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

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

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

Western blots of PrP^Sc from infected mouse brains.

<p>10% w/v brain homogenates (n = 3 per group) were digested with proteinase K and immunoblotted with anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, UK). (A) Transmission of Chandler/RML prions to <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT) litter mate controls. (B) Transmission of ME7 prion strain to <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT) litter mate controls. (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT) litter mate controls. No differences were seen between the two groups regardless of prion strain.</p

RML histology.

<p>Histological features of Chandler/RML prion transmission to <i>Sod1^−/−</i> (A–C) and wild type control (D–F) mice. Panels A and D show distribution of disease-associated PrP by immunohistochemistry using anti-PrP monoclonal antibody ICSM35. Panels B, C, E and F show detail from the hippocampus and are stained with haematoxylin and eosin (H&E) to visualise spongiform change and neuronal loss. There is almost no neuronal loss in the <i>Sod1^−/−</i> mice but mild neuronal loss is seen in the wild type animals. Overall, the pattern of spongiosis, gliosis and PrP distribution are similar between the two groups, however, the distribution of disease-associated PrP is patchier in the knockouts especially in the cortex. Scale bar corresponds to 3 mm (A, D), 660 µm (B, E) or 160 µm (C, F).</p

Kaplan-Meier survival curves.

<p>Data are shown as % of animals (female) surviving (y-axis) plotted against the number of days post inoculation (x-axis). (A) Transmission of Chandler/RML prion strain to <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT) litter mate controls (B) Transmission of ME7 prion strain to <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT). (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT). A reduction in mean incubation time of 20%, 13%, and 24% was seen in A-C respectively. This reduction in survival was statistically significant for each transmission (P<0.001, Kaplan-Meier log-rank survival test).</p

Major strain distribution patterns genotyped for HS mice.

<p>The number of SNPs is taken from genomic sequence generated by the Sanger Institute (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>) and spans 5 kb upstream of the 5′UTR start site to the end of the 3′UTR (NCBI Build 37). Ambiguous SNPs have been excluded. The strain distribution pattern (BALB, CBA); (A, AKR, C3H, C57, DBA, LP) was also seen for <i>App</i> (239/978) but this was not genotyped in the HS mice. Other individual strain distribution patterns were seen ≤5 times.</p

SNP genotyping in HS mice.

<p>Genomic location is given based on mouse genome assembly NCBI build 37. Details of the <i>Sod1</i> and <i>Il1-r1</i> SNPs are available from the Sanger Centre (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>). All polymorphisms were analysed by allele discrimination using a 7500 Fast real time PCR system (Applied Biosystems). For probe details see Table S1. For all genotypes, the statistical test used was the Kruskal-Wallis non-parametric ANOVA. The allelic test used was the Mann-Whitney test.</p

Quantification of Sod and PrP^C

<p>. 10% weight/volume brain homogenates immunoblotted with rabbit polyclonal anti-human SOD1 (Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (A) Brains from <i>Sod1^+/+</i> wild type mice inoculated with PBS compared with end stage Sod1^+/+ wild type mice inoculated with RML, ME7 and MRC2 prion strains. No differences were seen between the groups. (B) Uninfected mice. (C) Total SOD enzymatic activity in 10% (w/v) <i>Sod1^+/+</i> (WT) brains. Brains from terminally sick mice infected with RML, ME7 and MRC2 were compared with uninfected mice. Samples were run in triplicate with n = 6 for each group. Data are shown normalised by total protein content (µg/ml) as determined by a Bradford protein assay (mean ± standard deviation). No significant difference was seen between the groups. (D) PrP^c levels in <i>Sod1^−/−</i> and <i>Sod1^+/+</i> (WT) litter mate control mice by ELISA. PrP^c levels (µg/ml) were determined in triplicate using 10% (weight/volume) brain homogenate for <i>Sod1^−/−</i> (n = 3) and <i>Sod1^+/+</i> (n = 3) in a PrP specific ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054454#pone.0054454-Wadsworth2" target="_blank">[32]</a>. Data are shown normalised by total protein content (µg/ml and x 1000) as determined by a BCA assay (mean ± standard deviation). No significant difference was seen between the two groups.</p