Neuronal Cell Death: Methods and Protocols

395 Pages·2015·18.506 MB·English

by  Laura Lossi, Adalberto Merighi (eds.)

Methods in
Molecular Biology 1254
Laura Lossi
Adalberto Merighi Editors
Neuronal
Cell Death
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire

Hat fi eld, Hertfordshire, AL10 9AB, UK
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Neuronal Cell Death
Methods and Protocols
Edited by
Laura L ossi and Adalberto Merighi
Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Italy
Editors
Laura Lossi Adalberto M erighi
Department of Veterinary Sciences Department of Veterinary Sciences
University of Torino University of Torino
Grugliasco, Torino, Italy Grugliasco, Torino, Italy
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-2151-5 ISBN 978-1-4939-2152-2 (eBook)
DOI 10.1007/978-1-4939-2152-2
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Prefa ce
The ongoning research on neuronal cell death is rapidly expanding after the recognition
that not only do neurons die as a consequence of external insults, but also following activa-
tion of stereotyped genetic programs/intracellular pathways leading to death. However, a
long time passed before the term “cell death” took over in the scientifi c literature: in the
nineteenth century, when Rudolf Virchow published his famous book entitled Lectures on
Cellular Pathology in its Grounds on Physiological and Pathological Histology fi rst reporting
on the occurrence of cell death in damaged tissues, the dominating idea was that only
injured tissues and cells underwent some sort of degeneration when damaged. Thus, the
concept that cell death took place also during the course of normal development or during
growth and aging was still very far from general acceptance, and required a considerable
amount of experimental work to convince the academic community. A signifi cant step for-
ward along this direction was done in the 1960s of the last century, when electron micros-
copists started describing the different modes of cell death. Since then the fi eld has
dramatically widened, particularly with the recognition of several forms of neuronal degen-
eration, such as necrosis, apoptosis, autophagy, pyroptosis, oncosis, etc. and of the close
relationship of many of these with cell proliferation and aging. Today, the ultimate frontiers
in neuronal cell death research lie in the development of novel approaches to monitor the
phenomenon by the use of in vivo and/or ex vivo preparations, such as organotypic cul-
tures, that are more closely related to the intact mammalian brain than primary cultures,
and to better exploit the use of non-mammalian model organisms. In parallel, there is a
need for understanding the type and role of cell death in neurodegenerative diseases, to
develop pharmacologically active compounds that are capable to exert their biological
role(s) in vivo, and to construct genetic vectors to be employed in gene therapy.
With such a wide array of exciting and rapidly expanding fi elds of research, this book,
from its initial conception, had obviously to be limited in the choice of subjects, but we
believe it represents a valuable and readily reproducible collection of established and emerg-
ing techniques for neuronal cell death research. Such a collection is preceded by a general
introductory chapter (Chapter 1 ) that recalls the history of cell death and, to put things
into perspective, discusses the main morphological features of the most diffuse types of cell
death in neurons, in parallel with relevant cellular pathways and current assays for a proper
recognition. The methods presented include immunocytochemical localization at light and
electronic levels, biochemical characterization, and functional analysis in vivo or ex vivo by
novel types of microscopy, as well as protocols for development and production of genetic
probes. Although this book is primarily devoted to approaches for analysis of the mamma-
lian brain, a few non-mammalian species are also taken into consideration to demonstrate
specifi c methodologies that are of great value to boost cell death research by taking advan-
tage from the use of less complex models.
As a general indication to the readers, the book is divided into four parts.
Part I (Chapters 2 – 12 ) is focused on a series of techniques for the molecular, structural,
functional, and genomic characterization of dying neurons. They cover a broad range of
protocols, such as epifl uorescence and digital holography to monitor the cell volume
v
vi Preface
(Chapter 2 ) ; a series of techniques to study DNA synthesis/alterations and the morpho-
logical signs of nuclear sufferance (Chapters 3 and 4 ); a number of approaches to monitor
parameters of primary importance in cell viability (Chapters 5 – 8 ), such as oxygen and cal-
cium concentration, mitochondrial function, and activation of caspase-3 in single alive cells;
and a series of molecular approaches for RNA silencing, genomic analysis, and high-
throughput cell death assays (Chapters 9 – 12 ) .
