Table Of ContentNEUROMETHODS q 29
Apoptosis Techniques and Protocols
NEUROMETHODS
Program Editors: Alan A. Boulton and Glen B. Baker
30 Regulatory Protein Modification: Tech- 15 Neurophysiological Techniques Appka-
moues and Protocols, edlted by Hugh tlons to Neural Systems, edited by Alan
C’Hemmmgs, Jr., 1997 - - A. Boulton, Glen B Baker, and Case
29 Apoptosis Technisues and Protocols, H. Vanderwolf, 1990
e&d by Judes Pkrier, 1997 14 Neurophysiological Techniques. 5a.w
28 Transgenic Xenopus: MIcroinJection Methods and Concepts, edited by Alan
Methods and Developmental Neuro- A. Boulton, Glen B. Baker, and Case
biology, by Shlomo Seidman and H. Vanderwolf, 1990
Hermona Soreq, 1997 13 Psychopharmacology, edlted by Alan
27 Voltammetric Methods in Brain Sys- A. Boulton, Glen B Baker, and Andrew
tems, edlted by Alan A Boulton, Glen J Greenshaw, 1989
B. Baker, and Ralph N. Adams, 12 Drugs as Tools in Neurotransmitter
1995 Research, edited by Alan A Boufton,
26 Patch-Clamn Aooiications and Proto- Glen B Baker, and August0 V Juorto,
cols, edlted by Klan A. Boulton, Glen 1989
B. Baker, and Wolfgang Walz, 1995 11 Carbohvdrates and Enerav Metabo-
25 Neurotrophic Factors, edlted by Alan lism, e&ted by Alan A B&ton, Glen
A. Boulton, Glen B. Baker, and Franz B. Baker, and Roger F. Butterworth,
Hefts, 1993 1989
24 Animal Models of Drug Addiction, 10 Analysis of Psychiatric Drugs, edited
edlted by Alan A Boulton, Glen B by Alan A Boulton, Glen B Baker, and
Baker, and Peter Wu, 1992 Ronald T Coutts, 1988
23 Practical Cell Culture Techniques, 9 The Neuronal Microenvironment, edited
edlted by Alan A. Boulton, Glen B by Alan A. Boulton, Glen B Baker,
Baker, and Wolfgang Walz, 1992 and Wolfaana Walz. 1988
22 Animal Models of Neurological Disease, 8 Imagini”andu Corrklative Physico-
II* Metabok EnceDhaloDathles and the chemical Techniaues. edlted bv Alan
Eptlepsles, edited ‘by Aian A. Boulton, A Boulton, Glen 6 Ba’ker, and bonald
Glen B. Baker, and RogerF. Buttenvotih, P. Boisvert, 1988
1992 7 Lipids and Related Compounds, edited
21 Animal Models of Neurological Dis- by Alan A. Boulton, Glen B Baker,
ease. I: Neurodeaeneratwe Disease, and Lloud A. Horrocks. 1988
edlteb by Alan d Boulton, Glen B. 6 Peptide;, edited by Alan A Boulton,
Baker, and Roger F. Butterworth, 1992 Glen B. Baker, and Quentm httman.
20 lntrac~llular fiessengers, edited by 1987
Alan A. Boulton, Glen B. Baker, and Neurotransmitter Enzymes, edlted by
Calm W. Taulor. 1992 Alan A Boulton. Glen B. Baker. and
19 Animal Modills in Psychiatry, II, edrted Peter H. Yu, 1986
by Alan A. Boulton. Glen B. Baker, Receptor Binding Techniques, edited
and Mathew T. Martin-Iverson, 199 1 by Alan A. Boulton, Glen B Baker,
18 Animal Models in Psychiatry, I, edlted and Pave1 D. Hrdina, 1986
by Alan A. Boulton, Glen B. Baker, Amino Acids, edlted by Alan A Boulton,
and Mathew T Martm-lverson, 1991 Glen B. Baker, and James D. Wood,
17 Neuropsychology, edlted by Alan A. 1985
Boulton, Glen B. Baker, and Merrill Amines and Their Metabolites, edited
Hlscock, 1990 by Alan A. Boulton, Glen B Baker, and
16 Molecular Neurobiological Techniques, Jbdlth M Baker, 1985
edlted by Alan A. Boulton, Glen B. General Neurochemical Techniques,
Baker, and Anthony T. Campagnom, edited by Alan A. Boulton and Glen B.
