Dopamina

Dopamine Analysis in Neuroscience Research 97
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the surface, a cross-linker (3) is commonly bound (glutaraldehyde, carbodiimide,

hydroxymethyl phosphine, etc.), which allows anchoring of an antibody. Such a prepared
immunoparticle can be used for the separation of biomarkers from complex matrices. The
produced antigen-antibody complex is separated from the solution using an external magnetic
field (Figure 7). The covalent bond between a magnetic particle and an antibody allows
carrying out the regeneration of immunoparticles without any significant loss of the binding
capacity.
By encapsulating antibodies to polyvinylalcohol (PVA) hydrogel (consisting of a mixture
of two polymers: PVA with the function of a supporting matrix and polyethylene glycol
(PEG) as a plasticizer) and producing porous immunolenses (Figure 8), a rapid, accurate and
very easy-to-handle tool is achieved for the separation of neurotransmitters from ACSF. It
was found experimentally that even after a repeated regeneration, no reduction occurred in the
binding capacity of the immunolenses.
Samples in the most of the above applied pre-treatment methods have been typically
processed under a room temperature, at which DA and its metabolites have been proved
unstable (Syslova et al., 2011). The method, minimizing the handling of the samples at the
room temperature, is the frost drying also known as lyophilization (LYO) (in this case,
samples, when stored, had to be defrosted and processed at laboratory temperature). This is
useful for concentrating non-volatile (or slightly volatile) substances dissolved in water.
However, in contrast to this temperature-related advantage, there was no selectivity at all for
the given analytes resulting in a deteriorated ionization during the mass-spectrometric
determination even with the preceding liquid chromatography step. Apparently, this was
caused by the presence of large quantities of substances including salts (Syslová et al., 2011).
Separation of DA and its metabolites/precursor from a brain tissue is more difficult than
from ACSF due to the brain tissue containing many proteins and lipids. For their removal,
following the tissue sonication, it is advisable to use an extraction by solvents in which
denaturation of proteins occurs while the analyzed substances are extracted into it. A range of
solvents can be used for the extraction of substances from a brain tissue. Namely, ethanol
(Liu et al., 2003), methanol (Hows et al., 2004) perchloric acid (Birgner et al., 2008) or a
mixture of solutions containing acetonitrile, HCl and EDTA water solution are suitable. The
extraction of analytes using methanol or ethanol is possible, however lipids from the brain
tissue are diluted as well. Such a sample could not be analyzed directly by any detection
methods and it was necessary to carry out further separation steps as e.g. SPE. Incorporating a
further separation step decreased the yield of neurotransmitters and their metabolites to 54 –
69 % (the method consisting of the extraction with methanol or ethanol and subsequent solid
phase extraction with a C18 stationary phase and detection by LC-MS). The use of perchloric
acid resulted in the complete denaturation of proteins, the obtained supernatant did not
contain dissolved lipids, but a low stability of neurotransmitters was described in this
U.S. or applicable copyright law.
environment (Birgner et al., 2008) resulting in the method being excluded as inaccurate.
Recovery values ranged in the interval of 63 - 72 %. Therefore, acetonitrile was selected as a
suitable extraction solvent, where none of the problems described above occurred and the
values of precision and accuracy ranged from 19.5 to 6.0 % (RSD parameter) and 82.0 - 94.4
% (recovery parameter). DA and its metabolites are unstable and oxidize rapidly, especially
in a strong alkaline medium. They can also be slowly oxidized in a neutral medium
(Verbiese-Genard et al., 1984). The addition of hydrochloric acid (0.4 mL; 0.1 M HCl) to the
brain tissue led to the stabilization of DA in its hydrochloride form. Furthermore, biogenic
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98 Kamila Syslová, Lukáš Rambousek, Věra Bubeníková-Valešová et al.
amines and their metabolites are sensitive to light. They are easily oxidized in the presence of
transition-metal cations such as Fe2+ (Kotake et al., 1985). Therefore, it was necessary to store
the samples in the dark with the use of the chelating agent EDTA.
Figure 7. Scheme of magnetic immunoparticles and their utilization for separation of DA and its
precursors/metabolites from complex matrices (e.g. ACSF, urine, plasma).
Figure 8. Polymeric immunoparticles with encapsulated antibodies.
6. DETECTION BASED ON MASS SPECTROMETRIC METHODS

AND COMPARISON WITH OTHERS
As it was mentioned, the following analytical methods are currently used for the
quantitative determination of DA and its precursor/metabolites in the biological samples: EIA
(ELISA), CE-EC, CE-FL, CE-MS, HPLC-EC, HPLC-FL, HPLC-MS and GC-MS. These
individual methods can be optimally compared on the basis of method validation parameters
(including pre-treatment methods and derivatization), i.e. precision, accuracy, limit of
detection (LOD) and limit of quantification (LOQ), which are listed in the table 1. The
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methods EIA (ELISA) was used in the most cases for monitoring DA in urine or plasma
(validation parameters for the matrices as ACSF and tissue brain have not at present been
determined). The concentration of DA and its metabolites/precursor in brain (tissue or CSF)
was determined in a few works (Eckstein et al., 2008; Khuhawar et al., 2008; Qiu et al., 2007;
Gao et al., 2010; Kaluzna-Czaplinska et al., 2010) by gas chromatography with mass
spectrometry or flame ionization detectore (FID). Nevertheless, this method is not suitable for
their determination because DA and its metabolites/precursors must be derivatized (analyzed
substances have a high boiling point as e.g. DA: 338 °C, DOPAC: 418 °C and HVA: 339 °C).
Using the detector FID (Khuhawar et al., 2008), qualitative identification of substances in a
sample is eliminated (substance identification in the sample is only based on comparison with
the retention time of the standard), which in complex matrices can cause co-elution with other
substances danger and thereby influence the amount of analyte detected. Derivatization
methods described are distinguished by a number of shortcomings that affect the validation
parameters of controlled substances: (1) the derivatization reagent, ethyl chloroformate or N,
O-bis (trimethylsilyl) trifluoroacetamide cannot monitor all levels of precursor (Phe) as the
reagent reacts only with the hydroxyl group; (2) entafluoropropionic acetic anhydride reacts
with hydroxyl groups as well as with the amino and carboxyl groups (Eckstein et al. 2008)
and allows analysis of DA and its metabolites and all precursors, however the reaction takes
place quantitatively only in an anhydrous environment (ACSF and extract from tissues
containing water, which ought to be removed using adequate separation steps); (3) reaction
with N, O-bis (trimethylsilyl) trifluoroacetamide and pentafluoropropionic acetic anhydride
proceeds under a high temperature (e.g. with N,O-bis trimethylsilyl) trifluoroacetamide, the
reaction conditions are -70 °C, 1h), which can cause significant degradation of controlled
substances. Thermal transfer of a substance that occurs in GC, is prevented using the
separation techniques as liquid chromatography (HPLC, UHPLC) or electrophoresis (CE).
These separation techniques allow an on-line connection with a number of detectors - FL, EC,
UV and MS. The methods used can be compared on the basis of: (1) limit of quantification
(LOQ), (2) sample requirements, (3) variety of detectable analytes, and (4) analysis time. The
capillary electrophoresis (CE) combined with FL detector generally showed good mass
detection sensitivity, typically approximately 300 amol of catecholamines.(Bert et al., 1996).
Nonfluorescent catecholamine analytes were successfully derivatized with naphthalene-2,3-
dicarboxaldehyde (NDA) before separation. Although the NDA derivatization technique is
highly selective, DOPAC cannot be derivatized due to the lack of a primary amine
(Desvignes, et al., 1999). Therefore, simultaneous detection of DA and its metabolites is not
possible. Other combinations of detectors (CE, UV and MS) with EC have led to worse limits
of quantification (see table 1). HPLC-EC is one of the most widely used techniques for
neurotransmitter detection because there is no need for the sample pre-treatment.
Simultaneous detection of various neurotransmitters and their metabolites is possible in 20
min depending on the separation conditions. Typical detection limits are 1 nM (Heidbreder et
al., 2001). The disadvantage of this detector type is the lack of qualitative information about
the detected substance. Most of the shortcomings of the methods described above can
eliminated using LC-MS method in a highly selective and accurate SRM mode (described in
section 4) affording both quantitative and qualitative information about the monitored
neurotransmitters. Liquid chromatography can be used in both HPLC mode (high
performance liquid chromatography) and UHPLC mode (ultra-high performance liquid
chromatography). In UHPLC, the separation of substances is carried out at higher flow rates
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of the mobile phase (1 mL/min) on LC columns with a smaller average particle size of the
stationary phase (diameter of particle ), shortening the time of LC-MS analysis. When
using the so-called "stable-isotope-dilution assay", the accuracy and precision of the LC-MS
method can be increased by suitable deuterated internal standards of DA and their metabolites
(Najmanova et al., 2011). Immediately after the collection, a known amount of isotopically
labelled internal standard is added to the analytical sample (in order to monitor the substance
amount from the very beginning and eliminate the error of neurotransmitters‟ disintegration
during the sample handling), which has the identical physico-chemical properties as the
endogenously present biomarkers with the only exception of a different molecular weight.
This is beneficially utilized in MS detection and the resolution of both substances (deuterium-
labelled DA and its metabolites/precursor are characterized by identical retention times in LC
separation, such as the unlabelled compounds, but they can be well separated in the mass
spectrometer). The inclusion of the pre-treatment step (SPE, immunoaffinity extraction),
during which the brain tissue or ACSF sample is exposed to room temperature, still remains
to be a disadvantage of the LC-MS analysis of DA and its metabolites/precursors. This
problem is resolved using 2D technology for liquid chromatography. In the first dimension, it
is possible to carry out an on-line SPE using the SPE column Hypersil GOLD (20x 2.1 mm,
particle size 12 m, Thermo Scientific, USA) and the subsequent dimension using the LC
chromatography.
7. EXPERIMENTAL AND CLINICAL EXPERIENCE

In the last decades, monoamine measurements have led to many improvements in our
understanding of the relationship between chemistry in the brain and the behavioral, cognitive
and emotional state of an organism. Dopamine and its major metabolites have been studied as
biomarkers in a clinical neuropsychiatric research in human cerebrospinal fluid, plasma and
urine. Development of diagnostic tests for neuropsychiatric disorders using biomarkers
obtained from peripheral blood or urine would be useful for diagnosis or predicting prognosis
in patients and for the evaluation of pharmacological treatment success. Indeed, ethological
and pathophysiological heterogeneity of disorders such as schizophrenia and Parkinson‟s
disease has been calling for a more complex multimarker screening rather than for a single
analysis of DA and its metabolites. In this sense, protein biomarker discovery from biological
fluids has been parallely utilized. Proteomic findings are reflective of complex gene-
environment interactions, which are increasingly applied in neuropsychiatry research (English
et al., 2011). Despite peripheral concentrations of DA and HVA have shown certain
relationships to schizophrenia and Parkinson‟s disease symptoms, the analytical significance
of monoamine as a biomarker has rather been limited. Reported results are often contradictory
and it is impossible to distinguish in plasma or urine whether DA and/or its metabolites
formed in the brain or at periphery (Huber et al., 1987; Markianos et al., 1976). Naturally, the
most promising body fluid for biomarkers‟ discovery studies is CSF. It directly reflects the
metabolism processes occurring particularly in the CNS and contains highest concentration
levels of endogenous compounds. Several significant biomarkers and findings have been
found in this fluid. Namely, a correlation between changes in DA turnover in CSF and
development of dyskinesia in Parkinson‟s disease has been found, and HVA/DA ratio
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increased with the disease progression (Lunardi et al., 2009). In next, increased levels of DA
terminal metabolite HVA have been described in children with ADHD (Solanto, 2002). Last
but not least, altered levels of dopamine and HVA have been reported in CSF of
schizophrenia patients (Faustman et al., 1993; Wieselgren & Lindström 1998). Despite the
fact that significant monoamine changes in CSF have been reported, it is advisable to
maintain a critical view when interpreting the data. Effects of sex, age, rostro-caudal spinal
gradient and circadian rhythm should be considered. Additionally, a sample pre-treatment and
detection methods may affect the results.
Alternative approach to the multimarker analysis of neuropsychiatric disorders‟
biomarkers, related to dopaminergic dysfunction including genetic profiling, is based on a
relatively novel technology, i.e. microarrays, or very recently, nanoarrays. Microarrays are
two-dimensional platforms, typically based on a glass microscope slide, upon which specific
biological probes are anchored, using either deposition or in-situ synthesis, in a high-density
(tens of thousands to millions of probes) matrix in a predetermined spatial order. The
analytical principle is based on a selective hybridization of the immobilized probes with
targets contained in the biological sample (urine, serum, plasma or CSF), using most
commonly fluorescence or color detection.
Table 1. Limit of quantification (LOQ) for detection methods for Dapomine
Methods LOQ References

Note
EIA 1 pM Kim et al., 2010 Blood
serum
LC-FL 300 fM Tsunoda et al., 2010 Brain
CE-UV 650 nM Kartsova et al., 2004 Brain
CE-FL 0.3 pM Bert et al., 1996
Microdialysate
LC-ECD 300 nM Rozet et al., 2006 Urine
LC-ECD 5 nM Lin, et al., 2006
Microdialysate
LC-MS 800 pM Uutela, et al., 2009
Microdialysat
LC-MS 3.7 pM Syslova et al., 2011,
Microdialysates
Measuring the changes in monoamine levels in discrete brain areas is currently

considered an important tool for identifying the neuronal systems involved in specific brain
processes, in pathophysiology of various diseases and in investigations of drug actions on the

dopaminergic system. The possibility of correlating animal behavior with changes in
monoamine neurotransmission would be a great advantage in the research of the
neurochemistry of important components of endogenous behavior such as reward system,
motivation and cognition (Wise et al 2004; Bruno et al., 1999). In these studies, rats are
usually examined in different tasks such as running wheels, mazes or in memory tests and
simultaneously, microdialysis with free-moving animals are used. For example, levels of
monoamines have been monitored during different types of learning (Young et al., 1998;
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Flagel et al., 2011), discrimination behavior (Stark et al., 2004), search behavior for food
(Philips et al., 2004) and sexual behavior (Pfaus et al., 1990). Likewise, the levels of
monoamines have been monitored during a treatment with drugs such as antipsychotics
(Kääriäinen et al., 2007; Ichikawa et al., 2002), antidepressants (Kitaichi et al., 2010;
Bymaster et al., 2002), neuroactive steroids (Sandri-Vakili et al., 2003) and psychostimulants
(Karoum et al., 1994; Gough et al., 2002).
However, the present value of dopaminergic metabolism analyses lays mainly in a
preclinical translational research of neuropsychiatric disorders in animals. Animal models can
be viewed as an experimental set-up developed for studying phenomena found in humans.
Genetic, neurodevelopmental or pharmacological manipulations are utilized in simulating the
pathogenesis or symptoms of neuropsychiatric disorders. DA metabolism has been studied in
animal models of Alzheimer‟s disease (Kushida et al., 2008), Parkinson‟s disease (Bazzu et
al., 2010; Muthian et al., 2010), schizophrenia (Schmidt & Fadayel 1996), drug addiction
(Chiara 2002; Bubenikova-Valesova et al., 2009) and many others. Liquid chromatography
based separation and electrochemical detection have been considered as the first choice
method. However, today‟s attention has been drawn to mass spectroscopic detection due to its
structural specificity and a decreasing cost of these analyses. MS detection has been applied
in pharmacokinetics, biochemical and pharmacological studies (Bergquist et al., 2002). In our
study, we have developed and applied LC-MS method without any pre-analysis adjustment of
the sample such as pre-concentration or derivatization for the consequent analysis of
dopamine and its first phase metabolites in the brain tissue after systemic administration of
MK-801 (Najmanova et al., 2011). This drug is non-competitive NMDA receptor antagonist
and induces symptoms similar to schizophrenia in both animals and humans. We found that
systemic administration of MK-801 (0.1 mg/kg; i.p.) increased DA turnover in PFC (+ 520%
compared to control group) and decreased it in the striatum and the hippocampus (-53 %, -
40%).
In next, we have successfully utilized LC-MS method for the analysis of DA and its
metabolites in the animal model of a prenatal exposure to MA (Syslova et al., 2011). The
assay incorporated a pre-treatment step for a rapid and effective pre-concentration
(lyophilization) of low analytes‟ concentrations in microdialysates. In the study, we have
measured the levels of metabolites of DA, DOPAC, 3-MT and HVA in microdialysis samples
obtained from nucleus accumbens of rats (Syslova et al., 2011; Bubenikova-Valesova et al.,
2009). Microdialysis probes (0.2 mm length cuprophane membrane, 6 kDA cut-off, MAB
4.15.2.Cu, AgnTho„s AB, Sweden) with the guide cannulae MAB 4.15 IC (Agn Tho„s AB,
Sweden) were implanted 2 mm above nucleus accumbens at the stereotaxic coordinates AP-
1.6, ML±1.5, DV-7.3. Then, we have compared their concentration levels after a challenge
dose of MA in prenatally exposed and control rats. A higher basal concentration of DA and
all its metabolites were found in the prenatally MA exposed group. The injection of the
challenge dose of MA elevated the dopamine levels by about 1000 %. The difference between
prenatally MA-exposed and saline-exposed rats at the maximum effect, after the injection of
the challenge dose, was 35 %. Interestingly, prenatally MA-exposed rats had higher basal
levels of dopamine by about 300 %. Further, we have observed that the injection of the
challenge dose of MA decreased the level of DOPAC in the nucleus accumbens by about
60% and increased levels of 3-MT by about 200 %. This finding is well documented in
existing publications (Periera et al., 2006). After the acute treatment of MA, a decrease of
HVA was published, which can be explained by the inhibition of MAO by MA (Lan et al.,
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2008; Tuomainen et al., 1996; Adachi et al., 2001). Selective MAO-A and MAO-B inhibitor
M30 increased tissue levels of DA and decreased levels of HVA and DOPAC in mouse
striatum (Gal et al., 2005). Contrary to previous publications, we found higher levels of HVA
(about 190 %) after the acute administration of MA. Moreover, we found a higher level
(about 120 %) of HVA in prenatally MA-exposed rats, as compared to controls. Using the
same analytical set-up, we have also found increased HVA concentrations in nucleus
accumbens after the acute administration of 4-bromo-2,5-dimethoxyphenethylamine (2-CB)
in rats (unpublished results Rambousek et al., 2011). Various microdialysis arrangements,
pretreatment and detection methods could have been the source of these contradictory results.
Further experiments must be carried out to clarify these contrast findings in HVA behavior
after the application of stimulant drugs. In addition, we believe that simultaneous quantitative
analysis of more dopamine metabolites should bring insight into this discrepancy. Methods
for analysis of aldehydic metabolites DOPAL (Mattammal et al., 1993; Loutelier-Bourhis et
al., 2004) and MOPAL (Kodaira et al.; 1984), alcoholic metabolites DOPET (Duncan et al.;
1984) and MOPET (Langlais et al; 1980; Racke et al.; 1987), as well as secondary metabolic
products such as glucuronides and sulfates of DA, DOPAC and HVA (Uutela et al., 2009)
have been developed. Relatively high levels of secondary metabolites may play yet unknown
role and may serve as an alternative source of dopamine and its first phase metabolites and
thus affect extracellular concentrations of neurotransmitters. Despite the fact that current
analytical methods allow monitoring nearly all dopamine products, glucoronide and sulfate
standards are unavailable and thus the majority of preclinical studies have focused only on
dopamine and its main metabolites.
8. FUTURE PERSPECTIVE OF DOPAMINE MONITORING

