Cytosolic caspases mediate mislocalised SOD2 depletion in an in vitro model of chronic prion infection

Abstract

SUMMARYOxidative stress as a contributor to neuronal death during prion infection is supported by the fact that various oxidative damage markers accumulate in the brain during the course of this disease. The normal cellular substrate of the causative agent, the prion protein, is also linked with protective functions against oxidative stress. Our previous work has found that, in chronic prion infection, an apoptotic subpopulation of cells exhibit oxidative stress and the accumulation of oxidised lipid and protein aggregates with caspase recruitment. Given the likely failure of antioxidant defence mechanisms within apoptotic prion-infected cells, we aimed to investigate the role of the crucial antioxidant pathway components, superoxide dismutases (SOD) 1 and 2, in an in vitro model of chronic prion infection. Increased total SOD activity, attributable to SOD1, was found in the overall population coincident with a decrease in SOD2 protein levels. When apoptotic cells were separated from the total population, the induction of SOD activity in the infected apoptotic cells was lost, with activity reduced back to levels seen in mock-infected control cells. In addition, mitochondrial superoxide production was increased and mitochondrial numbers decreased in the infected apoptotic subpopulation. Furthermore, a pan-caspase probe colocalised with SOD2 outside of mitochondria within cytosolic aggregates in infected cells and inhibition of caspase activity was able to restore cellular levels of SOD2 in the whole unseparated infected population to those of mock-infected control cells. Our results suggest that prion propagation exacerbates an apoptotic pathway whereby mitochondrial dysfunction follows mislocalisation of SOD2 to cytosolic caspases, permitting its degradation. Eventually, cellular capacity to maintain oxidative homeostasis is overwhelmed, thus resulting in cell death.

Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease

“…23,24,43,44 Also, oxidative stress has been identified as integral to prion-induced neurodegeneration. [9][10][11]45,46 Therefore, herein, we have evaluated PKCδ-mediated neuronal apoptosis in ex vivo cerebellar slice cultures and in RML-infected in vivo mice. Our data support the idea of PKCδ proteolytic activation during prion infection, as noted by the significantly increased native and cleaved PKCδ upon RML infection in COSCs.…”

Simplified Murine 3D Neuronal Cultures for Investigating Neuronal Activity and Neurodegeneration

“…[29,30]. Furthermore, using our NSC model, toxic changes are seen in differentiated neurones and astrocytes when exposed to prions [20,22].…”

“…Fixing and staining of 2D monolayers has been described previously [21,22]. Antibodies and concentrations were as described for 3D immuno-staining.…”

The prion protein regulates beta-amyloid-mediated self-renewal of neural stem cells in vitro

“…Aβ-amyloid peptides (China Peptides, China) were prepared as described previously [ 17 ]. NSC harvest and routine culture was as described previously [ 18 , 19 ]. For the neural colony-forming assay, cells were seeded in a semi-solid gel matrix made with a 2:1 solution of proliferation medium and collagen.…”