Part II (Chapters 1 3 – 18 ) groups together a number of protocols that are of primary
interest in neuropathology (Chapters 13 and 1 4 ) and in experimental neuropathology
(Chapters 15 – 18 ) by describing the current ameliorations to well-established diagnostic
techniques such as the Golgi method for study of neuronal and glial death in autopsy material
(Chapter 1 3 ) , the use of optimized protocols and image analysis algorithms for reliable
analysis of cell death in human and animal samples (Chapter 14 ) , some specifi c experimen-
tal approaches such as oxygen-glucose deprivation (Chapter 15 ) , single axon lesioning by
laser microbeam targeting (Chapter 16 ) , in vivo imaging of retinal apoptosis (Chapter 1 7 ) ,
and use of neurotoxins to model neuronal death in Parkinson’s disease (Chapter 1 8 ).
Part III (Chapters 1 9 – 22 ) is devoted to a series of gene engineering techniques to
obtain and manipulate neuronal stem cells and progenitors (Chapter 1 9 ) , to prepare HSV-1
vectors for the gene therapy (Chapters 20 and 21 ) , and to CNS transplantation of bone
marrow stem cells (Chapter 2 2 ) .
Part IV (Chapters 2 3 – 26 ) describes some very interesting protocols for study of cell
death in non-mammalian models, such as the analysis of caspase-3 activation in lamprey
(Chapter 23 ), the generation of zebrafi sh models by genome editing (Chapter 2 4 ), and the
assessment of cell death (Chapter 2 5 ) and phagocytosis (Chapter 2 6 ) in Drosophila .
All scientists who have excellently contributed to this book have a direct experience in
one or more fi elds of neuronal cell death research. We are very much indebted to all of
them for their time, the high standards of their contributions, and for successful effort in
emphasizing the description of the more common pitfalls in the techniques that they have
described, and of the hints to reduce the possibility of failure for beginners.
The collection of protocols that forms this book is surely not exhaustive of the wide
range of approaches that today can be employed in top-level cell death research. Yet it is
intended for a large audience of scientists, including histologists, biochemists, cellular and
molecular biologists, and electrophysiologists that are currently active in the fi eld or are
willing to enter such an exciting and still expanding area of neurobiology.
As the two of us have been the fi rst to benefi t from such an excellent assemblage of
information, we are confi dent that readers too will fi nd this book very useful for their
future work.
Grugliasco, Torino, Italy L aura L ossi, DVM, PhD
A dalberto Merighi, DVM, PhD
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
1 Neuronal Cell Death: An Overview of Its Different Forms
in Central and Peripheral Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Laura L ossi , C laudia C astagna , and Adalberto Merighi
PART I MOLECULAR, STRUCTURAL, FUNCTIONAL,
AND GENOMIC CHANGES IN DYING NEURONS
2 Cell Volume Regulation Monitored with Combined Epifluorescence
and Digital Holographic Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Nicolas Pavillon and P ierre M arquet
3 Flow Cytometric Analysis of DNA Synthesis and Apoptosis
in Central Nervous System Using Fresh Cell Nuclei . . . . . . . . . . . . . . . . . . . . 33
Noelia López-Sánchez and J osé M . Frade
4 N uclear Signs of Pre-neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3
Fernando C . B altanás , Jorge Valero , Jose Ramón A lonso ,
Maria T eresa B erciano , and Miguel L afarga
5 M ulti-parametric O Imaging in Three-Dimensional Neural Cell
2
Models with the Phosphorescent Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Ruslan I . D mitriev and Dmitri B . P apkovsky
6 C alcium Imaging in Neuron Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3
María Calvo , Carlos V illalobos , and Lucía N úñez
