14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins.

Mechanical stress and formation of protein aggregates in neurodegenerative disorders.

Misfolded protein aggregates and inclusion bodies have been associated with various protein conformation disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and prion diseases including bovine spongiform encephalopathy (BSE). Models have been proposed as plausible explanations for the extension and progression of protein aggregates; however, little is known about the initiation process of protein aggregation, particularly in sporadic neurodegenerative diseases. Epidemiological data have suggested a tight association between sporadic neurodegenerative diseases and history of mechanical stresses such as trauma, head injury, and occupational exposures, including professional soccer and boxer's brain that carries histological hallmarks of AD/PD. Here, we propose that mechanical stress is an environmental factor that provokes a disturbance in cellular quality control systems and molecular chaperones that target misfolded proteins. This subsequently initiates protein aggregation and results in sporadic neurodegenerative disorders. Further, continuous and repetitive exposure to environmental mechanical stress, mostly in an unrecognized manner, is inevitable in daily life and thus, it functions as a potential driving force for protein aggregation. In this regard, a recent identification of the fact that an intracellular mechanosensor actually exists may support our notion. Reduction in the mechanical stress in combination with other conventional aspects should facilitate the development of rational therapeutics for these neurodegenerative disorders.

Temporospatial patterns of COX-2 expression and pyramidal cell degeneration in the rat hippocampus after trimethyltin administration.

The temporospatial profile of cyclooxygenase-2 (COX-2) expression and neuronal degeneration following trimethyltin (TMT) administration was investigated in the rat hippocampus region. In the CA1 region, significant COX-2 expression was detected on day 3 after TMT administration but pyramidal cell degeneration was detected only on day 5 and thereafter. In the CA3 region, on the other hand, the constitutive COX-2 expression remained unchanged, and more severe pyramidal cell degeneration started on day 3. Concomitant with these observations, we observed that the coadministration of a COX-2 inhibitor prevented such neuronal degeneration only in the CA1 region and not in the CA3 region. In addition, COX-2 inhibition did not affect the increase in the plasma corticosterone concentration after TMT administration. Furthermore, the COX-2 inhibitor did not alleviate TMT-induced locomotor hyperactivity in rats, for which inhibitors of corticosterone synthesis are known to be effective. These data suggest that the COX-2-dependent pathway appears to assist TMT-induced degeneration of CA1 pyramidal cells but not CA3 pyramidal cells in a corticosterone-independent manner.

The possible role of protein X, a putative auxiliary factor in pathological prion replication, in regulating a physiological endoproteolytic cleavage of cellular prion protein.

The posttranslational conformational conversion of the cellular isoform of prion protein PrP(C) into its scrapie isoform PrP(Sc) is the fundamental process underlying the pathogenesis of prion disease. Based on several transgenic data, it has been postulated that a putative auxiliary factor denoted protein X functions as a molecular chaperone through its unfolding activity of PrP(C) during the formation of PrP(Sc). However, the assumption that protein X therefore exists exclusively in prion diseases appears improbable and thus, it should have some simultaneous physiological role. We, hereby, propose a novel concept - a characteristic role of protein X in supporting a physiological endoproteolytic cleavage of PrP(C). The events corresponding to the formation of the physiologically metabolized PrP(C) or the pathologically transformed PrP(Sc) are mutually exclusive. Amino acid residues that are critical in terms of the target site of protein X for the pathological alteration into PrP(Sc) overlap at the cleavage site. These amino acid residues tend to have a hydrophobic property and are most probably found buried inside the native protein structure. Therefore, a putative molecular chaperone identical to protein X may target the same hydrophobic residues in PrP(C) and work in conjunction with either PrP(Sc) in prion disease or PrP proteases during the physiological state. This postulation may help explain in a relatively simple manner these two mutually exclusive phenomena, viz. the physiological endoproteolytic cleavage of PrP(C) and its pathological conversion into PrP(Sc).

[Elusive function of prion protein].

