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Methods in
Molecular Biology 2322
Yuzuru Imai Editor
Experimental
Models
of Parkinson’s
Disease
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

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For over 35 years, biological scientists have come to rely on the research protocols and
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Experimental Models of
Parkinson’s Disease
Edited by
Yuzuru Imai
Department of Research for Parkinson’s Disease, Juntendo University Graduate
School of Medicine, Tokyo, Japan
Editor
Yuzuru Imai
Department of Research for Parkinson’s Disease
Juntendo University Graduate School of Medicine
Tokyo, Japan
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1494-5 ISBN 978-1-0716-1495-2 (eBook)
https://doi.org/10.1007/978-1-0716-1495-2
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
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Cover Illustration Caption: Dopaminergic neuron culture differentiated from iPS cells using the Chapter 8 protocol.
Image was taken by Dr. Risa Nonaka.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
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Preface
Parkinson’s disease (PD) is the second most common neurodegenerative disorder, charac-
terized by age-dependent motor dysfunction and degeneration of midbrain dopaminergic
neurons. Deposition of neuronal inclusions called Lewy bodies (LBs) in the affected regions
is a pathological feature of PD and related disorders, such as dementia with LB. LB
formation is thought to begin with α-synuclein aggregation and fibrillation. Experimental
studies based on the knowledge obtained from epidemiological and genetic studies continue
in the hopes of making PD risk predictable and surmountable.
Discovery of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) contributed to the development of experimental models of PD in the early stage
of studies and focused the spotlight on the roles of mitochondria in dopaminergic neurons.
MPTP is converted to 1-methyl-4-phenylpyridinium (MPP+) by glial monoamine oxidase B
and is transported to dopaminergic neurons, likely through the dopamine transporter, and
inhibits mitochondrial respiratory complex I subunits. Another neurotoxin,
6-hydroxydopamine, is a dopamine analog that produces selective damage to dopaminergic
neurons by generating reactive oxygen species. MPTP and 6-hydroxydopamine have been
widely used to create animal and cellular models of PD.
Advances in human genetics and genome-wide association studies have revealed some of
the genes involved in the etiology of PD, including α-synuclein, LRRK2, VPS35, ATP13A2,
PINK1, Parkin, and CHCHD2. The prion-like propagation of α-synuclein in neural circuits
is a very hot topic in this research field, while the dysregulation of mitochondrial function is
another important risk factor for PD development, which is characterized by studies on
PINK1, Parkin, and CHCHD2. Identification of several PD genes, including LRRK2,
VPS35, and ATP13A2, suggests dysregulation of the endolysosome pathway and exosomes
is an emerging topic. As invertebrate models, Drosophila, C. elegans, and budding yeast
Saccharomyces cerevisiae provide sophisticated genetics and have yielded a great deal of
information on protective and risk genes and factors, including lipids and chemicals.
This volume of Methods in Molecular Biology focuses on cutting-edge methods to
analyze the prion-like properties of α-synuclein, mitochondrial functions related to the
PINK1-Parkin pathway/CHCHD2, the endolysosome pathway related to LRRK2,
VPS35, and ATP13A2 using cultured cells (including patient iPS cells), deep brain stimula-
tion therapy, classic mitochondrial toxins related to PD, and genetic associations and screen-
ings using mammalian and invertebrate genetic models of PD. This volume intends to be an
introductory protocol book for basic research on PD pathogenesis.
Tokyo, Japan Yuzuru Imai
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I BIOCHEMICAL EXPERIMENTS AND CELLULAR MODELS

OF PARKINSON’S DISEASE
1 α-Synuclein Seeding Assay Using RT-QuIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Ayami Okuzumi, Taku Hatano, Takeshi Fukuhara, Shinichi Ueno,
Nobuyuki Nukina, Yuzuru Imai, and Nobutaka Hattori
2 Electron Microscopic Analysis of α-Synuclein Fibrils. . . . . . . . . . . . . . . . . . . . . . . . . 17
Airi Tarutani and Masato Hasegawa
3 α-Synuclein Seeding Assay Using Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Jun Ogata, Daisaku Takemoto, Shotaro Shimonaka,
Yuzuru Imai, and Nobutaka Hattori
4 Analysis of α-Synuclein in Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Taiji Tsunemi, Yuta Ishiguro, Asako Yoroisaka,
and Nobutaka Hattori
5 Measurement of GCase Activity in Cultured Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Yuri Shojima, Jun Ogata, Taiji Tsunemi, Yuzuru Imai,
6 Detection of Substrate Phosphorylation of LRRK2 in Tissues
and Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Kyohei Ito, Lejia Xu, Genta Ito, and Taisuke Tomita
7 Two Methods to Analyze LRRK2 Functions Under Lysosomal Stress:
The Measurements of Cathepsin Release and Lysosomal Enlargement. . . . . . . . . 63
Maria Sakurai and Tomoki Kuwahara
8 Differentiation of Midbrain Dopaminergic Neurons
from Human iPS Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Kei-Ichi Ishikawa, Risa Nonaka, and Wado Akamatsu
9 Monitoring PINK1-Parkin Signaling Using Dopaminergic Neurons
from iPS Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Kahori Shiba-Fukushima and Yuzuru Imai
PART II MAMMALIAN MODELS OF PARKINSON’S DISEASE
10 Generation of Mitochondrial Toxin Rodent Models of Parkinson’s Disease

Using 6-OHDA, MPTP, and Rotenone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Hiroharu Maegawa and Hitoshi Niwa
11 Midbrain Slice Culture as an Ex Vivo Analysis Platform
for Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Yuji Kamikubo, Keiko Wakisaka, Yuzuru Imai,
and Takashi Sakurai
vii
viii Contents
12 α-Synuclein Propagation Mouse Models of Parkinson’s Disease . . . . . . . . . . . . . . 119

Norihito Uemura, Jun Ueda, Shinya Okuda,
Masanori Sawamura, and Ryosuke Takahashi
13 Common Marmoset Model of α-Synuclein Propagation . . . . . . . . . . . . . . . . . . . . . 131
Masami Masuda-Suzukake, Aki Shimozawa,
Masashi Hashimoto, and Masato Hasegawa
14 Application of a Tissue Clearing Method for the Analysis
of Dopaminergic Axonal Projections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Kenta Yamauchi, Megumu Takahashi, and Hiroyuki Hioki
15 Deep Brain Stimulation Using Animal Models of Parkinson’s Disease . . . . . . . . . 151
Asuka Nakajima and Yasushi Shimo
PART III INVERTEBRATE MODELS OF PARKINSON’S DISEASE
16 Assessment of Cytotoxicity of α-Synuclein in Budding Yeast

Using a Spot Growth Assay and Fluorescent Microscopy . . . . . . . . . . . . . . . . . . . . 163
Masak Takaine
17 The Functional Assessment of LRRK2 in Caenorhabditis elegans
Mechanosensory Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Tomoki Kuwahara
18 Analysis of Dopaminergic Functions in Drosophila. . . . . . . . . . . . . . . . . . . . . . . . . . 185
Tsuyoshi Inoshita, Daisaku Takemoto, and Yuzuru Imai
19 Evaluation of Mitochondrial Function and Morphology in Drosophila . . . . . . . . 195
Yinglu Tang, Foozhan Tahmasebinia, and Zhihao Wu
20 Cytosolic and Mitochondrial Ca2+ Imaging in Drosophila
Dopaminergic Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Tsuyoshi Inoshita and Yuzuru Imai
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Contributors
WADO AKAMATSU • Center for Genomic and Regenerative Medicine, Juntendo University
TAKESHI FUKUHARA • Department of Neurology, Juntendo University School of Medicine,
Tokyo, Japan; Department of Research for Parkinson’s Disease, Juntendo University
Graduate School of Medicine, Tokyo, Japan
MASATO HASEGAWA • Department of Brain and Neurosciences, Tokyo Metropolitan Institute
of Medical Science, Tokyo, Japan; Dementia Research Project, Tokyo Metropolitan Institute
of Medical Science, Tokyo, Japan
MASASHI HASHIMOTO • Dementia Research Project, Tokyo Metropolitan Institute of Medical
Science, Tokyo, Japan
TAKU HATANO • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan
NOBUTAKA HATTORI • Department of Neurology, Juntendo University School of Medicine,
Tokyo, Japan; Department of Research for Parkinson’s Disease, Juntendo University
Graduate School of Medicine, Tokyo, Japan; Department of Diagnosis, Prevention and
Treatment of Dementia, Juntendo University Graduate of Medicine, Tokyo, Japan
HIROYUKI HIOKI • Department of Cell Biology and Neuroscience, Juntendo University
YUZURU IMAI • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan; Department of Research for Parkinson’s Disease, Juntendo University Graduate
TSUYOSHI INOSHITA • Department of Neurodegenerative and Demented Disorders, Juntendo
University Graduate School of Medicine, Tokyo, Japan
YUTA ISHIGURO • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan
KEI-ICHI ISHIKAWA • Department of Neurology, Juntendo University School of Medicine,
Tokyo, Japan; Center for Genomic and Regenerative Medicine, Juntendo University School
of Medicine, Tokyo, Japan
GENTA ITO • Social Cooperation Program of Brain and Neurological Disorders, Graduate
School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
KYOHEI ITO • Laboratory of Neuropathology and Neuroscience, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
YUJI KAMIKUBO • Department of Pharmacology, Juntendo University School of Medicine,
Tokyo, Japan
TOMOKI KUWAHARA • Department of Neuropathology, Graduate School of Medicine, The
University of Tokyo, Tokyo, Japan
HIROHARU MAEGAWA • Department of Dental Anesthesia, Osaka University Graduate School
of Dentistry, Osaka, Japan
MASAMI MASUDA-SUZUKAKE • Dementia Research Project, Tokyo Metropolitan Institute of
Medical Science, Tokyo, Japan
ASUKA NAKAJIMA • Department of Neurology, Juntendo University Nerima Hospital, Tokyo,
Japan
ix
x Contributors
HITOSHI NIWA • Department of Dental Anesthesia, Osaka University Graduate School of

Dentistry, Osaka, Japan
RISA NONAKA • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan; Center for Genomic and Regenerative Medicine, Juntendo University School of
Medicine, Tokyo, Japan
NOBUYUKI NUKINA • Laboratory of Structural Neuropathology, Graduate School of Brain
Science, Doshisha University, Kyoto, Japan
JUN OGATA • Department of Research for Parkinson’s Disease, Juntendo University
Graduate School of Medicine, Tokyo, Japan; Juntendo Advanced Research Institute for
Health Science, Juntendo University Graduate School of Medicine, Tokyo, Japan
SHINYA OKUDA • Department of Neurology, Kyoto University Graduate School of Medicine,
Kyoto, Japan
AYAMI OKUZUMI • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan; Laboratory of Structural Neuropathology, Graduate School of Brain Science,
Doshisha University, Kyoto, Japan
MARIA SAKURAI • Department of Neuropathology, Graduate School of Medicine, The
University of Tokyo, Tokyo, Japan
TAKASHI SAKURAI • Department of Pharmacology, Juntendo University School of Medicine,
Tokyo, Japan
MASANORI SAWAMURA • Department of Neurology, Kyoto University Graduate School of
Medicine, Kyoto, Japan
KAHORI SHIBA-FUKUSHIMA • Department of Neurodegenerative and Demented Disorders,
Juntendo University Graduate School of Medicine, Tokyo, Japan
YASUSHI SHIMO • Department of Neurology, Juntendo University Nerima Hospital, Tokyo,
Japan; Department of Research and Therapeutics for Movement Disorders, School of
Medicine, Juntendo University, Tokyo, Japan
SHOTARO SHIMONAKA • Department of Diagnosis, Prevention and Treatment of Dementia,
Juntendo University Graduate of Medicine, Tokyo, Japan
AKI SHIMOZAWA • Dementia Research Project, Tokyo Metropolitan Institute of Medical
Science, Tokyo, Japan
YURI SHOJIMA • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan
FOOZHAN TAHMASEBINIA • Department of Biological Sciences, Dedman College of
Humanities and Sciences, Southern Methodist University, Dallas, TX, USA
MEGUMU TAKAHASHI • Department of Cell Biology and Neuroscience, Juntendo University
Graduate School of Medicine, Tokyo, Japan; Department of Neuroscience, Graduate School
of Medicine, Kyoto University, Kyoto, Japan
RYOSUKE TAKAHASHI • Department of Neurology, Kyoto University Graduate School of
MASAK TAKAINE • Gunma University Initiative for Advanced Research (GIAR), Gunma
University, Maebashi, Gunma, Japan; Institute for Molecular and Cellular Regulation
(IMCR), Gunma University, Maebashi, Gunma, Japan
DAISAKU TAKEMOTO • Department of Research for Parkinson’s Disease, Juntendo University
YINGLU TANG • Department of Biological Sciences, Dedman College of Humanities and
Sciences, Southern Methodist University, Dallas, TX, USA
Contributors xi
AIRI TARUTANI • Department of Brain and Neurosciences, Tokyo Metropolitan Institute of

Medical Science, Tokyo, Japan; Laboratory of Neuropathology and Neuroscience, Graduate
School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
TAISUKE TOMITA • Laboratory of Neuropathology and Neuroscience, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; Social Cooperation
Program of Brain and Neurological Disorders, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo, Japan
TAIJI TSUNEMI • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan
JUN UEDA • Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto,
Japan
NORIHITO UEMURA • Department of Neurology, Kyoto University Graduate School of
SHINICHI UENO • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan; Department of Neurology, Fukushima Medical University, Fukushima, Japan
KEIKO WAKISAKA • Department of Research for Parkinson’s Disease, Juntendo University
Graduate School of Medicine, Tokyo, Japan; Department of Neurology, Juntendo University
ZHIHAO WU • Department of Biological Sciences, Dedman College of Humanities and
Sciences, Southern Methodist University, Dallas, TX, USA
LEJIA XU • Laboratory of Neuropathology and Neuroscience, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
KENTA YAMAUCHI • Department of Cell Biology and Neuroscience, Juntendo University
Graduate School of Medicine, Tokyo, Japan; Advanced Research Institute for Health
Sciences, Juntendo University, Tokyo, Japan
ASAKO YOROISAKA • Department of Neurology, Juntendo University School of Medicine, Tokyo,
Japan
Part I
Biochemical Experiments and Cellular Models of Parkinson’s

