Quantitative trait loci mapping in mice and GWAS in human have confirmed that multiple genes in addition to Prnp
influence prion disease incubation time and susceptibility 
. These studies have successfully identified candidate loci but no individual genes have yet been confirmed as functionally significant. Alternative strategies have included targeting genes from pathways implicated in prion disease and testing them directly in mouse models. This identified App
as interesting candidates, however, it did not address whether or not they contributed to the natural variation 
. In this study we carried out an association study for each of these genes in HS of mice. The mapping resolution available in the HS cross at generation 37 is approximately 1–2 cM which is too big to unambiguously ascribe an association to one gene although mapping to SDPs may help to narrow down the region 
is a very promising candidate gene particularly given its involvement in Alzheimer’s disease (AD), a neurodegenerative protein misfolding disease. There is also increasing evidence for an interaction with PrP where PrP has been implicated in post-translational processing of APP through the inhibition of BACE1 and in some mouse models PrP may bind amyloid-β and mediate aspects of its neurotoxicity 
. Despite its attractions as a candidate we did not find any evidence of an association with prion disease incubation time in this cross although it is possible that other SDPs not tested by us may show some association. App
may still have a role in prion disease although it does not appear to contribute to the natural variation observed in the parental lines of this cross.
Il1-r1 is a receptor for the pro-inflammatory cytokine, Il-1, produced by glia and implicated in the disease process as a result of extensive gliosis. Il1-r1
knockout mice have been previously studied confirming that deletion of Il1-r1
is protective in models of mouse scrapie 
. In this study we identify a non-synonymous SNP (Ala71Val) on a SDP that is linked to prion disease incubation time. It is not clear whether this SNP is the functional variant or whether it is carried within the same haplotype block.
Sod1 is an ubiquitously expressed cytoplasmic superoxide dismutase that protects cells from oxidative damage by removing superoxide through converting it to hydrogen peroxide. It has been associated with other neurodegenerative diseases notably amyotrophic lateral sclerosis where mutations in human SOD1 account for ~20% of familial ALS cases probably via a toxic gain of function 
. Sod1 has also been implicated in Alzheimer’s disease whereby Sod1
knockout accelerated amyloid-β oligomerisation and memory loss in an AD mouse model and conversely overexpression of Sod1 rescues the cerebral endothelial dysfunction in another AD model 
. In this study we have shown a highly significant association with the Sod1
locus and prion disease incubation time thereby confirming its effect on the natural variation within these inbred lines of mice. The functional SNPs remain unknown and we have not detected any evidence for differential mRNA or protein expression. We have also confirmed the role of Sod1 as a protective factor in prion disease as deletion of Sod1 significantly reduces incubation time in two different strains of mouse adapted scrapie (Chandler/RML and ME7) and a mouse passaged BSE prion strain, MRC2. These data broadly agree with previously published data that suggested a protective effect of overexpressing human SOD1 in mice challenged with RML but not 301 V prions 
. This difference may be explained by the presence of the human protein or by differences in the mouse adapted BSE strains caused by passage in Prnpb
rather than Prnpa
mice. We cannot exclude the possibility that the targeted knockout of Sod1 in these mice may inadvertently affect the expression of adjacent loci which themselves influence prion disease incubation time and that this would explain the absence of detectable differences in Sod1 expression.
Oxidative stress has been implicated in neurodegenerative diseases and is caused by excess reactive oxygen species such as superoxide accumulating with the cell which may be the result of excess production by malfunctioning mitochondria and depletion of catalytic enzymes. In mouse models of prion disease damaged mitochondria have been described as well as evidence for increased superoxide generation and free radical damage 
. Markers of oxidative damage have also been described in CJD brains 
. We did not detect a change in Sod1 protein expression or total Sod activity in response to prion infection with any of our prion strains, however, the presence of normal Sod1 activity is clearly protective as Sod1 deficient mice show a significantly reduced incubation time across all prion strains tested.
In conclusion, we have shown that both Il1-r1 and Sod, but not App, are associated with the natural variation in prion disease incubation time measured in the Northport HS mice. In addition, Sod1 deficient mice show highly significant reductions in incubation time of 20, 13 and 24% with Chandler/RML, ME7 and MRC2 prion strains, respectively. Although no differences in Sod1 expression or activity were detected our data highlight the importance of oxidative damage in prion disease and the protective role played by endogenous Sod1 protein.