We believe that monitoring of the whole dopaminergic pathway should bring more
information about neurochemical processes taking place in the background of brain
functioning and explain many discrepancies occurring in the existing literature. Mass
spectrometry allows a parallel detection of dozens of analytes with a high structural
selectivity and sensitivity. This pathway consists of dopamine precursors (L-DOPA, tyrosine,
phenylalanine), further products (norepinephrine, epinephrine), products of the first and
second phase of enzymatic metabolism including glucuronides and sulfates of the first phase
metabolites (DOPAC, HVA, 3-MT) as well as its reactive products. In addition, arrays
utilization for monitoring of expression of related enzymes, receptors and intracellular
messengers would be beneficial for better clinical and pre-clinical data understanding.
ACKNOWLEDGMENTS
The authors wish to acknowledge with gratitude the financial support by the Grant
Agency of the Czech Republic (Grant GACR 305/09/0126, P303/10/0580 and 203/08/H032),
Institutional Research Concept (AV0Z50200510) and the Ministry of Education of the Czech
Republic (Grant CEZ: MSM 604 613 7301).
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In: Dopamine: Functions, Regulation and Health Effects ISBN: 978-1-61942-147-9
Editors: Endo Kudo and Yuriko Fujii © 2012 Nova Science Publishers, Inc.
Chapter 4
DOPAMINE-RESPONSIVE ISOFORMS OF
ADENYLYL CYCLASE AS COINCIDENCE DETECTORS
IN DEVELOPMENT AND FUNCTION
OF DOPAMINERGIC NEURONS
Barbara Pavan1* and Alessandro Dalpiaz2†

1
Department of Biology and Evolution, General Physiology Section, University of
Ferrara, via L. Borsari 46, 44100 Ferrara, Italy
2
Department of Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara
19, 44100 Ferrara, Italy
ABSTRACT
Accumulating evidence on molecular mechanisms leading to the differentiation of
neurons with retained dopaminergic fate and function suggests the induction of such
differentiation as a potential form of treatment of many neurodegenerative disorders, such
as Parkinson's disease (PD) and schizophrenia. Signals conveyed by the effector enzyme
adenylyl cyclase (AC) appear to be important for survival or mature functioning of
neurons. Indeed, although drugs interfering with this pathway have been traditionally
considered to target membrane receptors coupled to G-proteins, the ACs can be thought
as new interesting “druggable” target, being known to work as signaling catalysts. We
discuss herein the advance of isoform-selective stimulator and/or inhibitor compounds for
AC that could lead to cell-specific pharmacotherapeutics for treating dopamine (DA)-
mediated disorders, including neuropsychiatric, neurodegenerative and neuroapoptotic
diseases. In this context, the calcium- and DA-sensitive isoforms of AC are considered as
potential key cues for dopaminergic neuronal patterning and maintenance. In particular,
cell lines differentiating dopaminergic properties and expressing selective DA- and
calcium-sensitive isoforms of AC are taken into account for new therapeutic and
experimental tools in inducing regenerative processes or to evaluate how cAMP signals
discriminate among sympatho-adrenal heterogenic lineages of neurons.
*
E-mail: pvnbbr@unife.it
†
E-mail: dla@unife.it
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114 Barbara Pavan and Alessandro Dalpiaz
INTRODUCTION
The enzyme adenylyl cyclase (AC) catalyses the conversion of ATP into the second
messenger cyclic adenosine-3′,5′-monophosphate (cAMP), which is universally involved in
the control of several physiological functions including learning, memory, cell replication and
differentiation in virtually all prokaryotic and eukaryotic cells [1-3]. cAMP is generated by
two families of enzymes from the class III AC superfamily (AC; E.C. 4.6.1.1) [4]. One family
consists of transmembrane ACs, which are encoded by nine distinct genes (tmACs AC1 to
AC9), and which play key roles in cellular responses to extracellular signals. They are
modulated through heterotrimeric G-proteins in response to activated G-protein coupled
receptors (GPCRs). The other family consists of cytoplasmic enzymes referred to as „soluble‟
ACs (sAC, AC10), which are directly activated by calcium and the cellular metabolites
bicarbonate and ATP [5]. Each of these AC molecules have distinct basal activity, tissue
expression patterns and regulatory properties during postnatal development [6, 7]. ACs are
regulated by α/βγ subunits of the heterotrimeric G-proteins, sensitivity to Ca2+, and
phosphorylation. In particular, there are the Ca2+/calmodulin-activated AC1, AC3 and AC8,
the calcium-insensitive AC2, AC4 and AC7, and the calcium-inhibited AC5 and AC6. cAMP
activates protein kinase A (PKA), leading to a phosphorylation cascade with multiple cellular
targets, and is ultimately broken down to 5′-AMP by cyclic nucleotide phosphodiesterases
(PDEs). The balance between the formation of cAMP by ACs and degradation by PDEs
determines cellular cAMP levels. Perturbation of cAMP system during development would
be therefore expected to have a significant impact on brain cell development in several human
diseases [1-3]. Indeed, a key aspect of this pathway is the tight spatio-temporal control of
signal propagation, which underlies the initiation, distribution, and termination of cAMP
mode of action. Evidence for a compartmentalization of cAMP signalling is provided by
recent studies related to the cAMP effectors other than the classic PKA. Among them, cAMP-
gated ion channels get attention, because of their crucial role in neuronal excitability and
signal transduction of sensory neurons, such as an example the Ca2+ entry through cAMP-
gated channels in olfactory neurons [8,9]. Depending upon the specific cellular environments
as well as their relative abundance, distribution and localization, the three cAMP effectors,
i.e. cAMP-gated ion channels, exchange protein activated directly by cAMP (Epac) and PKA,
may act independently, converge synergistically or oppose each other in differential
regulation of cellular targets. Transcriptional responses to increased cAMP occur through
activation of the cAMP response element–binding protein (CREB), cAMP response element
modulator (CREM), and activating transcription factor 1 (ATF1) [10]. Each of these
transcription factors contains a kinase-inducible domain containing a conserved site for
phosphorylation by PKA [10]. All these AC features lead us to focalize the attention on
several isoforms of AC as potential key cues for neuronal patterning and maintenance and
consequently as valuable therapeutic target against to the increasing neurodegenerative
disorders, such as Parkinson‟s disease (PD) and schizophrenia. Attempts to discover more
about the in vivo signaling molecules and mechanisms responsible for neuronal development
are of fundamental importance to the progress of cell replacement therapies targeting the
central nervous system (CNS) [11]. Neurogenesis, as the production of new neurons, was
firstly considered to occur only during the embryonic and early postnatal periods; however,
various degrees of adult neurogenesis have been recently recognized in selected regions of the
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Dopamine-Responsive Isoforms of Adenylyl Cyclase 115
mammalian brain, namely the hippocampus and olfactory bulb. By contrast, other brain
regions, including the cerebellum, brainstem, basal ganglia and spinal cord, appear to be non-
neurogenic [12]. Although neurogenesis can be stimulated in response to injury in neurogenic
as well as in non-neurogenic brain regions, it remains unclear whether any constitutive
neurogenesis occurs in these regions under normal conditions. This possibility has received
recent support from the finding that a resident population of neural progenitor cells exists in
adult brain, obviating the need for mature neurons to become mitotic [12]. It is known that
adult neurogenesis occurs within the unique local environment called “niche”, which provides
both supportive and instructive signals for the development of adult neural progenitors. The
molecular mechanisms leading to the differentiation of neural stem cells (NSCs) into neuronal
progenitors or precursors and finally into mature neurons are now gradually being discovered
[12]. The events that are responsible for the definition of the number and distribution of
neurons that compose the mature tissue in the CNS are detailed in Figure 1. NSCs, with their
intrinsic capacity to self-renew and differentiate, have sparked great interest as potential tools
for aiding recovery in patients with neurodegenerative disorders, such as PD and
schizophrenia. Although most clinical applications of NSCs are likely to rely on the in vitro
expansion and differentiation strategies before cell transplantation, the knowledge of the
mechanisms and methods required to differentiate NSCs into specialized cell types is still a
major limitation [13].
As the supply of fetal-derived neuroblastic tissue is limited, attaining a stable and
homogeneous population of dopaminergic neurons would provide a useful supply of neural
tissue for developmental studies and clinical applications. Neural differentiation in vivo as
well as in vitro is known to require elevation of the intracellular cAMP concentration [14-16].
AC–cAMP signaling has been identified as a key intracellular pathway activated by
environmental factors that lead to the induction of genes controlling differentiation in lineage-
committed progenitor cells [17]. It is also important in the generation of pure populations of
dopaminergic neurons from human NSCs in developing methodologies [18,19]. Therefore,
the different AC isoforms are of particular interest to researchers because of their regulatory
impact on cAMP as an ultimate determinant of neural cell fate [16,20]. Cumulative evidence
indicates that neuronal fate is determined by both intrinsic and extrinsic factors in a
coordinated way [21]. In this chapter, we discuss the potential role of DA-sensitive isoforms
of the AC enzyme as cues for dopaminergic neurons to retain their mature functional
differentiation. We suggest that such isoforms could be valuable pharmacological targets for
the treatment of aberrant or damaged neurons.
PHYSIOLOGICAL AND THERAPEUTICAL RELEVANCE

OF AC ISOFORMS IN THE CNS
The importance of AC in signal transduction of the CNS is highlighted by three special

properties of the enzyme. First, a number of indispensable neurotransmitters and
neuromodulators in the CNS are GPCRs, including the receptors for the classical
neurotransmitters, such as DA and other catecholamines, and AC is known to be the rate-
limiting component distal to GPCRs and to be involved in integrating multiple signaling
pathways into a single second messenger [6]. The second essential property of AC is the
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diversity of the AC superfamily which allows its members to function in different signal
transduction pathways of neurotransmitters, neuromodulators, and even neurotrophic factors.
Indeed, changes in the concentration of intracellular cAMP have also been identified as key
events in neuronal and glial cell migration modulated by neurotrophins, such as nerve growth
factor (NGF), and neuropeptides [23,24]. The coupling between neurotransmitter receptors
and different AC isozymes provides an important basis of AC-mediated multiple neuronal
signal transductions. Third, the regulation of AC activity by GPCRs represents an early stage
in which extracellular signals can be transduced and integrated for neuronal information
processing. Furthermore, AC isozymes are tightly modulated by signals other than G proteins,
including calcium/calmodulin-dependent kinase, protein kinase C, PKA, and even protein
phosphatase 2A. Members of the AC superfamily therefore serve as critical “coincidence”
detectors in integrating multiple signals [7]. This is particularly important for the CNS in
which a single neuron may receive simultaneous or sequential multiple inputs from other
neurons in a fraction of a second. The coincidence detector AC renders neurons capable of
detecting simultaneous stimulation of two or more neurotransmitters. The neuronal
computation of the multiple inputs will then be determined by the biochemical characteristic
of the AC that is expressed by that particular neuron [7]. To date, the AC signal transduction
cascade has been pharmacologically targeted through GPCR agonists or antagonists and
PDEs inhibitors. However, increasing therapeutic interest currently surrounds the
development of new approaches for the manipulation of GPCR signalling at steps distal to
receptors. The coincidence detector AC is an attractive pharmacological target in the CNS
[1]. On the basis of the efficacy (maximal response) and potency (affinity, sensitivity, EC50,
Ka), AC has been indicated as the most crucial than all the GPCR/Gs/AC signalling pathway
components [25]. In particular, AC has been referred to as a downstream „bottleneck‟ for the
GPCR/Gs signal, limiting the maximum efficacy of the system in terms of maximal cAMP
generation. This central role indicates the „signal strength‟, that is the stoichiometric
relationship between the components of this amplification cascade [26]. In addition, AC is not
subject to the desensitization occurring during long-term receptor agonist stimulation with
consequent loss of drug efficacy [27]. Moreover, the distinct regulatory properties and tissue
distribution of AC isoforms imply the feasibility of developing tissue-targeted isoform-
specific AC activators and inhibitors. Certainly, the generation of mice with targeted
disruption of genes for the various AC isoforms (AC knockout mice) has greatly enhanced the
understanding of their in vivo relevance [1]. Currently, several AC isoforms are known
potentially involved in therapeutics against brain and heart diseases.
STRUCTURE OF ACS
TmACs contain 12 transmembrane helices arranged in two sets of six separated by a

large hydrophilic domain formed by two intracellular lobes generally referred to as C1 and C2
(Figure 2) [28]. The nucleotide-binding site of the tmACs, which has been designated „P‟-site,
consists of two homologous cytoplasmic domains, C1a and C2a, within the intracellular
lobes. Its occupancy traditionally requires molecules characterized by the presence of an
intact adenine ring [29-31]. These domains comprise approximately 230 amino acids that
share at least 50% similarity across the AC family. Mutagenesis analysis has revealed that
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residues from both C1a and C2a domains contribute to binding and catalysis of ATP. X-ray
crystal structures of the catalytic site depict AC as a receptor characterized by three main
features: (i) a phosphate binding region containing two metal ions (normally Mg2+ or Mn2+),
(ii) a purine ring binding site possessing H-bond donating and accepting side-chains suitably
oriented for affixing adenosine and (iii) a hydrophobic pocket [28].
EMBRYONIC STEM CELL NEURAL STEM CELLS ADULT STEM CELL
Pluri/totipotent Multipotent
High self-renewal Adequate/limited
self-renewal
Asymmetry of division
Niche
PROGENITORS or PRECURSORS
Limited
self-renewal
Surrounding microenvironments
LINEAGE RESTRICTED
COMMITTED CELL
MATURE NEURONS
migration
differentiation
IMMATURE NEURONS
Figure 1. Neurogenesis degrees. Neural stem cells originate from both embryonic and adult tissues, but
embryonic tissues are the primary source of all adult stem and differentiated cells. A neural stem cell
gives rise by asymmetric division either to another daughter stem cell (self-renewal) or to a neural
progenitor or precursor with a more restricted proliferation and lineage potential. The progenitor is
committed to differentiate into a specific fate in response to cell intrinsic and/or extrinsic cues arising
from the stem cell microenvironment or niche. As a process that is necessary for neural differentiation,
the departure (migration) of progenitors and functional immature neurons from the stem cell niche
toward the surrounding microenvironments is also orchestrated by different internal and external
signals (From Pavan et al, 2011 [22]).
DOPAMINERGIC SYSTEM IN THE ADULT BRAIN