1990 Baker, 1985
NEUROMETHODS 0 29
Apoptosis Techniques
and Protocols
Edited by
Judes Pokier
Douglas Hospital Research Centre, Verdun, Canada
Humana Press Totowa, New Jersey
0 1997Humana Press Inc
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Cover illustration Figure 2 m “Detection of Apoptotic or Necrotic Death m Neuronal Cells
by Morphological, Biochemical, and Molecular Analysis,” by Martin Gschwmd and Gerda
Huber
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Preface
After many years of neglect, the study of normal cell death
and the so-called programmed cell death has finally gained some
needed momentum. Evidence obtained over the last five years
indicates that programmed cell death (also referred to as
apop tosis) occurs normally in most animal tissues at some well-
defined stages of their development and/or maturation. The
molecular mechanisms by which it is executed remains un-
known. However, key regulatory and metabolic steps have been
identified in the apoptotic cascade and a central concept has
progressively emerged: Apoptosis appears to occur by default
in many different cell types unless suppressed by signals origi-
nating from neighboring cells. As Martin Raff noted a few years
ago, this molecular cascade probably imposes a form of social
control on cell survival and cell death.
Since our understanding of the apoptosis phenomena has
grown significantly over the last few years, we have been em-
powered by recent technological advances to biochemically dis-
sect the key metabolic components of apoptotic and necrotic
cascades in normal and injured brains. Moreover, the develop-
ment of improved histochemical tools has allowed us to better
define the fundamental criteria of cell death, and transgenic tech-
nology has allowed scientists to test working hypotheses per-
taining to the mechanism of action of the so-called “cell death
gene.” Sophisticated molecular biological tools have also en-
abled us to determine the key elements of normal and abnor-
mal cell cycles, indicating novel functions for the so-called
cell-cycle regulated markers that appear to do much more than
just regulate cell division
The present volume of Apoptosis Techniques and Protocols
now offers neuroscientists at all levels of experience a com-
bination of theoretical and practical information for under-
standing and studying apoptosis. Topics include the practical
V
vi Preface
analysis of the concepts of necrosis and apoptosis in the CNS
as well as histological, biological, and molecular criteria for
investigating apoptosis and programmed cell death. Topics
range from cellular and invertebrate to animal and human
models of apoptosis in various pathological situations in the
brain, including Alzheimer’s disease, AIDS, and stroke.
Numerous techniques are described for examining the criti-
cal steps involved in the apoptotic process. These methods
range from PCR analysis of cell-cycle regulated proteins, his-
tochemical analysis of DNA degradation, and DNA laddering
analysis, to cytochemical alterations of living cells.
It is our hope that Apaptosis Technzques and Protocols will
prove useful not only as a technical reference, but also as an
introduction to the key notions associated with the phenom-
ena of apoptosis and necrosis.
