M E T H O D S I N M O L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Neuronal Cell Death

Methods and Protocols

Edited by

Laura Lossi and Adalberto MerighiDepartment of Veterinary Sciences, University of Torino, 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 Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014955570

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Editors Laura Lossi Department of Veterinary Sciences University of Torino Grugliasco , Torino , Italy

Adalberto Merighi Department of Veterinary Sciences University of Torino Grugliasco , Torino , Italy

v

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

Pref ace

vi

(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 13 – 18 ) groups together a number of protocols that are of primary interest in neuropathology (Chapters 13 and 14 ) 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 13 ), 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 17 ), and use of neurotoxins to model neuronal death in Parkinson’s disease (Chapter 18 ).

Part III (Chapters 19 – 22 ) is devoted to a series of gene engineering techniques to obtain and manipulate neuronal stem cells and progenitors (Chapter 19 ), to prepare HSV-1 vectors for the gene therapy (Chapters 20 and 21 ), and to CNS transplantation of bone marrow stem cells (Chapter 22 ).

Part IV (Chapters 23 – 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 24 ), and the assessment of cell death (Chapter 25 ) and phagocytosis (Chapter 26 ) 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 Laura Lossi, DVM, PhD Adalberto Merighi, DVM, PhD

Preface

vii

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Neuronal Cell Death: An Overview of Its Different Forms in Central and Peripheral Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Laura Lossi , Claudia Castagna , 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 Pierre Marquet

3 Flow Cytometric Analysis of DNA Synthesis and Apoptosis in Central Nervous System Using Fresh Cell Nuclei . . . . . . . . . . . . . . . . . . . . 33 Noelia López-Sánchez and José M. Frade

4 Nuclear Signs of Pre-neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Fernando C. Baltanás , Jorge Valero , Jose Ramón Alonso , Maria Teresa Berciano , and Miguel Lafarga

5 Multi-parametric O2 Imaging in Three-Dimensional Neural Cell Models with the Phosphorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Ruslan I. Dmitriev and Dmitri B. Papkovsky

6 Calcium Imaging in Neuron Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 María Calvo , Carlos Villalobos , and Lucía Núñez

7 Monitoring Mitochondrial Membranes Permeability in Live Neurons and Mitochondrial Swelling Through Electron Microscopy Analysis . . . . . . . . 87 Macarena S. Arrázola and Nibaldo C. Inestrosa

8 Real-Time Visualization of Caspase-3 Activation by Fluorescence Resonance Energy Transfer (FRET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Silvia Alasia , Carolina Cocito , Adalberto Merighi , and Laura Lossi

9 Design 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 Genomic Analysis Using Affymetrix Standard Microarray GeneChips (169 Format) in Degenerate Murine Retina . . . . . . . . . . . . . . . . . 129 Sook Hyun Chung , Weiyong Shen , and Mark Gillies

11 Genomic Analysis of Transcriptional Changes Underlying Neuronal Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Sebastiano Cavallaro

Contents

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12 High-Throughput Cell Death Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Matthew E. Pamenter and Gabriel G. Haddad

PART II CELL DEATH IN NEUROPATHOLOGY AND EXPERIMENTAL NEUROPATHOLOGY

13 Staining of Dead Neurons by the Golgi Method in Autopsy Material. . . . . . . . 167 Stavros J. Baloyannis

14 Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Stan Krajewski , Jeffrey Wang , Tashmia Khan , Jonathan Liu , Chia-Hung Sze , and Maryla Krajewska

15 In Vitro Oxygen-Glucose Deprivation to Study Ischemic Cell Death. . . . . . . . 197 Carla I. Tasca , Tharine Dal-Cim , and Helena Cimarosti

16 Laser Microbeam Targeting of Single Nerve Axons in Cell Culture . . . . . . . . . 211 Nicholas Hyun , Linda Z. Shi , and Michael W. Berns

17 Real-Time Imaging of Retinal Cell Apoptosis by Confocal Scanning Laser Ophthalmoscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Eduardo M. Normando , Mohammad H. Dehabadi , Li Guo , Lisa A. Turner , Gaia Pollorsi , and M. Francesca Cordeiro

18 Targeted Toxicants to Dopaminergic Neuronal Cell Death . . . . . . . . . . . . . . . 239 Huajun Jin , Arthi Kanthasamy , Dilshan S. Harischandra , Vellareddy Anantharam , Ajay Rana , and Anumantha Kanthasamy