The neurotransmitter DA is a member of the catecholamine family and it is related to

physiological functions such as motor control, cognition and reward, as well as to several
syndromes, including PD and schizophrenia [32]. DA is a precursor of noradrenaline and
adrenaline and it is synthesized from the essential amino acid L-tyrosine, which is converted
to L-dihydroxy-phenylalanine (L-DOPA) by the enzyme tyrosine hydroxylase (TH). L-DOPA
is subsequently decarboxylated by the enzyme aromatic l-amino acid decarboxylase (AADC)
to produce DA (Figure 3). Dopaminergic neurons (Figure 3) are therefore characterized by
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the expression of TH and AADC, the rate-limiting and key enzymes in the biosynthesis of
DA, together with the DA-uptake transporter (DAT) and the vesicular monoamine transporter
2 (VMAT2), by which DA is packaged in pre-synaptic vescicles [33]. DA neurons are found
in several areas of the mammalian brain [34]. However, the most prominent DA cell group
resides in the ventral part of the mesencephalon, which contains approximately 90% of the
total number of DA cells. The mesencephalic DA system includes the nigrostriatal and the
mesocorticolimbic pathways. The cell bodies of the nigrostriatal pathway reside in the
substantia nigra pars compacta (SNpc) and project to the dorsal striatum (caudate putamen),
the center of sensorimotor integration within the basal ganglia, mainly controlling voluntary
movement and body posture. The mesolimbic pathway originates in the ventral tegmental
area (VTA) and terminates mainly in the nucleus accumbens (NA); one function of this
system is the mediation of natural and drug-induced reward. The mesocortical DA pathway,
which also originates in the VTA but terminates in the prefrontal cortex (PFC), regulates
complex cognitive processes, such as selective attention and working memory [34]. These
systems have no direct role in the regulation of prolactin secretion from the anterior pituitary,
which is instead affected by the tuberoinfundibular dopaminergic system [35], where DA-
inhibited prolactin secretion was demonstrated as independent from the AC system [36].
Hence a supposed pharmacological treatment of striatal DA-sensitive AC would not involve
this last physiological process.
TM1 TM2
1 2 3 4 5 6 1 2 3 4 5 6
C1b
C1a
+H N FSK
3
ATP
C2a
COO-
Figure 2. Schematic diagram of the proposed structure and membrane topology of AC. TM1 and TM2
denote the two regions of the six transmembrane-spanning domains in the AC molecule. C1a and C2a
are the first and the second cytoplasmic catalytic domains, respectively, which, facing each other, form
the catalytic core inside a crevice. The best known AC direct activator, the diterpene forskolin (FSK)
and the endogenous substrate ATP bind the catalytic core at opposite sites. C1b is the first cytoplasmic-
linker domain and C2b is the second non-catalytic domain. The C2b domain is present only in AC1,
AC2, AC3 and AC8 isoforms.
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All five DA receptor isoforms cloned in mammals have been linked to the AC enzyme.
After activation, D1-type DA receptors (D1 and D5) stimulate AC, whereas D2-type DA
receptors (D2, D3 and D4) inhibit AC (Figure 3). In particular, AC is considered to be the key
effector of D1 receptor in neuronal function [36], which is the most abundant DA receptor in
the CNS and it is expressed at higher levels than any other DA receptor [36]. In the rodent
neostriatum the primary pathway for D1-like receptor signaling has been proposed as D1
receptor →protein Golf →AC5→ PKA. Protein Golf is a stimulant alpha-subunit of olfactory
neuron specific G protein coupled with the D1 receptor in the CNS. In the rodent neostriatum
the primary pathway for D1-like and D-like receptor signaling has been proposed as D1
receptor→proteinGolf→AC5→PKA, and D2 receptor→Gi/o→AC5→PKA signaling path
[37].
Figure 3. Dopaminergic pre- and postsynaptic neurons and opposed coupling of D1-like (D1/D5) and
D2-like (D2/D3/D4) DA receptors to DA-sensitive AC. Chemical structures correspond with tyrosine,
l-DOPA and DA, respectively. Abbreviations: HVA, homovanillic acid; Gαi, protein G inhibitory α
subunit; Gαs, protein G stimulatory α subunit; Gβ and Gγ, protein G β and γ regulatory subunits; MAO,
monoaminoxidase; DAT: dopamine transporter DARPP-32: dopamine and cyclic adenosine 3‟:5‟-
monophosphate-regulated phosphoprotein (a DAergic pattern molecule thought to be important in
positive feedback signaling); CREB: cyclic AMP response element binding protein; MAPK: Mitogen-
activated protein kinases (from Pavan et al. [22]).
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PKA phosphorylates a number of proteins involved in signal transduction and regulation

of gene expression. Gβγ has a permissive effect on the calcium-insensitive AC type 2 and 4,
so that the stimulatory effect of other activators, including Gαs and PKC, is enhanced in the
presence of free Gβγ. D2 and D4 receptors markedly increase the activity of AC2, whereas
the D3 receptor has little or no effect [37]. D2-like receptor activation of Gβγ-stimulated AC2
response has only been observed by using heterologously expressed receptors and AC. It is
not known if it contributes to D2-like receptor signaling in neurons, although it has been
speculated that Gβγ-stimulated AC2 contributes to synergistic activation of spike firing in
nucleus accumbens neurons by D1-like and D2-like receptors [37].
CA2+- AND DA-RESPONSIVE ISOFORMS OF AC AS A PROSPECTIVE

TRACK POINT FOR DA NEURON DEVELOPMENT AND SURVIVAL
The expression and the transcriptional control of specific AC isotypes tracking the
dopaminergic pattern in developing human neurons remain yet to be examined. The
specificity of the so-called DA-sensitive ACs was demonstrated just in the striatum from
autopsied human brain, where AC activity was selectively stimulated by low concentrations
of DA, whereas, under the experimental conditions used, noradrenaline was about three times
less potent than DA and histamine and serotonin were ineffective [38]. Another past but
intriguing study carried out in primary cultures of virtually pure mesencephalic neurons or
glials from mouse embryo indicated that all neuronal cells having dopamine receptors possess
also β1-adrenergic receptors, but β1-adrenergic and dopamine receptors were coupled with
independent and different AC isoforms localized either on two different domains of the same
cell or on different cells. Moreover, no dopamine-sensitive AC was detected on
mesencephalic glial cells [39]. These DA-sensitive ACs have been recently identified as type
5 and 6, that belong to the same subfamily and are inhibited by physiological concentrations
(0.1–1.0 μm) of Ca2+ [40]. AC5 and AC6 also share high degree of identity [41], but are
differentially regulated by the D2 and D3 receptors [42], since the activity of both isoforms is
inhibited by the D2 receptor, whereas only the activity of AC5 is inhibited by D3 receptor
[43]. AC5 is highly enriched in brain dopaminergic regions and largely localized in the
mammalian striatum as well as in other DA-innervated structures (i.e. the NA and olfactory
tubercle), where the cAMP pathway regulates diverse behavioral functions [44,45]. AC5
often is referred to as „striatal AC‟ and its importance in striatal functions involving
dopaminergic pathways has been further emphasized by the finding of cells expressing AC5
co-localized with D1 and D2 receptors [46]. All of this evidence opens the door to the
possibility that the role of individual ACs in neuronal differentiation could be successfully
clarified by evaluating the potential discrimination of cAMP signals among different lineages
of neurons. Indeed, AC5 distribution has been demonstrated to be limited to dopaminergically
innervated regions of brain [44]. In the matter of the role of DA-responsive AC isoforms as
permissive and/or instructive molecules during DAergic neuronal development, the study
carried out by Matsuoka et al. over the past 90 years has to be cited [47]. The Authors
demonstrated that mRNA for AC5 and the Ca2+/calmodulin-activated AC1, the other highly
expressed AC isoform in the CNS [6,7], showed opposite expression patterns during the
development of rat striatum. In particular, mRNA for AC1 was the major isoform expressed
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during the early phases of development, whereas mRNA for AC5 expression increased
dramatically during postnatal stages. Both expression patterns were reflected also by
comparable changes in Ca2+/calmodulin-sensitive AC activity (from stimulation to inhibition
of cAMP production) moving ahead in development. The progressive expression of AC5
mRNA was associated with the maturation of striatal neurons [47]. Interestingly, ontogenic
studies in heart have reported very low levels of AC5 mRNA expression at early stages of
cardiac development, which markedly increased as development progressed [48]. This
expression pattern requires changes in the regulation of cAMP formation throughout the
neurogenesis, perhaps incorporating distinct AC isoforms that are recruited according to the
type and timing of several extracellular signals. Therefore, a shift in calcium sensitivity of AC
could be regarded as a hallmark of striatal dopaminergic neurons maturation. Indeed,
spontaneous Ca2+ transients, observed in developing neurons, were reported to be sufficient
and necessary to drive normal differentiation and to regulate neurite extension [20], and the
possibility that ACs could be voltage-sensitive in the CNS has been also proposed [49].
However, as for all the AC subtypes, the tissue distribution and developmental expression of
AC5 and AC6 have only been determined at the mRNA level [48,50], as antibodies with the
requisite sensitivity for detection at the protein level are still lacking [51]. Interestingly, the
successful generation of an AC5 isoform-specific mouse monoclonal antibody has recently
been reported [52]. This novel AC5 antibody could lead to a better understanding of the tissue
distribution and ontogenic regulation of AC5 expression, as well as providing new insight
into the pathogenesis of AC5-related diseases.
The other Ca2+/calmodulin-activated type 3 isoform of AC (AC3) is known to be a
crucial element in the odorant-induced transduction cascade and a pivotal player in axonal
guidance during migration of dopaminergic neurons from the olfactory bulb toward the brain
[53]. In this link, olfactory tissues are also regarded as an open „window to the developing
brain‟, enabling, for example, early diagnosis of schizophrenia [54]. These tissues are also a
valuable route for drug delivery bypassing the inherent barriers associated with the CNS.
Therefore, AC3 could be an elective target for promoting olfactory neurogenesis as a
therapeutic resource for the CNS. The proposed pattern of localization of DA- and calcium-
sensitive AC isoforms in the CNS is illustrated in Figure 4. As reported above, the calcium-
insensitive AC2 is also dopamine-sensitive, being Gβγ-stimulated by D2-like receptor
activation [55] and well expressed in the CNS. However, developmental expression of AC2
mRNA has been reported similar in the neonatal and in the adult rat brain suggesting AC2 as
a constitutive AC enzyme in the brain contributing to cAMP-mediated metabolic regulation
of neuronal cells [47]. The lack of genetic models for AC2 does not moreover allow to test its
selective role. Therefore, only the calcium-sensitive AC1, AC3 and AC5/AC6, among the
DA-sensitive ACs, are considered in our scheme, because of their direct modulation by
calcium, which makes them particularly interesting for neural developmental mechanisms.
Indeed, together with cAMP, calcium is one of the major signals involved in cell growth,
migration, differentiation and synaptogenesis [56,57], and a spatiotemporal localization of the
calcium-modulated ACs, such as AC1, AC8, AC5 and AC6, during rodent brain
development has been manifold evidenced.
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(AC1) AC6 AC5

AC1
Striatum
AC5 Ventral
Pallidum
Substantia
nigra
Prefrontal
Cortex
Nucleus
accumbens
Ventral
Tegmental
Olfactory
Area cerebellum
tubercle
Brain Stem
Hippocampus
AC3
AC5 AC6 AC5
(AC1)
Figure 4. Proposed pattern of localization for DA- and calcium-sensitive AC isoforms in the different
areas of the three main dopaminergic inputs in the brain. The nigrostriatal pathway resides in the
substantia nigra pars compacta and projects to the dorsal striatum (caudate putamen). The mesolimbic
pathway originates in the ventral tegmental area and targets the nucleus accumbens and prefrontal
cortex. The mesocortical pathway also originates in the ventral tegmental area, but terminates in the
prefrontal cortex (from Pavan et al. [22]).
DOPAMINERGIC SYSTEM FAILURES

PD and schizophrenia are neuronal diseases characterized by altered dopaminergic
signals in the CNS. Both the disorders appear to share the D1–D2/AC5 pathway, as described
below.
PARKINSON'S DISEASE
PD is one of the most common late-life neurodegenerative diseases, affecting

approximately 2% of the population above 60 years of age. It is characterized by the
unilateral onset of resting tremor in combination with varying degrees of rigidity and
bradykinesia [58,59]. The etiology of PD is still not completely understood. It is likely to
result from a combination of several factors, among which the first is an age-related attrition
and death of the dopaminergic neuronal projections from the SNpc to the striatum [59].
Furthermore, PD might arise as a consequence of the ongoing aging process coupled with
environmental neurotoxins exposure that accelerates the process of nigral cell death [60].
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Another possibility is that some people might have a predetermined genetic susceptibility to
these environmental insults [61], suggesting an important role for genetic factors in the onset
of PD.
Levodopa (l-DOPA; 3,4-diidrossi-l-fenilalanina), which is converted to DA in the brain,
remains the gold standard for treating PD. However, long-term complications of this therapy
include dyskinesia [62] and cognitive difficulties altering the working memory [34]. The
positively AC-coupled D1 receptor is known to be involved in the anti-parkinsonian function
of l-DOPA as well as in inducing dyskinesia [63]. Studies in animal models of PD have
suggested an incoming supersensitivity of the D1 receptor after DA depletion in the striatum
and frontal cortex, which is likely to be further enhanced by l-DOPA treatment [64]. Tong et
al. [65] studied the status of D1-stimulated AC activity in the striatum and cerebral cortex of
patients with PD. Interestingly, the observed significant increase of DA-stimulated AC
activity in these patients is likely to be the result of enhanced coupling between the D1
receptor and stimulatory G-protein. Therefore, the supersensitivity of the D1 receptor as a
compensatory attempt to ameliorate the parkinsonism was also suggested to contribute to the
development of L-DOPA-induced dyskinesia [66].
Presynaptic dopaminergic deficiencies might also involve the so-called „Parkinson plus‟
syndromes, including multiple system atrophy, progressive supranuclear palsy, corticobasal
degeneration, dementia with Lewy bodies, Parkinson's disease dementia and frontotemporal
dementia. All these disorders are characterized by a reduction in the synthesis and storage of
DA, and alterations in its binding to DAT and to the D1 and D2 receptors. Among these
disorders, Huntington's disease is characterized by both pre- and postsynaptic dysfunctions
[67]. AC5 is known to be the major AC subtype in transducing D1 and D2 receptor signaling
in the adult striatum and its loss or inhibition is a crucial regulatory property for cAMP-
dependent motor control, especially in balancing and maintaining both coordination and
locomotion [68]. In the absence of AC5, the AC6 and AC1 isoforms are still present, but are
unable to compensate fully for the function of AC5 [68]. Accordingly, AC5-null mice were
found to exhibit parkinsonian-like motor dysfunctions that were only partially compensated
by selective D1 or D2 dopaminergic stimulation [68]. A pharmacological modulation of the
activity and/or expression of AC5 could be an alternative way of controlling the detrimental
effects of long-term l-DOPA treatment. It is interesting to observe that the effects of l-DOPA
against Parkinson's disease can be enhanced by the co-administration of adenosine (Gs-
coupled) A2A receptors antagonists. Such a strategy allows a reduction in l-DOPA dosage
with the consequent reduced intensity of unwanted effects that can result from long-term
treatment [69]. This phenomenon has been attributed to heteromeric receptor complexes
between the A2A and D2 receptors, where the activation of the A2A receptor induces a
reduction of the D2 receptor affinity for DA [70]. As a consequence, A2A antagonists enhance
the in vivo DA effects against PD by increasing the affinity of the D2 receptors. In such a
manner, DA and A2A antagonists converge to inhibit AC5 activity, inducing the appropriate
conditions for motor control of patients with PD. The same result can be obtained by using a
direct inhibitor of the activity of AC5, which could therefore be a key enzyme to target for
new clinical implications against PD.
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SCHIZOPHRENIA AS A PROTOTYPE OF DOPAMINERGIC OVERUSE

Schizophrenia is a complex brain disorder that induces cognitive impairment associated
with positive and negative psychotic symptoms. This disease affects one in 100 individuals,
who usually show a clinical schizophrenic onset during late adolescence or early adulthood
[71,72]. As for PD, the etiology of this disease is not fully understood [72], although several
pieces of evidence exist that suggest that perinatal brain disturbances underlie the initial risks
for the disease [73,74]. In particular, these disturbances can arise from genetic and
environmental factors that are able to affect the normal postnatal program of brain maturation,
leading to manifestation of the disease during late adolescence or early adulthood [75,76].
After disease manifestation, neurodevelopment continues to deviate further [77,78]. This
suggests that useful therapeutic approaches would still be possible after the onset of the overt
symptoms. Among the susceptible genes that might be responsible for the pathophysiology of
schizophrenia, those encoding neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia (DISC1)
appear to have important roles during neurodevelopment [76, 79]. A marked decrease in TH
expression and extracellular levels of DA was detected in the PFC of DISC1 knock-down
mice after puberty [80]. These data reflect a disturbed maturation of dopaminergic neurons
[81]. Interestingly, it has been demonstrated that the adult schizophrenic brain is characterized
by underexpression of D1 receptors in PFC and overexpression of D2 receptors in striatum,
compared with healthy subjects [82]. This D1–D2 imbalance leads to alterations in
dopaminergic transmission that could be related to the etiology of dopaminergic-based
neurodevelopmental disorders [83]. Accordingly, excessive stimulation of striatal DA D2
receptors and deficient stimulation of prefrontal D1 receptors have been recognized as
conditions associated with schizophrenia [84]. Pharmacological activation of the D1 receptors
has therefore been suggested as a potential adjunct in the usual treatment of schizophrenia
with D2-antagonists [85]. Given that D1 receptors stimulate AC activity via Gs proteins,
selective activators of the DA-sensitive isoforms of AC expressed in the PFC, such as AC5,
could be investigated as potential new drugs for the treatment of schizophrenic diseases.
The physiological consequences of D2 receptor overexpression have been studied by
generating mice with reversible increased levels of D2 receptors restricted to the striatum.
This tissue showed a reduction of DA-induced AC activity in the case of D2-transgenic mice
[86], a phenomenon consistent with an excess of D2 receptors that, in striatum, are coupled
with Gi proteins inhibiting AC activity [87]. The selective D2 receptor overexpression also
resulted in increased levels and decreased turnover of DA in striatum. Moreover, D2-
transgenic mice exhibited cognitive deficits even after the transgene was switched off,
suggesting that cognitive diseases could be attributed to the excessive expression of D2
receptors during neuronal development, rather than to their continued overexpression.
Interestingly, striatal D2 receptors alterations have also been associated with other mental and
affective disorders, such as bipolar affective illness, some inherited neuropsychiatric tics
(Tourette syndrome) and anxiety disorders [88]. Experiments carried out on AC5 knockout
mice revealed that the inhibitory effect of D2 activation on directly stimulated AC activity
was completely abolished in striatum, where AC5 is preferentially expressed under normal
conditions. Moreover, the administration of D2 antagonists to mice lacking AC5 did not
produce typical neuroleptic effects [89]. All these data suggest that AC5 is the physiological
relevant effector for the D2 receptors in striatum. In addition, AC5 deficiency in mice induced
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a consistent anxiolytic effect that was either increased or reduced by D1 agonists or

antagonists, respectively [90], probably as a result of the compounds acting on effectors that
were different from AC5.
Taken together, these studies indicate that an important role in the mechanism of anxiety
can be attributed to the AC5 isoform. As a consequence, selective AC5 targeting is likely to
be of great importance in the discovery novel drugs against aberrant neuronal development
involved in anxiety and schizophrenia. Accordingly, it is generally recognized that post-
receptor mechanisms can be considered as promising targets for the development of novel
drugs characterized by long-term clinical efficacy against schizophrenia [91].
EXPERIMENTAL TOOLS AND THERAPEUTIC IMPLICATIONS