Judes Pokier
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,
Contributors . . . . . *.. .*.. . . . . *. .* . . . .*1x
Gene Tools to Study Neuronal Apoptosis
lsabelle Martznou, Harald Frankowskz, Marc Mzssotten,
Remy Sadoul, and Jean-Claude Martinou . . . .., . .1
Detection of Apoptotic or Necrotic Death in Neuronal Cells
by Morphological, Biochemical, and Molecular Analysis
Martm Gschwlnd and Gerda Huber . . 13
Detection of Cell Death in Neural Tissue and Cell Culture
Thomas Mahalik, Malcolm Wood, and Gregory Owens 33
Cultured Cerebellar Granule Neurons as a Model
of Neuronal Apoptosis
Guang-Mez Yan and Steven M Paul 47
Optrmrzatron and Vahdatlon of RT-PCR as a Tool
to Analyze Gene Expression Durmg Apoptosls
Steven Estus . . . *. 67
p53 Induction Is a Marker of Neuronal Apoptosis
in the Central Nervous System
Shahm Sakht and Steven S Schrerber 85
Apoptosls and Ischemla m the Central Nervous System
Matthew D Ltnnzk 99
Analyses of Apoptosis-Associated DNA Fragmentation
In VIVO Durmg Neurodegeneration of the Perrpheral
Olfactory System m Adult Mammals
Emmanuel Moyse and Dents Mrchel . . . . . . . . . ,. . . . 133
Both Irreversible Neuronal Death and Reversible Neuronal Stress
Are Associated with Increased Levels of Statm, a Marker
of Cell Cycle Arrest
Patricia Boksa, Uwe Btffert, Dons Dea, Richard Alonso,
Dayan O’Donnell, and Judes Pomer . . .. . . .. . I 161
B-Amyloid-Induced Neuronal Cell Death
in Transgemc Mice and Alzhelmer’s Disease
Frank M LaFerla and Gdbert Jay . . . .* * 283
uii
Contents
Vlll1 ..
Use of TUNEL to Measure Retinal Ganghon Cell Apoptosls
LzsaA yn Kerr&an and Donald J Zack 197
Methods for Detection of Apoptosls in the CNS
Kathrln D Geiger, Floyd E Bloom, and Nora E Sarvetnlck 217
Techniques for Distinguishing Apoptosis from Necrosis
in Cerebrocortical and Cerebellar Neurons
Emanuela BoMfoco, Marta Ankarcrona, Dzmttrz Kraznc,
Plerluqi Ntcotera, and Stuart A Lzpton 237
The Nematode Caenorhabdztzs elegans
as a Model System to Study Neuronal Cell Death
Paul Deslardins and Stkphane Ledoux 255
Index . . . . . . ,... . . . . . . . . . . , ,.,, ,.., , . . . . . 278
Contributors
RICHARD ALONSO Douglas Hospital Reselzrch Centve, Centre
l
for Studies on Aging, Departments of Psychiatry and
Medicine, McGill University, Montreal, Canada
MARIA ANKARCRONA Department of Environmental Medicine,
l
Karolinska Institute, Stockholm, Sweden
UWE BEFFERT Douglas Hospital Research Centre, Centrefor
l
Studies on Aging, Departments @Psychiatry and
Medzcine, McGill University, Montreal, Canada
FLOYD E. BLOOM Department ofNeuropharmacology,
l
The Scripps Research Institute, La Jolla, CA
EMANUELA BONFOCO Department of Environmental Medicine,
l
Karolinska Institute, Stockholm, Sweden
PATRICIA BOSKA Douglas Hospztal Research Centre, Centre for
l
Studies on Agzng, Departments ofPsychiatry and
Medzcine, McGill University, Montreal, Canada
DORIS DEA Douglas Hospital Research Centre, Centre for
l
Studies on Aging, Departments ofPsychiatry and
Medicine, McGill University, Montreal, Canada
PAUL DESJARDINS Neuroscience Research Unit, HBpital
l
St.-Luc, Montreal, Canada
STEVEN ESTUS Department ofPhysiology, University of
l
Kentucky, Lexington, KY
HARALD FRANKOWSKI Glaxo Institute for Molecular Biology,
l
Geneva, Switzerland
KATHRIN D. GEIGER Edinger-lnstitut, Universitiit Frankfurt,
l
Germany
MARTIN GSCHWIND Pharma Division, Preclinical CNS
l
Research, F. H$j?nann La-Roche Ltd., Basel, Switzerland
GERDA HUBER Preclinical CNS Research, Pharma Division,
l
F Hoffmann La-Roche Ltd., Baser, Switzerland
GILBERT JAY Department of Virology, Jerome H. Holland
l
Laboratory, Rockville, MD
ix
