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 International
REVIEW OF

Neurobiology
Volume 102

SERIES EDITORS

R. ADRON HARRIS
Waggoner Center for Alcohol and Drug Addiction Research
The University of Texas at Austin
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PETER JENNER
Division of Pharmacology and Therapeutics
GKT School of Biomedical Sciences
King’s College, London, UK

EDITORIAL BOARD

ERIC AAMODT HUDA AKIL

PHILIPPE ASCHER MATTHEW J. DURING
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MARTIN GIURFA BARRY HALLIWELL
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NOBU HATTORI LEAH KRUBITZER
DARCY KELLEY KEVIN MCNAUGHT
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 CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Enrike G. Argandoña (317), Department of Medicine, Unit of Anatomy, Uni-

versity of Fribourg, Rue Albert Gockel 1, Fribourg, Switzerland
Daniela Belletti (207), Department of Pharmaceutical Sciences, University of
Modena and Reggio Emilia, Modena, Italy
Harkaitz Bengoetxea (317), Laboratory of Experimental Neuroscience
LaNCE, Department of Neuroscience, University of the Basque Country
(UPV/EHU), Sarriena Auzoa, Leioa, Spain
Lucia Bondioli (207), Department of Pharmaceutical Sciences, University of
Modena and Reggio Emilia, Modena, Italy
Cesario V. Borlongan (91), Center of Excellence for Aging & Brain Repair,
University of South Florida, Morsani College of Medicine, Tampa, Florida,
USA; Department of Neurosurgery and Brain Repair, University of South
Florida, Morsani College of Medicine, Tampa, Florida, USA
Susana Bulnes (317), Laboratory of Experimental Neuroscience LaNCE,
Department of Neuroscience, University of the Basque Country (UPV/EHU),
Sarriena Auzoa, Leioa, Spain
Rudy J. Castellani (47), Department of Pathology, University of Maryland,
Baltimore, Maryland, USA
Alessandro De Vita (207), Department of Pharmaceutical Sciences, University
of Modena and Reggio Emilia, Modena, Italy
Flavio Forni (207), Department of Pharmaceutical Sciences, University of Mo-
dena and Reggio Emilia, Modena, Italy
Svitlana Garbuzova-Davis (91), Center of Excellence for Aging & Brain
Repair, University of South Florida, Morsani College of Medicine, Tampa,
Florida, USA; Department of Neurosurgery and Brain Repair, University of
South Florida, Morsani College of Medicine, Tampa, Florida, USA; Depart-
ment of Molecular Pharmacology and Physiology, University of South Florida,
Morsani College of Medicine, Tampa, Florida, USA; Department of Patholo-
gy and Cell Biology, University of South Florida, Morsani College of Medicine,
Tampa, Florida, USA

xi
xii CONTRIBUTORS

Mathias Hallberg (189), Division of Biological Research on Drug Depen-

dence, Department of Pharmaceutical Biosciences, Uppsala University, P.O.
Box 591, Uppsala, Sweden
Diana G. Hernandez-Ontiveros (91), Center of Excellence for Aging &
Brain Repair, University of South Florida, Morsani College of Medicine,
Tampa, Florida, USA
Eugene A. Kiyatkin (147), Behavioral Neuroscience Branch, National Institute
on Drug Abuse-Intramural Research Program (NIDA-IRP), NIH, Baltimore,
Maryland, USA
José Vicente Lafuente (317), Laboratory of Experimental Neuroscience
LaNCE, Department of Neuroscience, University of the Basque Country
(UPV/EHU), Sarriena Auzoa, Leioa, Spain
Michael K. Louis (91), Center of Excellence for Aging & Brain Repair, Univer-
sity of South Florida, Morsani College of Medicine, Tampa, Florida, USA
Yu Luo (1), Department of Neurological Surgery, Case Western Reserve Univer-
sity, School of Medicine, University Hospitals Case Medical Center, Cleveland,
Ohio, USA
Herbert Mössler (249), Ever NeuroPharma, Oberburgau, Austria
Cecile Martijn (173), Laboratory of Cerebrovascular Research, Department of
Surgical Sciences/Anesthesiology and Intensive Care Medicine, Uppsala Uni-
versity, Uppsala, Sweden
Adriana Miclescu (173), Laboratory of Cerebrovascular Research, Depart-
ment of Surgical Sciences/Anesthesiology and Intensive Care Medicine, Upp-
sala University, Uppsala, Sweden
Dafin Fior Muresanu (107, 249), Department of Clinical Neurosciences, Uni-
versity Hospital, University of Medicine and Pharmacy, Cluj-Napoca, Romania
Fred Nyberg (189), Division of Biological Research on Drug Dependence,
Department of Pharmaceutical Biosciences, Uppsala University, P.O. Box
591, Uppsala, Sweden
Naiara Ortuzar (317), Laboratory of Experimental Neuroscience LaNCE,
Department of Neuroscience, University of the Basque Country (UPV/EHU),
Sarriena Auzoa, Leioa, Spain
Anand Kumar Pandey (107), School of Biomedical Engineering, Institute of
Technology, Banaras Hindu University, Varanasi, India
Ranjana Patnaik (107), School of Biomedical Engineering, Institute of Tech-
nology, Banaras Hindu University, Varanasi, India
Maria Carolina O. Rodrigues (91), Center of Excellence for Aging & Brain
Repair, University of South Florida, Morsani College of Medicine, Tampa,
Florida, USA; Department of Internal Medicine, Ribeirão Preto School of
Medicine, University of Sao Paulo, Sao Paulo, Brazil
Sten Rubertsson (173), Laboratory of Cerebrovascular Research, Department
of Surgical Sciences/Anesthesiology and Intensive Care Medicine, Uppsala
University, Uppsala, Sweden
 CONTRIBUTORS xiii

Barbara Ruozi (207), Department of Pharmaceutical Sciences, University of

Modena and Reggio Emilia, Modena, Italy
Paul R. Sanberg (91), Center of Excellence for Aging & Brain Repair, Univer-
sity of South Florida, Morsani College of Medicine, Tampa, Florida, USA;
Department of Neurosurgery and Brain Repair, University of South Florida,
Morsani College of Medicine, Tampa, Florida, USA; Department of Patholo-
gy and Cell Biology, University of South Florida, Morsani College of Medicine,
Tampa, Florida, USA; Department of Psychiatry, University of South Florida,
Morsani College of Medicine, Tampa, Florida, USA
Egidijus Semenas (173), Laboratory of Cerebrovascular Research, Depart-
ment of Surgical Sciences/Anesthesiology and Intensive Care Medicine,
Uppsala University, Uppsala, Sweden
Aruna Sharma (23, 47, 107, 249), Laboratory of Cerebrovascular Research,
Department of Surgical Sciences, Anesthesiology & Intensive Care Medicine,
University Hospital, Uppsala University, Uppsala, Sweden
Hari Shanker Sharma (23, 47, 107, 147, 173, 249), Laboratory of Cerebro-
vascular Research, Department of Surgical Sciences, Anesthesiology & Inten-
sive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
Stephen D. Skaper (277), Department of Pharmacology and Anesthesiology,
University of Padova, Largo ‘‘E. Meneghetti’’, Padova, Italy
Mark A. Smith (47), Department of Pathology, Case Western Reserve Univer-
sity, Cleveland, Ohio, USA
Giovanni Tosi (207), Department of Pharmaceutical Sciences, University of
Modena and Reggio Emilia, Modena, Italy
Maria Angela Vandelli (207), Department of Pharmaceutical Sciences, Uni-
versity of Modena and Reggio Emilia, Modena, Italy
Júlio C. Voltarelli (91), Department of Internal Medicine, Ribeirão Preto
School of Medicine, University of Sao Paulo, Sao Paulo, Brazil
Lars Wiklund (173), Laboratory of Cerebrovascular Research, Department of
Surgical Sciences/Anesthesiology and Intensive Care Medicine, Uppsala
University, Uppsala, Sweden
Alison E. Willing (91), Center of Excellence for Aging & Brain Repair, Univer-
sity of South Florida, Morsani College of Medicine, Tampa, Florida, USA;
Department of Neurosurgery and Brain Repair, University of South Florida,
Morsani College of Medicine, Tampa, Florida, USA; Department of Molecu-
lar Pharmacology and Physiology, University of South Florida, Morsani
College of Medicine, Tampa, Florida, USA; Department of Pathology and
Cell Biology, University of South Florida, Morsani College of Medicine,
Tampa, Florida, USA
 PREFACE

Recent Perspectives on Central Nervous System Injury and Neuroprotection

Recent expansion of new knowledge regarding the central nervous system (CNS)
and its reaction to injury resulted in the development of a variety of neurotherapeutic
agents. However, apart from exploration of new drugs to treat neurological dis-
orders, delivery of these agents in high quantities to the affected areas of the CNS is
also needed for better treatment options. Thus, further investigations on exploration
of new therapeutic agents and their delivery strategies to the CNS are highly needed.
The CNS is equipped with a blood–brain barrier (BBB) that restricts move-
ment of large molecules, unwanted substances, lipophilic drugs, and other ions or
proteins from blood to brain and vice versa to maintain a strict fluid microenviron-
ment around the neurons and glia cells (Rapoport, 1976; Sharma & Westman,
2004). The BBB largely resides within the endothelial cells of the cerebral
capillaries that are connected with tight junctions, a feature not seen in noncer-
ebral capillaries (Rapoport, 1976; Sharma, 1999). Although the BBB is a protec-
tive structure for healthy CNS, it also restricts drug transport into the brain for
therapeutic purposes in disease conditions (Sharma & Westman, 2004). On the
other hand, this barrier is invariably leaky in almost all neurological diseases or
following traumatic, ischemic, or metabolic insults to the CNS (Bradbury, 1979;
Sharma, 2009). Under such circumstances, proteins and water from the vascular
compartments enter into the brain causing edema that could compress neuronal,
glial, and vascular components within the cranium resulting in brain dysfunction
and/or damage (Sharma et al., 1998).
Thus, drugs that restore or attenuate BBB breakdown to proteins following
CNS insults are able to induce neuroprotection (Sharma, 2009; Sharma &
Westman, 2004; Sharma et al., 1998). This indicates that the BBB could be
regarded as a ‘‘gateway’’ to neurological diseases (Sharma, 1999, 2009). However,
drug delivery to the brain following tumor, localized infract, or ischemic region in
the CNS is a daunting task as most of the therapeutic agents could not reach brain

xv
xvi PREFACE

tissues in sufficient amount to treat these anomalies (Nair et al., 2011). Thus,
enhanced drug delivery to the CNS is also the need of the hour to induce better
therapeutic strategies in neurological diseases (Sharma et al., 2009). This suggests
that, on one hand, drug delivery across the BBB is required for better treatment
avenues for neurological diseases, and on the other hand, plugging of the leaky
barrier in neurological diseases is needed to achieve neuroprotection (Sharma,
1982, 1999). Thus, the treatment strategies of CNS diseases are always centered
on the modulation of the BBB function. This indicates that the BBB pays an
instrumental role in CNS injury and repair mechanisms (Sharma et al., 2009).
Although the term ‘‘neuroprotection’’ originally referred to the rescue of
nerve cells in the CNS following injuries, this has become clearly evident now
that, apart from neurons, the nonneuronal cells, for example, glial and endothelial
cells, that are 10–15 times higher in number than neurons are also equally
important in restoring the brain function (Sharma, 1999, 2007). Thus, the term
‘‘neuroprotection’’ in this volume is used to denote rescue of both neuronal and
nonneuronal cells in CNS injury or in neurodegenerative diseases.
There are reasons to believe that the BBB is altered in a variety of neurode-
generative diseases, for example, Parkinson’s disease (PD), Alzheimer’s disease
(AD), and amyotrophic lateral sclerosis (ALS). Moreover, psychostimulant abuse,
cardiac arrest, heat stroke, hypoxia, ischemia, spinal cord or brain injuries and/or
neurodevelopmental anomalies also affect the BBB function leading to CNS
injuries (Sharma et al., 2011). Thus, restoration of BBB in such situations is
needed to induce neuroprotection (Sharma, 2009; Sharma and Westman, 2004;
Zlokovic, 2011).
Interestingly, we still do not know whether pathogenesis of CNS injuries or
neurodegenerative diseases is affected by environmental factors, for example,
high ambient temperature, air pollution, or inhalation of microfine or nano-
particles (Sharma and Sharma, 2012). In addition, brain pathologies after CNS
insults may also be affected by other internal factors, that is, hypertension or
diabetes, often known as the comorbidity factors (Lafuente et al., 2012;
Muresanu and Sharma, 2007; Muresanu et al., 2010). It is imperative that the
standard drug dosage to treat CNS injuries or neurodegenerative disease
requires some adjustment when the CNS insults are complicated by these
disease-modifying agents or comorbidity factors (Sharma and Sharma, 2007,
2012; Sharma et al., 2011). To date, the researchers or clinicians alike do not
address these problems in details.
In clinical situations, these external and internal comorbidity factors often
play important roles in disease manifestation and/or the therapeutic outcome.
This could be one of the reasons of not getting optimal results during clinical trials
of even quite potent drugs (Sharma, 2008). Thus, new research is needed to
explore suitable neuroprotective strategies in such situations based on these
comorbidity factors, if any. In addition, new investigations are required to find a
common denominator, for example, BBB function that could be instrumental in
 PREFACE xvii