Neuronal replacement therapies can be performed by cell transplantation-based
approaches together with manipulation of endogenous or grafted precursors in situ. Hence, a
rapid and efficient experimental approach aimed at discriminating neuronal differentiation in
cell culture can support pharmacological studies directed at differentiating cells and screening
for new pro-neurogenic factors [92].
IN VITRO CELL LINES MODELS

Cell lines provide a powerful model for investigating the role and degree of regulation of
AC isoforms during neuronal development. Indeed, they can often undergo a de-
differentiation process leading to either the acquisition of an intermediate progenitor cell
phenotype or to a direct neuronal trans-differentiation process. Embryonic carcinoma P19
cells were the first recognized cell line expressing basal Ca2+/calmodulin-stimulated AC
activity, where inducted differentiation was accompanied by specific upregulation of AC2,
AC5 and AC8 mRNA and downregulation of AC3 mRNA [20]. Moreover, differentiation in
the catecholaminergic (CAD) mouse neural cell line, which expresses the TH enzyme and
accumulates l-DOPA, was accompanied by a significant increase of AC6 and a dramatic loss
of AC9 mRNA expression [16], showing, once again, a change in calcium-sensitive AC
expression during neural development. Furthermore, a simple and reliable method of
generating DA neurons has been recently provided by the mouse neural crest-derived cell line
Neuro2A, which was stimulated to enhance significantly both TH and DA levels in the
presence of a non-hydrolysable analog of cAMP [93]. Experimental evidence from rat
pheochromocytoma 12 (PC12) cells, a classic in vitro model for neuronal development, is
being used to help elucidate how neurotrophins, including NGF, facilitate AC–cAMP-
mediated commitment of neuronal stem cells to a dopaminergic phenotype [94]. As the
supply of fetal-derived neuroblastic tissue is limited, in vitro studies on cell lines, expressing
constitutive and/or induced DA/Ca2+-sensitive ACs during their differentiation, can offer an
easy-to-handle tool for performing new structure–differentiation relationships in AC drug
targeting. In this context, functional dopaminergic neurons have been recently generated
directly from mouse and human fibroblasts, without inducing the first cell undifferentiated
pluripotent state, which use might lead to the development of tumors if not properly
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controlled [95,96]. These induced dopaminergic (iDA) cells release dopamine and show
spontaneous electrical activity organized in regular spikes consistent with the pacemaker
activity featured by brain dopaminergic neurons. The three factors are able to elicit
dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and
Parkinson‟s disease patients. Direct generation of iDA cells from somatic cells might have
significant implications for understanding critical processes for neuronal development, in
vitro disease modelling and cell replacement therapies. The genetic reprogramming erases the
majority of the evident expression hallmarks of the cell of origin, while specifically inducing
the dopaminergic neuronal phenotype and not those of other closely related neuronal
subtypes. However, it should be noted that iDA expression profiling is close but
distinguishable from that of mDA neurons, with 160 genes differently expressed with a ≥5-
fold change. Whether this might indicate the presence of residual fibroblast gene expression
in iDA cells remains to be addressed [96]. TH and VMAT2 promoter regions were reported
highly demethylated in dopaminergic neuronal cells whereas they were fully methylated in
parental fibroblasts, indicating their epigenetic reactivation during dopaminergic neuronal
conversion [96]. This new approach mirrors increasing evidence on the epigenetic stability of
differentiation, where distinct states of pluripotency can interconvert through the modulation
of both cell intrinsic and exogenous factors. Interestingly, the direct activator of ACs
forskolin (FSK) has been reported to be one of the crucial exogenous factors involved in the
stabilization of naïve pluripotent state in human fibroblasts in vitro [97]. Similarly, it has been
reported that neural induction was achieved in mouse bone marrow (BM)-derived
mesenchymal stem cells by exposing cells simultaneously to inhibitors of DNA methylation
and histone deacetylation, and to pharmacological agents increasing cAMP levels, such as
FSK [98]. Concerning to the cell transdifferentiation approach, i.e. the phenomenon by which
an adult differentiated cell switches to another differentiated cell, the adult human retinal
pigment epithelium (HRPE) cells, a cell monolayer constituting the blood-retinal barrier, have
been recognized to express neural progenitor properties suggesting a neural predisposition
[99,100]. Moreover, an established HRPE cell line showed a circadian profile in the
expression of three AC isoforms, the Ca2+-inhibitable AC5 and AC6 and the Ca2+-insensitive
AC7, providing evidence for an intertwine between AC/[Ca2+]i signalling pathways in the
circadian clockwork of these cells [101]. We would speculate that this data might also be
correlated with the existence of spontaneous cAMP transients in embryonic spinal neurons,
where this endogenous AC activity regulates the natural Ca2+ spike frequency [102]. All these
features encourage the possibility that adult HRPE cells, exposed to appropriate conditions,
should be promising candidates for cell-based therapy of DA-neurodegenerative diseases.
DICTYOSTELIUM DISCOIDEUM: A POTENTIAL MODEL OF

DEVELOPMENTAL ACS EXPRESSION IN DA NEUROGENESIS?
Beside cultured in vitro cell models, the social amoeba Dictyostelium Discoideum, being
highly accessible at the biochemical and genetic levels, provides a simple and valuable
biomedical model to study determinant events of neural development, including polarisation,
migration and chemotaxis, as well as cell-fate determination and maturation. Indeed, few is
still known about the mechanisms underlying morphogenetic movements necessary to allow
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neuronal cells to move into appropriate positions for subsequent signaling pathways which
regulate cell-fate decisions. On the contrary, it is well known that throughout the
developmental program of D. discoideum, a multitude of signaling components are expressed
with specific temporal and spatial characteristics, among them cAMP plays a key role. In the
presence of nutrients, these amoebas live a solitary life, independently hunting and ingesting
various food sources. Upon starvation however, this egoistic behavior is drastically changed
and the cells start to communicate and aggregate in a cellular slime mold, forming mounds
that differentiate into a fruiting body composed of resistant spores atop a stalk of vacuolated
cells [103] (Figure 5). D. discoideum cells start to synthesize and secrete cAMP a few hours
after the initiation of starvation. The secreted cAMP binds and activates a family of four
GPCRs cAMP receptors subtypes (cAR1-cAR4) and 11 Gα subunits, which are sequentially
expressed throughout development, leading to the activation of multiple downstream
effectors. Chemotaxis, control of gene expression, synthesis and secretion of additional
cAMP are consequent. Three class III ACs, ACA, ACB, and ACG, with different topologies
and regulatory mechanisms, account for the synthesis of cAMP. D. discoideum cells that are
mutant in AC do not develop. The AC expressed during aggregation, ACA, is related to the
mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of
cAMP that is required for cell-cell signaling in early development. ACB harbors histidine
kinase and response-regulator domains and is required for terminal differentiation. Finally,
the AC expressed during germination, ACG, is similar to mammalian sACs, acts as an
osmosensor and is involved in controlling spore germination.
Therefore, D. discoideum uses cAMP as an extracellular chemoattractant and as an
intracellular signal for differentiation. As long as ACA is fully active, ACR is not required
until culmination but then plays a critical role in sporulation and construction of the stalk.
ACA is expressed at low levels in growing cells and at more than 25-fold higher levels during
development [104] (Figure5). Together, all these ACs generate the cAMP gradients that are
required for D. discoideum to provide positional information necessary to transition from uni-
to multi-cellularity [103]. Structure/function studies in D. discoideum may play an important
role in fleshing out the details of this model and further they would be valuable to track the
role of ACs in the timing of cell developmental events. To this end, a recent study performed
a systematic investigation of the effects of caffeine, ribose-modified adenosine analogues and
other known regulators of mammalian ACs on the activities of ACA, ACB and ACG. This
study added a large number of traits that distinguish these ACs from each other and that prove
useful really for enzyme identification in specific cell types or during specific stages in
development of D. discoideum or to recognize similar activities in other organisms [105].
Moreover, from the multicellular fruit body of D. discoideum, two novel amino sugar
analogues, furanodictine A and B have been isolated and with potent ability to induce neurite
outgrowth in rat PC-12 cells [106], the classical in vitro model for neuronal development.
D. discoideum could be furthermore functional to investigate the intriguing link between

developmental ACs expression and dopaminergic pattern, since it has been also proposed as a
powerful model system to study signaling processes related to the Wnt pathway of animals
[107]. The Wnts are highly conserved glycoproteins important for development. Three
specific Wnt proteins, Wnt1, Wnt3a, Wnt5a, have been implicated in the development of
midbrain DA neurons and were shown to influence the expression of all the three critical
transcription factors En-1, Ptx3, and Nurr1 that must be present in the neuron to be
considered a true midbrain DA neuron. Wnt3a promoted the proliferation of precursor cells
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Account: ns017152
expressing Nurr1 but did not increase the number of TH-positive neurons. Wnt1 and Wnt5a
increased the number of TH-positive neurons. Wnt1 predominantly increased the proliferation
of Nurr1 precursors, whereas Wnt5a increased the proportion of Nurr1-positive precursors
that acquired the DA phenotype and up-regulated Ptx3 [108].
ACB
NEURONAL
DIFFERENTIATION
ACG
FUNCTIONAL
ACA MATURATION
Cell streams in
response to cAMP
NEURAL
COMMITMENT
STEM CELLS
TO LINEAGE
PROGENITORS
Figure 5. Dictyostelium discoideum differentiation steps. The top drawing shows a montage of pictures
illustrating the morphological changes, accompanied by the three AC isoforms expression, that take
place during the development of this organism. The scheme below highlights the separation between
the two phases of unicellular or vegetative growth (comparable to the neural stem cells storage) and of
multicellular development (analogous to the commitment, migration, differentiation and maturation of
neurons).
DRUG-LIKE DIFFERENTIATING AGENTS:

MAKING DA NEURONS FROM NEURAL STEM CELL NICHE

Several small molecules are known to promote NSC differentiation (reviewed in [109]
and [110]) and some might target the AC pathway, such as ascorbic acid (AA; vitamin C;
Figure 6), retinoic acid (RA; vitamin A; Figure 6) and trophic factors. Discovery of AA as a
competitive inhibitor of AC activity has opened new fascinating research areas investigating
its potential novel therapeutic properties [111]. In developing rat striatum, RA, through its
nuclear receptors, is known to establish the infrastructure of DA neurotransmission by
upregulating the expression of striatum-enriched DA signaling molecules, including the D1
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Account: ns017152
receptor, Golf, AC5 and DA- and cAMP-regulated phosphoprotein (DARPP-32) [112]. This
suggests that their promoters contain RA response elements (RAREs) or responsive elements
for the RA-activated transcription factors. This feature could be used to investigate RA or its
more selective derivatives as feasible candidates in PD therapy. The best-known direct
activator of AC, the di-terpene forskolin (FSK; Figure 6), has been reported to act in concert
with RA to promote neuronal differentiation of adult NSCs [110]. This information led to the
revival of the idea that an endogenous FSK-like small molecule activator of AC might exist
[113]. The activity of some AC isoforms is also known to be regulated by receptors of trophic
factors, such as the epidermal growth factor (EGF), which specifically activates AC5 only via
tyrosine phosphorylation of the stimulatory protein G [7], and NGF, which increases the
responsiveness of PC12 cells to the cAMP-elevating property of FSK [94]. These results
expand management of cAMP production during neural differentiation to several small drug-
like molecules and neurotrophins. Therefore, a microenvironment containing these factors
with selective modulators toward DA-sensitive ACs, such as AC3 and AC5/AC6, and also
toward the Ca2+/calmodulin-activated AC1, as crucial link between neuronal activity and
intracellular cAMP, could be hypothesized. Detailed structure–activity relationships for these
selective modulators has been recently reviewed [6] and [114]. We highlight some of them
here for their potential use in streamlining the maturation and retained functionality of the
dopaminergic network. Currently, the best-known AC5-selective stimulators are the FSK-
derivative 6-[3-(dimethylamino)propionyl]FSK (NKH477; Figure6), in use clinically against
acute heart failure [6], and 6-[3-(dimethylamino)propionyl]-14-15-dihydroFSK (DMAP;
Figure 6). These compounds show AC5 selectivity with respect to AC2 and AC3 [6, 115-
118]. Together with their ability to discriminate between the two DA-sensitive AC3 and AC5,
the molecular framework of these derivatives could be further engineered for crossing the
blood–brain barrier (BBB). The most potent AC1 stimulator BODIPY-FSK (Figure 6), which
is selective with respect to AC5 but not AC2 [6, 119,120], as well as a new lead candidate
AC1 inhibitor, NB001 [121], could be valuable tools for probing developing striatal neurons
in culture [47]. Two other FSK-derivatives, 7-deacetyl-7-hydroxaminoFSK and 5,6-
dehydroxy-7-deacetyl-7-nicotinoylFSK (FD3 and FD4, respectively, Figure 6), are selective
activators for AC3 with respect to AC2 and AC5 [115], and could be tested in dopaminergic
neuronal migration.
Among the P-site inhibitors with metal chelating properties (PMC), the derivative 1R,4R-
3-(6-aminopurin-9-yl)-cyclopentane-carboxylic acid hydroxamide (PMC-6, Figure 6) has
been identified as a potent AC5 inhibitor that is selective with respect to AC2 and AC3 [122,
123]. Moreover, the 2′(3′)-O-(N-methylanthraniloyl) (MANT) nucleotides, such as MANT-
guanosine 5′-[γ-thio]triphosphate (MANT-GTPγS) and MANT-inosine 5′-[γ-
thio]triphosphate (MANT-ITPγS, Figure 6) have also been found to be potent AC inhibitors
[124], showing weak AC5 selectivity with respect to AC2, but not with respect to AC1 and
AC6 [125]. However, the selectivity of the compounds reported here has not yet been tested
for all AC isoforms [6, 22]; therefore, it is currently not possible to make definitive
statements about isoform selectivity. A drawback of MANT-nucleotides is their inability to
penetrate the BBB, so they do not appear to be suitable drug candidates for neurological
diseases. By contrast, appropriate devices could be prepared to manage the uptake of MANT-
nucleotides into the CNS, taking into account the fact that polymeric micro- and nanoparticles
can be useful for the brain targeting of drugs unable to cross the BBB [126-128]. Similarly, as
for NKH477 and DMAP, PMC-6 and MANT-nucleotides could be adapted to cross the BBB
and be used potentially to counteract the D1 supersensitivity effect in the l-DOPA treatment of
PD. Furthermore, it should be pointed out that the same devices may be useful also to avoid
IBEROAMERICANA
Account: ns017152
that peripheral organs having dopamine innervation and expressing AC5, such as heart,
kidney, pituitary and retina, may be also impacted by the DA-responsive ACs therapeutic
targeting.
O O O
OH 11 12 13 CH2 OH CH2 OH CH2
C C C
9 H H H
1 O
2 10 8
OH OH OH
3 5 7 OH
4 6
O N
H H H
OH Forskolin OH FD3 FD4
N
O O
OH CH2 OH O
C
H
O O OH
OH OH
Retinoic acid
O O
H H
O N O N
NKH477 DMAP
O O
O
OH CH2
C
H
O O O
OH H2
C 2
O C N N C
H H2 2 H H H2 2 N
OH
S
BODIPY-forskolin F
F
N OH
NH
O O OH
N N R
S O O
NH2
P P P
HO O O O O HO OH
N
O O O N Ascorbic acid
N N
O O
R= H MANT-ITPS PMC-6
H O NHOH
R = NH2 MANT-GTPS
NH O
CH3
Figure 6. Chemical structures of AC activators [forskolin; FD3; FD4; NKH477; DMAP; and BODIPY-
FSK], AC inhibitors [MANT-ITPγS and MANT-GTPγS; PMC-6; and ascorbic acid] and RA, acting in
concert with forskolin to promote neuronal differentiation.
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Account: ns017152
CONCLUSION, INSIGHTS AND THE FUTURE