PART III NEURAL STEM CELLS, PROGENITORS, AND GENE THERAPY STRATEGIES

19 Stem Cells, Neural Progenitors, and Engineered Stem Cells . . . . . . . . . . . . . . 255 Raj R. Rao and Shilpa Iyer

20 Herpes Simplex Virus Type 1 (HSV-1)-Derived Recombinant Vectors for Gene Transfer and Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Peggy Marconi , Cornel Fraefel , and Alberto L. Epstein

21 Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon Vectors for Gene Transfer and Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Cornel Fraefel , Peggy Marconi , and Alberto L. Epstein

22 Bone Marrow Transplantation for Research and Regenerative Therapiesin the Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 David Díaz , José Ramón Alonso , 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) . . . . . . . . . . 329 Antón Barreiro-Iglesias and Michael I. Shifman

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24 Generation of Zebrafish Models by CRISPR/Cas9 Genome Editing . . . . . . . . 341 Alexander Hruscha and Bettina Schmid

25 In Vivo Assessment of Neuronal Cell Death in Drosophila . . . . . . . . . . . . . . . . 351 Pierre Dourlen

26 Drosophila Model for Studying Phagocytosis Following Neuronal Cell Death. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Boris Shklyar , Flonia Levy-Adam , and Estee Kurant

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

Contents

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SILVIA ALASIA • Department of Veterinary Sciences University of Turin , Turin , Grugliasco , Italy

JOSE RAMÓN ALONSO • Laboratory of Neuronal Plasticity and Neurorepair, Institute for Neuroscience of Castilla y León (INCyL) , Universidad de Salamanca , Salamanca , Spain ; IBSAL – Institute of Biomedical Research of Salamanca , Universidad de Salamanca , Salamanca , Spain ; Instituto de Alta Investigación , Universidad de Tarapacá , Arica , Chile

VELLAREDDY ANANTHARAM • Parkinson’s Disorder Research Laboratory, Department of Biomedical Sciences, IA Center for Advanced Neurotoxicology , IA State University , Ames , IA , USA

MACARENA S. ARRÁZOLA • Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas , Pontifi cia Universidad Católica de Chile , Santiago , Chile

STAVROS J. BALOYANNIS • Thessaloniki and Institute for Research on Alzheimer’s Disease and Ageing , Aristotelian University , Iraklion, Langada , Greece

FERNANDO C. BALTANÁS • Institute for Molecular and Cell Biology of the Cancer , CSIC – Universidad de Salamanca , Salamanca , Spain

ANTÓN BARREIRO-IGLESIAS • Centre for Neuroregeneration, School of Biomedical Sciences , University of Edinburgh , Edinburgh , UK

MARIA TERESA BERCIANO • Department of Anatomy and Cell Biology, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) , Universidad de Cantabria-IDIVAL , Santander , Spain

MICHAEL W. BERNS • Beckman Laser Institute , University of California , Irvine , CA , USA ; Department of Bioengineering, Institute for Engineering in Medicine , University of California , San Diego, La Jolla , CA , USA

MARÍA CALVO • Instituto de Biología y Genética Molecular (IBGM) , Consejo Superior de Investigaciones Científi cas y Universidad de Valladolid , Valladolid , Spain

NADIA CANU • Department of System Medicine , University of “Tor Vergata” Rome , Rome , Italy ; National Council Research , Institute of Cell Biology and Neurobiology , Rome , Italy

CLAUDIA CASTAGNA • Department of Veterinary Sciences , University of Turin , Turin , Italy SEBASTIANO CAVALLARO • Functional Genomics Center, Institute of Neurological Sciences ,

Italian National Research Council , Catania , Italy SOOK HYUN CHUNG • Macular Research Group, Clinical Ophthalmology and Eye Health ,

The University of Sydney , Sydney , NSW , Australia HELENA CIMAROSTI • Reading School of Pharmacy , University of Reading , Reading , UK CAROLINA COCITO • Department of Veterinary Sciences , University of Turin , Turin , Italy M. FRANCESCA CORDEIRO • Glaucoma and Retinal Degeneration Research Group,

Visual Neurosciences , UCL Institute of Ophthalmology , London , UK ; Imperial College Healthcare Trust , The Western Eye Hospital , London , UK

THARINE DAL-CIM • Departamento de Bioquímica, Centro de Ciências Biológicas , Universidade Federal de Santa Catarina , Florianópolis , SC , Brazil

