scispace - formally typeset
Search or ask a question
Author

Ryuichiro Atarashi

Bio: Ryuichiro Atarashi is an academic researcher from University of Miyazaki. The author has contributed to research in topics: PrPSc Proteins & Scrapie. The author has an hindex of 28, co-authored 64 publications receiving 8141 citations. Previous affiliations of Ryuichiro Atarashi include Nagasaki University & National Institutes of Health.


Papers
More filters
Journal ArticleDOI
Daniel J. Klionsky1, Kotb Abdelmohsen2, Akihisa Abe3, Joynal Abedin4  +2519 moreInstitutions (695)
TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

5,187 citations

Journal ArticleDOI
TL;DR: A new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg ofPrPSc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate, and indicates the promising enhanced diagnostic capacity of RT- QUIC in the antemortem evaluation of suspected CJD.
Abstract: Definitive diagnosis of prion disease currently requires postmortem brain tissue. Now, Ryuichiro Atarashi and colleagues have discovered an assay that can identify prions in human cerebrospinal fluid. This assay could allow for diagnosis of disease in living humans.

488 citations

Journal ArticleDOI
TL;DR: End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity and can be accomplished in 2 days or less.
Abstract: A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

396 citations

Journal ArticleDOI
TL;DR: A much faster seeded polymerization method (rPrP-PMCA) is described which detects ≥50 ag of hamster PrPSc (≈0.003 lethal dose) within 2–3 d and should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrP Sc formation.
Abstract: The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects ≥50 ag of hamster PrPSc (≈0.003 lethal dose) within 2–3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-μl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

322 citations

Journal ArticleDOI
TL;DR: A new prion assay, abbreviated QUIC for quaking-induced conversion, which uses rPrP-sen as a substrate and automated tube shaking rather than sonication is developed, which can detect about one lethal prion dose within a day, and is faster and simpler than previous described PMCA6 and rPrp-PMCA5 assays.
Abstract: To the editor: A key problem in managing prion diseases is the lack of a rapid, practical assay for prions (infectivity) at low-level infectious, or sub-infectious, amounts. Prion diseases involve the accumulation of a pathological, typically protease-resistant form of prion protein, termed PrPSc, which appears to propagate itself in infected hosts by inducing the conversion of its normal hostencoded precursor, PrP-sen, into additional PrPSc (refs. 1–4). In crude brain homogenates, PrPSc and infectivity can be amplified from endogenous PrP-sen during multiple rounds of intermittent sonication and serial dilution into fresh normal brain homogenate2,4. This ultrasensitive assay, termed PMCA, allows detection of ~1 ag of PrPSc in ~3 weeks5. To improve the speed and practicality of prion detection assays, we recently developed a cell-free conversion reaction that supports sustained PrPSc-seeded conversion of recombinant PrP-sen (rPrP-sen) to specific protease-resistant (rPrP-res) forms. This method (which we previously reported in Nature Methods), called rPrP-PMCA, uses periodic sonication and serial reaction rounds of the PMCA method, but is faster6. To circumvent problems associated with sonication in the PMCA and rPrP-PMCA methods (see Supplementary Results online), we have now developed a new prion assay, abbreviated QUIC for quaking-induced conversion, which uses rPrP-sen as a substrate and automated tube shaking rather than sonication. This assay can detect about one lethal prion dose within a day, and is faster and simpler than previous described PMCA6 and rPrP-PMCA5 assays. Initial testing of QUIC reaction conditions revealed that periodic shaking enhanced PrPSc-seeded conversion of hamster rPrP-sen (residues 23–231) into PK-resistant conversion products (rPrP-res(Sc), where (Sc) refers to seeding by PrPSc; Supplementary Fig. 1 and Supplementary Methods online). Consistent with our previous observations with rPrP-PMCA reactions6, the rPrP-res(Sc) reaction products had 17-, 13-, 12and 11-kDa fragments, which represented different C-terminal PrP fragments (Supplementary Fig. 2 online). These results showed that periodic shaking could substitute for sonication in promoting rPrP-res(Sc) formation. Additional experiments revealed that rPrP-res(Sc) generation was also sensitive to rPrP-sen concentration, reaction volume (Supplementary Fig. 1), reaction time (Supplementary Fig. 2), number of serial reactions (Supplementary Fig. 3 online), temperature (Supplementary Fig. 4 online) and shaking cycle (Supplementary Results). In QUIC reactions performed at 45 °C, we observed rPrP-res(Sc) formation in single 46-h QUIC reactions seeded with ≥100 ag of PrPSc (Fig. 1a). In contrast, 21 negative control reactions seeded with comparable dilutions of normal brain homogenate or buffer alone produced no rPrP-res (Fig. 1b). We obtained results similar to those shown in Figure 1a,b in an independent repeat experiment done in triplicate (data not shown). When we diluted products of PrPSc-seeded reactions 1,000-fold into fresh rPrP-sen to seed the subsequent reaction rounds, we observed strong propagation of rPrP-res(Sc) through at least 4 serial reactions (Supplementary Fig. 5 online). Elevation of QUIC reaction temperatures accelerated rPrP-res(Sc) formation. At 55 °C, we detected rPrP-res(Sc) in single 8-h reactions seeded with as little as 10 fg PrPSc (~2 lethal intracerebral doses; Supplementary Fig. 4). We detected 1 fg in 18-h reactions (Supplementary Fig. 6 online). At 65 °C, we detected 100 fg PrPSc seed with a 4-h reaction (Supplementary Fig. 4). However, at 65 °C, there was also 25 20

295 citations


Cited by
More filters
28 Jul 2005
TL;DR: PfPMP1)与感染红细胞、树突状组胞以及胎盘的单个或多个受体作用,在黏附及免疫逃避中起关键的作�ly.
Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1(PfPMP1)与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用,在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员,通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

18,940 citations

Journal ArticleDOI
Lorenzo Galluzzi1, Lorenzo Galluzzi2, Ilio Vitale3, Stuart A. Aaronson4  +183 moreInstitutions (111)
TL;DR: The Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives.
Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

3,301 citations

Journal ArticleDOI
TL;DR: A functional classification of cell death subroutines is proposed that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic programmed cell death, regulated necrosis, autophagic cell death and mitotic catastrophe.
Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

2,238 citations

Journal ArticleDOI
TL;DR: A way forward is suggested for the effective targeting of autophagy by understanding the context-dependent roles of autophile and by capitalizing on modern approaches to clinical trial design.
Abstract: Autophagy is a mechanism by which cellular material is delivered to lysosomes for degradation, leading to the basal turnover of cell components and providing energy and macromolecular precursors. Autophagy has opposing, context-dependent roles in cancer, and interventions to both stimulate and inhibit autophagy have been proposed as cancer therapies. This has led to the therapeutic targeting of autophagy in cancer to be sometimes viewed as controversial. In this Review, we suggest a way forwards for the effective targeting of autophagy by understanding the context-dependent roles of autophagy and by capitalizing on modern approaches to clinical trial design.

1,606 citations

Journal ArticleDOI
TL;DR: It is shown that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle.
Abstract: Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradati...

1,178 citations