Prion Pathogenesis is Independent of Caspase-12

Abstract

The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer's disease and many others. The ER stress response and up-regulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.

Acknowledgements

We are grateful to Artur Topolszki (WIBR) for providing expert technical assistance with all aspects of mouse work and to Marie Hardwick (Johns Hopkins) for providing the Caspase-12 knockouts. Funding for this work was provided by the US Department of Defense (DAMD17-00-1-0296), Ellison Medical Research Foundation, and the Howard Hughes Medical Institute (to S.L.) and FONDAP (15010006), FONDECYT (1070444) and the HighQ foundation (to C.H.).

Figures and Tables

Figure 1 ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD₅₀ RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.

Figure 2 The survival after prion inoculation of C12 WT (either +/+ or +/− for C12) and KO. The median survival for C12 WT was 169 days and median survival for C12 KO was 171 days (p = 0.13, log rank test).

Figure 3 Behavioral analysis of prion inoculated C12 WT and KO mice (n = 6–9 per group). Mice were single housed and video recorded for 24 hours and their behaviors were quantitated by Homecagescan 2.0 software. A similar decrease in the amount of time spent grooming (A) and hanging (B) was observed and a similar dramatic increase in activity as measured by the percent of total time spent walking (C) was observed in C12 WT and KO mice inoculated with prions. Data is shown as mean values plus standard error of the mean. No statistically significant differences (at a threshold of p < 0.05) were observed for grooming, hanging and walking (Wilcoxon rank sum test).

Figure 4 Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.