Disease
Chapter 1
α-Synuclein Seeding Assay Using RT-QuIC

Ayami Okuzumi, Taku Hatano, Takeshi Fukuhara, Shinichi Ueno,
Nobuyuki Nukina, Yuzuru Imai, and Nobutaka Hattori
Abstract
Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of
α-synuclein (αSyn). They include Parkinson’s disease, dementia with Lewy bodies, and multiple system
atrophy. In each disease, it has been proposed that aggregates of αSyn represent different conformational
strains of αSyn, leading to self-propagation and spreading from cell to cell. It has been considered that αSyn
aggregates grow by seeded polymerization mechanisms. Previously, the mechanism of seed conversion in
prion protein aggregation has been exploited by real-time quaking-induced conversion (RT-QuIC) assay. It
was further refined by incorporating the fluorescent dye thioflavin-T, which enabled the real-time monitor-
ing of kinetic changes with a highly sensitive detection of seed aggregates present at an extremely low level.
In an application for diagnostics, it has been reported that αSyn RT-QuIC exhibits specificity between 82%
and 100%, while its sensitivity varies between 70% and 100%, on the basis of a study in which this assay was
performed at multiple different laboratories. Furthermore, it has been suggested that the αSyn RT-QuIC
method can be applied to study the biochemical characteristics of different αSyn strains among synuclei-
nopathies. In this article, we describe the detailed protocols for αSyn RT-QuIC assays.
Key words α-Synuclein, Synucleinopathy, Biomarker, Cerebrospinal fluid, Brain, Seed, Diagnosis,
Strain, β-Sheet, RT-QuIC
1 Introduction
α-Synucleinopathies, such as Parkinson’s disease (PD), dementia

with Lewy bodies (DLB), and multiple system atrophy (MSA), are a
class of neurodegenerative diseases characterized by the presence of
misfolded, fibrillary α-synuclein (αSyn). In patients with PD or
DLB, αSyn is misfolded and accumulates in neurons, while MSA
patients show a different pattern of oligodendroglial cytoplasmic
inclusions (GCI). Recent studies have suggested that the aggrega-
tion and propagation of misfolded αSyn play key roles in disease
initiation and progression [1–5]. One of the most effective strate-
gies to develop disease-modifying therapies is to prevent the
Yuzuru Imai (ed.), Experimental Models of Parkinson’s Disease, Methods in Molecular Biology, vol. 2322,
https://doi.org/10.1007/978-1-0716-1495-2_1, © The Editor(s) (if applicable) and The Author(s), under exclusive license to
Springer Science+Business Media, LLC, part of Springer Nature 2021
3
4 Ayami Okuzumi et al.
accumulation of misfolded αSyn aggregates. Toward the develop-

ment of such therapies, it is important to first establish an accurate
diagnostic method at the earliest stage of the disease. However,
accurate diagnosis remains difficult, especially for patients at a stage
prior to the prodromal phase of synucleinopathies. There is thus an
urgent need to develop a detection system sensitive enough to
discriminate the protein aggregates that circulate in peripheral
blood, resulting in accumulation in the brain to cause neurological
dysfunction. In fact, several investigations of pathological biomar-
kers in accessible specimens such as cerebrospinal fluid (CSF) sug-
gested arranging an appropriate cohort for clinical trials and the
value of serially monitoring the therapeutic efficacy in patients.
As for the biophysical mechanism of αSyn aggregation, the
propagation of misfolded proteins requires the initial formation of
a homotypic complex between the two conformers of a protein
[6, 7], which leads to the conversion of the native form into the
pathogenic form. The de novo β-sheet-enriched molecule may act
as a seed, resulting in contact with other native molecules (typically
referred to as substrates) and inducing their conversion into
β-sheets [7]. Currently, the most practical and widely implemented
method to detect prion seeds is real-time quaking-induced conver-
sion (RT-QuIC) assay. RT-QuIC is an in vitro cell-free assay that
amplifies small numbers of pathogenic seeds and enables the detec-
tion of aggregates from the fluorescent intensity mediated by
thioflavin-T (ThT) binding to cross-β structures typically found in
amyloid deposits [8–10]. Thus, RT-QuIC technology has been
modified to apply it to the detection of αSyn seeds that are derived
from CSF, brain homogenate samples [7, 8, 11–17], submandibu-
lar glands [18], and olfactory mucosa [19] of patients with synu-
cleinopathies. The results showed that RT-QuIC could detect
extremely low levels of αSyn seeds in a highly sensitive manner.
The term “strain” was initially introduced in the field of prions
to discriminate the pathological phenotypes associated with the
intracerebral inoculation of prion proteins in goats [20, 21]. Strains
hence refer to different conformations of the same protein. In some
cases of prion RT-QuIC assays, characteristic distinctions among
seed strains were confirmed by results based on either SDS-PAGE
analyses of protease-mediated cleavage sensitivity of RT-QuIC
amplicons or morphological analysis of the ultrastructure of these
aggregates using electron microscopy [8, 19]. Taking these find-
ings together, RT-QuIC is a powerful tool to determine the specific
characteristics of protein seed strains as well as the susceptibility to
diseases in diagnostics. In this chapter, we introduce the detailed
method of performing αSyn RT-QuIC.
RT-QuIC for α-Synuclein 5
2 Materials (See Note 1)
1. αSyn expression vectors: Wild-type full-length human αSyn

cDNA in bacterial expression plasmid pRK172 was used
[22]. Codon 136 was changed from TAC to TAT by site-
directed mutagenesis [23] (see Note 2). The plasmids were
transformed into Escherichia coli BL21(DE3) (EMD
Biosciences).
2. LB-M broth medium (sterilized by autoclaving).
3. Ampicillin (Sigma): 100 mg/mL in stock solution, stored at
20 C.
4. 1 M DTT, stored at 20 C.
5. Purification buffer: 50 mM Tris–HCl (pH 7.4), 1 mM EGTA,
and 1 mM DTT. Add DTT stock solution to the buffer imme-
diately before use. Make up the volume to 1 L and prepare and
sterilize by filtration.
6. Wash buffer: 50 mM Tris–HCl (pH 7.4), 1 mM EGTA, 1 mM
DTT, and 0.1 M NaCl.
Add DTT stock solution to the buffer immediately
before use.
7. Elution buffer: 50 mM Tris–HCl (pH 7.4), 1 mM EGTA,
1 mM DTT, and 0.35 M NaCl.
Add DTT stock solution to the buffer immediately
before use.
8. 30 mM Tris–HCl (pH 7.5). Prepare 2 L.
9. Ammonium sulfate.
10. 1 M isopropyl β-D-thiogalactoside (e.g., Sigma).
11. BioSpectrometer (Eppendorf).
12. 50-mL high-g-force-resistant polypropylene tubes (e.g.,
Himac, Item#: 328353A).
13. 500-mL high-g-force-resistant polypropylene tubes (e.g.,
Himac, Item#: 330437A).
14. Sonicator (e.g., MISONIX).
15. 2-Mercaptoethanol.
16. 0.22-μm filter (Millex).
17. Q-Sepharose ion exchange chromatograph (GE).
18. Dialysis membrane: 10 kDa MWCO (e.g., Pierce Thermo
Scientific).
19. 100-kDa cut-off filter (Millipore, Amicon Ultra).
20. Seed spoon (Biomedical Science Inc., Item#: SS200).
21. 96-Well optical black-bottom plate (Nunc Item#: 265301).

22. Plate sealer EASYseal (Greiner Item#: 6760x1).
23. Zirconium/silica beads, 0.5 mm in diameter (BIOSPEC
Item#: 11079105Z).
24. Fluorescence plate reader (BMG Labtech, FLUOstar
OPTIMA).
25. RT-QuIC Reaction Buffer: 100 mM phosphate buffer
(pH 8.2), 10μM ThT.
26. Spectrophotometer (ThermoFisher Scientific, NanoDrop).
27. Brain homogenate buffer: 1 PBS containing 1 mM EDTA,
0.5% Triton X-100, and 1 protease inhibitors (Roche, cOm-
plete protease inhibitor cocktail).
3 Methods
3.1 α-Synuclein 1. Purify recombinant human αSyn protein from Escherichia coli
Purification from BL21(DE3) harboring pRK172-αSyn (Y136-TAT). If the
Bacteria transformed bacterial cells express the target gene with a
good yield of the recombinant protein, the cells can be stored
in glycerol stock at 80 C until use.
2. Prepare 50 mL LB-M broth culture containing 50μg/mL
ampicillin in a 200 mL breathable culture tube as a starter
culture.
3. Inoculate BL21 harboring αSyn expression vector into 50 mL
LB-M broth starter culture from a frozen bacterial glycerol
stock previously transformed with pRK172-αSyn (Y136-TAT)
(see Subheading 2, item 1). Alternatively, this starter culture
may be inoculated with colonies grown on an agarose plate for
bacterial culture.
4. Grow the starter culture in a shaking incubator at 37 C with
vigorous shaking overnight.
5. Prepare and warm 250 mL autoinduction medium (LB-M
broth medium with 50μg/mL ampicillin) at 37 C in a
500-mL culture flask.
6. Inoculate the prepared 250 mL autoinduction medium with
the entire 50 mL starter culture.
7. Culture at 37 C for several hours with vigorous shaking until
the optical density (OD600) measured by the BioSpectrometer
reaches 0.6.
8. Add isopropyl 1-thio-β-D-galactopyranoside at a final concen-
tration of 0.5 mM into 250 mL autoinduction medium and
incubate for 6 h with vigorous shaking.
9. Collect the cultured medium in 500-mL high-g-force-resistant

polypropylene tubes.
10. Centrifuge for 30 min at 3000 g and 4 C.
11. Decant and discard the supernatant.
12. Lyse the cell pellet by adding 10 mL αSyn buffer with gentle
resuspension using a 10-mL serological pipette.
13. Sonicate the resuspended bacterial cells on ice under the opti-
mized conditions.
14. Centrifuge the sonicated cell suspension at 20,000 g and
4 C for 10 min.
15. Transfer the clear supernatant to a 50-mL high-g-force-resis-
tant polypropylene tube and add 2-mercaptoethanol at a final
concentration of 1%.
16. Incubate in boiling water for 5 min.
17. Centrifuge the solution at 20,000 g and 4 C for 15 min and
transfer the supernatant to a new tube.
18. Prepare 5 mL Q-Sepharose ion exchange beads (50% slurry in
20% EtOH) in a 15-mL tube.
19. Wash and equilibrize the beads three times using 10 mL αSyn
purification buffer.
20. Load the clear supernatant carefully onto the beads in the
column and rotate for 1 h at 4 C.
21. Wash the column with 10 mL αSyn purification buffer at 4 C
three times. Then, wash the column with 4.5 mL αSyn wash
buffer two times.
22. Collect the eluate by loading 9 mL αSyn elution buffer to elute
αSyn.
23. Precipitate by adding solid ammonium sulfate powder at the
level of 50% saturation.
24. Centrifuge at 20,000 g for 15 min at 4 C and discard the
supernatant.
25. Resuspend the pellet with 1 mL of 30 mM Tris–HCl (pH 7.5)
buffer prior to filtrating with a 0.22-μm filter (Millex).
26. Dialyze pooled fractions using a 10-kDa MWCO dialysis mem-
brane against 2 L of 30 mM Tris–HCl, pH 7.5, at 4 C for 1 h
twice and then overnight.
27. Determine the concentration of dialyzed protein using a BCA
assay.
28. Snap-freeze the protein solution in aliquots appropriate for
single experiments. Store the aliquots at 80 C.
3.2 Measurement 1. Open the “Omega” software of the fluorescent plate reader
of the Aggregation (FLUOstar OPTIMA microplate reader).
Using αSyn 2. Enter the following settings into your script: target tempera-
RT-QuIC Assay ture, 30 C; shaking speed, 200 rpm; gain, 30%; cycle time,
900 min; read every 15 min; total measurement time as
required, typically for a week.
3. Set the temperature to 30 C and warm up the machine prior to
starting measurement.
4. Thaw frozen test samples (including brain homogenate and
CSF) on ice.
3.3 Preparation 1. Thaw αSyn substrate on ice.

