A Highly Toxic Cellular Prion Protein Induces a Novel,Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(ΔCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(ΔCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.Mechanisms of neuronal death have been studied intensively to gain insight into the pathological processes associated with acute and chronic neurological illnesses. Prion diseases are fatal neurodegenerative disorders of humans and animals that are accompanied by conversion of the cellular prion protein (PrP^C) into a conformationally altered isoform (PrP^Sc) that is infectious in the absence of nucleic acid.¹ Although the basic principles of prion propagation are understood, the mechanism by which abnormal forms of PrP cause neuronal death remains obscure. Membrane-anchored PrP^C is required to transduce neurotoxic signals elicited by pathogenic forms of PrP, suggesting that a normal biological activity of PrP^C may be altered during the disease process.^2,3,4,5 However, the cellular pathways and molecular components involved in this mechanism have yet to be identified.A window into the neurotoxic potential of PrP comes from transgenic mice that express PrP molecules carrying deletions within the unstructured N-terminal half of the protein. It was originally reported that mice expressing PrPΔ32-121 or Δ32-134 (collectively referred to as PrPΔN) spontaneously develop a neurodegenerative illness characterized by massive degeneration of cerebellar granule neurons (CGNs) and by white matter abnormalities.^6,7 Remarkably, this phenotype was exhibited only in the absence of endogenous PrP, and introduction of even a single Prn-p allele encoding wild-type PrP was sufficient to completely prevent the disease.⁶To further define the sequence determinants of neurotoxicity, we previously generated Tg(ΔCR) transgenic mice expressing PrP with a smaller deletion (residues 105-125) within the highly conserved central region of the protein.⁸ Tg(ΔCR) mice die within the first week of life on the Prn-p^0/0 background, and supraphysiological (5X) expression of wild-type PrP is necessary to confer survival beyond 1 year.⁸ Like Tg(PrPΔN) mice, Tg(ΔCR) animals display dramatic degeneration of CGNs and vacuolation of white matter regions.⁸ Importantly, PrP(ΔCR) is identical to PrP^C in terms of its solubility, protease sensitivity, and localization in cultured cells.^8,9 Thus, we hypothesize that deletion of critical residues in the central region of PrP^C alters a physiological activity of the protein rather than converting it to a misfolded state. Other PrP deletion mutants encompassing this region are likely to act via a similar mechanism.¹⁰To categorize the type of neuronal death induced by deleted forms of PrP, we have performed a combined biochemical, histological, and ultrastructural analysis of the brains of Tg(ΔCR) mice. We discovered that neuronal loss in these animals does not occur through either apoptosis or autophagy. By electron microscopy, we observed a novel morphology in degenerating CGNs that is reminiscent of certain forms of excitotoxic neuronal death. The same morphology was present in mice expressing PrPΔ32-134, suggesting that a common nonapoptotic mechanism may underlie the neurotoxic activity of PrP proteins lacking the critical central region. Our study has implications for understanding PrP-related cell death pathways, and it represents a starting point for designing therapeutic strategies.