Abstract
Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.
Highlights
Transmissible spongiform encephalopathies (TSE) are inexorably fatal neurodegenerative disorders caused by prions [1] which consist of PrPSc, a protease-resistant isoform of the normal cellular prion protein PrPC
We find that the proteolytic processing of the calpain substrate is induced by prion infection and blocked by calpain inhibitors without prion replication being affected
In a first series of experiments, we confirmed our published finding that Rocky Mountain Laboratory (RML) prions replicate efficiently in cultured organotypic cerebellar slices (COCS) and, in addition, we found that infectivity could be serially passaged by slice-to-slice transmission (Figure S1)
Summary
Transmissible spongiform encephalopathies (TSE) are inexorably fatal neurodegenerative disorders caused by prions [1] which consist of PrPSc, a protease-resistant isoform of the normal cellular prion protein PrPC. Prnpo/o mice lack PrPC, cannot generate PrPSc, and withstand prion inoculation [2]. PrPSc forms aggregates that grow by recruiting PrPC and whose breakage underlies prion replication [3]. The hallmarks of TSEs include PrPSc deposition and progressive brain damage. Prnpo/o mice show mild phenotypes and no TSE [4,5,6,7], indicating that TSEs are not caused by loss of PrPC function. Several observations suggest that extracellular deposition of PrPSc is intrinsically innocuous [8,9,10], whereas neurotoxicity is driven by unknown secondary triggers. A mechanistic dissection of prion neurotoxicity necessitates faithful, experimentally versatile in vitro models – yet such models have proven difficult to generate [11,12]
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