of αSyn Substrate 2. Centrifuge the substrate at 100,000 g and 4 C for 20 min
for RT-QuIC and transfer it to a new tube (see Note 3).
3. Filter the supernatant through a 100-kDa MWCO filter imme-
diately prior to use (see Note 4).
4. Measure the absorbance at 280 nm (A280) with a NanoDrop
spectrophotometer and calculate the concentration of the
substrate.
3.4 Preparation 1. RT-QuIC reactions are performed in black 96-well plates (see
of αSyn Reactions Subheading 2, item 21) (see Note 5). Preload plates with
37 3 mg of 0.5 mm zirconium/silica beads per well (see
Note 6).
2. The RT-QuIC reaction buffer is composed of 100 mM phos-
phate buffer (pH 8.2) and 10μM thioflavin T (ThT). Add
0.1 mg/mL filtered αSyn substrates (final concentration) (see
Note 7).
3. For brain homogenate samples: Prepare initial 10% (w/v) brain
homogenates using phosphate-buffered saline (PBS) contain-
ing 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, and
cOmplete protease inhibitor cocktail. Each well should contain
95μL reaction buffer. For reactions, seed 5μL brain homoge-
nate diluted with PBS appropriately to a final volume of 100μL.
4. For CSF samples: Place 90μL reaction buffer in each well. Seed
reactions with 10μL undiluted CSF to a final reaction volume
of 100μL (see Note 8).
5. For pluripotent stem cell (iPSC)-derived dopaminergic neuron
samples: Place 95μL reaction buffer in each well. Seed reactions
with 5μL iPSC-derived dopaminergic neuron samples to a final
reaction volume of 100μL.
6. Centrifuge the plate at 300 rpm for 5 min at 4 C.
7. Seal the plate with EASYseal.
8. Place the prepared plate in the fluorescence plate reader and

incubate it at 30 C for 120 h with intermittent shaking cycles:
double-orbital mode with 1 min of shaking at 200 rpm fol-
lowed by 14 min of resting.
9. Monitor the kinetics of fibril formation by the bottom reading
method for the fluorescence intensity every 15 min using exci-
tation light at 450 nm and emission fluorescence at 480 nm.
Set instrument gains to 30% of the detection limit of the
instrument (260,000).
10. Data analysis: For the general strategy, see below.
3.5 RT-QuIC Data 1. Analyze the relative fluorescent intensity measured by

Analysis: Parameters RT-QuIC and plot it using the built-in program Omega
of Kinetic Change Mars. Calculate the area under the curve (AUC), the time to
(Fig. 1, See Note 9) reach half maximum fluorescence, and the speed of aggregation
(Figs. 1 and 2, see Note 10).
3.6 RT-QuIC Data 1. Using a FLUOstar OPTIMA microplate reader (BMG Lab-
Analysis for Sensitivity tech), a positive response is defined when ThT fluorescence
and Specificity (See reaches 260,000 RFU (maximum fluorescence) at 120 h.
Note 11)
Fig. 1 Diagram of the potential seeding conversion mechanism of αSyn in RT-QuIC assay. The seeds trigger
the aggregation of monomeric αSyn. This conversion causes conformational modification into misfolded
oligomers that elongate into fibrils. After the detection of fibrils, a quaking event breaks the longer fibrils into
shorter, reactive oligomers, which further act as seeds for the conversion of monomeric αSyn. The typical
shape of the kinetic curve is shown during the aggregation process
Fig. 2 Amyloid formation is accelerated in a seed dose-dependent manner
2. Each sample is typically set in duplicate. If only one of two

samples is positive, the analysis should be repeated in quadru-
plicate. A positive response in at least two of the replicates is
considered as a positive response overall [17].
3. Positive or negative response data sets (see Subheading 3.6,
step 2) are subjected to multiple logistic regression analyses
to describe the appropriate receiver operating characteristic
(ROC) curves with area under the curve (AUC) by using
GraphPad Prism 6 or other graphing software for calculating
sensitivity and specificity.
4 Notes
1. Buffers and reagents are filtered through a pre-rinsed 0.22-μm

filter into pre-rinsed vessels that are sterilized and subjected to
minimization of lint contamination. Filters, syringes, and tubes
also need to be pre-rinsed [24].
2. To avoid cysteine misincorporation at codon 136 in bacterially
expressed αSyn protein, the expression constructs with site-
directed mutagenesis at codon 136 (Y136-TAC to Y136-
TAT) can be generated and used as αSyn constructs [23].
3. It is common to store recombinant αSyn monomer at 80 C
until use. However, a freezing and thawing cycle might pro-
mote αSyn aggregation or seed formation. Therefore, we
always centrifuge αSyn monomer at 100,000 g and 4 C
for 20 min and collect the supernatant for use.
4. A small portion of the substrate might be eliminated during the
filtration step.
5. Do not use the outermost wells of a 96-well plate due to the

risk of uneven heating. The temperature on a plate reader is
unstable, especially in these parts. Apply 100μL water to the
outermost wells.
6. Add two seed spoons full of zirconium/silica beads to wells to
fill approximately 37 mg beads per well.
7. Never vortex after the substrate has been added to the plate.
Vortexing or vigorous mixing might promote aggregate (arti-
ficial seed) formation [24].
8. The applied protocols differ widely in terms of the physical and
chemical conditions of the reaction and the starting seed and
substrate used for the reaction. As previous reports have
described, multiple approaches may be used to detect αSyn
seeding activity. For example, a different microenvironment
of the reaction, different physical settings for the aggregation,
a different source of the seed, a different method of preparing
substrate, or any combination of these factors may yield differ-
ent results [8]. The addition of further compounds such as SDS
detergent can change the seeding kinetics and the sensitivity of
αSyn RT-QuIC of CSF [7] (see Table 1).
9. RT-QuIC assay is a relatively qualitative method. The seeding
activity might differ slightly in each assay due to the differences
in substrate lot or reaction environment. This variation should
be especially noted when you compare the results among
experiments by using quantitative parameters (e.g., lag time
or half-maximum fluorescence).
10. RT-QuIC assay takes advantage of the seeding conversion
mechanism that underlies prion aggregation. In this assay,
small amounts of misfolded αSyn seeds recruit single recombi-
nant αSyn substrate molecules and induce their conversion by
integrating them into a growing amyloid aggregate concomi-
tant with a conformational change of the substrate into a
seeding-competent state (Fig. 1) [7, 26, 27]. RT-QuIC con-
sists of cycles of (a) incubation steps to control the size of the
amyloid product because αSyn fibrils increase exponentially,
and (b) vigorous shaking steps, which make fragments of
αSyn aggregates into smaller seeds to promote the conversion
(Fig. 1). The growth of the αSyn fibrils follows a sigmoidal
curve (Fig. 1). It displays a lag phase, during which the nucle-
ation process occurs, followed by the exponential phase repre-
senting elongation of the fibrils, and then a plateau phase once
the monomers have been converted into β-sheet-enriched spe-
cies, which saturates the signal (Fig. 1) [7]. The lag phase or
time to threshold depends on the initial concentration of seeds
(Fig. 2). Over a long period, αSyn substrates will eventually
form fibrils spontaneously under various RT-QuIC conditions
Table 1
12
Comparison of different αSyn RT-QuIC protocols
Shaking/
Ref Sample Seed Sn (%) Sp (%) Substrates incubation T pH SDS Beads
[11] 42 DLB, 22PD, 35 neurol 5–15μL CSF; 2μL 95% 100% 0.1 g/L human 1 min 30 C 8.2 No 37 3 mg of
controls, 35 controls BH 1:20,000 in αSyn double 0.5 mm
PBS orbital, zirconium/
200 rpm/ silica beads
15 min
Ayami Okuzumi et al.
[12] 7 DLB, 6 neurol controls 5μL 10% w/V BH ND 100% 0.1–0.15 g/L 40 s circular, 40 C 7.5 No No
in PBS S129Ah αSyn 432 rpm/
140 s
[13] 17 DLB, 12PD, 16 neurol 2μL 10 % w/V BH 93% 100% 0.1 g/L filtered 1 min 42 C 8 0.0015% 6 silica of
controls, 15 controls in PBS; 15μL human αSyn; double for 0.8 mm
CSF 0.1 g/L orbital, CSF
K23Q aSyn 400 rpm/
1 min
[14] 15 PD, 11 controls 15μL CSF; 2μL 100% 100% 0.1 g/L human 1 min 42 C 8 0.0015% 6 silica of
BH 1:104 αSyn double for 0.8 mm
orbital, CSF
400 rpm/
1 min
[17] 118 synucleinopathies, CSF of uncertain 75% 85–98% 0.1 g/L human 1 min 30 C 8.2 No 37 3 mg of
52 controls synucleinopathy αSyn double 0.5 mm
patients orbital, zirconium/
15 min
[15] 105 PD, 79 controls 15μL CSF; 2μL 96% 82% 0.1 g/L human 1 min 30 C 8.2 No 37 3 mg of
BH 1:20,000 in αSyn double 0.5 mm
PBS orbital, zirconium/
15 min
[8] 10 DLB, 10PD, 10 controls from 3 μL BH 70% for 95% for 0.14 g/L 1 min 37 C 8.4 2% No
FCx; 8 DLB, 6 PD, 5 controls FCx; FCx, human αSyn double
from SNc 75% for 100% orbital,
SNc for 600 rpm/
SNc 29 min
[16] 51 LRRK2 PD, 10 idiopathic 15μL CSF 90% for 80% 0.1 g/L human 1 min 30 C 8.2 No 37 3 mg of
PD, 10 controls PD; αSyn double 0.5 mm
40% for orbital, zirconium/
LRRK2 200 rpm/ silica beads
PD 15 min
[19] BH from 1 FTDP, 1 PSP, 1 CBD, 2μL diluted 10 3 65.50% 84.30% 70μM human 1 min single 42 C 8 No 1 glass bead
1 PD, 1 MSA, and 1 control from 10% BH αSyn orbital, of 3 mm
200 rpm/ beads
29 min
[25] Definite NP; 21 DLB, 15μL CSF 95.30% 98% 0.1 g/L filtered 1 min 42 C 8 0.0015% 6 silica of
101 neurol controls. Clinical human αSyn double for 0.8 mm
cohort; 34 DLB, 31 MSA, orbital, CSF
71 PD, 119 neurol controls, 400 rpm/
62 controls 1 min
BH brain homogenate, CBD corticobasal degeneration, DLB dementia with Lewy bodies, FCx frontal cortex, FTDP frontotemporal dementia and parkinsonism, MSA multiple
system atrophy, ND not determined, OM olfactory mucosa, PD Parkinson’s disease, PSP progressive supranuclear palsy, Ref reference, Sn sensitivity, Sp specificity, SNc substantia
nigra pars compacta, T temperature
RT-QuIC for α-Synuclein
13
in the absence of preformed seeds. Thus, we usually distinguish

between appropriately matched samples with differing seed
concentrations and characteristics by comparing kinetic curves
or times to reach the threshold [9, 10, 24]. The slope obtained
by differential processing of plotted curves indicates the speed
of aggregation. To distinguish different types of αSyn seed
strain, the end-products of RT-QuIC were indicated to be
resistant to proteinase K (PK) by western blotting [8, 19].
11. Sensitivity and specificity: To obtain accuracy in terms of speci-
ficity and sensitivity, we make positive/negative decisions using
samples from synucleinopathy patients and healthy controls,
respectively. The relative level of seeding activity can be
assessed using end-point dilution [13] or kinetic analyses, as
has been described elsewhere for αSyn RT-QuIC reactions.
Acknowledgments
We thank Edanz (https://en-author-services.edanzgroup.com/ac)

for editing the English text of the draft of this manuscript. This
work was supported by Japan Agency for Medical Research and
Development (AMED), Strategic Research Program for Brain
Sciences (20dm107156 to T.H.), and Grants-in-Aid for Scientific
Research (19K16928 to A.O., 19K07831 to T.F.) from the Japan
Society for the Promotion of Science (JSPS).
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Chapter 2
Electron Microscopic Analysis of α-Synuclein Fibrils

Airi Tarutani and Masato Hasegawa
Abstract
α-Synuclein (α-syn) is a major component of abnormal protein deposits observed in the brains of patients
with synucleinopathies, including Parkinson’s disease, dementia with Lewy bodies, and multiple system
atrophy (MSA). The synaptic protein α-syn is water-soluble under normal physiological conditions, but in
these patients’ brains, we see accumulation of insoluble amyloid-like α-syn fibrils with prion-like properties.
Intracerebral accumulation of these fibrils is correlated with disease onset and progression. Recombinant
α-syn protein also forms amyloid-like fibrils that are structurally akin to those extracted from patients’
brains. Recent cryo-electron microscopic studies have identified the core structures of synthetic α-syn fibrils
and α-syn fibrils extracted from the brains of patients with MSA at the atomic level. In this chapter, we
describe negative staining and immunoelectron microscopy protocols for ultrastructural characterization of
synthetic α-syn fibrils and pathological α-syn fibrils.
Key words α-Synuclein, Amyloid-like fibrils, Negative staining, Immunoelectron microscopy, Par-
kinson’s disease, Dementia with Lewy bodies, Multiple system atrophy
1 Introduction
Accumulation of filamentous α-synuclein (α-syn) in neurons

and/or glial cells is the neuropathological hallmark of synucleino-
pathy [1]. α-Syn accumulates in neurons as Lewy bodies and Lewy
neurites in Parkinson’s disease (PD) and dementia with Lewy bod-
ies (DLB) and is observed in oligodendrocytes as glial cytoplasmic
inclusions in multiple system atrophy (MSA). Electron microscopic
studies of detergent-insoluble fractions extracted from brains of
synucleinopathy patients show that α-syn forms amyloid-like struc-
tures with a width of 5–10 nm (Figs. 1f and 3c–e) [2–4]. In
addition, more than 90% of this accumulated α-syn is phosphory-
lated at serine 129 and partially ubiquitinated [5, 6]. Missense
mutations and multiplications of the SNCA gene encoding α-syn
have been reported to cause familial synucleinopathy [7].
α-Syn is a water-soluble, natively unfolded protein composed of
140 amino residues and is localized in synaptic terminals at
17
18 Airi Tarutani and Masato Hasegawa
Fig. 1 Electron microscopic analysis of negatively stained α-syn fibrils. (a) Carbon-coated 300-mesh copper
grid. (b) Holding the grid with the cross-lock tweezers. (c) Dropping the sample and staining solutions on the
grid. (d) Blotting the sample and staining solutions with filter paper. (e) Negatively stained synthetic α-syn
fibrils. Scale bar, 100 nm. (f) Negatively stained α-syn fibrils extracted from the brain of a patient with MSA.
Scale bar, 100 nm
Ultrastructural Observation of α-syn Fibrils 19
relatively high concentration [8]. In patients, α-syn in the brain

structurally changes from the normal form rich in α-helix to an
abnormal form with β-sheet structure. Recombinant α-syn protein
purified from bacterial cells also forms amyloid-like fibrils closely
resembling those observed in patients upon shaking at 37 C for
several days (Figs. 1e and 3a, b) [9, 10]. Furthermore, different
misfoldings of α-syn caused by mutations and various cellular con-
ditions lead to the formation of α-syn strains with distinct confor-
mations [11]. Synthetic α-syn fibrils containing familial mutations
and synthetic α-syn fibrils formed under various conditions have
been reported to exhibit distinct ultrastructural properties and
cytotoxicities [12–14]. Cryo-electron microscopy (EM) of α-syn
fibrils extracted from MSA patients has revealed that MSA-derived
α-syn fibrils are conformationally distinct from synthetic α-syn fibrils
and DLB-derived α-syn fibrils [15]. These distinct α-syn strains
exhibit heterogeneity of prion-like properties in vitro and in vivo
[4, 16–18].
Transmission electron microscopy can visualize samples at
nanoscale by irradiating them with electron beams and obtaining
an interference image from the transmitted electrons. EM analysis
makes it possible to observe filamentous α-syn structures and quan-
titatively evaluate structural differences using the fibril width and
the periodicity as indicators. In this chapter, we describe how to
prepare a grid bearing α-syn fibrils for EM analysis. Negative stain-
ing is a method to examine easily and quickly all structures
contained in the sample solution. Heavy metals in the staining
solution occupy the gaps and surrounding parts of the structure
and reveal the morphology of the structure. On the other hand,
immuno-EM can specifically observe the ultrastructural features of
the target protein even in the presence of contaminants.
2 Materials
2.1 Negative 1. Carbon-coated 300-mesh copper grid (see Note 1).

