models of prion disease.

Prion infections, causing neurodegenerative conditions such as Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep and BSE in cattle are characterised by prolonged and variable incubation periods that are faithfully reproduced in mouse models. Incubation time is partly determined by genetic factors including polymorphisms in the prion protein gene. Quantitative trait loci studies in mice and human genome-wide association studies have confirmed that multiple genes are involved. Candidate gene approaches have also been used and identified App, Il1-r1 and Sod1 as affecting incubation times. In this study we looked for an association between App, Il1-r1 and Sod1 representative SNPs and prion disease incubation time in the Northport heterogeneous stock of mice inoculated with the Chandler/RML prion strain. No association was seen with App, however, significant associations were seen with Il1-r1 (P = 0.02) and Sod1 (P<0.0001) suggesting that polymorphisms at these loci contribute to the natural variation observed in incubation time. Furthermore, following challenge with Chandler/RML, ME7 and MRC2 prion strains, Sod1 deficient mice showed highly significant reductions in incubation time of 20, 13 and 24%, respectively. No differences were detected in Sod1 expression or activity. Our data confirm the protective role of endogenous Sod1 in prion disease.

Sex effects in mouse prion disease incubation time.

Prion disease incubation time in mice is determined by many factors including PrP expression level, Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrP(C) levels nor in the neuropathological markers of prion disease: PrP(Sc) distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used.

The retinoic acid receptor beta (Rarb) region of Mmu14 is associated with prion disease incubation time in mouse.

In neurodegenerative conditions such as Alzheimer's and prion disease it has been shown that host genetic background can have a significant effect on susceptibility. Indeed, human genome-wide association studies (GWAS) have implicated several candidate genes. Understanding such genetic susceptibility is relevant to risks of developing variant CJD (vCJD) in populations exposed to bovine spongiform encephalopathy (BSE) and understanding mechanisms of neurodegeneration. In mice, aspects of prion disease susceptibility can be modelled by examining the incubation period following experimental inoculation. Quantitative trait linkage studies have already identified multiple candidate genes; however, it is also possible to take an individual candidate gene approach. Rarb and Stmn2 were selected as candidates based on the known association with vCJD. Because of the increasing overlap described between prion and Alzheimer's diseases we also chose Clu, Picalm and Cr1, which were identified as part of Alzheimer's disease GWAS. Clusterin (Clu) was considered to be of particular interest as it has already been implicated in prion disease. Approximately 1,000 heterogeneous stock (HS) mice were inoculated intra-cerebrally with Chandler/RML prions and incubation times were recorded. Candidate genes were evaluated by sequencing the whole transcript including exon-intron boundaries and potential promoters in the parental lines of the HS mice. Representative SNPs were genotyped in the HS mice. No SNPs were identified in Cr1 and no statistical association with incubation time was seen for Clu (P = 0.96) and Picalm (P = 0.91). Significant associations were seen for both Stmn2 (P = 0.04) and Rarb (P = 0.0005), however, this was only highly significant for Rarb. This data provides significant further support for a role for the Rarb region of Mmu14 and Stmn2 in prion disease.

Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice.

Prion diseases are fatal neurodegenerative disorders of humans and animals, which include bovine spongiform encephalopathy (BSE) and its human form, variant Creutzfeldt-Jakob disease (vCJD). They are characterized by a prolonged incubation period, which is known to be influenced by polymorphisms in the prion protein gene. Previous studies of inbred mice have demonstrated that additional genetic loci also contribute to the observed variation in incubation period. However, a substantial transmission barrier between cow and mouse complicates studies using BSE. As a result, primary transmissions display large variations in incubation period and not all animals develop clinical signs of disease. To identify quantitative trait loci for BSE without the presence of a transmission barrier, we analysed 124 animals from an F2 intercross between CAST/Ei and NZW/OlaHsd mice and challenged them intracerebrally with a strain of BSE that was passaged twice through C57BL/6OlaHsd mice. Interval mapping identified two highly significant linked regions on chromosomes 2 and 11 with peak lod scores of 6.34 and 4.77, respectively. Composite interval mapping suggests that chromosome 2 includes three linked quantitative trait loci. Loci in the same position on chromosomes 2 and 11 were also identified in a previous study using the same mouse cross but infected with Chandler/RML scrapie prions. If these are the same loci, it suggests that these loci may be influencing incubation time independently of prion strain. This provides hope that it may be possible to identify human quantitative trait loci for prion incubation time using mouse models that may allow identification of at-risk individuals and the discovery of novel therapeutic targets.

