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

Abstract

Several in vitro and in vivo studies addressed the identification of molecular determinants of the neuronal death induced by PrP(Sc) or related peptides. We developed an experimental model to assess PrP(Sc) neurotoxicity using a recombinant polypeptide encompassing amino acids 90-231 of human PrP (hPrP90-231) that corresponds to the protease-resistant core of PrP(Sc) identified in prion-infected brains. By means of mild thermal denaturation, we can convert hPrP90-231 from a PrP(C)-like conformation into a PrP(Sc)-like structure. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing caspase 3 and p38-dependent apoptosis, while in the native alpha-helix-rich conformation, hPrP90-231 did not induce cell toxicity. The aim of this study was to identify drugs able to block hPrP90-231 neurotoxic effects, focusing on minocycline, a tetracycline with known neuroprotective activity. hPrP90-231 caused a caspase 3-dependent apoptosis via the blockade of ERK1/2 activation and the subsequent activation of p38 MAP kinase. We propose that hPrP90-231-induced apoptosis is dependent on the inhibition of ERK1/2 responsiveness to neurotrophic factors, removing a tonic inhibition of p38 activity and resulting in caspase 3 activation. Minocycline prevented hPrP90-231-induced toxicity interfering with this mechanism: the pretreatment with this tetracycline restored ERK1/2 activity and reverted p38 and caspase 3 activities. The effects of minocycline were not mediated by the prevention of hPrP90-231 structural changes or cell internalization (differently from Congo Red). In conclusion, minocycline elicits anti-apoptotic effects against the neurotoxic activity of hPrP90-231 and these effects are mediated by opposite modulation of ERK1/2 and p38 MAP kinase activities.

Global transcriptional profiling of the postmortem brain of a patient with G114V genetic Creutzfeldt-Jakob disease

“…PrP106-126 has been demonstrated to activate p38 MAP kinase in human microglia accompanied by upregulation of NF-κB (24), and to induce a p38 MAP kinase-dependent apoptosis in SH-SY5Y neuroblastoma cells independently from the amyloid fibril formation (25). The decrease of ERK in our microarray is also in line with the observation that PrP fragment (aa 90-231) activates p38 MAP kinase by inhibiting the activation of extracellular-regulated kinases 1/2 (ERK1/2), followed by the caspase-3-dependent cell apoptosis in SH-SY5Y cells (26).…”

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

“…To evidence the differential effects of the compounds, we used a relatively mild PK treatment. In agreement with previous studies (Corsaro et al , 2009Chiovitti et al 2007), the a-helix structured peptide was significantly sensitive to PK treatment (about 90% for a hPrP90-231/PK ratio of 100:1, w/w), but upon conversion in a b-sheet-rich conformer hPrP90-231 was much more resistant to the protease being only partially digested (about 40% of the protein input) at the hPrP90-231/PK ratio (100:1 w/w) ( Fig. 9a and quantified in Fig.…”

“…These data well match the observation that in vivo quinacrine affects the formation of new PrP Sc molecules but it is unable to interfere with the protease resistance of pre-existing PrP Sc (Gayrard et al 2005;Barret et al 2003). Interestingly, minocycline, that was also proposed as antiprion agent (De Luigi et al 2008) and was shown to revert the cytotoxic effects of hPrP90-231 in vitro (Corsaro et al 2009), acts by a completely different mechanism. Minocycline binds with low affinity hPrP90-231 (K D = 2.6 lM) and exerts its neuroprotective effects acting downstream the proapoptotic pathways activated by this peptide, regulating ERK1/2 and p38 MAP kinase activities (Corsaro et al 2009).…”

“…Importantly, the K D value we calculated for hPrP90-231 was very similar to the EC 50 of quinacrine in the inhibition of PrP Sc replication in cell cultures (0.3 lM) (Korth et al 2001), further showing the reliability of our cell model. We previously demonstrated that the amyloidophilic compound Congo red that possesses higher affinity for hPrP90-231 than quinacrine (K D = 0.3 lM) (Corsaro et al 2009), inhibits hPrP90-231-induced SH-SY5Y cell death by preventing hPrP90-231 acquisition of a b-sheet-rich configuration ). According to these results, we can propose that quinacrine and Congo red cytoprotective activities rely on similar mechanisms: the interaction with hPrP90-231 to prevent the conversion in a structural conformer responsible of the toxic effects.…”

“…More importantly, we demonstrated that such structural refolding determines hPrP90-231 gain of toxicity, in vitro . The toxic effects of denatured hPrP90-231 are mediated by b-rich monomers or small oligomers but not by macroaggregates or amyloid fibrils, being dependent on their ability to be internalized ) and to revert the MAP kinase-mediated survival signaling (Corsaro et al 2009). Noteworthy, hPrP90-231 reproduced in vitro some alterations of astrocytes (proliferation) and microglia (activation) that in the brain of TSE patients are involved in the neurodegenerative process .…”