Contributors

xii

MOHAMMAD H. DEHABADI • Glaucoma and Retinal Degeneration Research Group, Visual Neurosciences , UCL Institute of Ophthalmology , London , UK

DAVID DÍAZ • Laboratory of Neuronal Plasticity and Neurorepair, Institute for Neuroscience of Castilla y León (INCyL) , Universidad de Salamanca , Salamanca , Spain ; IBSAL – Institute of Biomedical Research of Salamanca , Salamanca , Spain

RUSLAN I. DMITRIEV • School of Biochemistry and Cell Biology , University College Cork , Cork , Ireland

PIERRE DOURLEN • Unité de Santé Publique et d’Epidémiologie moléculaire des maladies liées au vieillissement , Institut Pasteur de Lille, INSERM UMR744 , Lille , France

ALBERTO L. EPSTEIN • Centre International de Recherche en Infectiologie (CIRI), INSERM U1111 – CNRS UMR5308 , Ecole Normale Supérieure Lyon , Lyon , France

JOSÉ M. FRADE • Department of Molecular, Cellular and Developmental Neurobiology , Cajal Institute, IC-CSIC , Madrid , Spain

CORNEL FRAEFEL • Institute of Virology , University of Zurich , Zurich , Switzerland MARK GILLIES • Macular Research Group, Clinical Ophthalmology and Eye Health ,

The University of Sydney , Sydney , NSW , Australia LI GUO • Glaucoma and Retinal Degeneration Research Group, Visual Neurosciences ,

UCL Institute of Ophthalmology , London , UK GABRIEL G. HADDAD • Division of Respiratory Medicine, Departments of Pediatrics

and Neuroscience , University of California , San Diego, La Jolla , USA ; The Rady Children’s Hospital-San Diego , San Diego , USA

DILSHAN S. HARISCHANDRA • Parkinson’s Disorder Research Laboratory, Department of Biomedical Sciences, IA Center for Advanced Neurotoxicology , Iowa State University , Ames , IA , USA

ALEXANDER HRUSCHA • German Center for Neurodegenerative Diseases (DZNE) , Munich , Germany

NICHOLAS HYUN • Department of Bioengineering and Institute of Engineering in Medicine , University of California , San Diego, La Jolla , CA , USA

NIBALDO C. INESTROSA • Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas , Pontifi cia Universidad Católica de Chile , Santiago , Chile ; Center for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine , University of New South Wales , Sydney , NSW , Australia

SHILPA IYER • Department of Chemical and Life Science Engineering , Virginia Commonwealth University , Richmond , VA , USA

HUAJUN JIN • Parkinson’s Disorder Research Laboratory, Department of Biomedical Sciences, IA Center for Advanced Neurotoxicology , Iowa State University , Ames , IA , USA

ANUMANTHA KANTHASAMY • Parkinson’s Disorder Research Laboratory, Department of Biomedical Sciences, IA Center for Advanced Neurotoxicology , Iowa State University , Ames , IA , USA

ARTHI KANTHASAMY • Parkinson’s Disorder Research Laboratory, Department of Biomedical Sciences, IA Center for Advanced Neurotoxicology , Iowa State University , Ames , IA , USA

TASHMIA KHAN • Sanford-Burnham Medical Research Institute , La Jolla , CA , USA MARYLA KRAJEWSKA • Sanford-Burnham Medical Research Institute , La Jolla , CA , USA ;

Cellestan-Immunoquant, Inc. , Oceanside , CA , USA STAN KRAJEWSKI • Sanford-Burnham Medical Research Institute , La Jolla , CA , USA ;

Cellestan-Immunoquant, Inc. , Oceanside , CA , USA

Contributors

xiii

ESTEE KURANT • Department of Anatomy and Cell Biology, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine , Technion – Israel Institute of Technology , Haifa , Israel

MIGUEL LAFARGA • Department of Anatomy and Cell Biology, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) , Universidad de Cantabria-IDIVAL , Santander , Spain

FLONIA LEVY-ADAM • Department of Anatomy and Cell Biology, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine , Technion – Israel Institute of Technology , Haifa , Israel

JONATHAN LIU • Sanford-Burnham Medical Research Institute , La Jolla , CA , USA NOELIA LÓPEZ-SÁNCHEZ • Department of Molecular, Cellular and Developmental

