Intracellular accumulation of a mild‐denatured monomer of the human PrP fragment 90–231, as possible mechanism of its neurotoxic effects

Abstract

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a prefibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric milddenatured hPrP90-231 (incubated for 1 h at 53°C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death. Keywords: intracellular internalization, neurotoxicity, protein aggregation, PrP90-231. Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of neurodegenerative diseases that recognize as etiopathologic agent an aberrant isoform of the cellular prion protein (PrP C ), named PrP Sc (Prusiner 1998). PrP C is a ubiquitous membrane-bound protein, highly concentrated in the central nervous system in both neurons and glial cells, whose physiological function is still largely unknown. In PrP Sc large portions of the protein N-Terminus adopt a b-sheet configuration, being this feature the main difference between PrP c and PrP Sc (Prusiner 1991). In the past few years a large number of data has been published to better characterize both the molecular mechanisms involved in the conversion of PrP C into PrP Sc (Aguzzi 2006) and the intracellular signaling by which PrP Sc (Muller et al. 1993;Hetz et al. 2003), or some cytotoxic fragments derived from its sequence, may induce cell death (Forloni et al. 1993;Brown et al. 1996;Florio et al. 1998; Thellung et al. 2000a,b). To explain prion infectivity it was postulated that the pathogenic PrP Sc self-propagates in an autocatalytic manner using PrP C as a substrate (Prusiner 1982).A novel concept emerging in prion diseases research is represented by the dissociation between prion transmissibility/ These authors equally contributed and should be both considered as first author.2 Joint last and corresponding authors.Abbreviations used: CD, circular dichroism; hPrP90-231, human PrP fragment 9...

Dual Modulation of ERK1/2 and p38 MAP Kinase Activities Induced by Minocycline Reverses the Neurotoxic Effects of the Prion Protein Fragment 90–231

“…We recently demonstrated that a b-sheet-rich conformer of hPrP90-231 determines SH-SY5Y apoptotic cell death, via the activation of p38 MAP kinase and caspase 3 Chiovitti et al 2007). Moreover, hPrP90-231 cell exposure induces a time-dependent intracellular accumulation of the toxic isoform of this prion fragment into endolysosomal vesicles and that this phenomenon correlates with cell apoptosis ).…”

“…The first hypothesis was assessed using thioflavin-T binding and protein intrinsic fluorescence assays. hPrP90-231 toxic conformer possesses higher thioflavine T binding (due to increased b-sheet content) and reduced intrinsic fluorescence, (due to increased aggregability) Chiovitti et al 2007) in comparison to the native, a-helicalrich isoform. These structural changes were responsible for hPrP90-231 cell internalization and activation of the apoptotic cascade ).…”

“…SH-SY5Y (Pahlman et al 1995) human neuroblastoma cell line due, to the neuroectodermal origin, is widely used to study intracellular events linked to neurotoxic insults such as trophic factors withdrawal, oxidative stress, DNA damage, and neurodegenerative disorders Corsaro et al 2003). PrP-derived peptides, including hPrP90-231, are able to induce SH-SY5Y cell death through the activation of pro-apoptotic cascades (O'Donovan et al 2001;Thellung et al 2002;Bate et al 2004;Corsaro et al 2006;Chiovitti et al 2007). To develop more effective anti-prion pharmacological approaches, a wide number of molecules have been screened for their ability to counteract biochemical and biological properties of PrP Sc (Trevitt and Collinge 2006).…”

Efficacy of Novel Acridine Derivatives in the Inhibition of hPrP90-231 Prion Protein Fragment Toxicity

“…It was proposed that the higher hydrophobicity of the toxic conformer might favor the interaction of this PrP fragment with cell membranes, its internalization, and the activation of apoptotic pathways Chiovitti et al 2007). Thus, we evaluated the effects of quinacrine on the increased hydrophobicity of hPrP90-231, as assessed by the binding of the fluorescent probe TNS.…”

Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

“…live animals (79,84). It is therefore possible that the increased neurotoxicity and decreased protease resistance of the A116V-PrP Sc are due to increased levels of smaller oligomeric species.…”