Prion protein is a highly conserved glycoprotein tethered to cell membranes by a glycosylphosphatidylinositol(GPI) anchor that is expressed in many tissues including brain, heart, and muscle. Although misfolding of the cellular prion protein (PrP(c)) into alternative form, denoted (PrP(Sc)), is a key event in prion infections, the normal function of PrPc remains to be clearly defined. Many PrP(c)-binding proteins have been identified, and several roles for PrP(c) have been suggested, including oxidative stress, cell adhesion, copper uptake, cell survival, protection against oxidative stress, but authentication of these interactions in functional assays is incomplete. In this article, we pick out some researches that pertain to the biology of mammalian prion protein functions.

Three-repeat Tau 69 is a major tau isoform in laser-microdissected Pick bodies.

By utilizing a novel combinatorial method of a Laser Microdissection System and Western blot analysis, we demonstrate that a distinct isoform of abnormally phosphorylated tau (69 kDa, Tau 69) predominantly aggregated in laser-microdissected Pick bodies (PBs) in sporadic Pick's disease. By contrast, tau migrated as two major bands of 60 and 64 kDa (Tau 60 and 64) in total brain homogenates as previously reported. Comparative immunohistochemical analysis with anti-4-repeat antibody revealed that a major component of the abnormally phosphorylated tau in these PBs was 3-repeat tau (3R-tau). Whether 29 amino acid repeat encoded by exons 2 and 3 in the Tau 69 might accelerate the formation of PBs remains to be further investigated. Such a combination of morphological and biochemical techniques significantly complements the existing histopathological methods.

Intracerebroventricular delivery of dominant negative prion protein in a mouse model of iatrogenic Creutzfeldt-Jakob disease after dura graft transplantation.

We have developed a novel procedure in which a small collagen sheet (3 mm x 3 mm) absorbing prion-infected brain homogenates was transplanted onto the brain surface of highly prion-susceptible transgenic mice (Tg(MoPrP)4053/FVB), as an animal model of iatrogenic Creutzfeldt-Jakob disease (iCJD) caused by prion-contaminated cadaveric dura graft transplantation. Using the iCJD model, we further investigated the in vivo efficacy of dominant negative recombinant prion protein with lysine substitution at mouse codon 218 (rPrP-Q218K), which is known to inhibit prion replication in vitro (H. Kishida, Y. Sakasegawa, K. Watanabe, Y. Yamakawa, M. Nishijima, Y. Kuroiwa, N.S. Hachiya, K. Kaneko, Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro, Amyloid, vol. 11, 2004, pp. 14-20.). Following 7-day intracerebroventricular administration of the rPrP-Q218K via an indwelling catheter connected to the implanted osmotic pump, the median incubation period of Tg(MoPrP)4053/FVB was prolonged considerably from 117 days to 131 days (p=0.016, log-rank test) in the rPrP-Q218K-treated group, even after a lengthy latency period of as long as 30 days by starting the rPrP-Q218K injection. Whether wild-type rPrP, other mutant rPrPs, or the combination of rPrP-Q218K with other anti-prion compounds might extend the survival period in that condition must be further investigated.

The alternative role of 14-3-3 zeta as a sweeper of misfolded proteins in disease conditions.

Here, we propose a novel hypothesis that 14-3-3 zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates, which are referred to as inclusion bodies. Studies on the localization of the 14-3-3 proteins in different types of inclusion bodies in the brain including neurofibrillary tangle in Alzheimer's disease, pick bodies in Pick's disease, Lewy body-like hyaline inclusions in sporadic amyotrophic lateral sclerosis, prion/florid plaques in sporadic/variant Creutzfeldt-Jakob disease, nuclear inclusions in spinocerebellar ataxia-1, and possibly Lewy bodies in Parkinson's disease suggest a close association of these diseases with 14-3-3 zeta. The highly conserved hydrophobic surface of the amphipathic groove in 14-3-3 zeta represents a general mechanism with diverse cellular proteins, and it may also allow for the molecular recognition of misfolded proteins by hydrophobic interaction in disease conditions. When the abnormal processing of misfolded proteins overwhelms the quality control systems of the cell, it is likely that 14-3-3 zeta is recruited to form deposits of protein aggregates with nonnative, misfolded proteins in order to protect the cell against toxicity. Hence, 14-3-3 zeta may be considered as an auxiliary therapeutic tool in the protein aggregation disorders.