precipitating brain pathologies in CNS injuries. Further, exploration of nano-

technologies for enhanced drug delivery to the CNS is also needed.
This volume is a refereed collection of 12 invited reviews by the leading experts
in the world engaged in the cutting edge of advanced research on neuroprotection
and neuroregeneration. Yu Luo (Cleveland, OH, USA) describes new roles of
orphan nuclear receptor Nurr1 in neuroprotection related to PDs. Aruna Sharma
and Hari Shanker Sharma (Uppsala, Sweden) present new evidences to treat brain
and spinal cord injuries using monoclonal antibodies directed against nitric oxide
synthase (NOS), dynorphin A, tumor necrosis factor a, and serotonin. Rudy J.
Castellani (Baltimore, MD, USA) and his team discusses the importance of BBB in
AD and further suggests that nanowired drug delivery of cerebrolysin, a mixture of
various neurotrophic factors in animal model of AD, attenuated BBB dysfunction
and brain pathologies. Neurovascular aspects of ALS are described in detail by Paul
R. Sanberg (Tampa, FL, USA) and his team. New roles of quercetin, an antioxidant
compound, are discussed in inducing neuroprotection in animal models of ischemic
injuries by Ranjana Patnaik (Varanasi, India) and her coworkers. Although the role
of quercetin is still controversial, new results suggest that the compound may be
beneficial in reducing brain edema and cell injury effectively in ischemia, indicating
the need for further research in this area.
That environmental high temperature influences methamphetamine neurotox-
icity is meticulously shown by Eugene A. Kiyatkin (Baltimore, MD, USA) and Hari
Shanker Sharma (Uppsala, Sweden). Administration of methamphetamine at high
ambient temperature exacerbated brain injury and behavioral dysfunctions. Thus,
it remains to be seen whether neuroprotective drugs attenuating methamphetamine
addiction require additional adjustments in hot environments in clinical situations.
Lars Wiklund (Uppsala, Sweden) and coworkers demonstrate massive brain
pathologies following cardiac arrest that is accompanied with upregulation of
neuronal and endothelial NOS. Treatment with antioxidants reduced these brain
pathologies. However, cardiac arrest-induced brain pathologies and drug effects are
different in male piglets as compared to females of similar age group (Sharma et al.,
2011b). This suggests that outcome of brain injuries and drug effects could vary due
to sex differences.
Development of addictive behavior to alcohol or other substances of abuse could
stimulate neurodegenerative changes. Fred Nyberg and Mathias Hallberg (Uppsala,
Sweden) describe a potential interaction between opioids and anabolic steroids in the
development of addiction and subsequently alterations in the brain function.
To treat brain injuries and related disorders, high quantities of drug should
reach the affected areas of the CNS. Giovanni Tosi (Modena, Italy) and his team
discuss the role of nanodrug delivery of neurotrophic factors for the effective
treatment strategies of neurodegenerative diseases in animal models.
Dafin Fior Muresanu (Cluj-Napoca, Romania) and his coworkers presents
new evidences showing that heat-induced neurotoxicity are exacerbated in ani-
mals with hypertension or diabetes, or following nanoparticle intoxication
xviii PREFACE

(Lafuente et al., 2012; Sharma et al., 2011a). Further, in the presence of these
comorbidity factors, to induce considerable neuroprotection, substantially high
dose of the drug is needed. In cases of nanoparticles intoxication, either a double
dose of the compound or nanowired drug delivery is required to achieve effective
neuroprotection following identical heat exposure. This suggests that comorbidity
factors markedly influence brain pathologies and thus drug dosages are required
profound adjustment to treat such conditions in clinical conditions.
Stephen D. Skaper (Padova, Italy) revisited the role of beta amyloid in causing
brain pathologies in AD. Since overproduction of Ab peptides in the brain of
transgenic mouse models fails to cause overt neurodegeneration, it appears that
several other factors, for example, AD-related genes, that is, microtubule-asso-
ciated protein tau, polymorphisms of apolipoprotein E4, as well as inflammation
and oxidative stress, also contribute to AD pathogenesis.
Finally, new roles of vascular endothelial growth factor are discussed by José
Vicente Lafuente (Bilbao, Spain) and coworkers in the developmental anomalies
of brain pathologies in an animal model.
The new concepts and recent advancements in neurotherapeutics presented
in this volume will benefit neurologists, neuropharmacologists, neurosurgeons,
neuropathologists, neurophysiologists, neuropsychiatrists, nanotechnologists,
immunologists, clinicians, medical students, researchers, healthcare providers,
military experts, and policymakers alike.
We firmly believe that this volume will stimulate further research in these
novel areas of neuroprotection and neuroregeneration for the benefit of mankind.

Hari Shanker Sharma, PhD (BHU),

Dr Med Sci (UU), FAIS (New York, USA)
Uppsala University
Sweden

References

Bradbury, M.W.B. (1979). The Concept of a Blood-Brain Barrier. John Wiley & Sons, New York. pp.
1–380.
Lafuente, J.V., Sharma, A., Patnaik, R., Muresanu, D.F., and Sharma, H.S. (2012). Diabetes exacer-
bates nanoparticles induced brain pathology. CNS Neurol. Disord. Drug Targets 11(1), 26–39.
Muresanu, D.F., and Sharma, H.S. (2007). Chronic hypertension aggravates heat stress induced
cognitive dysfunction and brain pathology: an experimental study in the rat, using growth
hormone therapy for possible neuroprotection. Ann. N.Y. Acad. Sci. 1122, 1–22.
Muresanu, D.F., Sharma, A., and Sharma, H.S. (2010). Diabetes aggravates heat stress-induced blood-
brain barrier breakdown, reduction in cerebral blood flow, edema formation, and brain pathology:
possible neuroprotection with growth hormone. Ann. N.Y. Acad. Sci. 1199, 15–26.
 PREFACE xix

Nair, B.G., Varghese, S.H., Nair, R., Yoshida, Y., Maekawa, T., and Kumar, D.S. (2011). Nanotech-
nology platforms; an innovative approach to brain tumor therapy. Med. Chem. 7(5), 488–503.
Review.
Rapoport, S.I. (1976). Blood-Brain Barrier in Physiology and Medicine. Raven Press, New York.
Sharma, H.S. (1982). Blood-Brain Barrier in Stress, Ph D Thesis. Banaras Hindu University, Varanasi,
India, pp. 1–85.
Sharma, H.S. (1999). Pathophysiology of blood-brain barrier, brain edema and cell injury following
hyperthermia: New role of heat shock protein, nitric oxide and carbon monoxide. An experimental
study in the rat using light and electron microscopy, Acta Universitatis Upsaliensis 830, 1–94.
Sharma, H.S. (2007). Neurotrophic factors in combination: a possible new therapeutic strategy to
influence pathophysiology of spinal cord injury and repair mechanisms. Curr. Pharm. Des. 13(18),
1841–1874. Review.
Sharma, H.S. (2008). New perspectives for the treatment options in spinal cord injury. Expert Opin.
Pharmacother. 9(16), 2773–2800. Review.
Sharma, H.S. (2009). Blood–central nervous system barriers: the gateway to neurodegeneration,
neuroprotection and neuroregeneration. In: Lajtha, A., Banik, N., and Ray, S.K. (Eds.), Handbook
of Neurochemistry and Molecular Neurobiology: Brain and Spinal Cord Trauma. Springer Verlag,
Berlin, pp. 363–457.
Sharma, H.S., and Westman, J. (2004). Blood-Spinal Cord and Brain Barriers in Health and Disease.
Elsevier Academic Press, San Diego. pp. 1–607.
Sharma, H.S., and Sharma, A. (2007). Nanoparticles aggravate heat stress induced cognitive deficits,
blood-brain barrier disruption, edema formation and brain pathology. Prog. Brain Res. 162,
245–273. Review.
Sharma, H.S., and Sharma, A. (2012a). Nanowired drug delivery for neuroprotection in central
nervous system injuries: modulation by environmental temperature, intoxication of nanoparticles,
and comorbidity factors. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 4(2), 184–203.
Sharma, H.S., and Sharma, A. (2012b). Neurotoxicity of engineered nanoparticles from metals. CNS
Neurol Disord Drug Targets 11(1), 65–80.
Sharma, H.S., Westman, J., and Nyberg, F. (1998). Pathophysiology of brain edema and cell changes
following hyperthermic brain injury. Prog. Brain Res. 115, 351–412. Review.
Sharma, H.S., Ali, S., Tian, Z.R., Patnaik, R., Patnaik, S., Lek, P., Sharma, A., and Lundstedt, T.
(2009). Nano-drug delivery and neuroprotection in spinal cord injury. J. Nanosci. Nanotechnol. 9(8),
5014–5037.
Sharma, H.S., Muresanu, D.F., Patnaik, R., Stan, A.D., Vacaras, V., Perju-Dumbrav, L., Alexandru, B.,
Buzoianu, A., Opincariu, I., Menon, P.K., and Sharma, A. (2011a). Superior neuroprotective
effects of cerebrolysin in heat stroke following chronic intoxication of Cu or Ag engineered
nanoparticles. A comparative study with other neuroprotective agents using biochemical and
morphological approaches in the rat. J. Nanosci. Nanotechnol. 11(9), 7549–7569.
Sharma, H.S., Miclescu, A., and Wiklund, L. (2011b). Cardiac arrest-induced regional blood-brain
barrier breakdown, edema formation and brain pathology: a light and electron microscopic study
on a new model for neurodegeneration and neuroprotection in porcine brain. J. Neural. Transm.
118(1), 87–114.
Zlokovic, B.V. (2011). Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other
disorders. Nat. Rev. Neurosci. 12(12), 723–738. Review.
xx PREFACE

Excerpts

‘‘This volume brings together authors working on cutting edge research on neuroprotection and
neuroregeneration. It will be a must-read for anyone interested in these fields. I commend the editor
and authors on a well thought-out and exciting contribution to the literature’’

Paul R. Sanberg, Ph.D., D.Sc.