Because of their involvement in PD and other disorders in the CNS, mesencephalic DA
neurons are a focus of clinical interest and a subject of intensive studies. For PD, a potential
therapy has been so far to replace the lost DA neurons with healthy DA neurons generated in
vitro through the differentiation of stem cells and to integrate them appropriately into the host
brain. It is clear that a comprehensive understanding of the cell fate determinants to obtain
functional midbrain specific DA neurons is required. In this regard, it is known that DA as
well as other neuron types, as they differentiate and migrate, encounter different cues that are
spatially and temporally regulated. The exact sequence of encountered cues can be crucial for
the retained functionality of neurons. Alternatively, therapies based upon the manipulation of
endogenous precursors in situ might have the most obvious advantage over transplantation-
based approaches in that they work without an external source of cells. However, there are
also potential limitations. First, such an approach might be limited to particular regions of the
brain, because multipotent neural precursors are more densely distributed in particular
subregions of the adult brain. As a consequence, it is possible that there simply are not
adequate numbers of precursor cells to bring about functional recovery. In addition, the
potential differentiation fates of endogenous precursors might be too limited to allow their
integration into varied portions of the brain. However, the more relevant difficulty is that it
could be hard to provide the precise combination and sequence of molecular signals necessary
to induce endogenous precursors to proliferate efficiently and differentiate precisely into
appropriate types of neuron deep in the brain. Therefore, knowledge of the sequence of
biochemical events is essential not only for the therapeutic use of exogenous NSCs, but also
for human adult neurogenesis, with the aim of stimulating endogenous neuronal repair during
brain failure, including PD and schizophrenia. A rapid and efficient approach oriented to
discriminate neuronal differentiation in cell culture can support pharmacological studies
directed at differentiating cells and screening for new pro-neurogenic factors.
This field is only now beginning to understand the complex interplay between neural
precursor potential and signals in the local microenvironment; much remains to be learned
about precursor heterogeneity and how to take advantage of what might be partial cell-type
restriction, permissive and instructive developmental signals, and modulation of specific
aspects of neuronal differentiation and survival. These goals could be approached by
modulating promising post-receptor targets, therefore bypassing unwanted phenomena such
as the under- and overexpression or supersensitivity of receptors. The potential of AC as
object of drug therapy has been already reviewed elsewhere [6, 22, 114], highlighting the
different AC isoforms as specific and integrative detectors for environmental signals.
Therefore, ACs could be a site of convergence for extrinsic and intrinsic factor signaling
during neurogenesis. Recruitment of DA/Ca2+-sensitive AC1, AC3 and AC5/6 in a subtype-
specific manner could provide both instructive and permissive cues for the production of
dopaminergic-specified neurons. In other words, it would be interesting to know whether the
change in Ca2+-regulated ACs is either a consequence or the driving cue of dopaminergic
differentiation. Moreover, the role of individual ACs in neural differentiation should be
clarified by evaluating the potential discrimination of cAMP signals among different lineages
of neurons. An answer to these questions requires not only potent ligands whose selectivity
has to be systematically examined across all the ACs, but also antibodies with the requisite
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Account: ns017152
sensitivity for each isoform [6, 114]. As promising investigative tool, a selective AC5
antibody has recently been identified [52]. However, evidence that only the catalytic activity
of an isoform is usually predominant in a specific tissue, such as the striatal AC5 or the
olfactory AC3, in combination with the recent approach using specific AC knockout mice [6],
encourages AC-selective drug targeting. For example, AC1 and AC5 targeting might enable
researchers to discern the relevance of the shift in calcium- and voltage-sensitive AC
expression and activity during development of mesencephalic DA neurons.
Besides the cell culture models, D. discoideum may be also an ideal organism for
studying how ACs and other regulatory molecules, such as neurotrophic factors, are
integrated to control cell fate decisions. The insights provided should be directly applicable to
all eukaryotes. Moreover, the rapidity with which gene function can be examined in D.
discoideum should facilitate to identify pathways that may pioneer studies in other systems.
The close link between migration and differentiation observed in D. discoideum could be also
useful for testing olfactory AC3-discerning ligands, framed potentially on the structure of the
two 7-deacetyl-7-hydroxaminoFSK and 5,6-dehydroxy-7-deacetyl-7-nicotinoylFSK
derivatives, in studies aimed to enhance neuronal migration performance. In addition, nasal
route may result in an addition route for drug delivery that obviates the inherent barriers
associated with the CNS or even for cell delivery as one of the new incoming cell
replacement therapies [129]. One of the most important challenges for the future will be to
find the highest efficient method to generate functional dopaminergic neuronal cells by direct
reprogramming differentiated cells without reverting them to a progenitor cell stage. These
procedures will help to provide DA neurons in a patient without the risk of carcinogenesis,
which is currently associated with pluripotent stem cells replacement therapies. However, the
degree of the global genetic reprogramming remain to be properly characterized.
Furthermore, it should understand if the functional maturation in DA neurons may be better
retained using the direct conversion of somatic cells by a transfectionally forced expression of
lineage-specific factors or maybe favoring an intrinsic transition of these cells into DA
neurons through an exogenous cocktail of differentiation agents.
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Chapter 5
MOLECULAR AND GENETIC BASIS OF THE

REGULATION OF DOPAMINE TRANSPORTER
Shigeo Kitayama, Chiharu Sogawa and Norio Sogawa

Department of Dental Pharmacology,
Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
ABSTRACT
Dopamine transporter (DAT) is a member of the solute carrier (SLC) gene family,
such as the Na+/Cl- -dependent transporter subfamily, which participates in the reuptake
of released neurotransmitter, thereby controlling the fine-tuning of neurotransmission.
DAT as well as noradrenaline transporter and serotonin transporter are associated with
psychiatric and neurological disorders, and are targets for antidepressants and drugs of
abuse. Recent molecular, pharmacological and genetic approaches to the regulation of
DAT have established its importance in the dopaminergic neurotransmission. These
studies achieved the identification of intracellular signaling cascades leading to the
control of DAT expression, function, trafficking, and degradation. In this chapter, we
review the current knowledge about the role of DAT-associated proteins in the regulation
of DAT expression and function.
In addition, we discuss possible implication of DAT regulation to the vulnerability of
dopaminergic neurons to the putative toxicity of certain drugs like amphetamine and
factors involved in dopaminergic neurodegeneration. Understanding the molecular and
genetic basis of the regulation of DAT is essential for contributing the identification of
potential new targets for the treatment of various brain diseases including Parkinson‟s
disease.

Corresponding to: Shigeo Kitayama Department of Dental Pharmacology, Okayama University Graduate School
of Medicine, Dentistry and Pharmaceutical Sciences Shikata 2-5-1, Kita-ku, Okayama 700-8558, Japan, (Tel)
+81-86-235-6660, (Fax) +81-86-235-6664, (E-mail) shigeok@md.okayama-u.ac.jp
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Account: ns017152
142 Shigeo Kitayama, Chiharu Sogawa and Norio Sogawa
INTRODUCTION
Neurotransmission takes place at synapse, a highly specialized structure, and requires
machineries controlling neurotransmitter clearance at synaptic cleft. The most important key
player is neurotransmitter transporter, which acts to terminate synaptic transmission by
reuptake of released neurotransmitters [1]. The plasma membrane neurotransmitter
transporters belong to the solute carrier (SLC) gene family, and consist of the Na+/Cl- -
dependent transporter subfamily (SLC6). Dopamine transporter (DAT, SLC6A3) is a member
of this subfamily along with other monoamine transporters, such as noradrenaline transporter
(NET, SLC6A2) and serotonin transporter (SERT, SLC6A4). Among members of this family,
these monoamine transporters have drawn much attention, since they are targets for
antidepressants and drugs of abuse [2].
Brain dopamine (DA) has been known to play an integral role in cognition, affect,
behavioral reinforcement, and motor function, thereby their pathologies are suspected to
associate with disorders, such as depression, attention deficit-hyperactivity disorder (ADHD),
Parkinson‟s disease (PD), and addiction [3, 4]. There are two major projections of
dopaminergic pathways: nigrostriatal DA neurons from the substantia nigra pars compacta to
the striatum, and mesocorticolimbic DA neurons from the ventral tegmental area to the
medial prefrontal cortex and nucleus accumbens. Because of its participation to the clearance
of released DA at synaptic cleft, the DAT plays a key role in dopaminergic neurotransmission
[3, 4].
In relation to the diseases mentioned above, the DAT is a target of several drugs, such as
the psychostimulants methylphenidate and amphetamine clinically used for ADHD, and
modafinil for narcolepsy, as well as the antidepressant bupropion [2]. Psychostimulants
cocaine and methamphetamine produce the reinforcing and euphoric effects through the
interaction with the DAT [5]. This has been supported by analysis of DAT knockout mice that
show an attenuated response to cocaine and amphetamine [6, 7].
Members of SLC6 family are glycoproteins with 12 transmembrane-spanning domains
and intracellular N- and C-termini [2]. This membrane topology was supported by the recent
success of the X-ray crystallography of bacterial homolog of the neurotransmitter transporter,
leucine transporter (LeuT) [8]. It opened avenues to understanding the structure-function
relationship of the neurotransmitter transporters including DAT, resulting in the fruitful
achievement of the molecular and genetic basis of the regulation of DAT.
DAT regulation can occur through functional modifications at the plasma membrane that
include (1) altered substrate/Na+/Cl- affinity, (2) kinetic activation, and (3) altered
conductance states. In addition, expressional regulation of the DAT has been extensively
investigated at different stages; (4) trafficking of newly synthesized DAT to the plasma
membrane, (5) endocytic trafficking of DAT from the plasma membrane, and (6) changes in
protein turnover. At each step, a series of protein-protein interaction is known to occur.
Furthermore, regulation of the DAT gene expression is important processes for DAT
availability to promote DA signaling, which include transcriptional and post-transcriptional
regulations.
According to these issues, we review the current knowledge about the regulation of DAT
expression and function that indicates a fruitful achievement of the molecular and genetic
basis of the importance of DAT. In addition, we discuss possible implication of DAT
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Account: ns017152
Molecular and Genetic Basis of the Regulation of Dopamine Transporter 143
regulation to the vulnerability of dopaminergic neurons to the putative toxicity of certain

drugs like amphetamine and factors involved in dopaminergic neurodegeneration like PD.
1. CELL SURFACE EXPRESSION AND TRAFFICKING OF DAT

De novo synthesis rate of DAT protein was examine using covalently binding ligand
specific to DAT, indicating a relatively slow; the half-life of striatal DAT protein was
approximately 2-3 days [9, 10]. Therefore, rapid modification of DAT function necessary for
prompt response to intra- and extracellular stimuli occurs via posttranslational regulatory
processes without de novo protein synthesis or transcriptional and/or posttranscriptional
modulation.
That could be achieved by affecting the membrane trafficking of the transporter,
redistributing the DAT between the plasma membrane and intracellular endosomal
compartments [11, 12].
DAT is synthesized at the endoplasmic reticulum (ER) membrane, packaged into COPII
vesicles, and transported to the Golgi apparatus, where the transporter is proposed to be N-
glycosylated in the extracellular loop 2 [13]. Then DAT traffics to the plasma membrane. The
mechanisms that control movement of newly synthesized DAT from Golgi to specific cell
surface sites are not known.
Electron microscopy analysis revealed that DAT present in the plasma membrane of
axonal varicosities, axons in striatum and nucleus accumbens, and the distal dendrites in the
substantia nigra pars compacta and ventral tegmental area, but not in the neuronal soma [14-
16]. In axonal varicosities DAT was not detected in the active zone of the synapses, but rather
it was located extrasynaptically [14].
The molecular mechanisms of the anterograde trafficking of DAT have been investigated
mostly in heterologously expressed systems and primary cultures of embryonic
mesencephalic neurons. However, site-directed mutagenesis studies for exploring a possible
functional interaction by amino acid substitution of the transporter protein at the putative
tranmembrane domains often resulted in its reduced expression at the plasma membrane, due
probably to ER retention [e.g. 17, 18]. Such ER retention could be explained by assuming that
the disruption of the integrity of the membrane spanning -helices yields an unfolded
molecule that is incorrectly incorporated into the ER membrane.
Once the transporter has been delivered to the plasma membrane through the biosynthetic
pathway as mentioned above, it undergoes constitutive and regulated endocytosis. Logically it
is expected that constitutive endocytosis of DAT is much slower than the endocytosis of
synaptic vesicle membrane proteins like vesicular monoamine transporter-2 (VMAT-2) that
must be efficiently internalized and sorted to these vesicles after exocytotic neurotransmitter
release.
However, early studies found that loss of cell surface DAT in accordance with the
downregulation of DAT function can occur within minutes after stimulus, such as activation
of protein kinase C (PKC) [19, 20]. These findings emphasized subsequent investigation
regarding the elucidation of mechanisms of DAT endocytosis, as described below.
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Account: ns017152
2. CELLULAR SIGNALING CASCADES REGULATING DAT EXPRESSION

2.1. Protein Kinase C
Members of the nurotransmitter transporter family including DAT possess multiple

consensus amino acid sequences for phosphorylation, many of which lie on the N- and C-
termini. The most extensively investigated mechanism for DAT regulation is the effect of
PKC. Numerous studies in various cell lines heterologously expressing DAT have shown that
the activation of PKC by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA),
decreases DA transport activity [eg. 21]. A similar result of DAT down-regulation was also
observed in a various studies using synaptosomal preparations from the brain [e.g. 22]. The
important early studies by two groups independently reported that activation of PKC resulted
in the internalization of DAT into lysosomes for degradation [19], or into early and recycling
endosomes [20].
PKC-induced down-regulation of DAT was associated with dynamin-dependent
endocytosis [19, 23], although trafficking-independent and initial rapid inactivation of the
DAT occurred at the plasma membrane was observed [24]. PKC-induced DAT
phosphorylation might not be directly coupled to DAT down-regulation. Indeed, PKC
phosphorylated serines at the N-terminus of DAT protein [25]. However, truncation of the
distal N-terminal region of DAT containing these serines, or mutations of these serines to
alanines, did not affect the down-regulation of DAT by PMA [26]. These results suggested a
possible phosphorylated protein(s) involved upstream of the DAT down-regulation. In
addition, nonclassical, distinct endocytic signals located in C-terminus of the DAT were
identified, which were necessary and sufficient to drive constitutive and PKC-regulated DAT
internalization [27].
2.2. Ubiquitination
Clues to clarify the mechanism underlying the PKC-regulated DAT internalization were
obtained from recent studies by Miranda et al., in which involvement of ubiquitination in the
PKC-activated DAT down-regulation was indicated [28]. Ubiquitination has emerged as the
major mode of regulation of the subcellular localization and turnover of membrane proteins
[29]. Miranda et al. demonstrated that DAT was constitutively ubiquitinated and that
ubiquitination was enhanced by PMA stimulation [28]. The mutagenesis analysis suggested
that three lysine residues (Lys19, Lys27, and Lys35) clastered in the DAT N-terminus were
the major ubiquitin-conjugation sites [30]. Furthermore, mutations of these residues to
arginine abolished PKC-dependent internalization, suggesting that the ubiquitin moieties

conjugated to DAT may serve as a molecular interface of the transporter interaction with the
endocytic machinery [30].
Large-scale RNA interference analysis of PKC-dependent internalization of the DAT
revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2, as well as the
involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15, and
Eps15R [31]. Deletion of Nedd4-2 resulted in a reduction of PKC-dependent ubiquitination of
DAT. DAT and Nedd4-2 co-localized in heterologous cells transfected with DAT and in
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primary culture of rat dopaminergic neurons. Furthermore, endogenous Nedd4-2, epsin, and
Eps15 were coimmunoprecipitated with heterologously expressed human DAT and
endogenous DAT isolated from rat striatum. Based on these results, authors proposed a new
mechanistic model of DAT endocytosis that the PKC-induced ubiquitination of DAT
mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and
acceleration of DAT endocytosis [31].
2.3. PI3K, MAPK, other Protein Kinases
Additional kinase pathways have been shown to affect DAT function and cell surface
expression. Carvelli et al. reported the involvement of phosphatidylinositol 3-kinase (PI3K), a
downstream effector of insulin signaling, in DAT regulation [32]. Inhibition of PI3K by
LY294002 induced internalization of DAT, thereby reducing transport activity. Conversely,
insulin caused an increase in DA uptake. Expression of a dominant negative mutant of
dynamin1 inhibited the LY294002-induced reduction of DA uptake and DAT surface
expression, suggesting a possible modulation of transport capacity of DAT by dynamin-
dependent trafficking. Akt is a protein kinase effector immediately downstream of PI3K.
Garcia et al. demonstrated that both over-expression of a dominant-negative mutant of Akt
(K179R) and the addition of ML9, a pharmacological inhibitor of Akt, decreased cell-surface
expression of DAT, suggesting a role of basal Akt signaling in the homeostasis of DAT [33].
A physiological significance of this signaling pathway in the DAT regulation was further
examined in rats treated with the diabetogenic agent streptozotocin [34]. Rat depleted of
insulin by streptozotocin showed that striatal Akt function, as well as DAT cell-surface
expression, were significantly reduced.
Moron et al. provided evidence suggesting that mitogen-activated protein kinase (MAPK)
regulates DAT surface expression and DA transport capacity [35]. They demonstrated that
MAPK kinase (MEK) inhibitors decreased DA uptake both in rat striatal synaptosomes and in
HEK293 cells transfected with epitope-tagged human DAT. MAPK inhibition promoted the
clathrin-associated redistribution of DAT from the plasma membrane to the cytosol in
heterologously DAT-expressed cells, suggesting that activity of MAPK appears to be
important for maintaining the DAT surface expression maximally. Interestingly, using a
genetic complementation screen, a MAPK phosphatase, MKP3, was identified as a molecule
that inhibits PKC-induced internalization of transporter, resulting in enhanced DAT activity
[36]. However, the effects of MKP3 on DAT internalization were observed to be independent
of classical MAPK cascades [36]. There is also evidence indicating the DAT regulation by
tyrosine kinases [37]. It was suggested that striatal DAT function and surface expression is
constitutively upregulated by tyrosine kinase activation and that brain-derived neurotrophic
factor can mediate this type of rapid regulation.
3. SCAFFOLDING OF DAT
Early studies on the truncation at C-terminus have indicated the diminished cell surface
expression of DAT protein [38, 39], and the subsequent investigations suggested not only
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important role of that region for ER export [40], but also scaffolding of DAT at the plasma
membrane through its PSD-95/Discs-larger/ZO-1 (PDZ) homology motif at C-terminus [41].
Scaffolding proteins serve to organize macromolecular complexes to ensure special and
temporal control of cellular processes, resulting in the connection of membrane proteins such
as DAT to their downstream signaling partners or anchoring to the microdomains of cellular
contexts. Studies in Caenorhabditis elegans showed that DAT localizes dopaminergic
synapses precisely probably through the unknown assemble mechanisms [42, 43]. Little is
known about DAT scaffolding and microdomain localization.
Using yeast two-hybrid screen system with DAT C-terminus as bait, Torres at al.
identified the PDZ domain protein interacting with C-kinase 1 (PICK1) as a DAT interacting
protein [41]. Human DAT has a canonical class II PDZ-binding sequence at its C-terminus
(LKV618-620), and its interaction with the PDZ motif of PICK1 was validated by co-
immunoprecipitation experiments from heterologous expression and rat brain tissues. This
interaction promoted DAT surface expression and induced a clustering phenotype in
transfected cells [41]. However, subsequent study by Bjerggaard et al. demonstrated that,
although the extreme C-terminus is indispensable for directing the transporter to the cell
surface, canonical PDZ domain interactions are not required [44]. Based on the mutagenesis
data, they proposed dual roles of the DAT C-terminus: a role independent of PDZ interaction
for ER export and surface targeting, and a not fully clarified role involving PDZ interactions
with proteins such as PICK1. Thus, the functional significance of the DAT-PICK1 interaction
remains to be elucidated.
A second protein identified by yeast two-hybrid screen was Hic-5 [45]. Hic-5 is a
member of the focal adhesion family of adaptor proteins, which contain multiple Lin11/Isl-
1/Mec-3 (LIM) domain. Co-immunoprecipitation and GST-pulldown assay showed the
interaction mediated by the membrane proximal part of the DAT C-terminus and the LIM
domain-containing half of Hic-5. In heterologous cells, overexpression of Hic-5 reduced DAT
transport activity through reduction of DAT surface expression. However, the mechanism
underlying this effect of Hic-5 was not further elucidated, and its physiological relevance was
not examined in the native tissues.
Using the split ubiquitin system, the synaptic vesicle protein synaptogyrin-3 was
identified as another scaffolding protein interacting with DAT [46]. This interaction was
confirmed through co-immunoprecipitation experiments using heterologous cell lines and
mouse brain. DAT and synaptogyrin-3 co-localized at presynaptic terminals from mouse
striatum. In catecholaminergic cell lines synaptogyrin-3 over-expression with DAT increased
DA uptake while synaptogyrin-3 siRNA decreased DAT transport activity. These changes
were not attributed to changes in transporter surface expression or to direct effect of the
protein-protein interaction. Instead, the effect of synaptogyrin-3 on DAT activity was reduced
by the VMAT-2 inhibitor reserpine, suggesting a dependence on the vesicular DA storage
system. This attractive hypothesis should be challenged in the future.
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4. SYNTAXIN1A MODIFICATION OF DAT

