DNA glycosylase Neil2 contributes to genomic responses in the spleen during clinical prion disease.

The DNA glycosylase Neil2 is a member of the base excision repair (BER) family of enzymes, which are important for repair of oxidative DNA damage. Specifically, Neil2 participates in repair of oxidized bases in single-stranded DNA of transcriptionally active genes. Mice with genetic ablation of Neil2 (Neil2-/-) display no overt phenotypes, but an age-dependent accumulation of oxidative DNA damage and increased inflammatory responsiveness. In young mice intra-cerebrally inoculated with prions, vigorous prion propagation starts rapidly in the germinal follicles of the spleen due to inoculum spillover. Here, we compare experimental prion disease in Neil2-/- mice with that in wild-type mice at disease onset and end-stage. Specifically, we investigated disease progression, accumulation of DNA damage, and mitochondrial respiratory complex activity in brain and spleen. We used genome-wide RNA sequencing of the spleen to compare the immune responses to prion propagation between the two groups of mice, at both onset and end-stage prion disease. The Neil2-/- mice deteriorated more rapidly than wild-type mice after onset of clinical signs. Levels of DNA damage in brain increased in both mouse groups, slightly more in the Neil2-/- mice. Transcriptome data from spleen at disease onset were similar between the mouse groups with moderate genomic responses. However, at end-stage a substantial response was evident in the wild-type mice but not in Neil2-/- mice. Our data show that Neil2 counteracts toxic signaling in clinical prion disease, and this is separate from gross pathological manifestations and PrPSc accumulation.

Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

Neil3 induced neurogenesis protects against prion disease during the clinical phase.

Base excision repair (BER) is the major pathway for repair of oxidative DNA damage. Mice with genetic knockout of the BER enzyme Neil3 display compromised neurogenesis in the sub-ventricular zone of the lateral ventricle and sub-granular layer of the dentate gyrus of the hippocampus. To elucidate the impact of oxidative DNA damage-induced neurogenesis on prion disease we applied the experimental prion disease model on Neil3-deficient mice. The incubation period for the disease was similar in both wild type and Neil3-/- mice and the overall neuropathology appeared unaffected by Neil3 function. However, disease in the Neil3-/- mice was of shorter clinical duration. We observed a mildly reduced astrogliosis in the hippocampus and striatum in the Neil3-deficient mice. Brain expression levels of neuronal progenitor markers, nestin (Nestin), sex determining region Box 2 (Sox2), Class III beta-tubulin (Tuj1) decreased towards end-stage prion disease whereas doublecortin (Dcx) levels were less affected. Neuronal nuclei (NeuN), a marker for mature neurons declined during prion disease and more pronounced in the Neil3-/- group. Microglial activation was prominent and appeared unaffected by loss of Neil3. Our data suggest that neurogenesis induced by Neil3 repair of oxidative DNA damage protects against prion disease during the clinical phase.

Accelerated clinical course of prion disease in mice compromised in repair of oxidative DNA damage.

The detailed mechanisms of prion-induced neurotoxicity are largely unknown. Here, we have studied the role of DNA damage caused by reactive oxygen species in a mouse scrapie model by characterizing prion disease in the ogg1(-/-)mutyh(-/-) double knockout, which is compromised in oxidative DNA base excision repair. Ogg1 initiates removal of the major oxidation product 8-oxoguanine (8-oxoG) in DNA, and Mutyh initiates removal of adenine that has been misincorporated opposite 8-oxoG. Our data show that the onset of clinical signs appeared unaffected by Mutyh and Ogg1 expression. However, the ogg1(-/-)mutyh(-/-) mice displayed a significantly shorter clinical phase of the disease. Thus, accumulation of oxidative DNA damage might be of particular importance in the terminal clinical phase of prion disease. The prion-induced pathology and lesion profile were similar between knockout mice and controls. The fragmentation pattern of protease-resistant PrP as revealed in Western blots was also identical between the groups. Our data show that the fundamentals of prion propagation and pathological manifestation are not influenced by the oxidative DNA damage repair mechanisms studied here, but that progressive accumulation of oxidative lesions may accelerate the final toxic phase of prion disease.

The PrP(C) C1 fragment derived from the ovine A136R154R171PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrP(C) protein in vitro.

Expression of the cellular prion protein (PrP(C)) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrP(C) comprised of 25% more C1 fragment than PrP(C) from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrP(C) variants. We propose that the increased α-cleavage of ovine ARR PrP(C) contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrP(C) ß-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.