Executive Director of the Center of Excellence for Aging and Brain Repair
Distinguished University Professor
Senior Associate Vice President for Research & Innovation
University of South Florida

‘‘A couple of decades ago, neuroprotection was largely limited to empiric interventions of
modest benefit, such as steroids for acute spinal cord injury. Technical advances in neuroimaging,
nervous system physiology, chemistry, and electrical activity have enhanced our understanding of the
nervous system—and how we can protect it—in ways virtually unimaginable twenty years ago.
Quantum dots allow us to map the firing patterns of ensembles of neurons, optogenetic techniques
allow us to select which neuron ensembles to stimulate, and carbon nanotube ‘‘endoscopes’’ allow
us to peek inside the cells of the nervous system to see what really ‘‘makes them tick’’—both in
health and in disease. Professor Sharma is to be congratulated for assembling this intellectual
‘‘delight’’ of papers on current issues in neuroprotection. Exogenously administered drugs are no
longer the only ‘‘main course’’ of neuroprotection: we are learning that the blood vessels and the
blood-brain barrier, neurotrophic factors and neurotransmitters—plus a myriad of receptors—all
have increasingly amazing roles in the neuroprotection!’’

Russell J Andrews
NASA Ames Research Laboratory, Moffet Field, CA, USA

‘‘Although the study of neuroprotection has been advanced by the continuing meeting series, the
International Conferences on Neuroprotective Agents, cofounded by Bruce Trembly, MD and
William Slikker, Jr, PhD in 1991, progress focused on studies of the blood-brain-barrier (BBB)
as a central mediator of neurotoxicity and neuroprotection has not been clearly defined until this
contribution entitled ‘‘New Perspectives of Central Nervous System Injury and Neuroprotection’’
International Review Neurobiology (Vol. 102) 2012. The Editor, Dr. Hari Sharma and the
contributing authors have captured the excitement of the new understanding of the BBB and its
importance to blocking neurotoxicity elicited via numerous pathways. Students of neuroprotection,
regardless of experience, will benefit from reading this well structured and comprehensive review’’

William Slikker, Jr.

Director, National Center for Toxicological Research/FDA
 ACKNOWLEDGMENTS

I am deeply indebted to Johannes Menzel and Lisa Tickner from Elsevier

London, UK, for their constant encouragement and patience during the develop-
ment of this project, which resulted in the compilation of this volume in the
present form. Ben G. Davie’s (Elsevier, London) help is gratefully acknowledged
for his untiring work during the acquisition and revision process of the chapters.
I want to express my sincere gratitude to Shellie Bryant and Paul Milner (Elsevier
London, UK). They worked very hard for the production of this volume at every
stage. Without their continuous and excellent help, the publication of this volume
in the present form would not have been possible. I extend my sincere thanks
to Aruna Sharma (Uppsala University) in assisting me during the editing process
and Suraj Sharma (Uppsala University) for helping me with graphic design and
development.

xxi
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION IN
MIDBRAIN DOPAMINERGIC NEURONS, FROM DEVELOPMENT
AND MAINTENANCE TO SURVIVAL

Yu Luo
Department of Neurological Surgery, Case Western Reserve University, School of Medicine,
University Hospitals Case Medical Center, Cleveland, Ohio, USA

Abstract
I.Introduction
II.The Midbrain Dopamine System: Neurochemistry
III.The Midbrain Dopamine System: Development
IV. Dopaminergic Neurons and Parkinson’s Disease
V. Nurr1, A Protein Whose Function Is Important in the Life Cycle of VM DANs
A. Nurr1 and the Development of Midbrain Dopaminergic Neurons
B. Nurr1 and the Maintenance of Dopaminergic Neurons
C. Nurr1 Expression Levels Affect the Vulnerability of Dopaminergic Neurons in Response
to Dopaminergic Cytotoxic Reagents
VI. The Mechanisms of Nurr1 as a Nuclear Receptor
A. Downstream Target Gene of Nurr1
B. Regulation of Nurr1 Activity
VII. Most Recent Development in Application of Nurr1 in Dopaminergic Differentiation
and Implications in Future Treatment for PD
Acknowledgment
References

Abstract

Nurr1 is critical for the development and maintenance of midbrain dopami-

nergic (DA) neurons in mouse. Loss of Nurr1 function early during development
in mice leads to the absence of midbrain DA neurons. Reduction of Nurr1
function in adulthood leads to a slowly progressive loss of striatal DA and markers
for DAergic neurons, supporting its selective roles in the maintenance of DAergic
neuronal survival and function. To understand the molecular mechanisms of
Nurr1 action, our group has identified VIP as a potential target gene of Nurr1.
Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP
promoter through Nurr1-responsive cis elements. Nurr1 loss of function leads to

INTERNATIONAL REVIEW OF 1 Copyright 2012, Elsevier Inc.

NEUROBIOLOGY, VOL. 102 All rights reserved.
DOI: 10.1016/B978-0-12-386986-9.00001-6 0074-7742/12 $35.00
2 YU LUO

the decrease of VIP mRNA level in developing midbrain, suggesting that Nurr1 is
involved in the in vivo regulation of VIP expression in midbrain. Our group has
also cloned a novel protein interactor for Nurr1. We identified a family of gene
products that interact and regulate the activity of Nurr1 by screening yeast two-
hybrid library and termed the longest splicing form, NuIP. In vivo NuIP protein is
largely colocalized with Nurr1 in adult midbrain dopaminergic neurons. NuIP
interacts and positively regulates the activity of Nurr1 protein and could also
possibly mediate cross talk between Nurr1 and GTPase mediated signaling
pathways. Other recently identified potential target genes and interacting proteins
of Nurr1 are also summarized and discussed in this review.

I. Introduction

Dopamine is one of the major neurotransmitters in the brain, and dopaminergic

projections are involved in the regulation of several essential functions and behaviors
such as voluntary movement control and reward-related behaviors. The midbrain
dopaminergic neurons form several important projection pathways including the
nigrostriatal (NS) pathway (substantia nigra, SN, to striatum), the mesolimbic
pathway (ventral tegmental area, VTA, to nucleus accumbens), and the mesocortical
pathway (VTA to prefrontal cortex). The NS pathway is involved in regulation of
voluntary movement and degeneration of the NS pathway causes Parkinson’s
disease in humans. The mesolimbic pathway and the mesocortical pathway have
essential roles in reward-related behavior as well as other higher cognitive functions
such as memory and goal maintenance. Dysregulation in the mesolimbic pathway
and mesocortical pathway has been implicated in pathological conditions such as
schizophrenia and addiction. Due to its importance, the midbrain dopaminergic
system has been extensively studied and many genes have been identified to play a
key role in the development and regulation of midbrain dopaminergic neurons. One
of the genes that have essential function in midbrain dopaminergic neurons is the
nuclear receptor Nurr1 gene. In this chapter, we summarize the past findings on the
development and maintenance of midbrain dopaminergic neurons and recent
progress on the role and molecular mechanisms of Nurr1 in these cells.

II. The Midbrain Dopamine System: Neurochemistry

Dopamine (DA) was first identified as a neurotransmitter by Arvid Carlsson in

1958 (Aarnisalo et al., 2002; Carlsson et al., 1958), and in the brain, several groups
of neurons use DA as their neurotransmitter. DA is made in the cells by converting
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 3

the amino acid, L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) by the

cytosolic enzyme tyrosine hydroxylase (TH), the rate-limiting step. Following
hydroxylation of tyrosine, L-DOPA is converted into DA by another enzyme
L-aromatic amino acid decarboxylase. The newly made DA is then concentrated
into storage vesicles via the vesicular amine transporter located within the mem-
brane of the synaptic vesicle. Depolarization of DA neurons (DANs) leads to the
release of DA from these vesicles into the synaptic cleft in the striatum. In the
synaptic cleft, DA acts through the DA receptors on the postsynaptic membrane.
Termination of transmitter action is carried out mainly by the reuptake of DA into
presynaptic cells via the dopamine transporter (DAT) which is unique to midbrain
DANs and is often used as DA cell marker. DA can also be catabolized in the cleft
by catechol-O-methyltransferase. Recycled DA is either repackaged into vesicles
or catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetic acid and
hydrogen peroxide. To function as a DA cell, neurons need to have this entire
set of ‘‘machinery’’ for synthesis, storage, and release of dopamine.
The localization of DANs was initially characterized by the Falck-Hillarp
histofluorescence method (Dahlstrom and Fuxe, 1964a) which was based on the
visualization of fluorescent monoamines upon formaldehyde treatment. By this
method, it was shown that DA cells in the central nervous system (CNS) are
localized in the forebrain, midbrain, and olfactory bulb. Within the ventral
midbrain, the DANs are located in the lateral groups of the substantia nigra pars
compacta (SNc) and the medially located VTA, which are referred to the A9 and
A10 DANs, respectively (Dahlstrom and Fuxe, 1964a). Among different groups of
DANs, the midbrain DANs are the most prominent both in the terms of cell
numbers (> 70%) and DA content (Dahlstrom and Fuxe, 1964b).

III. The Midbrain Dopamine System: Development

The development of midbrain DANs is initiated at embryonic day 9 in the

mouse ventral mesencephalon (VM) through combined signaling mediated by
sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8) (Hynes and
Rosenthal, 1999; Rosenthal, 1998), derived from the floor plate of the ventral
midline and the mid/hindbrain border, respectively. SHH is a diffusible factor
determining the ventral–dorsal position of the neuraxis and FGF8 is locally
produced in the precise location of midbrain–hindbrain boundary (MHB). The
combination of these two factors in the ventral MHB leads to the induction of DA
progenitor cells at this particular location (Hynes and Rosenthal, 1999). The first
expressed marker specific for DAN is TH, the rate-limiting enzyme for the
synthesis of dopamine. TH is expressed in mouse DAN at about E11.
4 YU LUO

As development proceeds, the midbrain DA progenitor cells differentiate into a

more mature DA phenotype characterized by the expression of other DA cell
markers such as VMAT, DA receptors, and DAT (Fig. 1). Before the expression of
DA-specific markers, multiple transcription factors are expressed in the DA
progenitor cells. These include Nurr1, Lmx1b, Pitx3, and En1/En2 (Wallen and
Perlmann, 2003). The role of these transcription factors in the development of
midbrain DAN was established by gene targeting studies. Currently, there are at
least two independent pathways for the specification of a midbrain DA phenotype.
One of them is the Lmx1b–Ptx3 pathway and the other is the Nurr1–TH pathway
(Burbach et al., 2003). Lmx1b and Ptx3 are both expressed in midbrain DANs.
Lmx1b is a member of the LIM homeodomain family and is an essential regulator
of dorsoventral patterning of the developing limbs (Dreyer et al., 1998). In the
ventral midbrain, Lmx1b is first expressed in midbrain DA progenitor cells at E7.5
and its expression is maintained throughout adulthood in these neurons (Smidt
et al., 2000). In contrast to the broader and earlier expression pattern of Lmx1b,
Ptx3, another homeobox gene, is exclusively expressed in the midbrain DANs at a
later stage (E11.5) (Smidt et al., 1997). Loss of Lmx1b function in knockout mice
reveals an absence of Ptx3 expression in midbrain DANs, suggesting that Ptx3
expression is downstream of Lmx1b and is dependent on Lmx1b function (Smidt
et al., 2000). In contrast, Nurr1 and TH expression are still detected at E12.5,
suggesting that Nurr1 and TH expression is not Lmx1b dependent. From E16.5,
however, no midbrain DANs can be detected in these mice, suggesting that Lmx1b
is required to sustain the dopaminergic cell fate (Smidt et al., 2000). The role of Ptx3
remains elusive since knockout mice for Ptx3 have not been generated. However,