Staining 2. Cross-lock stainless steel tweezers.
3. Filter paper.
4. Sample solution: synthetic α-syn fibrils (see Note 2), sarkosyl-
insoluble fractions extracted from brains of patients with synu-
cleinopathy (see Note 3).
5. Staining solution: 2% phosphotungstic acid, pH 7.5 (see
Note 4).
6. Transmission electron microscope.
2.2 Immunolabeling 1. Carbon-coated 300-mesh copper or nickel grid (see Note 5).
2. Cross-lock stainless steel tweezers.
3. Parafilm.
4. Humidified chamber.
5. Filter paper.
6. Sample solution.
7. Blocking and diluent solution: 0.1% gelatin in sterile
phosphate-buffered saline (PBS), 0.1% sodium azide.
8. Primary antibody recognizing α-syn (see Note 6).
9. Wash buffer: sterile PBS.
10. Secondary antibody conjugated to gold particles (see Note 7).
11. Staining solution: 2% phosphotungstic acid, pH 7.5.
12. Transmission electron microscope.
3 Methods
3.1 Negative 1. Hold the carbon-coated 300-mesh copper grid with cross-lock
Staining of α-Syn stainless steel tweezers (Fig. 1a, b).
Fibrils 2. Place 2–4μL of the sample solution on the grid mesh (Fig. 1c).
Wait for 1 min.
3. Blot the sample solution with filter paper or a Pipetman
(Fig. 1d, see Note 8).
4. Place a few drops of 2% phosphotungstic acid on the grid mesh
before the grid dries. Blot the staining solution on the grid with
filter paper (Fig. 1c, d).
5. Repeat step 4 twice.
6. Dry completely.
7. Set the prepared grid in the holder of the transmission electron
microscope.
3.2 Immuno-EM 1. Hold the carbon-coated 300-mesh copper or nickel grid with
of α-Syn Fibrils cross-lock stainless steel tweezers (Fig. 1a, b).
2. Place 2–4μL of the sample solution on the grid mesh (Fig. 1c).
Wait for 1 min.
3. Blot the sample solution with filter paper or a Pipetman
(Fig. 1d).
4. Dry completely.
5. Put 50μL of the blocking solution onto the parafilm in the
humidified chamber and place the grid on top of the drop with
the side to which the sample solution is fixed facing down
(Fig. 2a–e). Incubate for 10 min at room temperature.
Fig. 2 Immunoelectron microscopy of α-syn fibrils. (a) The parafilm in the humidified chamber. (b) Solutions
on the parafilm. (c) Placing the grid face-down on the drop. (d, e) Image of the grid during incubation. (f)
Blotting surplus solutions on the grid with filter paper
6. Blot the blocking solution remaining on the grid surface with

filter paper (Fig. 2f, see Note 9).
7. Put 50μL of the primary antibody (1:50–1:100 dilution,
diluted in the diluent solution) onto the parafilm and place
the grid on top of the drop (Fig. 2b–e). Incubate for 1 h at
room temperature or 37 C.
8. Drop 50μL of wash buffer onto the parafilm and wash the grid
surface in it (Fig. 2b, c).
9. Wash twice as in step 8.
10. Blot the wash buffer remaining in the grid surface with filter
paper (Fig. 2f).
11. Put 50μL of the second antibody conjugated to gold particles
(1:50–1:100 dilution, diluted in the diluent solution) onto the
parafilm and place the grid on top of the drop (Fig. 2b–e).
Incubate for 1 h at room temperature.
12. Wash three times as in step 8.
13. Blot the wash buffer remaining in the grid surface with filter
paper (Fig. 2f).
14. Negative stain the grid as in Subheading 3.1.
4 Notes
1. Hydrophilization treatment of the grid is effective for disper-

sion of fibrils. The hydrophilized grid prepared with an
ion-sputtering device is used within a few hours.
2. Purification of recombinant α-syn protein from Escherichia coli
is performed as previously reported [19]. For fibrillization,
2–10 mg/mL recombinant α-syn protein is incubated under
shaking (200 rpm) at 37 C for 3–5 days. The degree of
fibrillization is evaluated by means of fluorescence assay with
thioflavin, which specifically binds to ß-sheet structure. Dilute
the formed α-syn fibrils to 1/10 with 30 mM Tris–HCl
(pH 7.5) and apply the diluted α-syn fibrils to the grid. If the
sample solution contains only a small number of fibrils, the
sample solution is ultracentrifuged at 113,000 g for 20 min
at 25 C, and the resulting pellet is suspended in a small
amount of 30 mM Tris–HCl (pH 7.5).
3. Preparation of the sarkosyl-insoluble fraction from the brain of
a patient with synucleinopathy is performed as previously
reported [20]. Add an appropriate amount of 30 mM Tris–
HCl (pH 7.5) to the sarkosyl-insoluble fraction and suspend by
pipetting. If large amounts of contaminants are contained in
the sarkosyl-insoluble fraction, centrifuge at 5000 g for
10 min at 25 C and use the supernatant as a sample solution.
The morphology of α-syn fibrils accumulated in cultured cells
or mouse brain can also be observed by electron microscopic
analysis of the sarkosyl-insoluble fraction prepared in the same
way as described for patients’ brains.
4. Store in the dark at room temperature. Gently centrifuge to
remove debris before use, and then use the supernatant. Uranyl
acetate (2%) can also be used as a staining solution.
5. If the incubation time with the primary antibody is prolonged,
a nickel grid can be used to prevent rusting.
Fig. 3 Immunolabeled synthetic α-syn fibrils and patient-derived α-syn fibrils.

(a, b) Synthetic α-syn fibrils positive for α-syn 131–140 antibody, immunola-
beled with secondary antibody conjugated to 5 nm (a) and 10 nm (b) gold
particles. Scale bar, 200 nm (a), 50 nm (b). (c, d) PS129-positive α-syn fibrils
extracted from the brain of a patient with DLB, immunolabeled with secondary
antibody conjugated to 5 nm gold particles. Arrowheads indicate ferritins. Scale
bar, 200 nm (c), 50 nm (d). (e) PS129-positive α-syn fibril extracted from the
brain of a patient with MSA, immunolabeled with secondary antibody conjugated
to 5 nm gold particles. Scale bar, 50 nm
6. Antibodies directed against α-syn phosphorylated at serine

129 can differentiate normal and abnormal α-syn and are useful
for immuno-EM of patient-derived α-syn fibrils. Antibodies
that recognize the C-terminal of α-syn are used for immuno-
EM of synthetic α-syn fibrils. The C-terminal of α-syn is located

outside the filamentous α-syn structure and is easily recognized
by immuno-EM.
7. The fibril width of α-syn fibrils is 5–10 nm. Gold particles with
a diameter of 5–20 nm are suitable to label α-syn fibrils. In
general, ferritin, approximately 10 nm in diameter, is one of the
major contaminants in the sarkosyl-insoluble fraction extracted
from the patient’s brain (Fig. 3d) [21]. Care is needed to
distinguish ferritins from gold particles.
8. Place the filter paper on the edge of the grid and take care not
to let the filter paper touch the grid mesh.
9. Stand the grid perpendicular to the filter paper.
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Chapter 3
α-Synuclein Seeding Assay Using Cultured Cells

Jun Ogata, Daisaku Takemoto, Shotaro Shimonaka, Yuzuru Imai,
Abstract
α-Synuclein, a presynaptic protein, is involved in synaptic vesicle dynamics in response to neuronal activity.
Mutations of the α-synuclein gene and the neuronal deposition of α-synuclein, called Lewy bodies, are
linked to the development of Parkinson’s disease. α-Synuclein has a prion-like property that converts its
physiological protein conformation to a pathogenic one, forming disease-causing fibrils. Aggregation of
these fibrils and subsequent inclusion formation are suggested to interfere with vesicular trafficking and
organelle function in neurons. Thus, detection of a prion-like property of α-synuclein and the evaluation of
its modifying factors are required to understand the pathogenesis of Parkinson’s disease and to develop new
therapies. In this chapter, we describe a cell-based assay for detecting α-synuclein propagation.
Key words α-Synuclein, Recombinant protein, Protein purification, Transfection, Cultured cells
1 Introduction
Several neurodegenerative diseases, including Parkinson’s disease,

Parkinson’s disease dementia, dementia with Lewy bodies, and
multiple system atrophy, are classified as synucleinopathies, which
are characterized by the progressive accumulation of fibrillized
α-synuclein in the affected regions [1, 2]. The deposition of Lewy
bodies is a pathological feature of synucleinopathies [1, 2]. The
Lewy body consists of fibrillized α-synuclein, which is often phos-
phorylated at Ser219 [3], organelles such as mitochondria and
lysosomes, and lipid membranes [4]. The formation of
α-synuclein inclusions interferes with intercellular trafficking and
organelle functions, leading to neuronal death [5]. Pathological
analyses have shown that Lewy body deposition extends from the
dorsal motor nucleus of the vagus to the upper brain stem and from
Jun Ogata and Daisaku Takemoto contributed equally to this work.
27
28 Jun Ogata et al.
the olfactory bulb to the limbic system [6, 7], suggesting that
pathogenic α-synuclein exhibits prion-like seeding activity to con-
vert the physiological form of α-synuclein to pathogenic fibrils.
The formation of α-synuclein cellular inclusions by α-synuclein
fibrils as ‘seeds’ has been experimentally demonstrated using
α-synuclein inclusions extracted from brain autopsies with synuclei-
nopathies, including Parkinson’s disease, as well as fibrils prepared
from recombinant α-synuclein [8, 9]. Depending on disease-
causing mutations, the brain environment, and conditions for the
preparation of recombinant α-synuclein fibrils, α-synuclein fibrils
form distinct structures, which could affect their propagation effi-
ciency and pathological effects [10–13].
To analyze the mechanism of α-synuclein aggregation and cell–
cell propagation using cultured cells, we describe a modified assay
protocol based on a method established by Hasegawa et al.
[9, 14]. The preparation of ‘competent seeds’ is a critical step in
this protocol. This assay can evaluate the efficiency of α-synuclein
aggregation and the effect of modifiers in the intracellular environ-
ment in a short time without protein transfection reagents. We
hope this protocol contributes to elucidating the pathogenesis of
synucleinopathies, modifier genes, and chemicals [15].
2 Materials
2.1 α-Synuclein 1. BL21 (DE3) competent cells: 20 μl aliquot with competency

Purification from >5 107 cfu/μg with pUC19.
Bacteria 2. LB medium: autoclaved.
3. 50 mg/ml ampicillin stock in 70% ethanol.
4. 2 l shake flask with 3 baffles.
5. LB agar plates: standard LB agar plates (100 mm in diameter)
with 50 μg/ml ampicillin.
6. Isopropyl ß-D-1-thiogalactopyranoside (IPTG): 0.1 M stock in
distilled water. Store at 20 C.
7. Bacterial expression vector: pRK172-human α-synuclein (see
Note 1).
8. Large-capacity refrigerated centrifuge (Himac, CR21N).
9. α-Synuclein purification buffer: 50 mM Tris–HCl, pH 7.4,
1 mM EGTA, 1 mM DTT. Prepare 500 ml and store at 4 C.
Add 1 M DTT stock just before use.
10. α-Synuclein wash buffer: α-synuclein buffer containing 0.1 M
NaCl. Add 0.29 g NaCl to 50 ml α-synuclein buffer.
11. α-Synuclein elution buffer: α-synuclein buffer containing
0.35 M NaCl. Add 1.0 g NaCl to 50 ml α-synuclein buffer.
α-Synuclein Seeding Assay Using Cultured Cells 29
12. High powered sonicator (Taitec, VP-050 N, see Note 2).