BY ALTERED CONDUCTANCE STATES
Rapid alteration of affinity for substrate, Na+ and Cl-, changes in the kinetics, and the
altered conductance states can modify the plasmalemmal DAT activity. In this section, we
focus the DAT regulation by its conductance states, which is associated with the binding
partner, syntaxin 1A.
DAT, as well as other neurotransmitter transporters, displays electrogenic and can be
analyzed electrophysiologically. Patch-clamp recording in dopaminergic neurons from
Caenorhabditis elegans suggested that UNC-64, a Caenorhabditis elegans homolog of the
soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein
syntaxin1A, binds the N-terminus of Caenorhabditis elegans DAT and suppresses the
channel-like activity [47].
They also observed that the loss of interaction between DAT and UNC-64 caused
swimming-induced paralysis, a behavioral phenotype previously observed in the DAT-
deficient nematodes [48].
There is a line of evidence suggesting the involvement of syntaxin1A in the regulation of
transporter conductance states, such as rat GABA transporter-1 (GAT-1) [49], rat SERT [50],
and human NET [51]. It was demonstrated that through the interaction with N-terminus of the
transporter syntaxin1A caused a switch from an electrogenic transport with non-
stoichiometric way to an electroneutral transport with a mode in which substrate and ion
transport is strictly coupled [50].
Syntaxin1A has been shown to bind the N-terminus of human DAT [52]. Binda et al.
demonstrated that perfusion of the patch pipette with the purified soluble cytosolic domain of
syntaxin1A enabled amphetamine-induced DA efflux at resting membrane potentials, and that
amphetamine increased the interaction between DAT and syntaxin1A in a Ca2+/calmodulin-
dependent protein kinase II (CaMKII) -dependent manner [53]. Based on these results, they
suggested a mechanism in which amphetamine activation of CaMKII strengthened the DAT-
syntaxin1A interaction, resulting in a mode of DAT in which efflux is possible [53].
Influence of amphetamine on DAT trafficking is discussed in the next section of this
chapter.
5. SUBSTRATES AND INHIBITORS REGULATING DAT TRAFFICKING

Amphetamine, substituted amphetamines and cocaine are psychostimulants widely
abused, and their stimulatory actions are well characterized as increasing in DA
concentrations at synaptic cleft through acting on the DAT. Amphetamine is a substrate that
is transported by DAT, resulting in the reverse transport of DA, while cocaine binds to DAT,
and acts as an uptake inhibitor [54]. They have been known to modify the regulation of DAT
expression in a different manner, as discussed below.
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5.1. Substrates Dopamine and Amphetamine
There is accumulating evidence indicating that substrates can produce down-regulation of

DAT activity and cell surface expression. Firstly systemic administration of D-
methamphetamine was observed to produce a reduction of DA uptake into striatal
synaptosomes prepared from rats 1h after administration [55]. Further analysis demonstrated
that the observed reduction of DA uptake was due to the decrease in Vmax without changing
the total DAT protein levels [56]. These results led to the hypothesis that methamphetamine
affects either DAT activity at cell surface or DAT trafficking to/from the plasma membrane.
Saunders et al. addressed this issue using fluorescent microscopy to visualize tagged
human DAT heterologously expressed in HEK293 cells [57]. They observed that
amphetamine-induced loss of human DAT activity was mediated by a decrease of cell surface
DAT. Rapid loss of DAT from the plasma membrane in response to amphetamine was
internalization-dependent and cocaine-sensitive. Further studies by Chi and Reith confirmed
and extended the effect of substrate DA like amphetamine on DAT trafficking using the
biochemical techniques [58]. Cell surface biotinylation assays demonstrated that both
HEK293 cells expressing DAT and rat striatal synaptosomes exposed to DA revealed
decreased surface DAT expression in association with DA uptake capacity. Sorkina et al.
visualized amphetamine-induced DAT internalization in living cells by FRET [59].
Amphetamine induced the endocytosis of oligomerized DAT to early and recycling
endosomes, identified by Rab5 and other well-characterized markers.
Methamphetamine-induced downregulation of DAT was observed in synaptosomes
prepared from striatum but not from nucleus accumbens of rat brain [56]. Similar anatomical
difference has been reported for amphetamine in rat dorsal striatum and nucleus accumbens in
vivo by high-speed chronoamperometric measurements [60]. A recent study by Richards and
Zahniser re-examined the difference in terms of DAT function and surface expression [61].
Brief (15min) in vitro exposure to amphetamine decreased maximal velocity of DA uptake by
synaptosomes prepared from striatum but not from nucleus accumbens. However, 45 min
after systemic administration of amphetamine, maximal velocity of ex vivo DA uptake was
reduced in both regions. These results suggest that relatively brief exposure to amphetamine
results in greater down-regulation of DAT activity in striatum than in nucleus accumbens.
Further investigations are needed to elucidate region-specific differences in DAT regulation.
Recent work by Kahlig et al. has shown that amphetamine acts intracellularly to regulate
DAT trafficking [62]. Using an uptake-impaired mutant DAT (Y335A), they demonstrated
that amphetamine did not induce cytosolic redistribution of this mutant DAT that still binds
amphetamine. Direct intracellular application of amphetamine stimulated the trafficking of
the mutant. Taken together with other results, they concluded that the DAT transport cycle is
not required for amphetamine-induced down-regulation, while an increase in intracellular

amphetamine is an essential component of DAT redistribution. As mentioned in the section
2.3, Akt is essential for insulin modulation of amphetamine-induced DAT trafficking [33].
Again, it was shown that the cell surface expression of DAT is regulated by insulin signaling
pathway, and that Akt plays a key role in the hormonal modulation of amphetamine-induced
DAT trafficking and in the regulation of basal DAT cell surface expression. Further study by
this group provided additional findings that amphetamine decreases Akt activity through a
CaMKII-dependent pathway, thereby regulates insulin signaling and DAT trafficking [63].
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Many studies have shown that substrates can increase cytosolic redistribution of cell surface
DAT, as mentioned above. However, these studies performed relatively long term treatment
with substrates, from several minutes to a few hours. Johnson et al. have examined the effects
of brief application of substrates, a scale of seconds to minutes, on the DAT trafficking, and
demonstrated biphasic effects of amphetamine on DAT redistribution; initial rapid increase in
surface DAT within 1 min followed by returning to control levels at 2.5 min [64]. Reversible
biotinylation assays showed that amphetamine-stimulated increase in surface DAT was due to
an increase in the delivery of DAT to the plasma membrane rather than a decrease in the
endocytotic process. More recently, this group demonstrated that both DA and amphetamine
rapidly increase the DAT trafficking to the cell surface by means of live-cell imaging using
total internal reflection fluorescence microscopy [65].
5.2. Cocaine
In contrast to the case of amphetamine, there are few investigations on cocaine in aspect
to its influence on DAT trafficking. One of the reasons appears that early studies showed
controversial results from DA uptake examinations or ligand binding assays. Studies in
postmortem brain samples from chronic cocaine users have shown that DA uptake function
assayed in cryopreserved human brain synaptosomes revealed an elevated maximal velocity
of DA uptake in association with an increase in cocaine analogue binding, suggesting the
possibility of cocaine-induced DAT expression [66]. However, it is unknown whether these
changes may reflect compensatory response to cocaine. Cellular analysis of the effects of
cocaine has been performed using heterologous cell expression system. Daws et al.
demonstrated that in HEK293 cells expressing human DAT brief exposure to cocaine
followed by extensive washing resulted in an increase in DA uptake as well as an increase in
cell surface DAT in biotinylation experiments [67]. DA uptake assays ex vivo in synaptosome
from cocaine-treated rat and in vivo assessment of DA clearance in the striatum of
anesthetized rats revealed consistant results with cocaine-induced up-regulation of DAT.
Little et al. also demonstrated that exposure to cocaine not only increased maximal velocity of
DA uptake and cocaine analogue binding, but also cell surface DAT expression in stably
DAT-transfected N2A cells [68]. The mechanism by which cocaine regulates DAT trafficking
and its relevance to the cocaine‟s addictive action remain to be elucidated.
6.  SYNYCLEIN, PARKIN AND GPR37 REGULATING

DAT TRAFFICKING
Parkinson‟s disease (PD) is a neurodegenerative disorder with characteristics of motor

deficits, such as resting tremor, muscle rigidity and slowed movement. Its pathology is based
on a selective loss of dopaminergic neurons in the nigrostriatal pathway, and the treatment
with levodopa, a precursor of DA, has been established [69]. However, the cause is still
unknown. Familial PD suggests genes involved in the disease.
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6.1. -Synuclein
Aggregation of -synuclein in protein inclusion, known as Lewy body, is characteristic

for the pathology of PD [70]. However, its physiological function is not known.
Lee et al. [71] first reported the interaction of -synuclein with DAT. Physical interaction
between these proteins was demonstrated by the finding that non-amyloid beta component of
-synuclein binds to the last 22 amino acid of DAT C-terminus. When Ltk-mouse fibroblasts
were co-transfected with DAT and -synuclein, an increase in DA uptake was observed as
compared to cells expressing DAT alone. Since parallel increase in DA-induced cell toxicity
was observed in association with enhancement of DAT recruitment to the plasma membrane,
they concluded that these interaction may participate in the mechanism by which the selective
vulnerability of dopaminergic neuron in PD is in part due to the oxidative stress, resulting
from the cellular accumulation of DA or DA-like molecules by the DAT [71]. This possibility
was supported by the findings that expression of mutant -synuclein enhanced DAT-
mediated MPP+ toxicity in HEK293 cells co-expressing -synuclein isoform and human
DAT [72].
In contrast, Wersinger and Sidhu detected a decrease in DA uptake by over-expression of
-synuclein in DAT expressing cells [73]. Additional studies by these authors established a
large body of evidence demonstrating negative modulation of DAT by -synuclein, while a
physical interaction between DAT and -synuclein was confirmed [74, 75]. The discrepancy
might reflect differencies in the level of -synuclein over-expression, as they demonstrated in
the study on NET, where -synuclein was found to regulate the activity and surface
expression of the NET, depending on its expression levels [76].
Recently a decrease in DAT activity by siRNA knockdown of -synuclein was observed
in human neuronal cell lines [77]. The decrease in Vmax of DA uptake accompanied a similar
decrease in WIN35,428 (cocaine analogue) surface binding without any change in total DAT
expression levels. Furthermore, immunoelectron microscopy demonstrated a marked
reduction of cell surface DAT upon -synuclein knockdown [78]. These results suggest that
under native conditions, endogenous -synuclein promotes DAT activity through
enhancement of DAT trafficking.
However, it seems likely that -synuclein modulation of DAT is a homeostatic process
that is extremely context-dependent; effects of -synuclein on DAT can be modulated by the
cellular process interacting with -synuclein, endogenous levels of -synuclein, or the means
of regulation of -synuclein expression. It should be kept in mind that -synuclein knockout
mice have failed to show any alterations in DAT function [79, 80]. Further assessment of the
interaction between DAT and -synuclein is needed to resolve these discrepancies.
6.2. Parkin and Its Interaction with -Synuclein
Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to PD [81]. Although a

variety of parkin substrates have been identified, none of these is selectively expressed in
dopaminergic neurons [82]. Jiang et al. first demonstrated that parkin increased DA uptake by
enhancing the cell surface expression of DAT [83]. The enhancement of the ubiquitination
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and degradation of misfolded DAT by parkin so as to prevent it from interfering with the
oligomerization and cell surface expression of native DAT was suggested to underlie its
mechanism. This function of parkin would enhance the precision of dopaminergic
transmission, increase the efficiency of DA utilization, and reduce DA toxicity of neighboring
cells.
In accordance with these observations, it was demonstrated that parkin protects human
dopaminergic neuroblastoma SH-SY5Y cells against DA-induced apotosis [84]. This
protective effect of parkin is selective for DA- and 6-OHDA- but not for H2O2- or rotenone-
induced apoptosis, suggesting the involvement of the interaction with DAT. However, if cell
surface expression of DAT is increased by parkin as they demonstrated, it should enhance
rather than protect DA-induced apoptosis. There is a conflicting report that stable expression
of parkin did not attenuate cell death induced by DA in SH-SY5Y cells [85].
Studies have shown that parkin can affect -synuclein-mediated toxicity, suggesting
functional crosstalk between parkin and -synuclein [86]. The involvement of -synuclein,
parkin, and DAT in PD implicates the importance of understanding the potential functional
interactions between these proteins. Based on the previous observation that the -
synuclein/DAT complex facilitated the plasma membrane clustering of DAT, thereby
accerelating cellular DA uptake and DA-induced apoptosis [71], the functional role of parkin
in their interaction was investigated. Parkin disrupted the DAT/-synuclein interaction, and
abolished -synuclein-induced enhancement of DAT transport activity [87]. In addition,
parkin protected against DA-induced cell toxicity in dopaminergic SK-N-SH cell, suggesting
that this mechanism could account for the protective effects of parkin on dopaminergic
neurons in PD [87].
This report also demonstrated that parkin by itself decreased DA uptake without affecting
cell surface expression of DAT [87], in contrast to the results observed by Jiang et al. [83].
Again, as mentioned above for the case of -synuclein, it seems likely that the effect of
parkin on DAT is also context-dependent. It has been reported that DA covalently modifies
and functionally inactivate parkin [88]. Therefore, the efficacy of parkin depends on DAT
expression levels, which determine intracellular DA concentrations and subsequent
inactivation of parkin.
6.3. GPR37
Recently it has been reported that the orphan G protein-coupled receptor GPR37 interacts
with DAT [89]. GPR37 is a substrate of parkin, and it has been named parkin-associated
endothelin-like receptor (PAEL-R) [90]. An insoluble form of GPR37 is accumulated in brain
samples of PD patients, and the over-expression of GPR37 in cell cultures, in the absence of
parkin, can lead to unfolded protein-induced cell death [91, 92]. GPR37 and DAT were found
co-localized in mouse striatal presynaptic membranes and in transfected cells, and their
interaction was confirmed by co-immunoprecipitation assays [89]. In GPR37 knockout mice,
DAT function was increased through an increased DAT expression, and this was suggested to
involve an interaction between DAT and GPR37 [89].
In their previous report on GPR37 null mice, they showed that the lack of GPR37
resulted in the resistance to the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-
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tetrahydropyridine (MTPT) [93]. MPTP reveals toxicity through its toxic metabolite 1-
methyl-4-phenyl-pyridinium ion (MPP+), which is selectively accumulated in dopaminergic
neurons through DAT. In this context, the enhancement of cell surface expression of DAT by
GPR37 does not favor such scenario, since the increased DAT could facilitate the
accumulation of MPP+. They also observed that GPR37 null mice have reduced levels of
striatal DA, which may result from the increase of DAT-mediated DA uptake. Conversely,
the over-expression of GPR37 in transgenic mice has been demonstrated to be associated with
increased DA content in the striatum [92]. Further studies are required to assess the
significance of a putative DAT-GPR37 interaction regarding to the dopaminergic
neurodegeneration.
7. GENETIC BASIS OF THE DAT REGULATION

