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Transgenic mice harboring a high-copy-number of wild-type mouse (Mo) cellular prion protein (PrP^C) are known to develop a spontaneous neurological dysfunction in an age-dependent manner, even without inoculation of the scrapie isoform of prion protein (PrP^Sc). Here we first demonstrate mitochondria-mediated apoptosis in aged transgenic mice overexpressing wild-type MOPrP^C. These mice remained healthy, and no neuropathological abnormality was observed, however, exhibited an aberrant mitochondrial localization of PrP^C concomitant with decreased proteasome activity, while younger littermates did not. Such aberrant mitochondrial localization of PrP^C was accompanied by cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal pyramidal cells. Next, we reconstituted these results using cell culture system. When neuronal (N2a) culture cells expressing wild-type PrP^C were used in combination with proteasome inhibitors, PrP^C targeted to mitochondria and several apoptotic phenomenon was observed. Furthermore, using the assay system, we revealed a novel mitochondrial PrP-related apoptotic pathway as follows. First, we found that PrP residues 122–139 required for the mitochondrial targeting and 14-3-3 protein worked as a transporter of PrP^C to Tom70, a mitochondrial outer membrane receptor for mitochondrial precursor proteins. Second, we show for the first time that Tom70 is involved in mitochondrial apoptosis, and thus plays a pivotal role in cell survival and death. Third, PrP^C thereafter binds BCL-2 on the mitochondrial outer membrane and subsequently induces mitochondrial apoptosis. Our data indicate that cell survival and death signals share, at least in part, a common pathway in the mitochondria1 protein transport system.

To investigate intracellular trafficking of cellular prion protein (PrP^C), we performed a real-time imaging of fluorescent PrP^C (GFP-PrP^C) in living cells. Localization of GFP-PrP^C correlated with endogenous PrP^C, however, its localization was congregated in the cytosol after the treatment with a microtubule depolymerizer, nocodazole, suggesting that the microtubule-dependent transport of PrP^C. This microtubule-associated intracellular trafficking of PrP^C exhibited an anterograde movement towards the direction of plasma membranes at a speed of 140–180 nm/s, and a retrograde movement inwardly at a speed of 1.0–1.2 µm/s. The anterograde and retrograde movements of GFP-PrP^C were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Anti-kinesin antibody (α-kinesin) blocked its anterograde motility, whereas anti-dynein antibody (α-dynein) blocked its retrograde motility. These data showed that the kinesin family-driven anterograde and the dynein-driven retrograde movements of GFP-PrP^C. Mapping of the interacting domains of PrP^C identified amino acid residues indispensable for interactions with kinesin family: NH2-terminal mouse (Mo) residues 53–91 and dynein: NH2-terminal Mo residues 23–33, respectively. Our findings argue that the discrete N-terminal amino acid residues are indispensable for the anterograde and retrograde intracellular movements of PrP^C. Furthermore, by utilizing double-labeled fluorescent cellular PrP^C, we revealed that the NH2-terminal and COOH-terminal PrP^C fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells. The double-labeled fluorescent cellular PrP^C was successfully processed in the cell and the processing was clearly inhibited by metalloprotease inhibitors, such as EDTA, and calpain inhibitor, calpastain.