13. 50 ml polypropylene centrifuge tubes.
14. 2-Mercaptoethanol.
15. Dialysate: 30 mM Tris–HCl, pH 7.5. Prepare 2000 ml and
store at 4 C.
16. Ammonium sulfate (biochemical grade).
17. Q Sepharose Fast Flow (GE Healthcare, 17051010).
18. Empty chromatography column (Bio-Rad, Econo-Pac
#7321010, 20 ml bed volume).
19. Vortex mixer.
20. Stirrer.
21. Cellulose dialysis tubing (MWCO 14,000).
22. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) system: a 12.5% gel is appropriate to separate
α-synuclein.
23. Coomassie Brilliant Blue (CBB) staining solution.
24. Bicinchoninic acid (BCA) assay kit.
2.2 α-Synuclein 1. Purified recombinant α-synuclein in dialysate from Subheading

Seed Preparation 3.1.
2. 1.5 ml safe-lock microcentrifuge tube (Eppendorf, see Note 3).
3. NaN3 solution: 1% in distilled water.
4. Dialysate: 30 mM Tris–HCl, pH 7.5. Filter it through a
0.22 μm filter.
5. 0.01% NaN3/PBS: Phosphate-buffered saline (PBS) contain-
ing 0.01% NaN3. Filter it through a 0.22 μm filter.
6. 1.5 ml tube incubator shaker with a lid (Eppendorf Thermo-
Mixer) or bacteria shaker.
7. High-powered sonicator (Taitec, VP-050 N, see Note 4).
8. Microultracentrifuge (Himac, CS150GXL).
9. BCA assay kit.
2.3 Cell Culture 1. Cultured human cells (see Note 5).

and Transfection 2. Cell culture medium: DMEM (Sigma-Aldrich) containing 10%
fetal bovine serum (Gibco) and 1 penicillin-streptomycin
solution (Gibco).
3. X-tremeGENE 9 (Roche).
4. Opti-MEM (Gibco).
30 Jun Ogata et al.
5. pcDNA3-human α-synuclein: 1 mg/ml in Tris–EDTA buffer,

pH 8.0 (see Note 6).
6. α-Synuclein fibrils (see Note 7).
2.4 Biochemical 1. PBS.

Fractionation 2. A68 buffer: 10 mM Tris–HCl, pH 7.4, 0.8 M NaCl, 1 mM
of α-Synuclein EGTA, 5 mM EDTA, 10% sucrose.
3. Triton-X100/A68 buffer: A68 buffer containing 1% Triton-
X100.
4. Sarkosyl/A68 buffer: A68 buffer containing 1% sarkosyl.
5. 3 Sodium dodecyl sulfate (SDS) sample buffer: 188 mM Tris-
Cl, pH 6.8, 6% (w/v) SDS, 30% glycerol, 0.03% bromophenol
blue, 150 mM DTT. Add 1 M DTT stock just before use.
6. High-powered sonicator (Taitec, VP-050 N, see Note 2).
7. Microultracentrifuge (Himac, CS150GXL).
2.5 Western Blot 1. SDS-PAGE system: A 12.5% gel is appropriate to separate

Analysis α-synuclein.
2. Polyvinylidene difluoride (PVDF) membranes were used for
western blots (Millipore).
3. Methanol.
4. Transfer buffer: 48 mM Tris, 39 mM glycine, 20% methanol,
1.3 mM (0.0375%) SDS.
5. Paraformaldehyde/PBS: 4% paraformaldehyde in PBS. Prepare
just before use.
6. TBS-T: 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.05%
Tween 20.
7. Blocking solution: TBS-T containing 5% bovine serum albu-
min (BSA).
8. Anti-phospho-Ser129 α-synuclein (Abcam, EP1536Y).
9. Anti-α-synuclein (BD Biosciences, 42/α-Synuclein).
10. Secondary antibody and detection solution (see Note 8).
3 Methods
3.1 α-Synuclein 1. Transform a 20 μl aliquot of BL21(DE3) with 80 ng pRK172-

Purification from human α-synuclein following standard procedures.
Bacteria 2. Streak bacteria on LB agar plates (see Note 9).
3. Collect all colonies of BL21 harboring α-synuclein from the LB
plate by pipetting with 5 ml of LB medium containing 50 μg/
ml ampicillin.
4. Add the bacterial suspension from step 3 to a 2 l shake flask

with baffles containing 500 ml LB medium and 50 μg/ml
ampicillin. The OD600 of the resultant culture will be approxi-
mately 0.2 (see Note 10).
5. Shake the LB culture at 200 rpm at 37 C for 3.5 h.
6. Add 1 ml of 0.1 M IPTG stock.
7. Shake the LB culture for 4 h more.
8. Harvest bacteria at 6000 g for 10 min at 4 C in a large-
capacity refrigerated centrifuge.
9. Completely remove the supernatant.
10. Store the centrifuge tube containing bacterial pellet at 80 C
(see Note 11).
11. Add 10 ml ice-cold α-synuclein purification buffer to the bac-
terial pellet and completely resuspend the pellet on ice.
12. Transfer the bacterial suspension to a 50-ml centrifuge tube.
13. Sonicate the suspension on ice until the solution is clear (see
Note 2).
14. Centrifuge the sonicated suspension at 20,000 g for 10 min
at 4 C in a refrigerated centrifuge.
15. Transfer the supernatant after centrifugation in step 14 to a
new 50-ml centrifuge tube, and add 2-mercaptoethanol to a
final concentration of 1%.
16. Incubate the tube in boiling water for 5 min (see Note 12).
17. Centrifuge the boiled tube at 20,000 g for 15 min at 4 C,
and transfer the supernatant to a new 50-ml centrifuge tube.
18. During centrifugation in step 17, step up the Q Sepharose
column (see Note 13).
19. Pour the supernatant into the Q Sepharose column.
20. Wash the Q Sepharose with 30 ml (>10 bead volume of beads)
of α-synuclein purification buffer.
21. Wash the Q Sepharose with 9 ml (3 bead volume of beads) of
α-synuclein wash buffer.
22. Elute α-synuclein bound to the Q Sepharose with 9 ml (3 bead
volume of beads) of α-synuclein elution buffer.
23. Add 2.82 g ammonium sulfate to the elution fraction on ice to
precipitate α-synuclein (see Note 14).
24. Centrifuge the elution fraction from step 23 at 20,000 g for
15 min at 4 C.
25. Aspirate the supernatant.
32 Jun Ogata et al.
Fig. 1 Recombinant α-synuclein purified from bacteria. Dialyzed α-synuclein

(16.4 mg/ml) was serially diluted at 1–16 dilutions and analyzed by SDS-PAGE/
CBB. An arrowhead indicates α-synuclein
26. Resuspend the α-synuclein pellet in 1–2 ml dialysate, and

transfer the suspension to dialysis tubing prehydrated in dis-
tilled water.
27. Dialyze the α-synuclein suspension against 1000 ml dialysate at
4 C for 1–2 h. Stir the dialysate gently with a stirrer.
28. Exchange the dialysate for another 1000 ml dialysate and
dialyze the α-synuclein solution at 4 C overnight.
29. Centrifuge the α-synuclein solution at 100,000 g for 20 min
at 4 C to remove the precipitate.
30. Collect the supernatant as purified α-synuclein (see Note 15).
31. Check the purity and concentration of α-synuclein by
SDS-PAGE/CBB staining and the BCA assay kit, respectively
(Fig. 1).
3.2 α-Synuclein 1. Add the NaN3 solution to 300 μl purified recombinant

Seed Preparation α-synuclein protein (4–10 mg/ml in dialysate) in a 1.5-ml
safe-lock microcentrifuge tube to a final concentration of
0.01%.
2. Shake the α-synuclein solution for 1–7 days at 37 C in a 1.5 ml
incubator shaker at 200 rpm (Fig. 2a, see Note 16).
3. Ultracentrifuge the α-synuclein solution at 100,000 g for
20 min at 4 C.
4. Remove the supernatant, and collect the α-synuclein fibrils
(Fig. 2b, c).
5. Resuspend the fibrils in 200–300 μl of 0.01% NaN3/PBS.
Fig. 2 α-Synuclein fibrils. (a) The appearance of homogenous gelled liquid (white arrowhead) and cloudy white
materials (orange arrowhead) after 7 days of incubation at 37 C. The latter contains poorly formed fibrils (see
e). (b) The appearance of fibrils formed (a, white arrowhead) just after ultracentrifugation. (c) Precipitates of
gelled fibrils (white arrowhead) and supernatant (orange arrowhead) after ultracentrifugation. (d) The mor-
phology of fibrils from highly viscous clear solution (a, white arrowhead) observed by electron microscopy. (e)
The morphology of fibrils from solution with white amorphous insoluble matter (a, orange arrowhead). (f)
Fragmented fibrils after sonication. The fibril length is approximately 50 nm, which is appropriate for the
propagation assay. Scale bars ¼ 100 nm
6. Sonicate the fibrils on ice using a Taitec high-powered sonica-

tor at a setting of pulse width modulation (PWM) 15–20% for
5 min to obtain α-synuclein fibrils approximately 50 nm in
length (Fig. 2d–f, see Note 4).
7. Ultracentrifuge the sonicated solution at 100,000 g for
20 min at 4 C.
8. Resuspend the sonicated fibrils in an optimal buffer (e.g. PBS).
9. Determine the protein concentration using the BCA assay kit.
10. Save the α-synuclein fibrils as seeds at room temperature (see
Note 17).
3.3 Introduction 1. Plate SH-SY5Y cells (or other cell lines) suspended in the cell
of α-Synuclein Seeds culture medium at 60–70% confluence in a six-well plate and
into Cultured Cells culture at 37 C in a humidified atmosphere containing 5%
CO2 for 12–16 h to reach 80–90% confluence.
2. Dilute 1 μg plasmid DNA (pcDNA3-α-synuclein) in 100 μl
Opti-MEM in a 1.5-ml tube, and add 3 μl X-tremeGENE
9. Mix the solution well, and incubate for 15 min at room
34 Jun Ogata et al.
temperature to allow DNA-X-tremeGENE 9 complex

formation.
3. Add the solution containing the DNA-X-tremeGENE 9 com-
plex dropwise to the cells.
4. Add 2.5 μg α-synuclein seeds from Subheading 3.2 diluted in
100 μl Opti-MEM to SH-SY5Y cells.
5. Incubate the cells for 1 day.
6. Replace the cell culture medium with fresh medium, and incu-
bate the cells for an additional 2 days.
3.4 Sequential 1. Remove the cell culture medium from the α-synuclein seed-
Extraction infected cells from Subheading 3.3.
of α-Synuclein from 2. Wash cells with 2 ml PBS.
Cultured Cells
3. Add 1 ml PBS and harvest cells with gentle pipetting.
4. Centrifuge the cell suspension at 16,900 g for 10 min at
4 C, and remove the supernatant.
5. Resuspend the cell pellet in 150 μl A68 buffer. Sonicate the cell
suspension on ice at PWM 25% for 1 min (see Note 18).
6. Ultracentrifuge the suspension at 100,000 g for 20 min.
7. Transfer the supernatant to a new 1.5-ml microcentrifuge tube.
8. Add 40 μl supernatant to a new 1.5-ml microcentrifuge tube
containing 20 μl 3 SDS sample buffer (this sample is the A68
buffer-soluble fraction). Estimate the protein concentration of
the A68 buffer-soluble fraction using the remaining superna-
tant from step 7.
9. Wash the pellets from step 7 with 500 μl A68 buffer and
subsequently ultracentrifuge at 100,000 g for 10 min.
10. Resuspend the A68 buffer-insoluble pellets in 100 μl Triton-
X100/A68 buffer.
11. Sonicate the suspension at PWM 25% for 40 s at room temper-
ature (see Note 18).
13. Collect the supernatant as the Triton X-100-soluble fraction
(see Note 19).
14. Wash the pellets in 500 μl Triton X-100/A68 buffer and
subsequently ultracentrifuge at 100,000 g for 10 min.
15. Resuspend the pellets in 100 μl Sarkosyl/A68 buffer.
16. Sonicate the suspension at PWM 25% for 40 s at room temper-
ature (see Note 18).
18. Collect the supernatant as the sarkosyl-soluble fraction (see
Note 19).
19. Wash the pellets with 500 μl Sarkosyl/A68 buffer and subse-
quently ultracentrifuge at 100,000 g for 10 min.
20. Dissolve the pellets in 50 μl of 3 SDS sample buffer.
21. Sonicate the suspension at PWM 25% for 10 s three times at
room temperature (this sample is the sarkosyl-insoluble
fraction).
22. Boil each sample at 100 C for 5 min.
3.5 Western Blot 1. Apply each fraction prepared from Subheading 3.4 on a 12.5%
to Monitor α-Synuclein SDS-PAGE gel. Two micrograms of protein per lane is appro-
Inclusion Formation priate for a standard 12-well gel.
2. After SDS-PAGE, transfer the separated proteins from the
SDS-PAGE gel onto a PVDF membrane prehydrated with
100% methanol and subsequent transfer buffer.
3. Incubate the PVDF membrane in 25 ml paraformaldehyde/
PBS at room temperature for 30 min (see Note 20).
4. Incubate the membrane in 25 ml blocking solution for 1 h at
room temperature.
5. Dilute anti-phospho-Ser129 α-synuclein (at 1:2000 dilution)
and/or anti-α-synuclein (at 1:3000 dilution) as primary anti-
bodies with 5 ml blocking solution.
6. Incubate the membrane with 5 ml primary antibody solution
overnight at 4 C.
7. Wash the membrane three times for 10 min each with 25 ml of
TBS-T.
8. Incubate the membrane with horseradish peroxidase (HRP)-
conjugated secondary antibody at RT for 1 h.
9. Wash the membrane three times for 10 min each with 25 ml of
TBS-T.
10. Incubate the membrane with a chemiluminescence reagent to
detect bands for α-synuclein (Fig. 3).
4 Notes
1. We used a modified α-synuclein gene (α-synuclein Y136-TAT),