Nurr1, Ptx3, En1/2

TH

SHH / FGF8
VMAT

DA receptor

DAT

E8 E9 E12 E14 E15 Adulthood

E10.5 E11.5 P1

FIG. 1. Representation of the temporal sequence of gene induction in developing DA cells. The
development of midbrain dopaminergic neurons is initiated at embryonic day 9 in the mouse VM
through a combined signaling mediated by SHH and FGF8. Nurr1 is turned on in these progenitor
cells at E10.5 one day before the appearance of the first DA cell marker TH. As development proceeds,
these cells mature into a more matured DA phenotype indicated by the induction of other DA cell
markers such as the VMAT, DA receptors, and DAT.
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 5

recent studies on a naturally occurring mutant form of Ptx3 in mice (aphakia) have
demonstrated that Ptx3 is responsible for the differentiation of the A9 group of
DANs (Nunes et al., 2003; van den Munckhof et al., 2003).
The second pathway leading to midbrain DAN development is the Nurr1–TH
pathway. Among the many transcriptional factors, Nurr1 has been most exten-
sively characterized and the role of Nurr1 in the development of VM DANs has
been unambiguously established. Since Nurr1 is the focus of this chapter we discuss
it in detail in the following section. Briefly, Nurr1 knockout mice fail to express the
TH gene in midbrain DA progenitor cells (E11.5) and rapidly lose other ventral
midbrain markers (Zetterstrom et al., 1997). By the time of birth no dopaminergic
markers are detected in Nurr1 knockout pups that die within 48 h of birth. Again,
Ptx3 expression is not affected in these mice confirming that Ptx3 expression is
regulated by a Nurr1-independent pathway (Wallen et al., 1999). En1 and En2 are
two other homeobox genes that are expressed in the ventral midbrain at an early
developmental stage (E9) (Smidt et al., 2003). While loss of function of either does
not seem to affect DAN development, double-null mutants of the two genes lead to
a smaller population of TH-positive cells in midbrain at E12 compared to wild-
type embryos and a rapid loss of these TH-positive neurons at E14 (Simon et al.,
2001). The role of En1/En2 in the Lmx1b–Ptx3 pathway or the Nurr1–TH
pathway is still unclear. The fact that disruption of multiple transcription factors
in different pathways such as En1/2, Lmx1b, Ptx3, and Nurr1 results in failure of
midbrain DAN development, suggests that the maintenance of the dopaminergic
phenotype even in early development requires complicated and delicate multiple
transcriptional regulations. Studies of these transcription factors have given us
insight about the mechanisms of DAN generation in vivo and enable the develop-
ment of new strategies for generating DANs in vitro. This has therapeutic potential
for cell replacement therapy in Parkinson’s disease (PD).

IV. Dopaminergic Neurons and Parkinson’s Disease

Degeneration of the NS dopaminergic pathway is one of the key characteris-

tics of pathology in PD. PD is one of the most common neurodegenerative
disorders. PD is named after the British physician James Parkinson who described
the disease almost 200 years ago (Parkinson, 1817). Clinically, it is diagnosed
primarily based on motor abnormalities including bradykinesia, resting tremor,
and cogwheel rigidity (Duvoisin, 1992). Although there is no known cause for
sporadic PD, there are some common pathological characteristics in PD patients,
including degeneration of DANs in the midbrain and diminished DA levels in the
striatum, which is due to the loss of the NS DA pathway. The NS pathway is one
6 YU LUO

of the most important DA pathways in the brain and contains about 80% of the
total brain DA. Interestingly, it is also the most vulnerable DA pathway that is
affected in PD since other DA pathways degenerate to a lesser extent in the
progress of the disease ( Jellinger, 1987).
The cause for PD is still unknown. Combinations of genetic factors and
environmental influences have been proposed to contribute to the etiology of PD
(Bowers et al., 1997; Duvoisin, 1992). The hypothesis that exposure to environ-
mental factors might contribute to the cause of PD is supported by the discovery
that intravenous injection of the compound 1-methyl-1,2,4,6-tetrahydropyridine
(MPTP) by drug addicts caused a condition that closely resembles the anatomic
and clinical features of PD (Kopin, 1987; Langston et al., 1983). In addition,
epidemiological studies during the 1970s and 1980s showed that the prevalence
of sporadic PD is high among farming communities ( Jenner and Olanow, 1998).
Multiple models have been developed in the laboratory in which the administra-
tion of a compound such as neural toxicant MPTP (Kopin, 1987; Langston et al.,
1983) or combination of herbicides and pesticides (Barbeau, 1986) can lead to a
pattern of cell death and DA loss similar to that of PD.
Genetic factors are also associated with familial PD. Many examples of familial
parkinsonism have also been reported. Mutations of several genes have been linked
to familial PD and parkinsonian syndromes (Kitada et al., 1998; Lincoln et al., 1999;
Polymeropoulos et al., 1996) including a-synuclein, Parkin, ubiquitin C-terminal
hydrolase isozyme L1 (UCH-L1), DJ-1, PINK1, and LRRK2 (Bialecka et al., 2005;
Bonifati et al., 2003; Iwatsubo et al., 2005; Lincoln et al., 1999). Other genes that are
involved in the development of DANs have also been of particular interest. Many of
these ‘‘developmental’’ genes are expressed in DANs throughout adulthood and
may play a role in maintaining the dopaminergic phenotype (Bowers et al., 1997;
Smith, 2000; Wallen et al., 1999). Understanding the development of these neurons
may give us insights into the requirement of maintaining functional and healthy
DANs later in life. Among these genes, Nurr1 has drawn much attention since
many studies have demonstrated that Nurr1 function is required for the develop-
ment and perhaps maintenance of adult midbrain dopaminergic neurons (Le et al.,
1999; Zetterstrom et al., 1997). Nurr1’s function and mechanisms of action will be
the focus of the rest of this chapter.

V. Nurr1, A Protein Whose Function Is Important in the Life Cycle of VM DANs

Nurr1 is a member of the orphan nuclear receptor family whose function is

important in the development of DANs (Zetterstrom et al., 1997). It is expressed in
many regions in the CNS including ventral midbrain (Xiao et al., 1996). In mouse
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 7

ventral midbrain, it is first expressed at E10.5, before the appearance of any

dopaminergic cell markers, and its expression is sustained in this region through-
out adulthood (Wallen et al., 1999). It was found that the distribution of Nurr1
mRNA in this area exactly parallels the distribution of DAN, as indicated by the
TH staining in these cells. Closer examination of the Nurr1 expression pattern
confirmed that in the ventral midbrain, 96% of the DANs are double labeled for
both TH and Nurr1 (Backman et al., 1999). In addition, exposure to 6-OHDA, a
DAN selective toxicant, leads to loss of both TH and Nurr1 expression in SN
(Zetterstrom et al., 1996a). Taken together, these data suggest that Nurr1 is
expressed in the NS DA system both during the development and adulthood
and may play a role in the generation and maturation of DAN.

A. NURR1 AND THE DEVELOPMENT OF MIDBRAIN DOPAMINERGIC NEURONS

Nurr1’s critical role in the development of DAN in the midbrain was further
demonstrated by knockout studies in mice (Saucedo-Cardenas et al., 1998;
Zetterstrom et al., 1997). Mice that lack functional Nurr1 were hypoactive and
died within 48 h of birth (Zetterstrom et al., 1997). Interestingly, loss of Nurr1
function in these mice led to the specific absence of DAN in the neonatal midbrain
while DAN in other regions of the brain were preserved, resembling the specific
pattern of DAN loss in PD. Another interesting observation is that Nurr1 does not
seem to be required for the induction of the dopaminergic progenitor cells as
indicated by the presence of other dopaminergic progenitor cell markers such as
aldehyde dehydrogenase 2 (AHD2) and a bicoid-related homeobox gene (Ptx3) at
early stages (Wallen et al., 1999). Instead, Nurr1 seems to play a critical role in the
maturation of midbrain dopaminergic progenitor cells. As development pro-
ceeded, those dopaminergic progenitor cell markers are lost and at birth all the
DA markers examined were absent in the Nurr1 (
/
) mice (Saucedo-Cardenas
et al., 1998; Zetterstrom et al., 1997).
What happens to DA progenitor cells lacking Nurr1 during development?
How and why do they lose their phenotype and show a failure to induce other
later markers? Answers to these questions may give us some clues about the key
steps in the induction of the dopaminergic phenotype and the conditions required
for these steps to occur. Studies have been carried out both in vivo and in vitro to
answer the question whether DAN progenitor cells degenerate during develop-
ment due to loss of Nurr1 function or if they are arrested at certain developmental
stages and retain the ability to be induced into DAN. The results have been
divergent. Zetterstrom et al. (1997) reported the agenesis of progenitor cells in
knockout mice while Castillo et al. (1998b) found no changes in neuron numbers
when stained with NeuN, a neuron specific marker. Furthermore, tracing studies
using either fluorogold or DiI showed opposite results in an effort to prove the
8 YU LUO

presence or absence of an intact nigrastriatal pathway in Nurr1 (
/
) mice

(Wallen et al., 1999; Witta et al., 2000). Two studies have also been carried out
to examine the development of Nurr1 (
/
) progenitor cells in vitro in primary
culture systems (Eells et al., 2001; Tornqvist et al., 2002). Using a roller-drum
method in which SNs from mice were cultured as a piece of tissue without cell
dissociation, Tornqvist et al. (2002) demonstrated that serum is needed to induce
TH-positive neurons in Nurr1 (
/
) tissue, suggesting that some factors in the
serum are able to compensate for the loss of Nurr1. In addition, recent data
showed TH-positive neurons in cultures from newborn Nurr1 (
/
) tissue (Eells
et al., 2001). Taken together, it suggests that dopaminergic progenitor cells in
Nurr1 (
/
) mice retain the capacity to be induced into mature DAN when
cultured in vitro. This makes primary culture a useful system to study the devel-
opment of dopaminergic progenitor cells in vitro, enabling us to test the function of
Nurr1 when introduced into Nurr1 (
/
) tissue. However, to address the issue of
cell survival versus arrested differentiation and to show unequivocally the origin
and fate of the dopaminergic progenitor cells in Nurr1 (
/
) mice, studies that
can more precisely label and track these progenitor cells are required in the future.

B. NURR1 AND THE MAINTENANCE OF DOPAMINERGIC NEURONS

In addition to its important role in the development of DAN, Nurr1 also plays
a role in the survival and maintenance of DAN throughout adulthood. In support
of this, Zetterstrom et al. (1996a) reported that Nurr1 expression begins at E10.5 in
mouse VM and is sustained throughout adulthood, suggesting that Nurr1 con-
tinues to influence the function of these cells during postnatal development and
adulthood. A recent study (Kadkhodaei et al., 2009) indicated that reduction of
Nurr1 function in adulthood leads to a slowly progressive loss of striatal DA and
markers for DAergic neurons, supporting its selective roles in the maintenance of
DAergic neuronal survival and function. Deficiency in Nurr1 expression results in
a PD like phenotype. For example, there were more dopaminergic neurons lost
in the substantia nigra compacta than in the VTA when Nurr1 was deleted in
maturing dopaminergic neurons (Kadkhodaei et al., 2009). The stoichiometry of
Nurr1 expression also seems to affect the dynamics and vulnerability of DANs
during aging and under variety of stress conditions. Newborn heterozygotes
(Nurr1 þ/
) show significantly reduced levels of Nurr1 protein and DA in the
striatum, indicating that NS DA levels are affected by Nurr1 mRNA dosage (Eells
et al., 2002; Zetterstrom et al., 1997). Studies have found that decreased Nurr1
level in heterozygous mice (Nurr1 þ/
) influenced the age-dependent decline in
the number of DANs and influenced DA signaling ( Jiang et al., 2005; Zhang et al.,
2011). Old Nurr1(þ/
) mice have decreased numbers of DANs in midbrain and
young Nurr1 (þ/
) mice exhibited a decrease in peak evoked DA release before
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 9

they showed significant DAN loss (Zhang et al., 2011). Recent experimental work
in constitutive heterozygous Nurr1 (þ/
) mice also showed a facilitation of the
development of schizophrenia-related behavioral abnormalities including motor
activity, sensorimotor gating, and responsiveness to the psychomimetic drug
MK-801 (Rojas et al., 2007; Vuillermot et al., 2011a,b). These data all suggest
that altered expression of Nurr1 might be a potential genetic risk factor for
dopamine-related disorders.