In mammals, prions reproduce by recruiting the normal cellular isoform of the prion protein (PrP^C) and by stimulating its conversion into the pathological conformational isoform (PrP^Sc). Since the conversion process is thought to require unfolding of PrP^C to cause prion infection, to identify such activities, we have tried to construct an in vitro protein-unfolding as-say using Trypsin-digestion susceptibility of E. coli-expressed recombinant mouse prion protein (recPrP). The highly purified recPrP was refolded as a soluble monomeric protein in 1 M arginine and a glutathione redox system-containing solution. We explored protein-unfolding activities of a mouse neuroblastoma cell line, Neuro-2a, which was widely used for prion infection experiments, and found that the protein unfolding activities existed in the cell extracts, particularly, in the Cytosolic fraction (100,000g sup). From the Cytosolic fraction we purified the activity to homogeneity by three steps of chromatography. The active polypeptide has a molecular weight of 90 kDa in denature condition (SDS-PAGE) and 200 kDa in native condition (Native-PAGE). The MALDI-TOF-MS/MS analysis revealed that the purified polypeptide was identical to Hsp90. The protein unfolding activity of the purified Hsp90 was inhibited by treatment of ATP-hydrolyzing enzyme, Apyrase. These results suggested that Hsp90 can modify the recombinant prion protein’s conformation in the presence of ATP and might be required for conformation change of PrP^C to PrP^Sc in prion propagation.

We have isolated a novel ATP-dependent robust protein-unfolding activity from S. cerevisiae and designated Unfoldin. ATP, but not its hydrolysis, promoted binding of Unfoldin to substrates and unfolded their conformation. Protein sequencing revealed that Unfoldin was identical to YDL178w identified as an actin interacting protein 2 (AIP2), the function of which is poorly understood. Gel-filtration and low angle shadowing electron microscopy revealed that Unfoldin formed a homo-oligomeric complex consisting of 10∼12 subunits arranged in an grapple-like structure with a ∼2 nm central cavity. Removal of the C-terminal coiled-coil region of Unfoldin led to dissociation of the oligomer concomitant with the loss of both substrate binding and protein-unfolding activity. Unfoldin bound to all substrates so far examined in vitro, and modified their conformation as determined by the trypsin susceptibility assay. It is worth noting that the robust protein-unfolding activity of Unfoldin modulated the conformation of several pathogenic, highly aggregated proteins such as prion protein in β-sheet form associated with prion disease, amyloid β(1–42) peptide with Alzheimer’s disease and α-synuclein with Parkinson’s disease, in the presence of ATP. Protein-unfolding activity of Unfoldin depends on the growth stage of yeast and the most significant activity was observed at the log phase, suggesting the presence of a cofactor/s. From the in vivo and in vitro experimental data, Unfoldin might have important roles in a development of new treatments for the neurodegenerative disorders.

The infectious agent of Transmissible Spongiform Encephalopathies (TSE) has been considered to be PrP^SC, a structural isoform of cellular prion protein PrP^C. PrP^SC can exist as oligomers and/or as amyloid polymers. Nucleic-acids induce structural conversion of recombinant prion protein PrP and PrP^C to PrP^SC form in solution and in vitro. Here we report that nucleic acids by interacting with PrP in solution produce amyloid fibril and fibres of different morphologies similar to those identified in the prion diseased brains. In addition, the same interaction produces polymer lattices and spherical amyloids of different dimensions (15–150 nm in diameters). The polymer lattices show apparent morphological similarity to the two-dimensional amyloid crystals obtained from linear amyloids isolated in vivo. The spherical amyloids structurally resemble ‘spherical particles’ observed in natural spongiform encephalopathy (SE) and in scrapie infected brains (TSE). We suggest that spherical amyloids, PrP^SC-amylospheroids, are probable constituents of the coat of the spherical particles found in vivo and the latter can act as protective coats of the SE and TSE agents in vivo.

The Sup35 protein of Saccharomyces cerevisiae is a eukaryotic polypeptide release factor (eRF3), which is necessary for terminating protein synthesis at stop codons. The aggregated form of Sup35, which is referred to as the [PSI^+] element, self-propagates and is transmitted cytoplasmically in the manner of the “protein-only” transmission of mammalian prion diseases. In [PSI^+] cells, most of the Sup35 is converted from a soluble, active state into an insoluble, inactive state similar to the mammalian prion amyloid. As with mammalian prions, a species barrier prevents prion transmission between yeast species. The N-terminal of Sup35 of Saccharomyces cerevisiae, necessary for [PSI^+], contains two species-signature elements — a Gln/Asn-rich region (residues 1–41; designated NQ) that is followed by oligopeptide repeats (designated NR).