in which the codon for Tyr136 is modified from TAC to TAT
to avoid mistranslation to a Cys residue instead of a Tyr in
bacteria [16].
2. We used a probe-type sonicator (Taitec, VP-050N) under an
operating condition of 1 min at PWM 50% on ice. The sonica-
tion conditions on different equipment should be determined
by the experimenters.
3. This tube should be ultracentrifuge-resistant.
36 Jun Ogata et al.
Fig. 3 Formation of sarkosyl-insoluble α-synuclein in cultured cells. (a) SH-SY5Y cells were transfected with
α-synuclein plasmid or pcDNA3 as a mock plasmid and infected with 2.5 μg α-synuclein fibrils in Opti-MEM
(+) or Opti-MEM alone (). Phospho-Ser219 α-synuclein and total α-synuclein were detected by western blot.
(b) A549 cells were transfected as in (a) and infected with 2.5 μg α-synuclein fibrils or 2.5 μg α-synuclein
monomer. A68, A68-soluble fraction; TX, Triton X-100-soluble fraction; Sar, sarkosyl-soluble fraction; Ppt,
sarkosyl-insoluble fraction
4. The sonication processes can be performed using a probe-type

ultrasonic homogenizer or an ultrasonic bath. The optimized
setting of the sonicator to obtain ~50 nm α-synuclein fibrils
should be determined by the experimenters.
5. We successfully performed this protocol using SH-SY5Y cells,
A549 cells, and HeLa cells.
6. Unmodified α-synuclein gene.
7. Fibrils as seeds prepared in Subheadings 2.2 and 3.2.
8. Any detection protocols for western blot can be used. We
routinely used horseradish peroxidase-conjugated secondary
antibodies and ECL solution (GE Healthcare).
9. Preparation of bacterial glycerol stocks can omit the
transformation step.
10. Alternatively, you can prepare 500 ml bacterial suspension with
OD600 0.2 by dilution of an overnight bacterial preculture. We
do not recommend adopting this protocol for high-yield pro-
tein production conditions (e.g. changing culture
medium or culture flasks). Under such conditions,
α-Synuclein could form insoluble aggregates in bacterial cells.
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The officials became so alarmed that just before the death of
Philip II. he was requested to prohibit any further enumeration of the
Moriscoes, because it acquainted them with their power and must
eventually prove prejudicial to the interests of the monarchy. Besides
their menacing increase, which no supervision, however effective,
could prevent, they possessed qualities that made them highly
obnoxious to their masters. Their frugality and thrift, their
shrewdness and enterprise, rendered competition with them
impossible. There was no profitable occupation in which they did not
excel. In agriculture they had no rivals. They monopolized every
industrial employment; all of the most useful trades were under their
control. They undersold the Castilian peasantry in their own markets.
Even the most opulent, instructed by previous experience,
sedulously avoided every exhibition of luxury; but the Moorish artisan
had not lost the taste and dexterity of his ancestors, and the splendid
products of the loom and the armory still commanded high prices in
the metropolitan cities of Europe. It was known that the Moriscoes
were wealthy, and popular opinion, as is invariably the case,
delighted in exaggerating the value of their possessions. While they
sold much, they consumed comparatively little and purchased even
less. Although the offence of heresy could no longer be consistently
imputed to them, specious considerations of public policy, as well as
deference to ineradicable national prejudice, demanded their
suppression. Their prosperity, secured at the expense of their
neighbors, and a standing reproach to the idleness and incapacity of
the latter, was the measure of Spanish decay. In the existing state of
the public mind, and under the direction of the statesmen who
controlled the actions of the King, a pretext could readily be found for
the perpetration of any injustice. The Moriscoes of Valencia, the
most numerous, wealthy, and influential body of their race, protected
by the nobles, had always shown less alacrity in the observance of
the duties of the Church than their brethren, and had thus rendered
themselves liable to the suspicion of apostasy. It was declared that
after a generation of espionage, prayer, and religious instruction they
were still secret Mussulmans. This opinion, perhaps in some
instances not without foundation, amounted to absolute certainty in
the narrow mind of Don Juan de Ribera, Archbishop of Valencia, a
prelate of vindictive temper, arbitrary disposition, limited abilities, and
violent prejudices. He owed much of his reputation for piety to the
fact that he had denounced to the Inquisition more than four
thousand alleged Moorish apostates. Knowing his feelings towards
them, the Moriscoes generally turned a deaf ear to his admonitions
and threats, and thus further incurred his displeasure. The energy of
Ribera was incessantly exerted for the ruin of these supposed
heretics, either by exile or by extermination. With this end in view he
addressed several memorials to Philip III., who had now ascended
the throne, in which the objects of his wrath were accused of every
crime against the civil and the moral law,—treason, murder,
kidnapping, blasphemy, sacrilege. In these appeals the Moriscoes
were called “the sponge that absorbed the riches of Spain.” He
enforced his arguments by the extraordinary statement that the
destruction of the Armada was a divine judgment for the indulgence
exhibited towards these enemies of the Faith, and that Philip II. was
aware of it, for he himself had informed him of that fact. The recent
occurrence of earthquakes, tempests, and comets was also sagely
attributed to the same cause. The Moriscoes were not ignorant of the
designs which the Archbishop was prosecuting to their injury, and
endeavored to obtain the assistance of France and England, both of
which countries were then hostile to Spain. They offered King Henry
IV. the services of a hundred thousand well-armed soldiers if he
would invade the Peninsula. The Duke of Sully says they even
signalized their willingness to embrace Protestantism in
consideration of support, it being a form of worship not tainted with
idolatry, like that of Rome. Negotiations were privately opened with
the courts of Paris and London, and commissions were even
appointed by the latter to verify the claims of the Moriscoes; but no
conclusion was arrived at, and the plot was eventually betrayed by
the very sovereigns whose honor was pledged to the maintenance of
secrecy. An embassy was also sent to the Sultan of Turkey by the
Moors, soliciting his aid and tendering him their allegiance. No plan
which promised relief was neglected. The furious Ribera again urged
upon the King the dangers that the toleration of such a numerous
and perfidious people implied; he alleged their prosperity and their
superior intelligence as crimes against the state; and as absolute
extermination did not seem to be feasible, he suggested expulsion
as of greater inconvenience, but of equal efficacy. Once more the
nobles interposed in behalf of their vassals, and while the King was
hesitating the Moriscoes endeavored to anticipate his decision by the
formation of an extensive conspiracy. Again they were betrayed, this
time by one of their own number. Public opinion, aroused by these
occurrences, and further inflamed by ecclesiastical malice and by the
pernicious influence of the Duke of Lerma, the all-powerful minister
of Philip III., now imperatively demanded their banishment. This
nobleman, of base antecedents and unprincipled character, and
whose dominating passion was avarice, was Viceroy of Valencia. His
brother was the Grand Inquisitor Their influence easily overweighed
the remonstrances of the Pope, whose voice was raised on the side
of mercy.
On the fourth of August, 1609, the royal decree which announced
the fate of the Moriscoes of Valencia was signed at Segovia. No
precaution which prudence could suggest was neglected to prevent
disaster consequent upon its enforcement. Great bodies of troops
were placed under arms. The frontiers of the kingdom were patrolled
by cavalry. Seventy-seven ships of war, the largest in the navy, were
assembled on the coast. In every town the garrison was doubled.
Several thousand veterans disembarked from the fleet and were
distributed at those points where the Morisco population was most
numerous. Such preparations left no alternative but submission, and
the Valencians, anticipating the final movement which would deliver
the unhappy Moors into their hands, began to rob and persecute
them without pity. Even after all had been arranged for the removal,
the nobles urged Philip to revoke an order which must cause
incalculable injury to his kingdom. The most solemn and binding
guarantees were offered for the public safety and for the peaceable
behavior of the Moriscoes. It was demonstrated that the
manufacturing and agricultural interests of the entire monarchy were
involved; that a population of a million souls, whose industry
represented of itself a source of wealth which could not be replaced,
would be practically exterminated; that the educational and religious
foundations of the realm alone received from Moorish tributaries an
annual sum exceeding a million doubloons of gold. It was also
shown that the vassals of the Valencian nobles paid them each year
four million ducats, nearly thirty-two million dollars. The alleged
conspiracies were imputed to the malice of the monks, who invented
them in the cloister; the heresies to ignorance of the clergy, too idle
or too negligent to afford their parishioners instruction. The evil
results of the iniquitous decree had already begun to manifest
themselves. The cultivation of the soil had almost ceased. The
markets were deserted. Commerce languished, and the Moriscoes,
to avoid the insults of the populace to which they were now
subjected, only appeared in the streets when impelled to do so by
absolute necessity. The Archiepiscopal See of Valencia, which
derived its revenues almost entirely from Morisco taxation, was
threatened with bankruptcy, and Don Juan de Ribera, realizing when
too late the disastrous consequences of the project he had so
sedulously advocated, now in vain endeavored to stem the tide of
public bigotry and official madness. While bewailing his unhappy
condition to his clerical subordinates, he was heard to plaintively
remark, “My brethren, hereafter we shall be compelled to live upon
herbs and to mend our own shoes.”
Philip refused to reconsider his determination, and the nobility
manifested their loyalty by the unflinching support of a measure
running directly counter to their interests. On the twenty-second of
September, 1609, the edict of expulsion was proclaimed by heralds
throughout the kingdom of Valencia. It represented that by a special
act of royal clemency “the heretics, apostates, traitors, criminals
guilty of lése-majesté human and divine,” were punished with exile
rather than with death, to which the strict construction of the laws
condemned them. It permitted the removal of such effects as could
be carried, and as much of their harvests as was necessary for
subsistence during their journey; all else was to be forfeited to their
suzerains. They were forbidden to sell their lands or houses. Three
days of preparation were granted; after that they were declared the
legitimate prey of every assailant. Dire penalties were denounced
against all who should conceal them or in any way assist in the
evasion of the edict. Those who had intermarried with Christians
could remain, if they desired; and six per cent. of the families were to
be reserved by the lords, that the horticultural and mechanical
dexterity which had enriched the country might not be absolutely
extinguished. These subjects of interested clemency refused to
accept this invidious concession, however, and hastened to join their
countrymen beyond the sea.
The wretched Moriscoes received the tidings of their expatriation
with almost the despair with which they would have listened to a
sentence of death. Astonishment, arising from the suddenness of the
notice and the inadequate time allotted them for preparation, was
mingled with their dismay. The traditions of centuries, the souvenirs
of national glory, the memory of their ancestors, contributed to
endear them to their native land. There were centred the most
cherished associations of a numerous and cultivated race. All around
were the visible signs of thrift and opulence and their results, won by
laborious exertion from the soil. The disfigured but still magnificent
monuments of fallen dynasties recalled the departed glory of Arab
genius and Moslem power. The loss of their wealth, the sacrifice of
their possessions, portended the endurance of calamities for which
they were ill-prepared, and of whose dreadful character their most
gloomy apprehensions could convey no adequate conception. In
every Moorish community appeared the signs of unutterable misery
and woe. The shrieks of frenzied women pierced the air. Old men
sobbed upon the hearthstones where had been passed the happy
days of infancy and youth. Overcome with grief, life-long friends met
in the streets without notice or salutation. Even little children, unable
to comprehend, yet awed by the prevailing sorrow, ceased their play
to mingle their tears with those of their parents.
As the disconsolate and sobbing multitude, urged on by the
ferocious soldiery taught by their religion to regard these victims of
national prejudice as the enemies of Christ, left their homes behind
forever, their trials and sufferings increased with their progress. The
government provided them with neither food, shelter, nor
transportation. The difficulties of the march were aggravated by
clouds of dust and by the pitiless heat of summer. Many were born
on the highway. Great numbers fell from exhaustion. Some, in
desperation, committed suicide. Every straggler was butchered by
the armed rabble which, equally ravenous for plunder or blood,
constantly hung on the flanks of the slowly moving column. Many
were assassinated by Old Christians, men of Moorish ancestry, the
conversion of whose forefathers dated before the Conquest, and
who told their beads and muttered prayers after each murder, as if
they had committed an action acceptable to God. The armed
brigands who composed the escort vied with the mob in their
atrocities. The men were openly killed, the women violated. Their
property was appropriated by force. Some died of hunger. Parents,
in their extremity, became so oblivious of the instincts of nature as to
barter their children for a morsel of bread. When they embarked for
Africa they fared even worse than they had done on land. On the sea
the opportunities for outrage were multiplied, the means of escape
and detection diminished. No pen can portray the horrors visited
upon the unhappy Moriscoes, helpless in the midst of savage
enemies who were insensible to pity, hardened by cruelty, and
dominated by the furious lust of beauty and gold.
The decree was not received everywhere with the same
submission as at the city of Valencia. There the exiles, overawed by
the large military force, yielded without disturbance. Half-crazed by
misfortune, they even feigned exultation, marched on board the
ships dressed in holiday costume and headed by bands of music,
and in token of delight gave themselves up to the most extravagant
exhibitions of joy. Some kissed the shore, others plunged into the
sea, others again quaffed the briny water as if it were a delicious
beverage. Before embarking they sold much of their property, and
articles of great elegance and beauty—curiously wrought vessels of
gold and enamel, silken veils embroidered with silver, magnificent
garments—were disposed of for a small fraction of their value.
During these transactions, and in settlement of their passage to
Africa, the Moriscoes succeeded in placing in circulation an immense
amount of counterfeit money which they had obtained in Catalonia,
thus literally paying the Spaniards in their own coin. The portable
wealth of which the kingdom was deprived by their banishment
cannot be estimated. It amounted, however, to many millions of
ducats. Some of the exiles were known to possess a hundred
thousand pieces of gold, an enormous fortune in those times. It was
ascertained after their departure that their lords, in defiance of law,
had purchased many of their estates, and had connived at the sale
or concealment of a great amount of their personal property. Those
who succeeded in reaching the cities were received with courteous
hospitality, but the desert tribes showed scant mercy to the
multitudes that fell into their hands.
Elsewhere in the kingdom the Moriscoes stubbornly resisted the
decree of expatriation. The Sierra de Bernia and the Vale of Alahuar
were the scene of the most serious disturbances, and at one time
twenty thousand insurgents were in the field. Armed for the most part
with clubs, their valor was ineffectual in the presence of veteran
troops. The women alone were spared; the men were butchered; the
brains of children were beaten out against the walls. The garrison of
the castle of Pop, which for a few weeks defied the Spanish army,
alone obtained advantageous terms. Of the one hundred and fifty
thousand Moors exiled from Valencia, at least two-thirds perished. A
large number had previously succumbed to persecution or had
escaped, and including these the total number of victims of the
inauguration of the insane policy of Philip III. was at least two
hundred thousand. The continuance of that policy until its aim had
been fully accomplished had already been determined on by the
councillors of the King. The secrecy which concealed their design did
not impose upon those who were the objects of it. They began by
tens of thousands to emigrate quietly to Africa. Then the decree,
which had been signed a month before, was published, with an
attempt to give the impression that it had been provoked by a
circumstance of which it was really the cause, namely, the agitation
of the Moriscoes. The latter were peremptorily commanded to leave
the kingdom within eight days. They were forbidden to take with
them money, gold, jewels, bills of exchange, or merchandise. They
were not permitted to dispose of their estates. In Catalonia their
property was confiscated, “in satisfaction of debts which they might
have owed to Christians,” and three days only were allowed them in
which to prepare for departure. Their little children were to be left
behind to the tender mercies of their oppressors, in order that their
salvation might be assured. Those of the northern provinces were
prohibited from moving southward; those of Andalusia were directed
to emigrate by sea. Within the allotted time all were in motion. The
embarkation of the exiles destined for Africa was effected without
difficulty. But their brethren of Castile and Aragon were refused
admission into France, by the direct order of Henry IV., to whose
agency was largely attributable their deplorable condition. His
opportune death somewhat relaxed official severity, and a great
number entered Provence. Although they were peaceable and
inoffensive, the French were anxious to be rid of their unwelcome
guests. Free transportation was furnished them by the city of
Marseilles, and they were distributed through Turkey, Italy, and
Africa. So many died during the passage by sea that their dead
bodies encumbered the beach, and the peasants refused for a long
time to eat fish, declaring that it had the taste of human flesh. The
progress of the unfortunates driven northward was marked by daily
scenes of persecution and agony. The commissioners appointed to
supervise the emigration connived at the evasion of the decree for
their own profit. They extorted enormous sums for protection, which
their duty required them to afford without compensation, and which,
even after these impositions, was insolently denied. Those things
which the ordinary dictates of humanity delight to bestow were sold
to the hapless wanderers at fabulous prices. For the shade of the
trees on the highway the grasping and unprincipled peasant exacted
a rental; and the water dipped from the streams in the trembling
hands of the sufferers commanded a higher price than that usually
paid for the wine of the country. The little which the commissioners
overlooked was seized by rapacious French officials, and the
condition of the Moriscoes was still further aggravated by the
absconding of those of their number to whom the common purse had
been intrusted.
In the merciless proscription thus imposed upon an entire people,
an insignificant number temporarily escaped. In the latter were
included young children torn from their parents to be educated by the
Church, and such persons “of good life and religion” as the clergy,
through interested or generous motives, chose to recommend to
royal indulgence. In 1611 the exemption enjoyed by these classes
was removed; searching inquiry was instituted throughout the
kingdom, and every individual of Moorish blood who could be
discovered was inexorably condemned to banishment or slavery. By
the persecution of the Moriscoes and the losses by war,
assassination, voluntary emigration, and enforced exile, Spain was
deprived of the services of more than a million of the most intelligent,
laborious, and skilful subjects in Christendom. Those who were
finally excluded were probably not more than half of the entire
Moorish population. No statistics are accessible in our day from
which an estimate can be formed of the vast number that perished
by famine, by torture, by massacre. Their trials were not at an end
even in Africa; they were pursued for sectarian differences, and
some who were sincere Christians returned to Spain, where they
were at once sentenced to the galleys. The skill and thrift of the
Moriscoes, qualities which should have made them desirable,
rendered them everywhere unpopular; they monopolized the trade of
the Barbary coast, even driving out the Jews; in Algiers the populace
rose against them, all were expelled, and large numbers were
remorselessly butchered. Hatred of their oppressors induced many
of hitherto peaceful occupations to embrace the trade of piracy, and
the southern coast of the Peninsula had reason to long remember
the exploits of the Morisco corsairs.
The ruthless barbarity, the blind and reckless folly of this measure,
was followed by an everlasting curse of barrenness, ignorance, and
penury. The sudden removal of enormous amounts of portable
wealth deranged every kind of trade. The circulation of counterfeit
money impaired public confidence. In Valencia four hundred and fifty
villages were abandoned. The absence of the most industrious and
prosperous class of its inhabitants was apparent in every community
of Castile. Catalonia lost three-quarters of its population. The
districts of Aragon rendered desolate by Moorish expulsion have
never been repeopled. Agricultural science and mechanical skill
disappeared. The hatred and disdain entertained by the Spaniards
for the conquered race had never permitted them to profit by the
experience and ingenuity of the latter. Intercourse with a Moor
brought moral and social contamination. Still less could the
admission of inferiority, which the adoption of his methods implied,
be tolerated by the haughty, the vainglorious, the impecunious
hidalgo.
The effects of the discouragement of all forms of art and industry
consequent upon war and persecution had been felt long previous to
the expulsion of the Moriscoes in every part of the Peninsula. For
many years after the capture of Cordova by Ferdinand III., it was
found necessary to bring provisions from the North, not only for the
support of the army, but to rescue from famine the sparse and
thriftless population of a province which under the Ommeyade khalifs
maintained with ease the great capital, as well as twelve thousand
villages and hamlets.
The decline in the number of inhabitants under Spanish rule
indicates the utter stagnation of trade and agriculture. In 1492 the
population of Castile was six and three-quarter million; in 1700 there
were in the entire kingdom of Spain but six million souls—such had
been the significant retrogression in two hundred years.
The combined revenues of the Spanish Crown at the close of the
fifteenth century amounted to a sum equal to three hundred
thousand dollars, about one-thousandth of the annual receipts of the
imperial treasury at the death of Abd-al-Rahman III., seven hundred
years before.
Fifty years after the banishment of the Moors, the combined
population of the cities of Cordova, Seville, Toledo, Granada, had
decreased by more than four-fifths; it is now about one-tenth of its
amount during the Moslem domination. In 1788 there were fifteen
hundred and eleven deserted towns in the Peninsula. Toledo,
celebrated for its silken fabrics, in the latter part of the fifteenth
century had sixty thousand looms; in 1651 it had five thousand; to-
day it has none. The same industry was pursued with great success
at Seville; in the seventeenth century the number of its looms had
decreased from sixteen thousand to sixteen. All other branches of
manufactures declined in the same proportion. Even a large part of
the kingdom of Valencia, the garden of Europe, was for years an
uninhabited wilderness. With the Moslem expulsion the knowledge of
many arts, once the source of great profit, was hopelessly lost.
To the pious Spaniard all these sacrifices were as nothing when
compared with the triumph of the Faith. The ports were unoccupied,
the quays grass-grown, but the armies of the Cross had conquered.
The manufactories had fallen into decay, the streets were silent, the
highways were deserted except by the timorous traveller and the
lurking robber, but not a Moslem or a Jewish heretic was to be
encountered in His Most Catholic Majesty’s dominions. At the close
of the seventeenth century, throughout the entire Peninsula, once
the centre of learning in Europe, the resort of scholars of every land,
the seat of the greatest educational institutions of the Middle Ages,
not a single academy existed where instruction could be obtained in
astronomy, natural philosophy, or any branch of mathematics. A
hundred years later no one could be found who understood even the
rudiments of chemistry. To-day, among the inhabitants of Spain,
according to the published tables of statistics, only one person in
every four can read. But what mattered the destruction of commerce,
the decay of production, the dearth of intelligence, if the land was
purged of false doctrines?, Was it not a source of national
congratulation that ecclesiastical authority was once more
paramount; that half of the able-bodied population, male and female,
were devoted to monastic life; that magnificent religious foundations,
such as the world had never before seen, arose on every side; that,
though the royal treasury was bankrupt, the annual revenues of the
Church amounted to nearly fifty-three million dollars? Surely these
manifold divine blessings were not to be weighed with the transitory
benefits derived from the labors of a mass of perverse and
unregenerate heretics!
The results, both immediate and remote, of this crime against
civilization thus proved fatal to Spain. Its principal sources of
subsistence removed, the kingdom was desolated by famine. It
became necessary to extend public aid to many noble families, once
affluent, but now impoverished by the suicidal course of the crown.
Popular sentiment, exasperated by distress, denounced in unsparing
terms the authors of the national calamity. The Archbishop of
Valencia, unable to endure the daily reproaches to which he was
subjected, and overcome by the sufferings for which he was
responsible, died of remorse. Silence and gloom occupied vast tracts
formerly covered by exuberant vegetation. In the place of the farmer
and the mechanic appeared the brigand and the outlaw. Deprived of
protection, the open country was abandoned; the peasantry sought
the security of fortified places, and all occupations whose pursuit
implied exposure to the danger of violence were necessarily
suspended. The conditions controlling every rank of society which
were established in the Peninsula by the blind and savage
prejudices of the seventeenth century are largely prevalent to-day. A
dreadful retribution has followed a tragedy whose example happily
no other nation has ventured to imitate; and which, from the hour of
its occurrence, has afflicted with every misfortune to the last
generation the people responsible for its hideous atrocities.
CHAPTER XXVII
GENERAL CONDITION OF EUROPE FROM THE VIII. TO THE XVI.
CENTURY
700–1500
Effects of Barbarian Supremacy on the Nations of Europe—Rise of