Neurobiology , Cajal Institute, IC-CSIC , Madrid , Spain LAURA LOSSI • Department of Veterinary Sciences , University of Torino, Grugliasco, Torino,

Italy ; Istituto Nazionale di Neuroscienze (INN) , Turin , Italy PEGGY MARCONI • Section of Applied Microbiology and Pathology, Department of Life

Sciences and Biotechnology , University of Ferrara , Ferrara , Italy PIERRE MARQUET • Département de Psychiatrie, Centre de Neurosciences Psychiatriques ,

Centre Hospitalier Universitaire Vaudois (CHUV) , Lausanne , Switzerland ; Ecole Polytechnique Fédérale Lausanne (EPFL) , Brain Mind Institute (BMI) , Lausanne , Switzerland

ADALBERTO MERIGHI • Department of Veterinary Sciences , University of Torino, Grugliasco, Torino , Italy ; Istituto Nazionale di Neuroscienze (INN) , Turin , Italy

EDUARDO M. NORMANDO • Glaucoma and Retinal Degeneration Research Group, Visual Neurosciences , UCL Institute of Ophthalmology , London , UK ; Imperial College Healthcare Trust , The Western Eye Hospital , London , UK

LUCÍA NÚÑEZ • Instituto de Biología y Genética Molecular (IBGM) , Consejo Superior de Investigaciones Científi cas y Universidad de Valladolid , Valladolid , Spain ; Departamento de Bioquímica y Biología Molecular y Fisiología , Universidad de Valladolid , Valladolid , Spain

MATTHEW E. PAMENTER • Department of Zoology , University of British Columbia , Vancouver , Canada

DMITRI B. PAPKOVSKY • School of Biochemistry and Cell Biology , University College Cork , Cork , Ireland

NICOLAS PAVILLON • Biophotonics Laboratory, Immunology Frontier Research Center (IFReC) , Osaka University , Suita, Osaka , Japan ; Microvision and Microdiagnostics Group (MVD), STI , Ecole Polytechnique Fédérale de Lausanne (EPFL) , Lausanne , Switzerland

GAIA POLLORSI • Glaucoma and Retinal Degeneration Research Group, Visual Neurosciences , UCL Institute of Ophthalmology , London , UK

AJAY RANA • Department of Molecular Pharmacology and Therapeutics , Loyola University Chicago , Maywood , IL , USA ; Hines Veterans Affairs Medical Center , Hines , IL , USA

RAJ R. RAO • Department of Chemical and Life Science Engineering , Virginia Commonwealth University , Richmond , VA , USA

BETTINA SCHMID • German Center for Neurodegenerative Diseases (DZNE) , Munich , Germany ; Munich Cluster for Systems Neurology (SyNergy) , Munich , Germany

WEIYONG SHEN • Macular Research Group, Clinical Ophthalmology and Eye Health , The University of Sydney , Sydney , NSW , Australia

Contributors

xiv

LINDA Z. SHI • Department of Bioengineering and Institute of Engineering in Medicine , University of California , San Diego, La Jolla , CA , USA

MICHAEL I. SHIFMAN • Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation) , Temple University School of Medicine , Philadelphia , PA , USA

BORIS SHKLYAR • Department of Anatomy and Cell Biology, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine , Technion – Israel Institute of Technology , Haifa , Israel

CHIA-HUNG SZE • Sanford-Burnham Medical Research Institute , La Jolla , CA , USA CARLA I. TASCA • Departamento de Bioquímica, Centro de Ciências Biológicas ,

Universidade Federal de Santa Catarina , Florianópolis , SC , Brazil LISA A. TURNER • Glaucoma and Retinal Degeneration Research Group, Visual Neurosciences ,

UCL Institute of Ophthalmology , London , UK JORGE VALERO • Center for Neuroscience and Cell Biology of Coimbra , University of Coimbra ,

Coimbra , Portugal CARLOS VILLALOBOS • Instituto de Biología y Genética Molecular (IBGM) , Consejo Superior

de Investigaciones Científi cas y Universidad de Valladolid , Valladolid , Spain JEFFREY WANG • Sanford-Burnham Medical Research Institute , La Jolla , CA , USA EDUARDO WERUAGA • Laboratory of Neuronal Plasticity and Neurorepair, Institute

for Neuroscience of Castilla y León (INCyL) , Universidad de Salamanca , Salamanca , Spain ; IBSAL – Institute of Biomedical Research of Salamanca , Salamanca , Spain

Contributors