How prions generate and propagate variation in both mammals and yeast is a topic of great interest and increasing evidence indicates that the tertiary or quaternary structure of prion conformations determines the strain phenotype. Whether or not the conformational information of specific prion ‘strains’ is transmissible to heterologous prion domains at the molecular level and whether this corresponds to a phenotypic change at the ‘strain’ level was investigated using the [PST^+] prion model of S. cerevisiae.

Human prion diseases occur in sporadic, genetic, and transmitted forms. It is associated with the conversion of normal cellular prion protein (PrP^C) into a protease-resistant isoform (PrP^res). We have shown previously that the human glioblastoma cell line T98G had no coding-region mutation of the prion protein gene (PRNP) which was of the 129 M/V genotype and produced a form of proteinase K-resistant prion protein (PrP) fragment following long-term culture and high passage number (Kikuchi et. al., 2004, J. Gen. Virol. 85, 3449–3457). In this study, we identified an unusual alternative splicing occurring within the exon 2, which resulted in the generation of mRNA laking a C-terminal glycosylphosphatidylinositol (GPI) anchor signal sequence. Only a low level of an alternative spliced form of PRNP was identified in T98G cells under normoxia. Under hypoxia, however, expression levels of the alternatively spliced mRNA were increased. Treatment with cobalt chloride, which mimics anoxia, also increased its expression levels in T98G cells. These results indicate that decrease in oxygen pressure may modulate the PRNP gene expression. The alternatively spliced mRNA encoded the 231 amino acids of polypeptide, which consisted of the amino-terminal portion of PrP and additional residues at its carboxy terminus.

Prion diseases appear as sporadic, inherited or acquired by an infection^1–2. In the inherited (familial) prion diseases over 20 point and insertional mutations were described, whereas in sporadic and acquired forms codon 129 homozygosity of the PRNP gene acts as a susceptibility factor. The etiology of sporadic CJD (sCJD) cases remains obscure. It is supposed that the expression level of prion protein gene may modulate susceptibility to sCJD. This idea comes from the results of the experiments with transgenic mice with additional Prnp copies. These mice show shorter incubation period after challenge with scrapie in comparison with that of with wild type mice. The level of prion protein expression depends on the PRNP gene regulatory elements; thus we suppose that polymorphisms in the regulatory region of the PRNP gene may be a risk factor for sCJD.

Tubulovesicular structures (TVS) are the only disease-specific structures found by electron microscopy in naturally occurring and experimentally induced prion diseases or transmissible spongiform encephalopathies (TSEs)^1–2, 4–5. We reported here the largest series of brain biopsy specimens of Creutzfeldt-Jakob disease (CJD), variant CJD and fatal familial insomnia (FFI) studied by thin-section electron microscopy. Our material includes two series of brain biopsies. The first, comprising 9 biopsy specimens collected at the Laboratoire de R. Escourolle, Hopital de la Salpetriere, Paris, France were coded by one of us (J-J.H) and examined blindly. This series has been completed between 1982 and 1986 as a part of epidemiology study of CJD in France. The second series consisted of brain (right prefrontal cortex) biopsies obtained between 1995–1999 by open surgery from 1 FFI case in a new French family, 1 vCJD case, 9 cases of sporadic CJD, and 2 cases of iatrogenic (human growth hormone) CJD. TVS were found in all specimens including cases of FFI and vCJD. They appeared as spheres approximately 30 nm in diameter or short tubules of helical appearance. They were of higher electron density than synaptic vesicles; occasionally they clustered as paracrystalline arrays. Their composition is unknown but they are not composed of PrP^3. Their constant presence in all TSEs studied so far, strongly suggests thair role in pathogenesis of these diseases. This study is supported by EC Neuro-Prion Network of Excellence.