Mitochondrial localization of cellular prion protein (PrPC) invokes neuronal apoptosis in aged transgenic mice overexpressing PrPC.

Recent studies suggest that the disease isoform of prion protein (PrPSc) is non-neurotoxic in the absence of cellular isoform of prion protein (PrPC), indicating that PrPC may participate directly in the neurodegenerative damage by itself. Meanwhile, transgenic mice harboring a high-copy-number of wild-type mouse (Mo) PrPC develop a spontaneous neurological dysfunction in an age-dependent manner, even without inoculation of PrPSc and thus, investigations of these aged transgenic mice may lead to the understanding how PrPC participate in the neurotoxic property of PrP. Here we demonstrate mitochondria-mediated neuronal apoptosis in aged transgenic mice overexpressing wild-type MoPrPC (Tg(MoPrP)4053/FVB). The aged mice exhibited an aberrant mitochondrial localization of PrPC concomitant with decreased proteasomal activity, while younger littermates did not. Such aberrant mitochondrial localization was accompanied by decreased mitochondrial manganese superoxide dismutase (Mn-SOD) activity, cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal neuronal cells. Following cell culture studies confirmed that decrease in the proteasomal activity is fundamental for the PrPC-related, mitochondria-mediated apoptosis. Hence, the neurotoxic property of PrPC could be explained by the mitochondria-mediated neuronal apoptosis, at least in part.

Prion protein with Y145STOP mutation induces mitochondria-mediated apoptosis and PrP-containing deposits in vitro.

A pathogenic truncation of an amber mutation at codon 145 (Y145STOP) in Gerstmann-Straussler-Scheinker disease (GSS) was investigated through the real-time imaging in living cells, by utilizing GFP-PrP constructs. GFP-PrP(1-144) exhibited an aberrant localization to mitochondria in mouse neuroblastoma neuro2a (N2a) and HpL3-4 cells, a hippocampal cell line established from prnp gene-ablated mice, whereas full-length GFP-PrP did not. The aberrant mitochondrial localization was also confirmed by Western blot analysis. Since GFP-PrP(1-121), as previously reported, and full-length GFP-PrP do not exhibit such mitochondrial localization, the mitochondrial localization of GFP-PrP(1-144) requires not only PrP residues 121-144 (in human sequence) but also COOH-terminal truncation in the current experimental condition. Subsequently, the GFP-PrP(1-144) induced a change in the mitochondrial innermembrane potential (DeltaPsi(m)), release of cytochrome c from the intermembrane space into the cytosol, and DNA fragmentation in these cells. Non-fluorescent PrP(1-144) also induced the DNA fragmentation in N2a and HpL3-4 cells after the proteasomal inhibition. These data may provide clues as to the molecular mechanism of the neurotoxic property of Y145STOP mutation. Furthermore, immunoelectron microscopy revealed numerous electron-dense deposits in mitochondria clusters of GFP-PrP(1-144)-transfected N2a cells, whereas no deposit was detected in the cells transfected with full-length GFP-PrP. Co-localization of GFP/PrP-immunogold particles with porin-immunogold particles as a mitochondrial marker was observed in such electron-dense vesicular foci, resembling those found in autophagic vacuoles forming secondary lysosomes. Whether such electron-dense deposits may serve as a seed for the growth of amyloid plaques, a characteristic feature of GSS with Y145STOP, awaits further investigations.

Oligomeric Aip2p/Dld2p modifies the protein conformation of both properly folded and misfolded substrates in vitro.