C. NURR1 EXPRESSION LEVELS AFFECT THE VULNERABILITY OF DOPAMINERGIC

NEURONS IN RESPONSE TO DOPAMINERGIC CYTOTOXIC REAGENTS

To investigate whether Nurr1 expression levels also affect dopaminergic

neurons in response to variety of toxic stresses, studies have been carried out in
Nurr1 heterozygous mice (þ/
) in different animal models. It has been shown
that Nurr1 heterozygous mice are more vulnerable to injury induced by the
dopaminergic toxin MPTP (Le et al., 1999). Our group has recently examined
the synergistic effects of repeated early exposure to methamphetamine in adoles-
cence and reduction in Nurr1 gene levels (Luo et al., 2010). METH binge
exposure in adolescence led to greater damage in the NS dopaminergic system
when mice were exposed to METH binge later in life, suggesting a long-term
adverse effect on the dopaminergic system. Compared to naı̈ve mice that received
METH binge treatment for the first time, mice pretreated with METH in
adolescence showed a greater loss of TH immunoreactivity in striatum, loss of
THir fibers in the substantia nigra reticulata (SNr) as well as decreased DAT levels
and compromised DA clearance in striatum. These effects were further exacer-
bated in Nurr1 heterozygous mice (Fig. 2) (Luo et al., 2010). Thus, early METH
binge exposure in young adulthood leads to long-term effects in the NS system
and results in a more marked dysfunction when animals are exposed to METH
again later in life. This adverse effect is further exacerbated in mice that have a
decreased level of Nurr1. Therefore, lowered Nurr1 levels may predispose
individuals to greater acute and/or long-term toxicity of METH in the nervous
system. It is possible that mutations or polymorphisms in the Nurr1 gene in
humans, which lead to lower levels of Nurr1 expression, may predispose certain
individuals to a greater susceptibility to neuronal disorders or greater susceptibil-
ity to neurotoxicants after repeated exposure. Small molecules that can regulate
Nurr1 function and activity might be a candidate for medication development for
METH toxicity and PD. In support of this hypothesis, Nurr1 has been implicated
in human pathological conditions. It has been reported that Nurr1 expression is
diminished in neurons with alpha-synuclein inclusions in postmortem PD brain
tissue (Le et al., 2008); Nurr1 mutations and polymorphisms have also been
identified in rare cases of PD (Grimes et al., 2006; Le et al., 2003, 2008;
10 YU LUO

A B
120

Striatum DAT immunoreactivity

+/+ +/- 100

(% of wt mean)
80

60

40
Control 20

0
+/+ +/-
120

Striatum DAT immunoreactivity

100

(% of wt mean)
80

1XMETH 60

40

20

0
+/+ +/-
120 *

Striatum DAT immunoreactivity

100

(% of wt mean)
80
2XMETH
60

40

20

DAT immunoreactivity 0
+/+ +/-

FIG. 2. Nurr1 heterozygous mice has decreased DAT expression levels in striatum after metham-
phetamine exposures. (A) Nurr1 þ/
mice showed decreased DATir after 2 METH treatment
compared to þ/þ animals, but not in saline-treated or 1 METH-treated groups, a result consistent
with decrease DA clearance in 2 METH-treated Nurr1 þ/
mice. (B) Quantitative analysis of
DATir represented as percentage of þ/þ mice * indicate that there is a significant difference
(p < 0.001, two-way ANOVA, post hoc Newman-Keuls test). (Figure published in Luo et al., 2010).

Xu et al., 2002). Furthermore, recent epidemiological studies have described a

Nurr1 exon mutation (Le et al., 2003) or intron polymorphism in families with
increased PD prevalence (Xu et al., 2002; Zheng et al., 2003). Taken together, these
data suggest that deficiency in Nurr1 expression may enhance susceptibility to
neuronal damage in dopaminergic neurons, which leads to PD-like symptoms in
animals and man.

VI. The Mechanisms of Nurr1 as a Nuclear Receptor

Given the important function of Nurr1 in the NS DA system, much effort has
been put into studying the mechanisms through which Nurr1 regulates the
development of DANs. Nurr1 is a member of the nuclear receptor superfamily
of ligand-activated transcription factors. The nuclear receptor family includes
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 11

AF-1 DBD Hinge LBD AF-2

FIG. 3. Domain structure of Nurr1 protein.

receptors for steroid hormones, retinoic acid, thyroid hormone, and vitamin D
(Mangelsdorf et al., 1995). NGFI-B, Nor1, and Nurr1 constitute a subfamily of
three highly homologous receptors, which like many other nuclear receptors, lack
identified ligands and are therefore referred to as ‘‘orphan receptors’’ (Law et al.,
1992). Nuclear receptors have a common structural organization, with a con-
served DNA-binding domain (DBD) and a somewhat less conserved ligand-
binding domain (LBD). As a member of the nuclear receptor family, Nurr1 has
a structure similar to other members in this family (Wansa et al., 2002). It is
composed of four independent but interacting functional modules, which from
N-terminal to C-terminal are the modulator domain, the DBD, the hinge region,
and LBD (Fig. 3). The modulator domain, which is also called the A/B domain,
displays the most variability both in terms of length and primary sequence. The
modulator domain contains a transcriptional activation function, referred to as
AF-1. The DBD recognizes specific DNA sequences which are termed the
hormone response elements. For Nurr1, one of the identified elements is the
NBRE (Wilson et al., 1991). The DBD of nuclear receptors is composed of two
zinc finger modules encoded by 66–70 aa and a carboxy-terminal extension
which is the most conserved domain of the nuclear receptor. The C-terminal
portion is the LBD of the nuclear receptor. Although it is called the LBD, it is a
multifunctional domain and it mediates ligand binding, dimerization, and ligand-
dependent transactivation (AF2 domain) in many nuclear receptors (Castillo et al.,
1998a; Castro et al., 1999; Giguere, 1999).
In general, nuclear receptor activity can be regulated by several different
mechanisms. It can be regulated by direct binding of small lipophilic ligands, by
protein–protein interactions with other transcription factors, and by posttransla-
tional modification such as phosphorylation after stimulation of cell surface
receptors or by cyclin-dependent kinases. Eventually, nuclear receptors bind to
DNA elements and regulate certain sets of downstream target genes that carry out
various biological functions (Giguere, 1999). Thus, understanding the down-
stream targets of Nurr1 is important for understanding its molecular action.

A. DOWNSTREAM TARGET GENE OF NURR1

As a transcription factor, it is reasonable to speculate that Nurr1 may regulate

the development of the dopaminergic phenotype by regulating the transcription
of certain groups of genes. A few studies have tried to identify the target genes
12 YU LUO

regulated by Nurr1. Sakurada et al. (1999) have reported that Nurr1 is able to
induce TH expression in a progenitor cell line derived from rat hippocampus,
possibly through the binding site of Nurr1 (NBRE) in the TH promoter (nucleo-
tides –873 to –866). Sacchetti et al. (2001) has reported that Nurr1 can regulate the
transcription of the DAT in an NBRE-independent manner in vitro. This has not
been confirmed in an in vivo model such as in knockout mice. Moreover, the
receptor tyrosine kinase signaling subunit Ret is absent in early stages of develop-
ment in Nurr1 mutant embryos, suggesting that Nurr1 regulates Ret gene expres-
sion in vivo (Wallen et al., 2001). Other recent discovered in vivo and in vitro target
genes for Nurr1 include BDNF (Volpicelli et al., 2007), Neuropilin1 (Hermanson
et al., 2006), Dlk1, Ptpru, and Klhl1( Jacobs et al., 2009b). To characterize the
Nurr1-regulated gene program, we undertook a series of studies to identify genes
whose expression was modulated by Nurr1 in the context of a cell with dopami-
nergic properties, the MN9D line. The parental clonal line of MN9D chosen for
these experiments, expressed virtually undetectable levels of Nurr1 and thus
represents a suitable line into which Nurr1 could be introduced. Using the
tetracycline autoregulated Nurr1–Big2i construct, we obtained a number of stable
transfectants that expressed Nurr1 mRNA and protein in response to doxycycline
administration.
To provisionally identify Nurr1-regulated genes we employed differential
display analysis. Amplified fragments that were reproducibly altered with Nurr1
induction were cloned, sequenced, and identified. Among this group of Nurr1
regulated genes was VIP, encoding the potent multifunctional neuropeptide
(Moody et al., 2003; Rostene, 1984).
In our study (Luo et al., 2007), VIP was induced at the mRNA and gene
product levels in MN9D cells expressing Nurr1. This could have been the result of
a direct or secondary transcriptional event. We first examined the possibility that
VIP was a primary transcriptional target of Nurr1. Nurr1 mediates its transcrip-
tional action through direct binding to cis elements known as NBREs (Perlmann
and Jansson, 1995; Wilson et al., 1991, 1992). Upon analysis of the mouse VIP
promoter, we discovered candidate NBREs at approximately
5 kb from the
transcription start site. These VIP NBREs share a core sequence (TGACCTTT)
identified in other studies (Wilson et al., 1992). The function of these cis elements
for Nurr1 binding and transcription enhancement were demonstrated by electro-
phoretic gel-shift and promoter–reporter gene transfection studies, respectively.
The magnitude of the effect of Nurr1 on the VIP promoter was highly
significant and NBRE-dependent. Basal transcriptional control of VIP is cell-
type specific and dynamic modulation is afforded by multiple signal transduction
pathways (Agoston et al., 1990; Hahm and Eiden, 1996, 1998a; Symes et al., 1995;
Tsukada et al., 1987; Waschek et al., 1988). Mutational analysis of the VIP
promoter has revealed a tissue-specifier element lying between
4.2 and

4.7 kb relative to the cap site (Hahm and Eiden, 1998a). This region restricts
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 13

cell-type transcriptional control through several octamer-like sequences that can

bind the POU homeodomain proteins Oct-1 and Oct-2. Stimulus promoted
transcription of the VIP gene is regulated by upstream transducing second
messenger cascades including PKA (Waschek et al., 1987), PKC (Adler and
Fink, 1993), JAK/STAT (Symes et al., 1994), and CREB (Fink et al., 1991). This
regulatory complexity allows the gene to be transactivated in response to cyto-
kines (Eskay and Eiden, 1992; Rao et al., 1992; Symes et al., 1993), electrical
depolarization (Girard et al., 2002), secretatogues (Waschek et al., 1987), and
growth factors (Hahm and Eiden, 1998b). Our data indicate that in addition
Nurr1 can directly activate VIP expression. The extent to which signal cross talk
emanating from other pathways may modulate Nurr1 dependent transactivation
is currently unknown.
The data presented in our study raise the issue as to the potential role of VIP
in the maturation and/or maintenance of the dopaminergic phenotype of A9
ventral midbrain neurons. While there are no reports linking VIP to dopaminer-
gic function, there are multiple studies demonstrating a robust neuroprotective
role for VIP when dopaminergic neurons are challenged with neurotoxicants.
In cultured PC12 and SY5Y neuroblastoma cells, VIP was neuroprotective in
nanomolar concentrations against challenge with 6-OHDA (Offen et al., 2000).
Similarly, in the mouse VIP delivery prior to MPTP challenge markedly protected
SNc dopaminergic neurons from injury (Delgado and Ganea, 2003). These
studies underscore the potency of VIP as a dopaminergic neuroprotective peptide
and suggest that Nurr1 transactivation of the VIP promoter would render dopa-
minergic neurons more resistant to oxidative stressors such as neurotoxicants.
A recent study by an independent group has confirmed that the VIP promoter
region is highly enriched by Nurr1 chromatin immunoprecipitation (ChIP). More
importantly, the same VIP promoter region was also enriched by Pitx3-ChIP,
suggesting that both Pitx3 and Nurr1 bind to the same promoter region of the
Nurr1 transcriptional target VIP ( Jacobs et al., 2009a). Identification of additional
target genes of Nurr1 will not only provide insights into the development of
midbrain dopaminergic neurons but also provide useful information in designing
strategies to delineate survival factors that might be helpful to PD patients.