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.

Association study of the chemokine, CXC motif, ligand 1 (CXCL1) gene with sporadic Alzheimer's disease in a Japanese population.

Inflammation is profoundly involved in the development of Alzheimer's disease (AD) and other neurodegenerative diseases. Chemokine, CXC motif, ligand 1 (CXCL1; or GRO1) is an inflammatory cytokine and appears to be implicated in the pathogenesis of AD. It is of interest and importance to see if the CXCL1 gene, mapped on chromosome 4q12-q13, has potential for conferring the predisposition to AD. Here we report on an association study of the CXCL1 gene with sporadic AD patients in a Japanese population; three single nucleotide polymorphisms (SNPs) in the CXCL1 locus were investigated in 103 AD patients and 130 healthy individuals. The results indicate that neither genotype frequencies nor allele frequencies of the examined SNPs attained statistical significance even after being stratified by the presence or absence of the Apolipoprotein E epsilon4 allele. Therefore, the data presented here suggests that the CXCL1 gene could not be associated with the susceptibility to AD in a Japanese population.

Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro.

Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication. In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated. When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrPSc) replication without reducing the viability of the cells. It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC50) = 0.20 microM, 99% effective concentration (EC99) = 0.86 microM vs. EC50 = 0.45 microM, EC99 = 1.5 microM. Besides, no apparent cell damage was observed at the concentration of up to 4.3 microM (100 micrograms/ml). In combination treatment with 0.43 microM (10 micrograms/ml) of rPrP-Q218K, EC99 of quinacrine was decreased from 1.5 microM to 0.5 microM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine. Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy. Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist.

Lack of association between TrkA single nucleotide polymorphisms and sporadic Alzheimer's disease in a Japanese population.

Nerve growth factor and its receptor with tyrosine kinase activity (TrkA) have been implicated in the development of Alzheimer's disease (AD). Entire coding regions of TrkA gene harboring exons 1 through 17 were sequenced in 114 patients with sporadic AD and 112 control subjects in a Japanese population, and six known and two novel single nucleotide polymorphisms were identified, but no mutation associated with sporadic AD was identified. Concurrently, case-control analysis of TrkA gene A1674G polymorphism in 534 patients with sporadic AD and 454 control subjects has revealed no significant differences in TrkA genotype or allele frequencies even after stratification for Apolipoprotein E epsilon4 carrier statuses. Thus, the TrkA genotype does not appear to influence the risk of developing sporadic AD in a Japanese population.

[Unfolding chaperone as a prion protein relating molecule].

Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPc and PrPSc are identical, but their conformations are rather different; PrPc rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPc and PrPSc. To examine the protein unfolding activities against collectively folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone. Unfolding, from S. cerevisiae. Unfolding consists of oligomeric ring-like structure with the central cavity and has an ATP-dependent protein Unfoldingg activity with broad specificity in vitro, of which targets included PrP in beta-sheet form, alpha-synuclein, and A beta protein. We have also found that mouse neuroblastoma N2a cells contained the activity. Treatment of this factor with an ATP-hydrolyzing enzyme, apyrase, caused the decrease in its protein Unfoldingg activity. It was suggested that the purified protein probably formed homo-oligomer consisting of 4-5 subunits and its activity was ATP-dependent.

Detection system based on the conformational change in an aptamer and its application to simple bound/free separation.

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.

Investigation of laser-microdissected inclusion bodies.

