Humanin rescues cortical neurons from prion-peptide-induced apoptosis
Introduction
Prion diseases are associated with a conformational transition of the cellular prion protein (PrPc) into a pathologically associated form commonly denoted PrPsc. Although the physiological role of PrPc remains obscure, its expression is required for the neurotoxicity and the accumulation of PrPsc-associated amyloids, one of the hallmarks of prion-associated diseases (for review, see Prusiner, 1998). More enigmatic at the present time are the biochemical and cellular events ensuring the pathophysiological progression of the neuronal and non-neuronal cell degeneration in the prion diseases (Chiesa and Harris, 2001). Thus, there is substantial neurodegeneration in the absence of PrPsc accumulation in some cases of natural and experimental prion disease (Hegde et al., 1999). This argues against the hypothesis presenting the PrPsc accumulation as the only cause of the pathology. Transmembrane topological variants of PrPc (named ctmPrP) have recently been described and associated with the pathology Hegde et al., 1998, Hölscher et al., 2001. An aberrant regulation of PrPc biogenesis at the endoplasmic reticulum results in the accumulation of aberrant topologies of PrPc that induce neurodegeneration Hegde et al., 1999, Stewart et al., 2001.
Although the relationships between ctmPrP forms and the accumulation of PrPsc remain unsolved, a recent report has demonstrated, using neuroblastoma cells, that fibrillar PrP(106–126) peptide induces the synthesis and accumulation of potentially neurotoxic transmembrane PrP (Gu et al., 2001). This suggests that the cell death in the prion disorders might be mediated by a complex pathway involving transmembrane PrP, and not only by the deposit of the aggregated and proteinase-K-resistant PrP. Thus, we and others have used the putative transmembrane domain of the ctmPrP—for example, amino acids 118–135 [PrP(118–135)]—to model and characterize the apoptotic neuronal death induced by these topological variants of PrPc Haı̈k et al., 2000, Pillot et al., 2000. By contrast to the 106–126 fragment of PrP, which requires its fibrillization and the presence of PrPc to exert its neurotoxicity (Brown et al., 1996), we have demonstrated that the nonfibrillar PrP(118–135) peptide induces the apoptosis of cortical neurons (Pillot et al., 2000), partly mediated by the membrane-destabilizing properties of this PrP fragment (Pillot et al., 1997; for review see, Brasseur et al., 1997). More importantly, these fusogenic properties of the soluble PrP(118–135) fragment might account for its neurotoxicity in vivo (Chabry et al., 2003).
A novel peptide termed humanin (HN) was recently reported to rescue neurons in vitro from the toxicity induced by the Alzheimer's amyloid-β peptide (Aβ) (Hashimoto et al., 2001; and for review, see Niikura et al., 2002). As we recently demonstrated that the PrP(118–135) and the Aβ peptides shared some biophysical and biological properties in common, we investigated whether HN and HN-derived peptides might modulate the neurotoxicity of soluble oligomers of PrP(118–135). Here we demonstrate that cortical neurons treated with HN in addition to PrP(118–135) could be rescued to more than 90% compared with PrP(118–135) treatment alone. Our results strongly suggest that the HN peptide modulates the neurotoxic properties of other insults than Alzheimer's-disease-related insults.
Section snippets
Humanin rescues rat cortical neurons from PrP(118–135)-induced cell death
To determine the effects of HN on PrP(118–135) neurotoxicity, cortical neurons were incubated with 5 μM PrP(118–135) peptide in the absence or presence of 50 nM–100 μM HN for 6–72 h. Cell viability was determined using the MTT assay. As previously demonstrated (Pillot et al., 2000), PrP(118–135) induced a 18% and 70% decrease in the MTT reduction after a 6 and 72 h of incubation, respectively (Fig. 1A). Interestingly, the presence of 10 μM and more HN dramatically reduced the PrP-induced cell
Discussion
Accumulative evidence highlights the perturbation of the cellular trafficking and degradation of PrPc, a process distinct from the self-propagating PrPsc isoform, as central events in diverse prion protein-associated neurodegenerative disorders Gu et al., 2003, Ma et al., 2002, Mishra et al., 2002. These studies emphasize the key role played by abnormal conformations of PrPc resulting in the intracellular accumulation of soluble PrPc and degradation products of PrPc in neuronal cell death
Materials
PrP(118–135), the caspase substrate peptides, were purchased from Bachem. Unless otherwise indicated, materials used for cell culture were obtained from Life Technologies (Grand Island, NY, USA). All other chemicals were of high purity grade from Sigma (St. Louis, MO, USA). Humanin peptides (HN, HNG, and HNA) were a generous gift of Dr. Laurent Pradier (Aventis-Pharma R&D, Vitry-sur-Seine, France). The amino acid sequences of the HN peptides used in this study are the following:
Acknowledgements
This work was supported in part by INSERM and by a grant from the Aventis French Network on Molecular Mechanism in Alzheimer's disease. Isabelle Sponne is supported by a postdoctoral fellowship from Aventis Pharma (Vitry-Sur-Seine, France).
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