B. REGULATION OF NURR1 ACTIVITY

Nurr1 could recognize DNA as a monomer, homodimer, and function as a

constitutively active transcription factor even in the absence of an identified ligand
(Zetterstrom et al., 1996b). However, this does not exclude the possibility that
Nurr1’s activity can be regulated. Indeed, it has been reported that Nurr1’s
transcriptional activity is highly regulated by unknown factors (Castillo et al.,
1998a; Castro et al., 1999). Transcriptional activity of the Nurr1 carboxyl-terminal
14 YU LUO

domain varies in different cell types and requires the integrity of the AF2 domain
(Castro et al., 1999). Also, Nurr1’s ability to induce DAN is cell-type dependent
(Sakurada et al., 1999) and can be regulated by secreted factors from midbrain glial
cells (Wagner et al., 1999). One possible explanation for the divergent activity is the
presence of a yet unknown endogenous ligand for Nurr1. This has been an open
question because on one hand Nurr1 does exhibit a constitutive activity which does
not seem to rely on a ligand, while on the other hand, it does have a conserved
domain that is structurally homologous to LBDs of other nuclear receptors. The
question was at least partially resolved when the crystal structure of the NLBD
revealed that the Nurr1 LBD contains no cavity due to the tight packing of side
chains from several bulky hydrophobic residues in the region normally occupied by
ligands (Wang et al., 2003). This finding suggests Nurr1 may be part of a unique
structural class of NRs, which show a ligand-independent NR function.
Another possible mechanism to regulate Nurr1 activity is through protein–
protein interactions. Nurr1 can be regulated by a protein interactor and works
coordinately to specify a midbrain DAN program, which is a common mechanism
for nuclear receptors (Giguere, 1999). There is some evidence supporting this
hypothesis. It has been reported that Nurr1 can form heterodimers with the
retinoid X receptor alpha (RXRa) and bind to certain retinoic acid-responsive
elements (Perlmann and Jansson, 1995). However, interaction of Nurr1 and
RXRa does not seem to be responsible for the induction of the DA phenotype
and the role of RXR seems to be dual. In the absence of a ligand for RXR, the
RXR–Nurr1 heterodimer has decreased activity on NBRE element compared to
a Nurr1 monomer or homodimer (Aarnisalo et al., 2002). It is possible that RXRa
binds to Nurr1, when there is no ligand for RXR, preventing it from binding with
other protein(s) that is (are) responsible for the induction of certain downstream
genes. Consistent with this speculation, Sakurada et al. has reported that over-
expression of RXRa decreased TH expression induced by Nurr1 in neural
progenitor cells (NPCs; Sakurada et al., 1999). However, when the ligand for
RXR is present, the Nurr1–RXR dimer can activate Nurr1’s transcription on
NBRE (Aarnisalo et al., 2002). This dual effect of RXR on Nurr1 activity,
depending on whether ligand is available, might be a mechanism to differentially
regulate Nurr1 activity in vivo depending on the synthesis and distribution of
retinoic acid. Recently, PIASg has been reported to be a Nurr1-interacting protein
which functions as a repressor of Nurr1’s transcriptional activity instead of as a
coactivator (Galleguillos et al., 2004). Interestingly, a target gene of Nurr1,
P57kip2, seems to cooperate with Nurr1 in promoting differentiation of DA
cells, possibly by a direct protein–protein interaction ( Joseph et al., 2003). Fur-
thermore, Nurr1 can also enhance the transcription of the hDAT gene through a
DNA-binding independent mechanism (Sacchetti et al., 2001), further suggesting
that Nurr1 might interact with other proteins to regulate gene expression as a
coactivator or repressor. Taken together, all of these studies suggest that
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 15

protein–protein interactions could be an important mechanism by which Nurr1’s

activity can be regulated, thus affecting the induction and maturation of midbrain
dopaminergic neurons. Unlike known regulators of Nurr1 transciptional activity
such as p57kip2 and PIASg which are both negative regulators that suppress
Nurr1 activity, we have identified a novel interacting protein of Nurr1 that might
enhance Nurr1 transcriptional activity (Luo et al., 2008).
Using the NLBD as bait in the yeast two-hybrid screen, we identified a Nurr1
interacting protein (NuIP). By alternative splicing, the NuIP gene generates a
variety of transcript products. The full-length NuIP is a protein of about 150 kDa
which is expressed mainly in the CNS in a pattern that is very similar to Nurr1
(Luo et al., 2008). The interaction of NuIP with Nurr1 in mammalian cells is
confirmed both by mammalian 2-hybrid assay and coimmunoprecipitation in a
dopaminergic cell line, MN9D cells. Furthermore, NuIP appears to be a Nurr1
transcriptional coregulator. When coexpressed in mammalian cells, it promotes
the transcriptional activity of Nurr1 both on NBRE driven reporters as well as the
endogenous TH promoter, a known target gene of Nurr1 (Luo et al., 2008). Our
results also suggest that NuIP can further promote Nurr1–RXR heterodimer
activity on NBRE driven reporters.
Apparently, NuIP protein interacts with Nurr1 in a different manner than
RXRa, since RXRa cannot form a heterodimer with the AF2-deleted NLBD
(Castro et al., 1999; Perlmann and Jansson, 1995) while NuIP interacts with both
the full-length and truncated forms of NLBD. This suggests that NuIP might
interact with Nurr1 at a different interface. Unlike other protein interactors that
negatively regulate Nurr1 activity, such as p57kip2 ( Joseph et al., 2003) and PIASg
(Galleguillos et al., 2004), NuIP potentiates Nurr1’s transcriptional activity on
both NBRE and endogenous TH promoter driven reporter constructs. In addi-
tion, NuIP further potentiates the activity of Nurr1 in Nurr1–RXR heterodimer
on NBRE driven reporter constructs. The mechanism underlying the ability of
NuIP to positively regulate the transcriptional activity of Nurr1 is not clear. As a
representative of a class of ligand-independent nuclear receptors, Nurr1 does not
need a ligand to activate its transactivation activity. Instead, its active conforma-
tion is maintained by intramolecular interactions between the side chains of
amino acids in the so-called LBD (Wang et al., 2003). In the same paper, Wang
et al. have exploited an assembly assay to evaluate the assembly of two LBD
fragments and have demonstrated that the assembly of the H1 and H3–12
domains of the NLBD correlates well with Nurr1’s transcriptional activity.
Using the assembly assay, we tested the hypothesis that NuIP protein promotes
Nurr1’s transcriptional activity by facilitating the assembly of domains of NLBD
and by doing so stabilizes the NLBD. Indeed, when NuIP was coexpressed, the
assembly of the two domains was potentiated almost twofold, suggesting that
NuIP protein does facilitate the assembly of NLBD. The mechanism underlying
this effect is not clear and needs to be further examined.
16 YU LUO

Another important question is whether NuIP is expressed in midbrain dopa-

minergic neurons, which will put NuIP into the right cell context for a Nurr1
coregulator. Both RT-PCR and immunohistochemistry results showed that NuIP
transcripts and protein are present in Nurr1 positive midbrain dopaminergic cells
during development as well as in adulthood (Luo et al., 2009). Sustained expres-
sion of NuIP in midbrain dopaminergic neurons suggests it might have an
important role in these cells. Although NuIP protein can be detected in midbrain
DANs, the expression of NuIP is not confined to this region. Rather, several
regions in the brain show NuIP and Nurr1 coexpression including cortex, hippo-
campus, and cerebellum. However, there are also regions that exclusively express
NuIP such as striatum, septum, globus pallidus, and the reticular thalamic
nucleus. We also find that NuIP protein expresses mainly in NeuN-positive
(Neuronal Nuclei) neurons but can be detected in GFAP-positive (glial fibrillary
acidic protein) glial cells in hippocampus. Interestingly, NuIP is expressed at high
levels in midbrain dopaminergic neurons including VTA and SN dopaminergic
neurons but is not expressed or expressed in low levels in other dopaminergic
neurons such as olfactory bulb and hypothalamus (Luo et al., 2009). Identification
of additional Nurr1-interacting proteins or small pharmacological molecules that
can enhance the function of Nurr1 stimulating interactors are needed to further
explore the potential therapeutic application of Nurr1.

VII. Most Recent Development in Application of Nurr1 in Dopaminergic Differentiation

and Implications in Future Treatment for PD

Exciting progress has been made recently regarding Nurr1 function and
dopaminergic differentiation. Lee et al. (2010) has reported that Foxa2 and
Nurr1 synergistically facilitated the generation of nigral (A9) specific midbrain
dopaminergic neurons and more importantly, the effects of Foxa2 and Nurr1 in
DA differentiation were observed regardless of the brain regions or species from
which NPCs were derived. This suggests that a combination of critical differen-
tiation factors are capable of ‘‘switching’’ the fate of progenitor cells even though
they originate from a different brain region. Remarkably, a recent study (Caiazzo
et al., 2011) has provided further support for this hypothesis, showing direct
generation of functional dopaminergic neurons from mouse and human
fibroblasts. A minimal set of three transcription factors—Mash1, Nurr1 and
Lmx1a—are able to convert both prenatal and adult fibroblast cells into func-
tional dopaminergic neurons. The authors reported that the induced dopaminer-
gic neurons (iDA) release dopamine and demonstrate the characteristic electrical
activity of dopaminergic neurons. Since the risk of tumor formation is one of the
 THE FUNCTION AND MECHANISMS OF NURR1 ACTION 17

major concerns of embryonic stem cell-derived transplantation, the direct gener-

ation of iDA cells from somatic cells without conversion back into a pluripotency
state have significant implications for future treatment of PD. Furthermore,
identification of small pharmaceutical molecules that can directly activate endog-
enous transcription factor (such as Nurr1) might offer more preferable options in
future clinical treatment which avoids the usage of transducing agents.

Acknowledgment

The author would like to thank Dr. Barry Hoffer for his helpful discussions and
suggestions.

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 MONOCLONAL ANTIBODIES AS NOVEL NEUROTHERAPEUTIC
AGENTS IN CNS INJURY AND REPAIR

Aruna Sharma and Hari Shanker Sharma

Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology &
Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden

Abstract
I. Introduction
II. Historical Perspectives on the Use of Antibodies as Therapy
III. Therapeutic Basis of Antibodies
IV. Antibodies Versus Receptor Antagonist Drugs
V. Antibodies Neutralize Effects of Endogenous Antigens
VI. Our Investigations on Monoclonal Antibodies to Induce Neuroprotection in CNS Injuries
VII. Neuroprotective Effects of Serotonin Antibodies in CNS Injuries
A. Serotonin Antibodies Induce Neuroprotection in Spinal Cord Injury
B. Serotonin Antibodies are Neuroprotective in Closed Head Injury
VIII. Neuroprotection by Dynorphin A Antibodies in CNS Injuries
A. Dynorphin A Antibodies Are Neuroprotective in SCI
B. Dynorphin Antibodies Modulate Endogenous NOS Activity in SCI
IX. Antibodies to nNOS Is Neuroprotective in CNS Injuries
X. TNF-a Antibodies Are Neuroprotective in CNS Injuries
A. Antibodies to TNF-a Is Neuroprotective in Hyperthermic Brain Injury
XI. Combination of nNOS and TNF-a Antibodies Enhances Neuroprotection in SCI
XII. Conclusion and Future Perspectives
Acknowledgments
References

Abstract

Central nervous system (CNS) injury is a complex in which numerous neuro-

chemicals and other vasoactive agents actively contribute towards the development
of posttraumatic brain pathology and/or repair mechanisms. A focal trauma to the
brain or spinal cord releases several endogenous neurodestructive agents within the
CNS, resulting in adverse cellular reactions. Our laboratory is engaged in identify-
ing these endogenous neurodestructive signals in the CNS following injury caused
by trauma or hyperthermia. Our observations show that serotonin (5-HT), dynor-
phin A (Dyn A 1–17), nitric oxide synthase (NOS), and tumor necrosis factor-a

INTERNATIONAL REVIEW OF 23 Copyright 2012, Elsevier Inc.