Genetic approach to understand the regulation of DAT has developed after molecular
cloning of DAT cDNA and gene. DAT gene consists of 15 exons spanning 60 kb, and reveals
specific polymorphic variants termed variable number of tandem repeats (VNTR) and single
nucreotide polymorphisms (SNPs). These might correlate with predisposition to a number of
neurological and psychiatric disorders, including attention-deficit hyperactivity disorder
(ADHD), bipolar disorder, autism, schizophrenia, drug abuse, and PD, in which DA systems
are implicated [94]. Therefore, it seems likely that these polymorphic variations affect the
regulation of DAT expression and function at multiple levels, for examples, transcriptional
and post-transcriptional regulation, and/or translational and post-translational modification.
7.1. VNTR
3’ UTR VNTR
Studies of the human DAT cDNA and gene identified a VNTR in the 3‟ untranslated
region (UTR), that composed of repeats of a 40 bp element containing 9 or 10, or more rare,
3, 5, 7, 8, and 11, copies of the repeat unit [95, 96]. Subsequent studies showed substantial
heterogeneity in allele frequencies among ethnic groups [97]. mRNAs at 3‟ UTR elements
bind regulatory proteins and form RNA-protein complexes that control mRNA transport,
translation and stability [98, 99]. Therefore, it is reasonable to anticipate that the 3‟
polymorphism in the SLC6A3 gene may affect DAT expression and function.
Several studies have attempted to investigate this hypothesis. Analysis of mRNA
expression in lymphocytes and brain regions from individuals with different DAT genotypes
by RT-PCR demonstrated that the highest levels of DAT mRNA were observed in individuals
with 10 repeat allele [100]. In accordance with this, it was demonstrated in the study of
abstinent alcoholics and controls that a 22 % increase in [123I]-CIT binding was observed in
the putamen of subjects with a 10/10 genotype, while no genotype difference between
controls and alcoholics [101]. On the other hand, no difference of [123I]-CIT binding
associated with the 9- or 10-repeat allele has been reported [102, 103]. In addition, recent
report demonstrated that an increased dopamine transporter availability was associated with
the 9-repeat allele of the SLC6A3 gene [104].
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Results from cell lines transfected with reporter gene have been equally controversial.
Fuke et al. showed that 10-repeat allele of SLC6A3 revealed higher luciferace expression than
7- or 9-repeat alleles in COS-7 cells transiently transfected with reporter gene constructs
[105]. In contrast, Miller and Madras demonstrated that vectors containing the 3‟ UTR
segments of a human DAT with 9-repeat allele downstream of the reporter gene resulted in
higher levels of luciferase expression than analogous vectors containing 10-repeat allele
[106]. Another study that employed similar constructs revealed no difference in expression of
reporter gene constructs containing the two alleles [107].
Taken together, it is not apparent that VNTR at 3‟ UTR of DAT gene has a role in
regulating the DAT gene expression. Conflicting results may result from different cell lines
used, which may express different factors involved in DAT expression through its 3‟ UTR
segment. For example, the electrophoretic mobility shift assay (EMSA) showed an interaction
of the DNA probe for VNTR sequence of DAT gene with proteins extracted from embryonic
stem cells [108]. Candidate protein that interacts with the 3‟ UTR segment of DAT gene has
been identified using a yeast one-hybrid system [109]. Further characterization by same group
showed that HESR1 binds directly to the region, as assessed by EMSA, and they suggested a
repression of the DAT gene expression by HESR1 [110]. However, role of VNTR in the
interaction of DAT and HESR1 is unclear. Recently, they reported the differential effects of
the HESR transcription factor family on DAT reporter gene expression through VNTR in SH-
SY5Y cells [111]. Although the results supported the hypothesis that HESR may interact with
3‟ UTR segment of DAT gene containing VNTR, the interpretation of results from
transfection studies is complicated. Further studies in vivo are required to establish the
functional role of VNTR in the regulation of DAT.
Intron 8 VNTR
Systematic analysis of the DAT gene from 5‟-flanking promoter to 3‟ UTR region has
identified multiple SNPs and VNTRs, including a novel VNTR located at intron 8 that
consists of 5 or 6 copies of a 30 bp repetitive element [112]. Subsequent studies have
demonstrated that VNTR in intron 8 was associated with ADHD in both English and
Taiwanese populations [113], as well as with cocaine abuse in a Brazilian population [114].
Functional analysis using reporter gene constructs demonstrated that the 6-repeat allele
displayed a lower expression compared with the 5-repeat allele at basal condition, while
increased responses when transfected cells were stimulated with cocaine, KCl, or forskolin
[114]. These results suggested that VNTR in intron 8 of the DAT gene might play a role in
cocaine dependence etiology [114]. Functional relevance of VNTR in intron 8 to ADHD was
investigated by measuring the expression levels of the DAT gene from post-mortem midbrain
tissue using TaqMan RT-PCR assays [115]. The 6-repeat of the intron 8 VNTR were
correlated with increased levels of the DAT transcript [115]. Recently Guindalini et al.
investigated a polymorphism within the intron 8 of the DAT gene in 27 healthy men using
SPECT image analysis [116]. No significant association was identified between the intron 8
VNTR and in vivo DAT availability in healthy subjects of Brazilian origin. Since the intron 8
VNTR has only recently been characterized, further studies are required to establish its
functional relevance to the DAT gene expression.
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7.2. SNPs
As mentioned above, resequencing of the DAT gene has identified SNPs as well as
VNTRs located in the promoter, introns, and 3‟ UTR regions throughout the gene [112].
Greenwood et al. reported that analysis of the linkage disequilibrium (LD) structure across the
DAT gene revealed the presence of two haplotype blocks defining the 5‟ (promoter through
intron 6) and 3‟ (exon 9 through exon 15) regions of the gene [117]. Little significant LD
between them may be interpreted by assuming a recombinant hot spot located near the middle
of the gene (introns 6-8), as suggested by Guindalini et al. [116]. Further investigation of the
functional relevance of these mutations by Greenwood and Kelsoe demonstrated that
promoter and intronic variants affected the transcriptional regulation of the human DAT gene,
as assessed by reporter gene expression assays [118]. Functional analysis of common SNPs in
a 2.8 kb region flanking the core promoter region of DAT gene was performed in the
neuroblastoma cell line SH-SY5Y using dual luciferase assays [119]. Haplotypes generated
by site-directed mutagenesis revealed varying impact of individual SNPs on promoter
activity. Although EMSA indicated DNA-protein interaction within this region, allele-
specific binding was not detected for any SNPs examined.
Several SNPs that produce missense variants of DAT have been reported [112, 120].
Initial study by Lin and Uhl reported the functional influences of variants V55A and V382A
in COS cell transient-expression assays [121]. V382A displayed decreased DA uptake
velocity Vmax in association with reduced cell surface expression, while V55A expressed
normally. Mazei-Robison and Blakely further characterized systematically the known DAT
variants, such as V55A, R237Q, V382A, A559V and E602G, using transiently transfected
COS-7 and SH-SY5Y cells [122]. All variants, except V382A, exhibited levels of surface
protein and DA transport activity comparable to the wild-type human DAT. In addition to the
decreased expression, V382A displayed a decrease in sensitivity to phorbol ester (PMA)-
induced internalization. Analysis of PMA-induced V382A internalization revealed a
trafficking-independent action of PMA, consistent with the existence of a surface-localized,
transport-inactive state.
Interestingly A559V variant was identified in two Caucasian male siblings with ADHD
and both subjects were homozygous for the ADHD-associated, 10-repeat 3‟ VNTR allele
[123]. The A559V variant was previously identified in a subject with bipolar disorder [120], a
psychiatric disorder that has a significant number of overlapping symptoms with ADHD.
Although functional analysis of the A559V variant revealed expression and function
comparable with human DAT [122], A559V may function in concert with the 10/10 VNTR
genotype to impact levels of DAT activity in vivo. More recent findings demonstrated that the
A559V variant exhibited anomalous DA efflux from DA-loaded cells, and that the two most
common ADHD medications, methylphenidate and amphetamine, both blocked the A559V
variant-mediated DA efflux, wheras these two drugs had opposite actions at wild-type DAT
[124]. Further study showed that tonic activation of DA D2 receptor (D2R) provided support
for DAT A559V variant-mediated anomalous DA efflux, in which a pertussis toxin-sensitive,
CaMKII-dependent phosphorylation mechanism was involved [125]. These results suggest a
signaling network downstream of D2R activation, normally constraining DA action at
synapse, that may be altered by DAT mutation to impact risk for DA-related disorders.
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7.3. Transcriptional Regulation of DAT: Transcription Factors
Multiple cis-acting elements regulating transcription are found within the DAT proximal
5‟-flanking sequences and several transcription factors involved in DAT gene expression
have been reported.
The importance of the nuclear receptor nurr1 for the appropriate development of
mesencephalic DA-synthsizing neurons has been clearly demonstrated through the targeted
disruption of the nurr1 gene. Nurr1 enhanced the transcriptional activity of human DAT gene
reporter constructs transiently transfected into cell line SN4741 that expresses a dopaminergic
phenotype. This action of nurr1 on hDAT transcription was independent on NGFI-B response
element (NBRE) [126]. In addition, two GC boxes at 5‟-flanking region of the DAT gene
were identified as important cis-acting elements mediating DAT promoter activity in
dopaminergic SK-N-AS cells [127]. Utilizing Sp-deficient Drosophila Schneider line (SL-2)
cells, both Sp1 and Sp3 were demonstrated as strong activators of DAT transcriptional
activity [127]. These results suggest that the Sp1 family of proteins plays an important role in
controlling the expression of the DAT gene.
Further study demonstrated that valproate, a drug used in the treatment of mania and
bipolar disorders, epilepsies, and addictions, increased endogenous DAT gene expression in a
human dopaminergic cell line and rat midbrain DA neurons in organotypic culture [128].
Valproate altered Sp protein abundance: it decreased Sp1, while unchanged Sp3 in SK-N-AS
cells or increased Sp3 in organotypic midbrain cultures. These findings favor the previous
observation that changes in the abundance or relative proportion of cellular Sp1 and Sp3
might regulate transcription of the DAT gene [127].
Analyzing the regulatory region upstream of human DAT gene core promoter by a series
of deletion mutant identified the activating cis-acting element for transcription in the
sequence between -2511 and 2492 termed HY1, and using the yeast one-hybrid screening the
zinc finger protein 161 (ZFP161) was cloned as a HY1-binding factor [129]. Both HY1
sequence and the downstream region were necessary for activation of the DAT promoter by
ZFP161, suggesting that the site of cofactor interaction with ZFP161 may exist downstream
of HY1.
By generating stable transgenic zebrafish lines expressing fluorescent reporters under
transcriptional control of fragments of slc6a3 genomic sequence, functional analysis of cis-
acting elements responsible for the expression pattern of slc6a3 identified the upstream
sequence between -6 kb and -2 kb that contains enhancer element, and drives slc6a3
expression in dopaminergic neurons of the pretectal region [130]. However, expression of
slc6a3 in dopaminergic neurons of the ventral diencephalon and telencephalon was shown to
be dependent on elements that lie outside the region -6 kb to +5 kb. Thus, it may be that the
expression of slc6a3 in different populations of dopaminergic neurons is regulated by discrete
enhancers, rather than by a single target sequence for a terminal factor involved in specifying
neurochemical phenotype. Recent study using Caenorhabditis elegans as a model for
development of dopaminergic neurons demonstrated that a transcription factor of the ETS-
family, encoded by the axon steering defect-1 (ast-1) gene, is a terminal selector gene for DA
cell phenotype including DAT [131]. Since Etv5, a member of ETS family, has been
proposed as a regulator of DA neuron differentiation in the mammalian midbrain, Wang and
Turner examined its role in comprising the DA neuron phenotype [132]. They showed that
Etv5 expression was not detectable until postnatal stages in the midbrain well after
IBEROAMERICANA
Account: ns017152
development of the DA system, and that Etv5 knockout and control mice revealed
comparable DAT expression in the embryonic and adult midbrain. Taken together with the
evidence with other known members of the ETS family, it was suggested that the ETS
factors, while required for the generation of the DA phenotype in nematodes, do not play such
a role in the mouse brain [132]. Ohyama et al. recently reported the nicotinic enhancement of
the transcription of DAT gene in SK-N-SH cells transiently transfected with reporter gene
construct [133]. Analysis of the 5‟-flanking region of the DAT gene containing the promoter
identified a novel nicotine responsive element in the human DAT gene between -3478 and -
1036. This enhanced promoter activity by nicotine would be consistent with the findings by
Li et al. that nicotine and cigarette smoking caused an increase in DAT mRNA expression in
the rat midbrain [134].
7.4. Post-Transcriptional Regulation of DAT: Alternative Splicing
Alternate promoters, differential splicing, 5‟- or 3‟-UTR variability, and SNPs in the
coding regions result in multiple mRNA species that regulate gene expression. In addition to
the VNTR and SNPs mentioned above, DAT mRNA isoforms produced by alternate splicing
have been reported recently [135, 136].
Fine-mapping the DAT genotype narrowed the potentially causal region to two correlated
clusters of associated SNPs localized predominantly to intron 3 and 4 [135]. Computational
analysis of these intronic regions predicted a novel cassette exon within intron 3, designated
E3b. Alternative splicing of E3b was confirmed in post-mortem human substantia nigra by
RT-PCR. As E3b introduces multiple in-frame stop codon, the SLC6A3 open reading frame
is truncated and the spliced product may undergo nonsense mediated decay, suggesting that
factors that increase E3b splicing could reduce the amount of unspliced product available for
translation.
We have also identified novel splicing variant of the human DAT in blood cells, which
lacked the region including a part of 2nd intracellular loop and 5th transmembrane domain
encoded by exon 6 [136]. RT-PCR analysis showed a differential expression of variants in the
brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous
expression of the truncated isoform revealed no transport activity, while expressed at least in
part in the plasma membrane. When transfected with the wild-type, the splice variant
displayed a dominant negative effect through the physical interaction, as assessed by
immunoprecipitation. Interestingly, immunocytochemical analysis using two different
antibodies, one specific to 2nd extracellular loop and other specific to the C-terminus of DAT,
showed unique membrane topology of the variant, suggesting the C-terminus to be located
extracellulary. These results suggest the importance of the DAT splice variant in
dopaminergic signaling in the brain and peripheral tissues.
IBEROAMERICANA
Account: ns017152
CONCLUSION
Molecular, pharmacological and genetic approaches to the regulation of DAT have
established its importance in the dopaminergic neurotransmission. These studies achieved the
identification of intracellular signaling cascades leading to the control of DAT expression,
function, trafficking, and degradation. DAT-associated proteins identified so far play an
important role in the regulation of DAT expression and function, expanding a fruitful
achievement of the molecular and genetic basis of the regulation of DAT. In addition, the
DAT regulation implicates the vulnerability of dopaminergic neurons to the putative toxicity
of certain drugs like amphetamine and factors involved in dopaminergic neurodegeneration.
These results provide clues to develop novel targets for manipulating the DAT regulation and
more effective therapeutics for the treatment of dopaminergic disorders.
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Chapter 6
DOPAMINERGIC AND GLUTAMATERGIC

DYSFUNCTIONS IN THE NEUROPATHOPHYSIOLOGY
OF SCHIZOPHRENIA
Wen-Jun Gao*
Department of Neurobiology and Anatomy, Drexel University College of Medicine,
Philadelphia, PA, US
ABSTRACT
Schizophrenia is a devastating mental disorder that affects almost 1% of the human
population. Although the etiology and fundamental pathological processes associated
with schizophrenia remain unclear, abnormalities of the dopamine and glutamate systems
have been implicated in their occurrence. The putative role of dopamine in the
pathophysiology and treatment of schizophrenia has been studied intensively in past
decades. The central importance of dopaminergic dysfunctions is clearly evidenced by
the efficacy of dopamine D2 receptor antagonists in the treatment of clinical symptoms.
Studies focusing on the dopamine system have purported the hypothesis of a dopamine
imbalance in mesocorticolimbic circuit, i.e., either excessive subcortical dopamine or
deficient prefrontal dopamine will cause schizophrenia symptoms. However, it is clear
that drugs specifically targeting dopamine receptors are not sufficient for the treatment of
schizophrenia. Novel approaches are urgently needed. Recent studies have proposed that
schizophrenia is closely associated with interacting abnormalities of both the dopamine
and glutamate systems. Indeed, besides dopamine, several lines of evidence support the
hypothesis of the persistent dysfunction of glutamatergic transmission, especially N-
methyl-D-aspartate (NMDA) receptors, in the pathogenesis of schizophrenia. In the past

decade, a number of studies have shown that the function of aberrant NMDA receptors
may underlie many aspects of molecular, cellular, and behavioral aberrations associated
with schizophrenia. We review the current literature and explain the onset and
pathogenesis of schizophrenia from basic and translational perspectives. Understanding
*
Correspondence: Wen-Jun Gao, Ph.D. Department of Neurobiology and Anatomy, Drexel University College of
Medicine, 2900 Queen Lane, Philadelphia, PA 19129; Phone: (215) 991-8907; Fax: (215) 843-9802. Email:
wgao@drexelmed.edu
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170 Wen-Jun Gao
the neurogenesis and neurobiological basis is important in the development of more

effective intervention strategies to treat or prevent this devastating disorder.
Keywords: Prefrontal cortex, dopamine, glutamate, NMDA receptors, psychiatric disorders,

schizophrenia
1. INTRODUCTION
Schizophrenia is a complex psychological disorder with neurological components first
described in 1887 as dementia praecox by German physician Emil Kraepelin. Schizophrenia
consists of a complex set of positive, negative, and cognitive symptoms. Positive symptoms
reflect an excess or distortion of normal functions. These active, abnormal symptoms include
delusions, hallucinations, thought disorder, and disorganized behavior. Negative symptoms
refer to a diminishment or absence of characteristics of normal function. They may appear
months or years before positive symptoms. They include loss of interest in everyday
activities, apparent lack of emotion, reduced ability to plan or carry out activities, neglect of
personal hygiene, social withdrawal, and loss of motivation. Cognitive symptoms, which
involve problems with thought processes, may be the most disabling in schizophrenia,
because they interfere with the ability to perform routine daily tasks. A person with
schizophrenia may be born with cognitive symptoms, but they may worsen when the disorder
starts. They include problems with making sense of information, difficulty paying attention,
and problems with memory. Schizophrenia can also affect mood, causing depression or mood
swings, that is, affective symptoms. In addition, people with schizophrenia often seem
inappropriate and odd, causing others to avoid them, which further leads to social isolation.
Given the serious nature of this disease, much research has been conducted over the past
century to better understand schizophrenia. One area of intense research involves
understanding how neuronal chemicals in the brain, such as dopamine and glutamate, impact
schizophrenia.
For more than 50 years, the dopamine hypothesis has dominated the theories of
schizophrenia. Hyperactivity in the mesolimbic dopamine pathway is specifically proposed as
the mediator of positive symptoms of schizophrenia. More recently, hypoactivity in the
mesocortical dopamine pathway is hypothesized to be the mediator of negative, cognitive,
and affective symptoms of schizophrenia. However, in the past two decades, hypotheses of
schizophrenia have progressed beyond the dopamine hypothesis of overactive mesolimbic
dopamine neurons and underactive mesocortical dopamine neurons. A major hypothesis of
schizophrenia proposes that numerous genetic risk factors converge on the N-methyl-D-
aspartate (NMDA) receptor for the neurotransmitter glutamate. Theoretically,
neurodevelopmental abnormalities in glutamate synapse formation result in the hypofunction

of NMDA receptors. Since NMDA receptors regulate dopamine neurons, the hypofunction of
NMDA receptors may be responsible for the abnormal dopamine activity associated with the
symptoms of schizophrenia. In this chapter, we review the current literature, elaborate the
formulations of hypotheses of schizophrenia, and explain what is known about the onset and
pathogenesis of schizophrenia from basic and translational perspectives.
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Dopaminergic and Glutamatergic Dysfunctions ... 171
2. THE DOPAMINE HYPOTHESIS