Characterization of two distinct prion strains derived from bovine spongiform encephalopathy transmissions to inbred mice.

Distinct prion strains can be distinguished by differences in incubation period, neuropathology and biochemical properties of disease-associated prion protein (PrP(Sc)) in inoculated mice. Reliable comparisons of mouse prion strain properties can only be achieved after passage in genetically identical mice, as host prion protein sequence and genetic background are known to modulate prion disease phenotypes. While multiple prion strains have been identified in sheep scrapie and Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (BSE) is thought to be caused by a single prion strain. Primary passage of BSE prions to different lines of inbred mice resulted in the propagation of two distinct PrP(Sc) types, suggesting that two prion strains may have been isolated. To investigate this further, these isolates were subpassaged in a single line of inbred mice (SJL) and it was confirmed that two distinct prion strains had been identified. MRC1 was characterized by a short incubation time (110+/-3 days), a mono-glycosylated-dominant PrP(Sc) type and a generalized diffuse pattern of PrP-immunoreactive deposits, while MRC2 displayed a much longer incubation time (155+/-1 days), a di-glycosylated-dominant PrP(Sc) type and a distinct pattern of PrP-immunoreactive deposits and neuronal loss. These data indicate a crucial involvement of the host genome in modulating prion strain selection and propagation in mice. It is possible that multiple disease phenotypes may also be possible in BSE prion infection in humans and other animals.

Identification and characterization of a novel mouse prion gene allele.

The major determinant of prion disease incubation time in mice is thought to be the amino acid sequence of the prion protein. Two alleles of the mouse prion gene ( Prnp) have been described, where Prnp(a) (Leu-108, Thr-189) and Prnp(b) (Phe-108, Val-189) are associated with short and long incubation times, to defined prion strains, respectively. As part of a survey of inbred mouse lines, the prion gene open reading frame was sequenced and revealed a new allele, Prnp(c) (Phe-108, Thr-189), in the strain MAI/Pas. To study the influence of Prnp(c) independently of the MAI/Pas genetic background, we generated a congenic line in which Prnp(c) was bred onto the C57BL/6JOlaHsd background. Following intracerebral inoculation with Chandler/RML scrapie prions, the congenic mice showed an increased mean incubation time relative to C57BL/6JOlaHsd, of over 100 days. However, no differences were observed in the intensity and pattern of PrP immunoreactivity deposition or spongiosis. We conclude that the new allele, Prnp(c), modulates incubation time but not neuropathology and that the previous classification of mice into two distinct groups based on incubation time and Prnp genotype should now be revised.

Human prion protein with valine 129 prevents expression of variant CJD phenotype.

Variant Creutzfeldt-Jakob disease (vCJD) is a unique and highly distinctive clinicopathological and molecular phenotype of human prion disease associated with infection with bovine spongiform encephalopathy (BSE)-like prions. Here, we found that generation of this phenotype in transgenic mice required expression of human prion protein (PrP) with methionine 129. Expression of human PrP with valine 129 resulted in a distinct phenotype and, remarkably, persistence of a barrier to transmission of BSE-derived prions on subpassage. Polymorphic residue 129 of human PrP dictated propagation of distinct prion strains after BSE prion infection. Thus, primary and secondary human infection with BSE-derived prions may result in sporadic CJD-like or novel phenotypes in addition to vCJD, depending on the genotype of the prion source and the recipient.

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein.

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.