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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.

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.

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.

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.

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.