the Papal Power—Character of the Popes—Their Vices and
Crimes—The Interdict—Corrupt Practices of Prelates and
Degradation of the Papacy—Institution of the Monastic Orders—
Their Great Influence—Their Final Degeneracy—Wealth of the
Religious Houses—The Byzantine System—Its Characteristics—
Power of the Eunuchs—Splendor of Constantinople—Destruction
of Learning—Debased Condition of the Greeks—The People of
Western Europe—Tyranny of Caste and its Effects—Feudal
Oppression—Life of the Noble—His Amusements—The Serf and
his Degradation—His Hopeless Existence—Treatment of the
Jews—Prevalence of Epidemics—Religious Festivals—General
Ignorance—Scarcity and Value of Books—Persecution of
Learning—The Empire of the Church—Its Extraordinary Vitality.
In order that the reader may thoroughly understand and properly
appreciate the moral and intellectual supremacy of the Spanish
Arabs and their prodigious advance in the domain of science and the
arts, I have thought it advisable, by way of contrast, to present to him
a short and superficial sketch of the religious, political, and domestic
conditions which prevailed in the society of contemporaneous
Europe. The extent of this vast and comprehensive subject—one
which has exhausted the erudition of many great historians, whose
works of themselves would constitute a considerable library—must,
therefore, excuse the incomplete and cursory character of this
chapter; while its importance as a standard of comparison will
account for an apparent deviation from the general plan embraced
by these volumes.
The elegant luxury and refined civilization of the Romans had
disappeared amidst the universal anarchy which followed the
dissolution of their empire. The boundaries of great states and
kingdoms had been obliterated. Provinces once famed for their
fertility were now the haunts of prowling beasts and truculent
barbarians. The despotic but generally salutary government of the
Cæsars had everywhere, save in the immediate vicinity of
Byzantium, been replaced by the capricious and irregular jurisdiction
of petty chieftains, whose violent passions were restrained only by
their weakness, and of marauding princes, ambitious to destroy
every vestige of that architectural magnificence and mental culture
whose monuments they despised, and whose example they had
neither the desire nor the capacity to emulate. Instead of a smiling
landscape, everywhere exhibiting the traces of agricultural skill and
laborious and patient industry, a prospect of universal desolation met
the eye of the anxious and hurrying wayfarer. Moss-grown heaps of
rubbish alone marked the site of many a once flourishing and
opulent city. The towering aqueducts,—those engineering marvels of
the ancient world,—whose majestic ruins still excite the admiration of
all mankind, were broken and fallen into decay. The peerless
temples and altars of the gods had been desecrated by the hands of
sacrilegious Goth, Hun, and Lombard. Bands of brigands, insensible
to pity, swarmed upon the highways. In the cities the equitable
decisions of the prætor had been supplanted by the extortions of
ecclesiastical fraud and barbarian insolence. The vices prevalent
during the most abandoned period of Roman licentiousness had
survived, and had been aggravated by the unfeeling cruelty of the
conquerors. No scruples of humanity or delicacy suggested the
concealment of the most revolting orgies. The streets of the Eternal
City exhibited enormities whose very mention the rules of modern
propriety do not tolerate. Banquets where the brutal propensities of
the turbulent and uncouth guests were indulged to the utmost
constantly afforded provocation for bloodshed and murder.
Knowledge of letters, understanding and appreciation of the arts,
had already wholly vanished. The literary masterpieces of classic
genius remained unknown or forgotten in the insignificant collections
of scattered libraries, or had been buried under the smoking ruins of
those institutions of learning which once adorned the capitals and
the provincial cities of Greece and Italy.
By the accident of geographical position, by the adoption of
familiar political maxims, and by the incorporation into its ritual of
many ceremonies long endeared to the votaries of Paganism, the
Church of Rome had secured an influence over the minds of men
which under any other circumstances it could scarcely have
acquired. The revered name and dignity of Supreme Pontiff imparted
authority to its decrees and gave prestige to its decisions on
questions of doctrine. The five Christian emperors, from Constantine
to Gratian, adopted without alteration the attributes and wore the
insignia of the sacred office established by Numa and usurped by
Augustus. The assumption of imperial power is shown by the extent
of Papal jurisdiction long sharply defined by the ancient limits of the
empire. The adoption of the Latin idiom enabled the Church to
communicate secretly with its servants in the most distant countries;
while at the same time it invested the proceedings of its worship with
a mystery which awed the ignorant and fanatic believer. The
splendid ceremonial, the imposing temples, the elaborate vestments,
the costly furniture of the altar enriched with gold and jewels, the
incense, the solemn chants, the consecration of the Host,—all
powerfully impressed the superstitious children of the slaves of
ancient mythology, in whose minds still lingered traces of those
traditions which had been received by their fathers with the implicit
faith due to the oracles of the gods.
In the course of centuries, the primitive simplicity of the Gospel
and the purity of life which distinguished the first Christians had been
lost in the complex theology, in the unseemly contests for
precedence, in the crimes and the licentiousness which distracted
the society of the Eternal City. From a simple priest, whose tenure of
office was dependent on the pleasure of his associates, the Bishop
of Rome had been exalted into a mighty sovereign, responsible only
to the powers of Heaven. The palace of the Vatican exhibited all the
vices of the most corrupt of courts. The assumption of infallibility,—
an inevitable result of the preposterous claims of the Papacy,—
through the contradictory interpretations of different individuals
whose interests were conflicting led to the most opposite
conclusions, often to results fatal to the peace and honor of the
Church. The faith of the populace was weakened. Infidelity in the
priesthood became too common to excite remark. The universal
depravity was incredible and appalling. The general demoralization
resulting from the example of the clergy, whose atheism and
debauchery were proverbial, threatened the existence of society, a
catastrophe which the thorough organization of the hierarchy alone
prevented. Even in the fifteenth century Machiavelli wrote, “The
nearer a nation is to Rome the more impious are the people.” When
the German Schopp called the famous scholar, Casaubon, an
atheist, the latter retorted: “If I were an atheist I should now be at
Rome, where I have often been invited.” The effects of this superb
ecclesiastical organization were not long in manifesting themselves.
The legitimate resources of power were aided by every device of
fraud, of oppression, of imposture, of forgery. A succession of able
and unprincipled pontiffs fastened on Christendom a yoke which the
intelligence and the science of subsequent generations have not
even yet been able to entirely remove. The temporal supremacy of
the Cæsars was re-established over Europe; the dogmas of
Catholicism were preached in distant continents unknown to the
ancient world; and a tyranny far more terrible in its consequences
than that experienced under the cruel rule of Nero and Domitian was
imposed upon the intellectual aspirations of mankind.
No branch of history affords such a significant illustration of
human craft and human weakness as the story of the ambition, the
intrigues, and the vices of the Popes. In its consideration, the fact
must never be lost sight of that the Holy Father was, as a necessary
consequence of his creed, the earthly embodiment of spiritual
perfection,—the vicegerent of Almighty God. Either the admission of
a single error of judgment, or a controversy involving the most
insignificant tenet sustained by one pope and disputed by his
successor, was fatal to the claim of infallibility, which was the
foundation of the entire ecclesiastical system. The omniscience
conferred by the apostolic succession, which traced its origin to the
Saviour Himself, could never be mistaken. The example of the
Supreme Pontiff, the relations he sustained to the great officials of
his court, his occupations, his diversions, his tastes, his habits, his
conversation, were of far greater importance in the eyes of the
meanest peasant of some remote kingdom who acknowledged his
mission than were the most glorious achievements of any temporal
sovereign. The possibilities for the attainment to positions of such
authority and influence as were offered by the Roman Catholic
hierarchy had been unknown to Paganism. These opportunities
enabled men of base origin, but of extraordinary talents, to reach the
chair of St. Peter, men whose faults were overlooked or palliated by
the indulgent spirit of the age on account of the successful
prosecution of their schemes and the veneration which attached to
their calling.
Thus, among the powers of the earth, highest in rank, greatest in
renown, supreme in influence, pre-eminent in infamy, was the
Papacy of Rome. The maintenance of an uniform standard of
orthodoxy was little considered by the spiritual potentate whose will
was the law of Christendom. It is well known to every student of
Church history that Jewish doctrines predominated in the early days
of Christianity and controlled the policy of its priesthood. The Pagan
ideas and ceremonies inherited from the Roman pontiffs it never laid
aside. Every form of heterodox belief was entertained at different
periods by the incumbents of the Holy See. St. Clement was an
Arian; Anastasius a Nestorian; Honorius a Monothelite; John XXII.
an unconcealed atheist. The contradictory dogmas, the acrimonious
disputes, the frightful anathemas, that resulted from the adoption of
these heretical principles of doctrine were the public reproach of the
Christian world. As the power of the Papacy increased, its
possession became more and more an object to ambitious and
unscrupulous adventurers. It was sought and obtained by arts
countenanced only by the vilest of demagogues. It was sold by one
Pope to another; and, like the imperial laurel appropriated by the
Pretorian Guards, it was put up at auction by cardinals and became
the property of the most wealthy purchaser. Some of the Holy
Fathers had not taken orders; others had not even received the
sacraments of baptism and communion before being invested with
the pontifical dignity. In some instances the tiara and the mitre were
placed upon the brows of children. Neither John XII. nor Benedict IX.
had attained the age of thirteen years when intrusted with the
direction of the spiritual affairs of Christendom. An infant of five years
was consecrated Archbishop of Rheims. Another who was only ten
was placed upon the episcopal throne of Narbonne. Alonso of
Aragon, the natural son of Ferdinand the Catholic, was made
Archbishop of Saragossa at the age of six. The origin of the vicars of
Christ was sometimes of the most obscure and often of the most
disgraceful character. Stephen VII., John X., John XI., John XII.,
Boniface VII., Gregory VII., were the sons of courtesans. In some
instances the infamy was further increased by the additional stigma
attaching to the crime of incest. The famous courtesan Marozia, who
for the greater part of her life disposed of the Papacy at her will, is
credited with the installation of eight Popes, all her lovers or her
children, one of whom was at once her son and grandson. The
empire she acquired by her talents and her beauty lasted almost a
quarter of a century. To that epoch is ascribed an occurrence that
many writers have designated as fabulous, but which is established
by evidence far more convincing than many events that have
successfully withstood the most formidable assaults of hostile
criticism. It was long asserted by chroniclers of the orthodox faith,
and universally credited, that in the capital of Christianity, hallowed
by the glorious deaths of countless martyrs, linked with the proud
associations of the rise and progress of the spiritual power of the
Papacy, and ennobled by the most signal victories of the Church, a
monstrous prodigy had occurred. It was said that Pope John VIII.,
whose sex had hitherto been unsuspected save by those favored
with her intimacy, while returning from the celebration of a solemn
festival, at the head of a procession of cardinals and bishops and
surrounded with the glittering emblems of pontifical power and
majesty, had been seized with the throes of parturition in one of the
most public thoroughfares of Rome.
The original acceptance of and belief in this portentous
catastrophe, and its subsequent denial, form one of the most curious
episodes in the annals of the Church. For five centuries it was
implicitly received as historic truth. The life of Pope Joan long
occupied a prominent place in the biographies of the successors of
St. Peter, dedicated to eminent prelates, often to the Pontiffs
themselves. The occurrence—whose locality was marked by the
statue of a woman wearing the Papal insignia and holding a child in
her arms—was minutely described in the works of learned and
respectable historians. This memorial was thrown into the Tiber by
the order of Sixtus V. Her bust, destroyed by Charles VIII. during the
French invasion of Italy, was long an ornament of one of the
churches of Sienna. Until the time of Leo X. certain ceremonies,
which cannot be described, were publicly instituted at the election of
every Pope to determine his sex. To these even the licentious Borgia
was forced to conform. John Huss, when arraigned before the
Council of Constance, amidst an unbroken silence, reproached the
ecclesiastical dignitaries assembled to condemn him, and whom the
slightest heretical assertion roused to tumultuous fury, with the
imposture which had so signally demonstrated the weakness of the
vaunted inspiration of the Papacy. More than five hundred writers,
whose interests were identical with those of the Vatican—among
them chroniclers, polemic divines, authorities on the history of the
Church and its discipline, all enthusiastic members of the Roman
Catholic communion—have confirmed the existence of a female
Pope.
But, whether true or false, the disgrace consequent upon this
gigantic scandal was insignificant when compared with the moral
effect of the long series of crimes which disfigure the annals of Papal
Rome. The shameless venality of the Princes of the Church had
from the most remote times disgraced the proceedings by which was
elevated to the throne of the apostles the immaculate Vicar of God.
So corrupt was the ecclesiastical society of the capital that no Pontiff
who endeavored to live a moral life was secure for a single hour.
Celestine was poisoned at the instance of the cardinals eighteen
days after receiving the tiara. Adrian V. was poisoned in the conclave
itself before his election. The partisans of antagonistic claimants of
the Papacy pursued each other with a vindictiveness scarcely
equalled by the most intense bitterness of political faction. Each
aspirant to the pontifical dignity denounced his opponent as an anti-
pope, and exhausted the rich vocabulary of clerical invective in
consigning him to the vengeance of Heaven. The defeated candidate
was subjected to every variety of torture; to the deprivation of his
nose, his eyes, his tongue; to the suffering of confinement in
noisome dungeons; to the pangs of prolonged starvation. The
temporal enemies of the Holy Father fared even worse than his rivals

