Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures

Abstract

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4–5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

DLP1‐dependent mitochondrial fragmentation and redistribution mediate prion‐associated mitochondrial dysfunction and neuronal death

“…PrP Sc is often found in neuropil deposits, which are referred to as ‘synaptic‐like’, as they appear to surround synaptic sites (Clinton et al ., 1993; Belichenko et al ., 2000; Cunningham et al ., 2003; Campeau et al ., 2013). In prion diseases, apart from neuronal loss, earliest and potentially most vital neuropathology changes occur at the level of synapses including synapse loss, morphological changes, and functional abnormality (Mallucci, 2009).…”

Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease

“…Prion-infected cerebellar slice cultures were established as previously described (17,18), using 10-to 12-day-old tga20/ϩ mice (19). Sagittal slices (350 m thick) of cerebellum were prepared and treated with 1.0 g of RML prion-infected or uninfected brain homogenates/ml as free-floating sections for 1 h on ice before washing and plating.…”

“…Given the partial efficacy of the bile acids in two cell culture models of prion infection, we examined their effect on the replication of PrP Sc in cerebellar slice culture. The prion organotypic slice culture assay (POSCA) allows prion replication ex vivo under conditions that closely resemble intracerebral infection (17,18). In addition, it has been demonstrated that this assay predicts in vivo efficacy of drug treatment more accurately than cell-based assays (30).…”

“…Another advantage of POSCA is that it not only propagates PrP res but also closely reproduces prion pathogenesis (18,30). To evaluate whether the antiprion propagation effect observed for the bile acids was accompanied by a neuroprotective effect, we assessed neuronal viability and overall levels of cell death in the cerebellar slice cultures over the course of the infection.…”

“…Although it is possible that the reduction in PrP res levels alone accounts for the neuroprotective effects seen in POSCA and the change in survival curves, TUDCA, and UDCA have been reported to have a number of downstream effects on the pathogenesis in other diseases (2). Using POSCA, we have previously observed early changes in postsynaptic density protein 95 (PSD95) induced by prion infection, occurring around the time of first PrP res detection (18). We therefore chose to examine whether this early marker is affected by TUDCA.…”