7 M onitoring Mitochondrial Membranes Permeability in Live Neurons
and Mitochondrial Swelling Through Electron Microscopy Analysis . . . . . . . . 87
Macarena S . A rrázola and Nibaldo C . I nestrosa
8 R eal-Time Visualization of Caspase-3 Activation by Fluorescence
Resonance Energy Transfer (FRET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 9
Silvia Alasia , Carolina C ocito , Adalberto M erighi , and Laura Lossi
9 D esign and Cloning of Short Hairpin RNAs (shRNAs)
into a Lentiviral Silencing Vector to Study the Function
of Selected Proteins in Neuronal Apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Nadia Canu
10 G enomic Analysis Using Affymetrix Standard Microarray
GeneChips (169 Format) in Degenerate Murine Retina . . . . . . . . . . . . . . . . . 1 29
S ook H yun Chung , Weiyong S hen , and Mark Gillies
11 Genomic Analysis of Transcriptional Changes Underlying
Neuronal Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 41
Sebastiano Cavallaro
vii
viii Contents
12 High-Throughput Cell Death Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Matthew E. Pamenter and G abriel G . H addad
PART II C ELL DEATH IN NEUROPATHOLOGY AND EXPERIMENTAL
NEUROPATHOLOGY
13 Staining of Dead Neurons by the Golgi Method in Autopsy Material. . . . . . . . 1 67
Stavros J. B aloyannis
14 Image Analysis Algorithms for Immunohistochemical Assessment
of Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 81
Stan K rajewski , Jeffrey Wang , Tashmia Khan , J onathan Liu ,
Chia-Hung S ze , and Maryla Krajewska
15 I n Vitro Oxygen-Glucose Deprivation to Study Ischemic Cell Death. . . . . . . . 1 97
Carla I . T asca , T harine D al-Cim , and Helena C imarosti
16 L aser Microbeam Targeting of Single Nerve Axons in Cell Culture . . . . . . . . . 2 11
Nicholas H yun , Linda Z . S hi , and Michael W . Berns
17 R eal-Time Imaging of Retinal Cell Apoptosis by Confocal Scanning
Laser Ophthalmoscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 27
Eduardo M. N ormando , M ohammad H . Dehabadi , L i Guo ,
Lisa A . T urner , G aia P ollorsi , and M. Francesca Cordeiro
18 Targeted Toxicants to Dopaminergic Neuronal Cell Death . . . . . . . . . . . . . . . 2 39
Huajun Jin , A rthi K anthasamy , Dilshan S . Harischandra ,
Vellareddy Anantharam , Ajay R ana , and Anumantha Kanthasamy
PART III NEURAL STEM CELLS, PROGENITORS,
AND GENE THERAPY STRATEGIES
19 Stem Cells, Neural Progenitors, and Engineered Stem Cells . . . . . . . . . . . . . . 2 55
Raj R . Rao and S hilpa I yer
20 H erpes Simplex Virus Type 1 (HSV-1)-Derived Recombinant Vectors
for Gene Transfer and Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Peggy M arconi , Cornel F raefel , and Alberto L. Epstein
21 H erpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon Vectors
for Gene Transfer and Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Cornel F raefel , Peggy Marconi , and Alberto L. Epstein
22 B one Marrow Transplantation for Research and Regenerative Therapies
in the Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 17
David D íaz , J osé R amón A lonso , and Eduardo Weruaga
PART IV NEURONAL DEATH IN NONMAMMALIAN MODELS
23 Detection of Activated Caspase-8 in Injured Spinal Axons
by Using Fluorochrome-Labeled Inhibitors of Caspases (FLICA) . . . . . . . . . . 3 29
Antón B arreiro-Iglesias and Michael I. S hifman
Contents ix
24 Generation of Zebrafish Models by CRISPR/Cas9 Genome Editing. . . . . . . . 3 41
Alexander Hruscha and B ettina S chmid
25 I n Vivo Assessment of Neuronal Cell Death in Drosophila. . . . . . . . . . . . . . . . 351
Pierre Dourlen
26 D rosophila Model for Studying Phagocytosis Following
Neuronal Cell Death. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Boris Shklyar , Flonia L evy-Adam , and Estee K urant
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 69