We established a novel combinatorial method of laser microdissection system and immunoblot analysis in combination with a novel unfolding chaperone (oligomeric Aip2p/Dld2p) that enables us to examine the molecular profile of proteins in the microscopic regions of interest. As a model system for analyzing inclusion bodies associated with various diseases such as Alzheimer's disease, Parkinson's disease, and prion diseases including bovine spongiform encephalopathy (BSE), we applied this novel method to examine brain samples of patients with Pick's disease, a type of progressive presenile dementia with intraneuronal lesions denoted as Pick bodies (PBs) whose major structural components are tau proteins. After boiling in Laemmli's sample buffer according to the established immunoblotting procedures, 500-2000 PBs were initially applied onto SDS-PAGE gels; however, only faint signals were obtained. Remarkably, only one Pick body was sufficient to illustrate an immunoblot signal; this indicates that pretreatment with oligomeric Aip2p/Dld2p enhances the immunoblot sensitivity by more than a 100-fold. This unprecedented property of laser microdissection combined with oligomeric Aip2p/Dld2p may have further potential applications. For example, a number of proteomic strategies for such inclusion bodies depend on liquid chromatography-tandem mass spectrometry (LC-MS/MS); however, sample preparation methods typically involve the use of detergents and chaotropic agents that often interfere with chromatographic separation and/or electrospray ionization. However, the use of oligomeric Aip2p/Dld2p would not interfere with the LC-MS/MS procedures. Therefore, it might significantly facilitate nanoscale analysis, which is often hindered by the aggregation property of the target proteins present under various analytical conditions, particularly, when the sample protein is present in minor quantities.

siRNA-mediated inhibition of endogenous Huntington disease gene expression induces an aberrant configuration of the ER network in vitro.

Huntingtin is a ubiquitously expressed cytoplasmic protein encoded by the Huntington disease (HD) gene, in which a CAG expansion induces an autosomal dominant progressive neurodegenerative disorder; however, its biological function has not been completely elucidated. Here, we report for the first time that short interfering RNA (siRNA)-mediated inhibition of endogenous Hdh (a mouse homologue of huntingtin) gene expression induced an aberrant configuration of the endoplasmic reticulum (ER) network in vitro. Studies using immunofluorescence microscopy with several ER markers revealed that the ER network appeared to be congregated in various types of cell lines transfected with siRNA directed against Hdh, but not with other siRNAs so far tested. Other subcellular organelles and structures, including the nucleus, Golgi apparatus, mitochondria, lysosomes, microtubules, actin cytoskeletons, cytoplasm, lipid rafts, and plasma membrane, exhibited normal configurations. Western blot analysis of cellular prion protein (PrP(C)) revealed normal glycosylation, which is a simple marker of post-translational modification in the ER and Golgi compartments, and immunofluorescence microscopy detected no altered subcellular distribution of PrP(C) in the post-ER compartments. Further investigation is required to determine whether the distorted ER network, i.e., loss of the huntingtin function, participates in the development of HD.

More than a 100-fold increase in immunoblot signals of laser-microdissected inclusion bodies with an excessive aggregation property by oligomeric actin interacting protein 2/D-lactate dehydrogenase protein 2.

We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/d-lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick's disease. Initially, 500 to 2000 pick bodies were applied onto SDS-PAGE gels after boiling in Laemmli's sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts.

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.

Microtubules-associated intracellular localization of the NH2-terminal cellular prion protein fragment.

By utilizing double-labeled fluorescent cellular prion protein (PrPC), we revealed that the NH2-terminal and COOH-terminal PrPC fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells and HpL3-4, a hippocampal cell line established from prnp gene-ablated mice [Nature 400 (1999) 225]. Of note, the NH2-terminal PrPC fragment, which predominantly localized in the intracellular compartments, congregated in the cytosol after the treatment with a microtubule depolymerizer (nocodazole). Truncated PrPC with the amino acid residues 1-121, 1-111, and 1-91 in mouse (Mo) PrP exhibited a proper distribution profile, whereas those with amino acid residues 1-52 and 1-33 did not. These data indicate the microtubules-associated intracellular localization of the NH2-terminal PrPC fragment containing at least the 1-91 amino acid residues.