Prion neurotoxicity

Abstract

Although the mechanisms underlying prion propagation and infectivity are now well established, the processes accounting for prion toxicity and pathogenesis have remained mysterious. These processes are of enormous clinical relevance as they hold the key to identification of new molecular targets for therapeutic intervention. In this review, we will discuss two broad areas of investigation relevant to understanding prion neurotoxicity. The first is the use of in vitro experimental systems that model key events in prion pathogenesis. In this context, we will describe a hippocampal neuronal culture system we developed that reproduces the earliest pathological alterations in synaptic morphology and function in response to PrP^Sc . This system has allowed us to define a core synaptotoxic signaling pathway involving the activation of NMDA and AMPA receptors, stimulation of p38 MAPK phosphorylation and collapse of the actin cytoskeleton in dendritic spines. The second area concerns a striking and unexpected phenomenon in which certain structural manipulations of the PrP^C molecule itself, including introduction of N-terminal deletion mutations or binding of antibodies to C-terminal epitopes, unleash powerful toxic effects in cultured cells and transgenic mice. We will describe our studies of this phenomenon, which led to the recognition that it is related to the induction of large, abnormal ionic currents by the structurally altered PrP molecules. Our results suggest a model in which the flexible N-terminal domain of PrP^C serves as a toxic effector which is regulated by intramolecular interactions with the globular C-terminal domain. Taken together, these two areas of study have provided important clues to underlying cellular and molecular mechanisms of prion neurotoxicity. Nevertheless, much remains to be done on this next frontier of prion science.

Keywords: antibody; cell culture; dendrite; glutamate; ionic current; mitogen-activated protein kinase (MAPK); neurodegeneration; neurotoxicity; prion; spine; synapse; transgenic mouse.

Figure 1

In vitro systems to study prion neurotoxicity. A. An organotypic brain slice system registers the effects of chronic prion infection, monitored by reductions in neuronal viability as PrP^Sc accumulates 34, 35, 36. Left: Cerebellar or hippocampal slices from WT or Tga20 (PrP‐over‐expressing) mice are prepared from neonatal pups and are treated with normal brain homogenate (NBH) or scrapie‐infected brain homogenate. Right: The slices become chronically infected, accumulating PrP^Sc (detectable by Western blotting for PK‐resistant PrP) and suffering neuronal loss over a matter of weeks. B. A hippocampal neuronal culture system allows analysis of the acute toxic effects of prions on synaptic structure and function 37, 38. Left: Neurons are isolated from hippocampi of neonatal pups and cultured at low density on coverslips that are suspended facedown, via wax dots, over a feeder layer of astrocytes. Right: Neurons in this system are susceptible to the synaptotoxic effects of prions within 24 hrs. Treatment with purified PrP^Sc, but not mock‐purified material, leads to retraction of dendritic spines, revealed by staining with fluorescent phalloidin, as well as local increases in p38 MAPK phosphorylation, visualized by staining with antibodies to phospho‐p38 (red) and total p38 (green). Electrophysiological abnormalities in synaptic transmission are also detectable using patch‐clamping techniques 38. Images of mice were taken from Servier Medical Art (http://smart.servier.com).

Figure 2

Model for a prion synaptotoxic pathway. The pathway is initiated by binding of PrP^Sc to endogenous PrP^C on the cell surface. This binding event itself, or the subsequent conversion of PrP^C to PrP^Sc, results in the activation of NMDA and AMPA receptors with influx of calcium ions. Calcium influx leads to subsequent activation of p38 MAPK and MK2/3, collapse of the dendritic spine cytoskeleton, spine retraction and decreases in synaptic transmission. Question marks indicate unknown components of the pathway. Figure was produced using Servier Medical Art (http://smart.servier.com).

Figure 3

Models for the neurotoxic effects of PrP. A. The C‐terminal domain of PrP^C negatively regulates the toxic effector function of the N‐terminal domain. +++, basic residues within the 23‐31 region at the extreme N‐terminus, which are essential for the toxic action of PrP. B. Binding of monoclonal antibodies to the C‐terminal domain disrupts this regulatory interaction, releasing the N‐terminal domain to produce toxic effects. C. Deletion of the central region, as in ΔCR PrP, produces a similar loss of regulation, with toxic consequences. D. When EGFP is substituted for the C‐terminal domain of PrP^C, regulation is also lost. E. Binding of ligands (PPS, antibodies, Cu²⁺) to the N‐terminal domain of ΔCR PrP blocks its ability to exert toxic effects. Reprinted from 131.