NEUROBIOLOGY, VOL. 102 All rights reserved.
DOI: 10.1016/B978-0-12-386986-9.00002-8 0074-7742/12 $35.00
24 ARUNA SHARMA AND HARI SHANKER SHARMA

(TNF-a) could be potential neurodestructive signals in the CNS injury. Thus,

neutralization of these agents using monoclonal antibodies directed against 5-HT,
NOS, Dyn A (1–17), and TNF-a in vivo will result in marked neuroprotection and
enhance neurorepair after trauma. In addition, a suitable combination of mono-
clonal antibodies, for example, NOS and TNF-a, when applied 60–90 min after
trauma, is capable to enhance neuroprotective ability and thwart cell and tissue
injury after spinal cord insult. Taken together, our novel observations suggest a
potential use of monoclonal antibodies as suitable therapeutic agents in CNS
injuries to achieve neuroprotection and/or neurorepair.

I. Introduction

Antibodies either monoclonal or polyclonal directed against several neuro-

transmitters, enzymes, or receptor proteins could mimic the biological function of
their physiological natural ligands in in vivo or in vitro situations (Prammer et al.,
1994; Sharma and Sharma, 2008; Taub et al., 1989; Waldmann, 2003). Thus,
specific antibodies raised against neurotransmitters, enzymes, and/or their re-
ceptors are able to antagonize their function much more precisely and effectively
than their pharmacologically active ligands (Frelinger et al., 1990; Garcia et al.,
1992a,b; Kasirer-Friede et al., 2007; Puzon-McLaughlin et al., 2000). This is
largely due to structural similarity between antibodies and the ligands that they
mimic (Frelinger et al., 1991; Prammer et al., 1994). These physicochemical and
biological properties of antibodies could thus be used for new therapeutic strat-
egies against structural and functional disorders of the central nervous system
(CNS) caused by various neurological diseases in clinical settings.

II. Historical Perspectives on the Use of Antibodies as Therapy

Antibodies as therapy for different diseases could be traced back as early as

1796, about two centuries ago (see Sharma and Sharma, 2008) (Table I). How-
ever, their use in patient care was first proposed in 1965, only 37 years ago (see
Sharma and Sharma, 2008), and the United States Food and Drug Administra-
tion (FDA) approved the use of antibodies to treat certain types of cancer and
autoimmune diseases in as late as 1998, just 14 years ago (Table II) (see Sharma
and Sharma, 2008). However, till date, only a few studies showed the usefulness of
antibodies therapy for neuropathic pain, Alzheimer’s disease, and stroke (see
 MONOCLONAL ANTIBODIES: NOVEL NEUROTHERAPEUTIC AGENTS 25

Table I
HISTORY OF ANTIBODIES THERAPY.

Year Scientist Details Disease

1796 Jenner Cowpox immunization Small pox

1879 Louis Pasteur Chicken-cholera Cholera
bacterium
1886 Louis Pasteur Rabies Rabies vaccination
1888 Emile Roux Diphtheria toxin Diphtheria
Alexandre Yersin
1890 Emil von Behring Diphtheria toxin Diphtheria
Shibasabo Kitasato
1900 Paul Ehrlich Molecules–tumor reaction Cancer therapy using antibody-
mediated immune therapy
1954–1955 Jonas Saik Attenuated polio virus Poliomyelitis
Albert Sabin
1975 George Köhler Production of monoclonal
Cesar Milstein antibodies

Table II
ANTIBODIES DEVELOPED AND USED FOR THERAPEUTIC PURPOSES.

Year Antibody Disease Possible use

1965 IgG, anti-D, anti-RH Erythroblastosis fetalis RH immunization

1982 Anti-idiotype antibody B-cell lymphoma Human neoplasm
1986–2000 IL-2, IFN-b, IFN-g Neoplasia, hepatitis, Approved by US FDA
multiple sclerosis
1998 TNF-a p75 TNF-receptor Rheumatoid arthritis, Antagonism of Fc of IgG1
Crohn disease

Data after Sharma and Sharma (2008).

Sharma and Sharma, 2008). Thus, further studies on antibodies therapy by

neutralizing the endogenous neurodestructive elements are needed to achieve
neuroprotection in several neurodegenerative disorders.

III. Therapeutic Basis of Antibodies

Antibodies are large heterodimeric molecules that are composed of two types
of polypeptide chains, the heavy and the light (see Frelinger et al., 1991; Sharma
and Sharma, 2008). Using specific ligands for particular polypeptide chain, the
antibodies could be targeted to specific cells or proteins to stimulate the body’s
26 ARUNA SHARMA AND HARI SHANKER SHARMA

immune system or to attack foreign or tumor cells (Frelinger et al., 1991). Binding
of antibodies to specific cell targets, for example, receptors, may block tumor
growth, for example, radioimmunotherapy (Table II) (see Waldmann, 2003).
Structure–function studies of antibodies and ligand interaction show that
three anti-GPIIb–IIIa murine monoclonal antibodies, PAC-1, LJ-CP3, and
OP-G2, and their RYD sequence in their H-CDR3 domain occupy the same
conformational space as RGD in conformationally constrained, bioactive,
GPIIb–IIIa cell-surface adhesion ligands (Frelinger et al., 1990; Garcia et al.,
1992a,b; Prammer et al., 1994; Sharma and Sharma, 2008). This suggests that
antibodies are more specific regarding their binding to certain cell targets or
receptors. These properties of the antibodies could be utilized as fine probes to
identify motifs of short adhesion stretches in the biomolecules needed for new
drug design and development (see Sharma and Sharma, 2008).

IV. Antibodies Versus Receptor Antagonist Drugs

Monoclonal antibodies directed against receptor proteins are often more power-
ful in antagonizing natural ligand of the receptor than the receptor antagonist drugs
(see Table II). This is largely due to their highly specific binding with the receptor
protein in vivo as compared with drug molecules (Garcia et al., 1992a,b; Puzon-
McLaughlin et al., 2000; Taub et al., 1989). This specific binding of anti-idiotypic
antibodies to the physiological membrane receptors could induce biochemical mod-
ulation like their natural ligands (Bentley et al., 1990; Frelinger et al., 1991; Jerne, 1974).
This suggests that the antibodies will ‘‘mimic’’ or act as ‘‘internal image’’ of the ligand
identified by the physiological receptor (see Puzon-McLaughlin et al., 2000).
In addition, antibodies could influence the receptor function by molecular
mimicry of the ligand to locate the receptor because of their particular structural
characteristics (Garcia et al., 1992a,b; Puzon-McLaughlin et al., 2000). Monoclo-
nal antibodies produced against the octapeptide hormone angiotensin II bind
with the hormone with very high affinity (association constant K2 > 109 M
1)
(Picard et al., 1986). The antibodies produced against angiotensin II or its receptor
(s) specifically bind to the target and sometimes interact with angiotensin analogs
responsible for the bioactivity (Couraud, 1987). The antibodies directed against
the carboxyl-terminal region of the angiotensin II bind to the same region that is
the most important for hormonal activity and sensitive to amino acids substitutes
(Couraud, 1986). Thus, an antibody can act as ‘‘surrogate receptor’’ for the
particular bound state of the peptide (Garcia et al., 1992a,b; Puzon-McLaughlin
et al., 2000; Reichmann et al., 1989). This suggests that antibodies binding to
receptors could be more strong and effective than the drug–receptor interaction
 MONOCLONAL ANTIBODIES: NOVEL NEUROTHERAPEUTIC AGENTS 27

in vivo (see Kasirer-Friede et al., 2007; Puzon-McLaughlin et al., 2000; Sharma and
Sharma, 2008; Taub et al., 1989).

V. Antibodies Neutralize Effects of Endogenous Antigens

Antibodies when administered in in vivo or in vitro situations neutralize the effects

of their physiological antigens (see Sharma and Sharma, 2008; Sharma et al., 1995).
This is evident from the studies on two monoclonal antibodies (7B1 and 14F11) that
bind to the LTB4-receptor BLT1 and influence agonist binding and/or receptor
activation (Sabirsh et al., 2003). Interestingly, only one antibody (7B1) specifically
inhibited ligand binding, while both are able to inhibit receptor activation. This is
because of the fact that the two antibodies are different isotypes and may recognize
two different portions of the receptor proteins present on the cell surfaces (Sabirsh
et al., 2003). Thus, the antibodies may act as pure antagonists and neutralize the
effects of their natural physiological ligands at the receptor level without activating it
(Gifford et al., 1987; Pettersson et al., 2000). This suggests that antibody–receptor
interaction is highly specific (Chung-a-on et al., 1996; Nguyen and Taub, 2002;
Prammer et al., 1994; Sabirsh et al., 2003) and could be used as a powerful tool to
neutralize the harmful effects of the physiological ligands in clinical situations.

VI. Our Investigations on Monoclonal Antibodies to Induce Neuroprotection in CNS Injuries

The potential therapeutic potential of select monoclonal antibodies directed

against neurodestructive factors (see Table III) was examined in our laboratory in
animal models of neurotrauma- or hyperthermia-induced brain damage. Our
observations showed that monoclonal antibodies might be used as suitable ther-
apeutic agents to achieve neuroprotection.

VII. Neuroprotective Effects of Serotonin Antibodies in CNS Injuries

Serotonin is one of the phylogenetically oldest neurochemical present in the

CNS (Sharma, 2009) that is often colocalized with many other neurotransmitters,
particularly, substance P, nitric oxide (NO), and calcitonin gene-related peptide
(CGRP) (Hökfelt et al., 1987). The amine is a known mediator of the blood–brain
barrier (BBB) breakdown and vasogenic edema formation (Sharma et al., 1990)
28 ARUNA SHARMA AND HARI SHANKER SHARMA

Table III
MONOCLONAL ANTIBODIES AGAINST ENDOGENOUS NEURODESTRUCTIVE ELEMENTS IN CNS INJURY
INDUCES NEUROPROTECTION.

Monoclonal
Antibodies Used Neuroprotection CNS Injury Model References

Serotonin Yes Brain and spinal cord injury Sharma et al. (1997, 2007)
Nitric oxide Yes Brain and spinal cord injury Sharma and Alm (2004)
Tumor necrosis Yes Spinal cord injury, heat stress Sharma et al. (2003)
factor-a
Dynorphin A Yes Spinal cord injury Sharma et al. (1995)

For details, see Sharma and Sharma (2008).

and capable to influence prostaglandin (PG) synthesis and its release in the CNS
(Sharma and Westman, 2004). Serotonin could act on the CNS through more
than seven kinds of receptors with multiple receptor subtypes. Thus, using a few
selective receptor blocker drugs will not give a clear view on its role in neuro-
trauma (see Sharma, 2004a,b; Sharma et al., 1990). Therefore, serotonin anti-
bodies could be more a powerful tool to antagonize the physiological functions of
the amine following CNS injuries (Frelinger et al., 1990; Garcia et al., 1992a,b).
A. SEROTONIN ANTIBODIES INDUCE NEUROPROTECTION IN SPINAL CORD INJURY

Serotonin antibodies (monoclonal 5-HT antiserum DAKO, Hamburg, Ger-

many, 1:20 in phosphate-buffered saline; 25 ml in 10 s) when applied over the
traumatized spinal cord 2 min after a rat model of focal spinal cord injury (SCI,
see Fig. 1) resulted in marked neuroprotection at 5 h (Sharma et al., 1997) as
compared with rabbit serum or neutralized 5-HT antibodies (Sharma et al., 1997).
In 5-HT antiserum-treated group, edema formation, microvascular perme-
ability disturbances, and cell injury at 5 h after trauma were considerably reduced
(Fig. 1) (Hökfelt et al., 1987; Sharma, 2004a,b; Sharma et al., 1997). These
observations provide strong evidences that 5-HT antiserum is neuroprotective in
SCI (Sharma, 2004a,b; Sharma et al., 1997).