Oligomeric actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377, Biochem. Biophys. Res. Commun. 295 (2002) 910] exhibits the unique grapple-like structure with an ATP-dependent opening [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which is required for the F-actin conformation modifying activity in vitro and in vivo [Biochem. Biophys. Res. Commun. 319 (2004) 78]. To further investigate the molecular nature of oligomeric Aip2p/Dld2p, the substrate specificity of its binding and protein conformation modifying activity was examined. In the presence of 1mM ATP or AMP-PNP, oligomeric Aip2p/Dld2p bound to all substrates so far examined, and modified the conformation of actin, DNase I, the mature form of invertase, prepro-alpha-factor, pro-alpha-factor, and mitochondrial superoxide dismutase, as determined by the trypsin susceptibility assay. Of note, the activity could modify even the conformation of pathogenic highly aggregated polypeptides, such as recombinant prion protein in beta-sheet form, alpha-synuclein, and amyloid beta (1-42) in the presence of ATP. The in vivo protein conformation modifying activity, however, depends on the growth stage; the most significant substrate modification activity was observed in yeast cells at the log phase, suggesting the presence of a cofactor/s in yeast cells, where F-actin is supposed to be a major target in vivo. These data further support our previous notion that the oligomeric Aip2p/Dld2p may belong to an unusual class of molecular chaperones [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which can target both properly folded and misfolded proteins in an ATP-dependent manner in vitro.

Oligomeric Aip2p/Dld2p forms a novel grapple-like structure and has an ATP-dependent F-actin conformation modifying activity in vitro.

In order to investigate the molecular mechanism of the F-actin conformation modifying activity [Biochem. Biophys. Res. Commun. 319 (2004) 78] of actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910], the ultrastructure and the regulatory mechanism of the activity were further examined. Interestingly, a novel oligomeric grapple-like structure of 10-12 subunits with an ATP-dependent opening was observed. ATP regulates the opening and closing of the "gate" that forms the opening within oligomeric Aip2p/Dld2p, where binding to the substrate occurs while in the open form. In the presence of ATP (open state of oligomeric Aip2p/Dld2p), oligomeric Aip2p/Dld2p bound the F-actin fiber within the opening, whereas in the absence of ATP (closed state of oligomeric Aip2p/Dld2p), no binding was observed. Simultaneously, the oligomeric Aip2p/Dld2p increased the trypsin susceptibility of F-actin in an ATP-dependent manner. Use of the non-hydrolyzable ATP analogue AMP-PNP yielded similar results to those observed with ATP, suggesting that ATP binding rather than ATP hydrolysis is required for the protein conformation modifying reaction of oligomeric Aip2p/Dld2p. Endogenous Aip2p/Dld2p purified from Saccharomyces cerevisiae also exhibited such protein conformation modifying activity, but monomeric Aip2p/Dld2p with a C-terminal coiled-coil region-truncation failed to exhibit the activity. These data suggest that the oligomerization of Aip2p/Dld2p, which exhibits the unique grapple-like structure with an ATP-dependent opening, is required for the F-actin conformation modifying activity.

Interaction of D-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p.

D-Lactate dehydrogenase protein 2 [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910] was initially identified as the actin interacting protein 2 (Aip2p) using a two-hybrid screen to search for proteins that interact with actin [Nat. Struct. Biol. 2 (1995) 28], but no other evidence indicating an interaction between Aip2p and actin cytoskeleton has been reported so far. During our search for the protein conformation modifying activity, we serendipitously identified Aip2p isolated from Saccharomyces cerevisiae as exhibiting an interaction with F-actin both in vitro and in vivo. Incubation with Aip2p facilitated the formation of the circular form of F-actin in vitro, which exhibited an aberrant trypsin susceptibility. Overexpression of Aip2p induced multi-buds in yeast cells, whereas reduced expression interfered with the formation of the cleavage furrow for the cell division, which was rescued by the introduction of wild-type Aip2p. While Aip2p-treated F-actin in the circular form was negligibly stained by rhodamine-labeled phalloidin (rhodamine-phalloidin) in vitro, rhodamine-phalloidin staining profiles in actin interacting protein 2 gene (AIP2)-modified cells suggested a correlation between the conformation of F-actin and the expression of Aip2p in vivo. AIP2-deleted cells became sensitive to osmotic conditions, a hallmark of actin dysfunction. Finally, immunoprecipitation of yeast cells using anti-Aip2p antibody demonstrated that Aip2p associates with actin. These properties suggest that Aip2p may interact with F-actin in vivo and play an important role in the yeast cell morphology.