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Experimental Models of Parkinson s

Disease Methods in Molecular Biology

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R Loops Methods and Protocols Methods in Molecular

Cancer Cell Biology Methods and Protocols Methods in

Rhodopsin Methods and Protocols Methods in Molecular

DNAzymes Methods and Protocols Methods in Molecular

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ISSN 1064-3745 ISSN 1940-6029 (electronic)

Tokyo, Japan Yuzuru Imai

PART I BIOCHEMICAL EXPERIMENTS AND CELLULAR MODELS

1 α-Synuclein Seeding Assay Using RT-QuIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

PART II MAMMALIAN MODELS OF PARKINSON’S DISEASE

10 Generation of Mitochondrial Toxin Rodent Models of Parkinson’s Disease

12 α-Synuclein Propagation Mouse Models of Parkinson’s Disease . . . . . . . . . . . . . . 119

PART III INVERTEBRATE MODELS OF PARKINSON’S DISEASE

16 Assessment of Cytotoxicity of α-Synuclein in Budding Yeast

HITOSHI NIWA • Department of Dental Anesthesia, Osaka University Graduate School of

AIRI TARUTANI • Department of Brain and Neurosciences, Tokyo Metropolitan Institute of

Biochemical Experiments and Cellular Models of Parkinson’s

α-Synuclein Seeding Assay Using RT-QuIC

α-Synucleinopathies, such as Parkinson’s disease (PD), dementia

accumulation of misfolded αSyn aggregates. Toward the develop-

2 Materials (See Note 1)

1. αSyn expression vectors: Wild-type full-length human αSyn

21. 96-Well optical black-bottom plate (Nunc Item#: 265301).

9. Collect the cultured medium in 500-mL high-g-force-resistant

3.3 Preparation 1. Thaw αSyn substrate on ice.

8. Place the prepared plate in the fluorescence plate reader and

3.5 RT-QuIC Data 1. Analyze the relative fluorescent intensity measured by

Fig. 2 Amyloid formation is accelerated in a seed dose-dependent manner

2. Each sample is typically set in duplicate. If only one of two

1. Buffers and reagents are filtered through a pre-rinsed 0.22-μm

5. Do not use the outermost wells of a 96-well plate due to the

Comparison of different αSyn RT-QuIC protocols

in the absence of preformed seeds. Thus, we usually distinguish

We thank Edanz (https://en-author-services.edanzgroup.com/ac)

Zerr I (2020) Effect of the micro-environment Neuroimmune Pharmacol 14:423–435.

Cysteine misincorporation in bacterially (1):49–62. https://doi.org/10.1007/

Electron Microscopic Analysis of α-Synuclein Fibrils

Accumulation of filamentous α-synuclein (α-syn) in neurons

relatively high concentration [8]. In patients, α-syn in the brain

2.1 Negative 1. Carbon-coated 300-mesh copper grid (see Note 1).

6. Blot the blocking solution remaining on the grid surface with

1. Hydrophilization treatment of the grid is effective for disper-

Fig. 3 Immunolabeled synthetic α-syn fibrils and patient-derived α-syn fibrils.

6. Antibodies directed against α-syn phosphorylated at serine

EM of synthetic α-syn fibrils. The C-terminal of α-syn is located

inhibit proteasome. Elife 9:e56825. https:// (2020) Discriminating alpha-synuclein strains

α-Synuclein Seeding Assay Using Cultured Cells

Several neurodegenerative diseases, including Parkinson’s disease,

Jun Ogata and Daisaku Takemoto contributed equally to this work.

2.1 α-Synuclein 1. BL21 (DE3) competent cells: 20 μl aliquot with competency

12. High powered sonicator (Taitec, VP-050 N, see Note 2).

2.2 α-Synuclein 1. Purified recombinant α-synuclein in dialysate from Subheading

2.3 Cell Culture 1. Cultured human cells (see Note 5).

5. pcDNA3-human α-synuclein: 1 mg/ml in Tris–EDTA buffer,

2.4 Biochemical 1. PBS.

2.5 Western Blot 1. SDS-PAGE system: A 12.5% gel is appropriate to separate