B. SEROTONIN ANTIBODIES ARE NEUROPROTECTIVE IN CLOSED HEAD INJURY

This neuroprotective effect of serotonin antiserum was also seen in a model

of closed head injury (CHI) in rats. CHI was produced by inflicting an impact of
0.224 N on the right parietal skull bone by dropping a weight of 114.6 g from
a height of 20 cm through a guide tube under anesthesia (Sharma et al., 2007)
(see Fig. 2).
 MONOCLONAL ANTIBODIES: NOVEL NEUROTHERAPEUTIC AGENTS 29
A
L

T9 T12
B
1 L

T12 untreated 5-HT abs-treated

E F

72 3 [131]-Iodine
T9 Water content % a T9 % b
T10–11
T10–11
70 T12 T12
2
68

66
1
64

62 0
Control SCI 5 h SCI+5-HT SCI+Neutr Control SCI 5 h SCI+5-HT SCI+Neutr
abs abs abs abs

FIG. 1. New model of spinal cord injury (a,b). A longitudinal lesion (L) on the right dorsal horn of
the T10–11 segment was made (a) and the tissue samples were collected from the adjacent rostral (T9)
and caudal (T12) segment for morphological analysis. The deepest part of the lesion is lying close to the
Rexed Lamina VIII (b). Tissue samples for electron microscopy were processed from contralateral
dorsal (1) and ventral (2) horns, respectively (b). Five-hour injury resulted in swelling and distortion of
the spinal cord (c) that was markedly attenuated by topical application of serotonin antiserum (d) over
the traumatized spinal cord. Treatment with serotonin antiserum also reduced significantly the water
content (e) and extravasation of radiotracer (f) across the spinal cord segments compared to the control
or neutralized antibodies. * P < 0.05 from control group, D p < 0.05 from spinal cord injury (SCI) 5 h.
Data modified after Sharma (2004a,b), Sharma et al. (1997), and Sharma and Sharma (2008).
30 ARUNA SHARMA AND HARI SHANKER SHARMA

A
a b 114.6 g

Left Right

Total impact
0.224 N

20 cm h
Impact injury model
B

FIG. 2. (A) Model of closed head injury (CHI) in rats. Under Equithesin anesthesia, silicon-coated
iron bar weighing 114.6 g was dropped over the right parietal skull bone (o) from a 20 cm height
through an aluminum guide tube (see Sharma et al., 2007). This impact induces concussive injury in
animals and do not normally induce fracture of the skull. After injury, the rats were allowed to survive
under anesthesia for 5 h (for details see text). (B) Nissl-stained nerve cells in the parietal cerebral cortex
of one untreated injured (a,b) and one serotonin (5-hydroxytryptamine, 5-HT) antibodies (abs)-treated
(c,d) rat. The impact injury (0.224 N) on the right parietal skull bone was made under Equithesin
anesthesia (for details, see text, Fig. 1). Five hours after closed head injury (CHI), profound edema (*),
sponginess, and neuronal damage (arrows) are seen in both the hemispheres (a,b). However, due to
counter-coup concussive insult, the injury severity in terms of edematous swelling and loss of nerve cells
is most pronounced in the contralateral hemisphere (left half, LH) compared to the injured right half
 MONOCLONAL ANTIBODIES: NOVEL NEUROTHERAPEUTIC AGENTS 31

The monoclonal serotonin antiserum (DACO, Hamburg, Germany; 30 ml

diluted to 1:20, 1:50, and 1:100) was administered at the rate of 3 ml/min for
10 min intracerebroventricularly into the left lateral cerebral ventricle either
30 min before or 30 or 60 min after CHI (see Sharma and Sharma, 2008;
Sharma et al., 2007). Serotonin antibodies administered either 30 min before or
after (but not 60 min after) CHI in high concentration (1:20) markedly attenuated
BBB permeability to Evans blue and radioiodine, and reduced edema formation
and cell injuries.
These observations demonstrate that intracerebroventricular administration
of monoclonal serotonin antibodies either 30 min before or 30 min after CHI
induces profound neuroprotection. Thus, an early intervention with serotonin
antiserum is neuroprotective in CHI.

VIII. Neuroprotection by Dynorphin A Antibodies in CNS Injuries

Dynorphin A is an endogenous opioid neuropeptide involved in inhibitory

neurotransmission in the CNS (Sharma and Westman, 2004; Sharma et al., 1995,
2006). However, high concentration of dynorphin in the spinal cord induces patho-
physiology (Sharma and Westman, 2004; Sharma et al., 1995). The major product of
dynorphin A (Dyn A 1–13 and Dyn A 1–17) causes neuronal injury through a
nonopioid mechanism (see Sharma et al., 1995). This is supported by the fact that
the neurotoxic effects of dynorphin A are prevented by N-methyl-D-aspartate
(NMDA)-glutamatergic receptor antagonist MK-801, but not by classical opioid
receptor antagonists (Sharma et al., 1995).

A. DYNORPHIN A ANTIBODIES ARE NEUROPROTECTIVE IN SCI

Monoclonal dynorphin A antiserum (1–17, Calbiochem, USA, dilution 1:20

or 1:200 in phosphate-buffered saline, 0.1 M, pH 7.0; 20 ml in 10 s) was applied
over the exposed spinal cord 2 min before or 5 min after injury (see Fig. 1) and the
animals were allowed to survive 5 h after trauma (Sharma et al., 1995). Spinal cord
conduction before and after injury was recorded using the spinal cord-evoked

(RH). Intracerebroventricular administration of monoclonal 5-HT abs (1:20) into the left cerebral
ventricle 30 min after CHI markedly attenuated neuronal loss (arrow heads) and reduced the devel-
opment of edema in the cerebral cortex (c,d). Thus, more healthy and dense nerve cell population can
be seen in the 5-HT abs-treated injured rat (c,d). This effect of 5-HT abs was most pronounced on the
left hemisphere. Probably, local administration of 5-HT abs in left side could be partially responsible for
this (for details, see text). Bars: a–d ¼ 50 mm.Data modified after Sharma et al. (2007) and Sharma and
Sharma (2008).
32 ARUNA SHARMA AND HARI SHANKER SHARMA

potentials (SCEPs) from the epidural spinal electrodes (Sharma, 2004a,b, 2010a,b;
Sharma and Westman, 2004). The mean negative amplitude (MNA), mean positive
amplitude (MPA), and their latencies were used to calculate spinal cord conduction
(Fig. 4). In addition, edema formation, cell injury, and blood–spinal cord barrier
(BSCB) breakdown was also monitored (see Sharma, 2004a,b, 2010a,b; Sharma
and Sharma, 2008).
Treatment with dynorphin A antibody prevented the decrease in the SCEP-
negative amplitude (Fig. 3), and the SCEP-positive amplitude did not develop in
this group (Fig. 4). However, an increase in SCEP-negative latencies after injury
was not reduced by the antibody treatment (Fig. 3). Dynorphin A antiserum

A a Untreated b Dyn A (1–17) antiserum E

(min)
a
-30 -30

-2 -2
0 0
2 2
4 4
10 10 1 µm
60 60
120
min 200 mV
120
100 mV b
2 msec 2 msec

B a b

SCI 5 h SCI+Dyn abs 1 µm

C Negative amplitude (%) Positive amplitude (%) Negative latency (%) Positive latency (%)
150 Controls 200 Controls 150 150
Dyn A (1–17) antiserum
a Dyn A (1–17) antiserum
b Controls
Dyn A (1–17) antiserum
c Controls
Dyn A (1–17) antiserum
d
150 125 125
100

100 100 100

50
50 75 75

0 0 50 50
-30 -2 0 2 4 10 60 120 -30 -2 0 2 4 10 60 120 -30 -2 0 2 4 10 60 120 -30 -2 0 2 4 10 60 120

D min
Spinal cord injury 5 h
3 6 Cord width mm 76 Water content %
[131]-lodine %
a T9 b T9 c
T9 T10–11 T10–11
5 75
T10–11 T12 T12
T12
2 4 74

3 73

1 2 72

1 71

0 0 70
Normal Dyn A 1–17 Normal Dyn A 1–17 Normal Dyn A 1–17
rabbit antiserum antiserum rabbit antiserum antiserum rabbit antiserum antiserum

FIG. 3. Effect of dynorphin A (1–17) antiserum on SCEP changes (A, C); gross spinal cord
pathology (B); blood–spinal cord barrier permeability, spinal cord swelling, edema formation (D),
 MONOCLONAL ANTIBODIES: NOVEL NEUROTHERAPEUTIC AGENTS 33

reduced visual cord swelling, microhemorrhages, edema, and structural changes

in the cord (Fig. 3). The effects of dynorphin A antiserum on spinal cord edema
formation, BSCB breakdown, and cell injuries were most pronounced when the
antiserum was given either 2 min before or 5 min after. Later application of the
antiserum was not effective (Fig. 3) (Sharma, 2004a,b). These observations strongly
indicate that dynorphin A antiserum when applied during the early phase of
trauma is capable to induce neuroprotection (see Sharma, 2010a,b; Sharma and
Sharma, 2008; Sharma and Westman, 2004; Sharma et al., 1995).

B. DYNORPHIN ANTIBODIES MODULATE ENDOGENOUS NOS ACTIVITY IN SCI

It appears that dynorphin neurotoxicity is mediated through mechanisms

involving NO in the brain or spinal cord (see Sharma, 2004a,b; Sharma et al.,
1990). Upregulation of neuronal nitric oxide synthase (nNOS) occurs following SCI
in the regions showing cell injury (Sharma, 2004a,b, 2010a,b; Sharma and Alm,
2004) and inhibitors of nitric oxide synthase (NOS) markedly attenuated dynorphin
A expression in the CNS during hyperthermia (Sharma, 2009; Sharma and Alm,
2004). We observed that treatment with dynorphin A antiserum significantly
attenuated NOS upregulation in the spinal cord at 5-h injury (Fig. 4) (Sharma,
2004a,b, 2009; Sharma and Alm, 2004). This observation clearly shows that
dynorphin A antiserum could attenuate nNOS expression in SCI. Furthermore,
our results also confirm that topically applied antiserum could penetrate deeper into
the spinal cord rapidly to influence cellular and molecular functions of the spinal
cord cells and tissues (Sharma, 2004a,b, 2010a,b; Sharma et al., 2006).

and ultrastructural changes (E) following spinal cord injury (SCI). Pretreatment with dynorphin A
(1–17) antiserum significantly attenuated trauma-induced SCEP changes (arrows). Thus, the depres-
sion of SCEP-negative amplitude seen in untreated injured rat (A:a, arrows) is completely absent in the
antiserum-treated rat (A:b, arrows; data modified after Winkler et al., 2002). Topical application of Dyn
antiserum even 5 min after SCI markedly reduced the spinal cord pathology (B:b) seen at 5 h in
untreated rat (B:a, bar ¼ 6 mm; data modified from Sharma et al., 1995b). In dynorphin antiserum-
treated rats, mean SCEP-negative amplitude is significantly increased (C:a) compared to the untreated
injured group. However, in antiserum-treated traumatized group, an increase in SCEP-negative
latency is significant (C:c), whereas the increase in SCEP-positive latency is reduced at 5 h (C:d; data
modified after Winkler et al., 2002). The Dyn antiserum is also capable to reduce extravasation of
radioiodine tracers in the injured as well as in the adjacent rostral (T9) and caudal (T12) segments
following SCI (D:a). The spinal cord width (D:b) and water content (D:c) are also significantly reduced.
The most pronounced effects of antiserum on reduction in BSCB permeability, spinal cord width, and
edema formation are seen in the T9 segment (D; data modified after Sharma et al., 1995b). At the
ultrastructural level, the antiserum effectively reduced the damage of myelin (arrowheads), edema (*),
and damage to neuropil (E:b) compared to the untreated traumatized rat (E:a; data modified after
Winkler et al., 2002). Values are mean  SD from 6 to 8 rats. * ¼ p < 0.05, ANOVA followed by
Dunnett’s test.Data reproduced after Sharma (2004a,b) and Sharma and Sharma (2008).
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