2.1. Evolution of Dopamine Hypothesis
The idea that dopamine and dopaminergic mechanisms are central to schizophrenia,
particularly psychosis, has been one of the most plausible and established hypothesis about
the illness. This hypothesis evolved from clinical observations, received empirical validation
from antipsychotic treatment, and gained support from direct imaging studies. Although the
dopamine hypothesis remains insufficient to clearly explain the complexity of this devastating
disorder, it offers a direct relationship to symptoms, particularly positive symptoms, and to
their treatment. Van Rossum in 1966 proposed the first dopamine hypothesis of
schizophrenia, i.e., hyperactivity of dopamine transmission was responsible for the disorder
[1]. This hypothesis was based largely on the observations that psychostimulants that increase
dopamine levels can activate dopamine receptors and cause psychosis, whereas antipsychotic
drugs that decrease dopamine levels can treat psychosis by blocking dopamine D2 receptors
[2]. Much of the evidence was derived from the original work of Arvid Carlsson, who
characterized the presence of dopamine in the brain and the effects of neuroleptics on
dopaminergic systems [3]. This classical dopamine hypothesis received further support from
the correlation between clinical doses of antipsychotic drugs and their potency to block D2
receptors [4, 5] and from the psychotogenic effects of dopamine-enhancing drugs [6-9].
Given the predominant localization of dopamine terminals and D2 receptors in subcortical
regions such as the striatum and the nucleus accumbens, this dopamine hypothesis of
schizophrenia mainly focused on subcortical regions.
Although this intriguing hypothesis was initially supported by a large body of evidence, it
was clear that dopamine metabolites, which reflect cortical dopamine metabolism, were not
universally elevated in the cerebrospinal fluid (CSF) or serum of patients with schizophrenia
[10]. In fact, dopamine metabolites were reduced in some patients with schizophrenia even
though the patients showed severe symptoms and responded to antipsychotic drugs. The focus
on D2 receptors was also brought into question by findings showing that clozapine had
superior efficacy for patients who were refractory to other antipsychotic drugs despite the fact
that clozapine has relatively low affinity and occupancy at D2 receptors [11-13]. Furthermore,
the postmortem studies of D2 receptors in patients with schizophrenia could not exclude the
confounding effects of previous antipsychotic treatment. Some positron emission tomography
(PET) studies of D2/3 receptors in drug-naive patients showed contradictory results [8].
Taken together, these findings were incompatible with the simple hypothesis of excess
subcortical dopamine transmission.
Furthermore, the importance of enduring negative and cognitive symptoms in this illness
and of their resistance to D2 receptor antagonism was becoming increasingly noteworthy.

This change in emphasis led to a reformulation of the classical or early version of the
dopamine hypothesis [7, 8, 14-16]. Many preclinical and clinical studies documented the
importance of prefrontal dopamine transmission at D1 receptors for optimal prefrontal cortex
performance [15, 17-21]. Functional brain imaging studies suggested that the cognitive and
negative symptoms might arise from altered prefrontal cortex functions [22]. Lesions of either
midbrain cell bodies in the ventral tegmental area or of frontal cortical areas directly, with
subsequent loss of dopamine terminals from the medial prefrontal cortex, induced
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172 Wen-Jun Gao
hyperactivity in rats and enhanced behavioral responses to amphetamine, whereas a lesion of

the subcortical dopamine pathways resulted in the opposite effects [23, 24]. These data
suggested that prefrontal dopamine activity exerts an inhibitory influence on subcortical
dopamine activity [23-27]. However, it was imaging studies, which showed reduced cerebral
blood flow in frontal cortex, that provided the best evidence of hypofrontality and regional
brain dysfunction in schizophrenia [28]. Because CSF dopamine metabolite levels reflected
cortical dopamine metabolism, the relationship between hypofrontality and low CSF
dopamine metabolite levels indirectly suggested a possible low frontal dopamine level. This
idea provided a mechanism for proposing that schizophrenia is characterized by frontal
hypodopaminergia that may cause striatal hyperdopaminergia.
Together, these observations led to the hypothesis that a deficit in dopamine transmission
at D1 receptors in the prefrontal cortex might be implicated in the cognitive impairments and
negative symptoms of schizophrenia [7, 29], whereas the excess dopamine transmission in the
striatum may be related to the positive symptoms. The major innovation of this hypothesis
was the move from a one-sided dopamine hypothesis explaining all facets of schizophrenia to
a regionally specific prefrontal hypodopaminergia and a subcortical hyperdopaminergia.
Consequently, an imbalance in dopamine with hyperactive subcortical mesolimbic
projections, causing hyperstimulation of D2 receptors and positive symptoms, and hypoactive
mesocortical dopamine projections to the prefrontal cortex, causing hypostimulation of D1
receptors, negative symptoms, and cognitive impairment, became the predominant hypothesis
in the past 20 years (Figure 1).
The main area of progress was the addition of regional specificity to the hypothesis to
account for the available postmortem and metabolite findings, imaging data, and new insights
from animal studies into cortical-subcortical interactions. Although it is a substantial advance,
the dopamine hypothesis has a number of weaknesses as well. Much of the evidence for the
hypothesis relied on the results from animal studies or other clinical conditions. There was
still no direct evidence of reduced dopamine levels in the frontal cortex and limited direct
evidence for elevated striatal dopaminergic function. In fact, recent evidence suggested that
the dopamine system may be "normal" in its configuration but instead is abnormally regulated
by modulatory processes [30]. It was also unclear how the dopaminergic abnormalities were
linked to the clinical phenomena. For example, there was no framework describing how
striatal hyperdopaminergia translates into delusions or how frontal hypodopaminergia results
in blunted affections. Furthermore, it has become clear that the cortical abnormalities are
more complicated than the proposed hypofrontality or cortical hypodopaminergia [31, 32], as
we discuss in Section 3 below. More importantly, this classical hypothesis ignored
neurodevelopmental and prodromal aspects of schizophrenia, did not describe the etiological
origins of the dopaminergic abnormality, did not pinpoint which element of dopaminergic
transmission was abnormal, and did not mechanistically address how cortical
hypodopaminergia and striatal hyperdopaminergia would occur.
2.2. Novel Evidence for an Integrated Hypothesis
Much has changed since the dopamine hypothesis was first formulated. Many critical
data, as exhibited below, have emerged to support a new integrated hypothesis for
schizophrenia, i.e., dopamine dysfunction as a final and common end point for the illness [8,
16].
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Figure 1. Dopamine imbalance hypothesis of schizophrenia. DA: dopamine, NAc: nucleus accumbens,
PFC: prefrontal cortex, VTA: ventral tegmental area.
2.2.1. Neurochemical Imaging of Schizophrenia

Although it is not possible to measure dopamine levels directly in human brains,
techniques have been developed to provide indirect indices of dopamine synthesis, release,
and putative synaptic dopamine levels. Presynaptic striatal dopaminergic function can be
measured using radiolabeled L-dopa, which is converted to dopamine and trapped in
dopamine nerve terminals. Using this technique, most of the studies conducted in patients
with schizophrenia have reported significantly elevated presynaptic striatal dopamine
synthesis capacity [33-40]. Evidence in line with this comes from a SPECT study using a
dopamine depletion technique by which it is reported that baseline occupancy of D2 receptors
by dopamine in the striatum is increased in schizophrenia [41-47]. In addition, by using
various radiotracers, PET and SPECT studies have provided imaging of dopamine D2/3
receptors in patients with schizophrenia, albeit the findings were inconsistent. Some reported
increased D2/3 receptor binding in the striatum [48-50], whereas others found no difference
from controls [51, 52] or a possible decrease in D2/D3 receptors in extrastriatal areas such as
the thalamus and anterior cingulate [53-56]. Further studies indicated that the D2 receptor
exists in two states, i.e., high and low, and that D2-high was particularly altered in patients
with schizophrenia [57-61]. Thus, dopamine D2 receptors continue to dominate and remain
necessary for antipsychotic treatment, at least until a nondopaminergic antipsychotic drug is
discovered [62, 63].
2.2.2. The Genetic Etiology of Schizophrenia

After many years of intensive investigations, it is unfortunate that no single gene was
found to be able to encode for schizophrenia. Rather, a number of high-risk genes are
associated with schizophrenia [64]. In fact, 4 of the top 10 gene variants most strongly
associated with schizophrenia are directly involved in dopaminergic pathways, including the
catechol-o-methyltransferase gene (COMT) [14, 65-70], neuregulin 1 (NRG1) [71, 72],
DISC1 [73, 74], and dysbindin [75-78]. Many of the other gene variants in the top list are
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involved in brain development, such as reelin, or influence more ubiquitous brain transmitters
such as glutamate or γ-aminobutyric acid (GABA) [64, 78-83]. Although recent findings have
generated great interest in the copy number variations in schizophrenia, they are rare and are
unlikely to account for the majority of cases of schizophrenia. Instead, most of them are likely
to be susceptibilities [64, 84, 85]. Of the ones that have been identified, some have already
been tied to altered dopamine transmission, but the functional relevance of most of them to
dopamine function is not known [86].
2.2.3. Environmental or Epigenetic Risk Factors for Schizophrenia

A large number of disparate environmental factors contribute to the risk for
schizophrenia. Markers of social adversity such as migration, unemployment, urban
upbringing, lack of close friends, and childhood abuse are all associated with a well-
established increased risk for schizophrenia that cannot readily be explained by genetic
factors [87-89]. Studies in animals of social isolation [90-92] and subordination [90, 93] find
that these factors lead to dopaminergic hyperactivity. Other environmental factors, such as
pregnancy/obstetric complications, act in early life to increase the subsequent risk of
schizophrenia [94-96]. There is now substantial evidence from animal models that pre- and
perinatal factors can lead to long-term hyperactivity in mesostriatal dopaminergic function
[30, 97-99]. For example, neonatal lesion of the hippocampus [100-102] or frontal cortex
[103, 104] increases dopamine-mediated behavioral responses in rats [105-108]. Neonatal
exposure to toxins also leads to increased dopamine-mediated behavioral responses [109] and
elevated striatal dopamine release [110]. Prenatal and neonatal stress, such as maternal
separation, also increases striatal dopamine metabolism [103] and release [111, 112].
A number of psychoactive substances also increase the risk of schizophrenia. The
relationship between psychostimulants and psychosis and their effects on dopaminergic
function has long been emphasized [113, 114]. For example, cannabis use has recently
emerged as a risk factor for schizophrenia [115, 116]. The main psychoactive component of
cannabis has been shown to increase striatal dopamine release via activation of cannabinoid
receptors [117-120]. Psychoactive drugs acting on other systems may also act indirectly on
the dopaminergic system by potentiating dopamine release. For example, the NMDA receptor
blocker ketamine, as well as phencyclidine (PCP) and dizocilpine (MK-801), was found to
increase amphetamine-induced dopamine release in healthy humans to the levels seen in
patients with schizophrenia [121-125].
2.2.4. Prodrome to Psychosis: Schizophrenia as a Neurodevelopmental Disorder

It is increasingly recognized that schizophrenia is a neurodevelopmental disorder that
involves disrupted alterations in brain circuits [29, 126, 127]. Although psychosis always
emerges in late adolescence or early adulthood, we still do not understand all of the changes
in normal or abnormal development prior to and during this period. It is particularly unclear
what factors alter the excitatory-inhibitory synaptic balance in the juvenile and what changes
induce the onset of cognitive dysfunction. Current studies suggest that problems related to
schizophrenia are evident much earlier. The emerging picture from genetic and epigenetic
studies indicates that early brain development is affected. Many of the structural variants
associated with schizophrenia indicate that neurodevelopmental genes or epigenetic factors
are involved with neuronal development [73, 128-130]. These findings, although intriguing,
are limited in that they do not reveal the changes that occur before psychosis. At present, the
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diagnosis of schizophrenia is based primarily on the symptoms and signs of psychosis. It has
recently been proposed that schizophrenia may progress through four stages: from risk to
prodrome to psychosis to chronic disability [131]. Obviously, the key to preventing or
forestalling the disorder is to detect early stages of risk and prodrome. Therefore, the need is
urgent to identify novel biomarkers and new cognitive tools as well as subtle clinical features
for early diagnosis and treatment [131, 132].
Significant neurobiological research in recent years has focused on the early signs or
prodrome of the illness and on the subtle manifestations of symptoms within family members
and the population in general [133-135]. These groups are at increased risk of schizophrenia
but have not yet developed the illness. Evidence from studying these groups therefore has the
potential to provide information about the causal chain of events leading to the development
of schizophrenia. Individuals meeting clinical criteria for a high risk of psychosis are likely to
develop schizophrenia within the following few years [135]. Before psychosis appears, they
have already shown elevated striatal dopamine transmission that is positively associated with
symptom severity in patients with schizophrenia [38]. Elevated presynaptic striatal
dopaminergic function is also seen in other groups with an increased risk of developing
psychosis [136] and in the relatives of people with schizophrenia. They also show higher
dopamine metabolite levels in response to stressors than healthy controls [137] and an
association between greater changes in dopamine metabolite levels with higher levels of
psychotic like symptoms following stress [138]. Overall, these findings indicate that
dopaminergic abnormalities are not just seen in people who are frankly psychotic but are also
seen in people with risk factors for psychosis, who often have signs and symptoms, although
at a less severe level. Furthermore, stress in these individuals has been linked to both an
increase in these symptoms and an increase in dopaminergic indices [88].
2.2.5. Interactions between or among Risk Factors and a Common Dopamine Pathway
for Schizophrenia
Genes and environmental factors, as discussed above, do not exist in isolation. Many
interact with each other, and some show synergistic effects on the risk of schizophrenia or
brain abnormalities associated with schizophrenia [14, 139-141]. Furthermore, animal studies
indicate that at least some of these factors interact to affect dopamine systems [142-144].
Interactions between gene variants, including those influencing dopaminergic function, and
environmental risk factors are another possible route to dopaminergic dysfunction. This idea
is illustrated by findings that variants of the COMT interact with early cannabis exposure to
increase the subsequent risk of psychosis [145] and, in other studies, to increase paranoid
reactions to stress [88]. Family history of psychosis also interacts with environmental factors
to increase the risk of schizophrenia [146, 147]. As reviewed above, animal studies indicate
that frontal dysfunction can increase striatal dopamine release. Animal studies, particularly
developmental models such as gestational exposure to methylazoxymethanol acetate (MAM)

and other high-risk genes (DISC1, NRG1, dysbindin, ), will certainly help to reveal the
neurodevelopmental trajectory of schizophrenia, yield disease mechanisms, and eventually
offer opportunities for the development of new treatments [30, 99, 148]. On the basis of
insights from animal research about normal brain development, it is proposed that the
appearance of diagnostic symptoms is linked to the normal maturation of brain areas affected
by the early developmental pathological processes [149-151]. The course of the illness and
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176 Wen-Jun Gao
the importance of stress and other risk factors may be related to normal maturation of
dopaminergic neural systems, particularly those innervating prefrontal cortex and striatum.
Although further work is clearly needed to investigate the nature and extent of all of these
possible interactions, the evidence indicates that many disparate, direct, and indirect
environmental and genetic factors may lead to dopamine dysfunction. As the dopamine
hypothesis evolves, the scientific challenge will be not just to find predisposing genes but also
to articulate how genes and environment interact to lead to dopamine dysfunction. In fact,
several recent studies of different high-risk genes for schizophrenia, including dysbindin,
DISC1, and NRG1, have consequently resulted in dopaminergic dysfunctions [71, 73, 75, 76],
although the exact mechanisms associated with these changes remain to be explored.
An attractive feature of the dopamine hypothesis is that it proposes a dysfunction in the
dopamine system as a complete explanation for schizophrenia: a prefrontal hypodopaminergia
leading to a subcortical hyperdopaminergia. However, there is little convincing evidence for
this sequence of events related to dopamine dysfunction. On the other hand, substantial
evidence indicates that multiple routes (genetic, neurodevelopmental, environmental, social)
lead to the striatal hyperdopaminergia, as discussed above. As a result, it has been proposed
that both dopamine imbalance models are mutually correlated because a deficiency in
mesocortical dopamine function may translate into disinhibition of mesolimbic dopamine
activity [29]. It should be noted that recent data suggest that these frontal/cognitive changes
need not necessarily be primary but instead may arise as a consequence of striatal dysfunction
[16, 152, 153]. Thus, in contrast to the classical dopamine hypothesis, recent advances
propose that changes in multiple transmitter/neural systems underlie the cognitive dysfunction
and negative symptoms of schizophrenia, and in many cases these dysfunctions precede the
onset of psychosis. It is when these pathways converge with other biological or environmental
influences and lead to striatal dopamine hyperfunction that psychosis becomes evident and
the diagnosis of schizophrenia is able to be confirmed.
Flowchart 1 Hypothesis of the pathophysiological process leading to schizophrenia

Etiology: multiple factors such as DNA, gene expression, viruses, toxins, birth injury, stress,
psychological experiences et al  Pathophysiology: brain development from conception to
early adulthood (e.g., neuron formation, synaptogenesis, synaptic pruning, activity-dependent
changes of enzymes, ion channels, and receptors)  Anatomical and functional disruption in
neuronal connectivity and communication  Dopaminergic dysfunction and impairment in
cognitive processes (e.g., attention, memory, emotion)  Symptoms of schizophrenia (e.g.,
cognitive, negative, positive, and affective symptoms).
In summary, as shown in Flowchart 1, multiple environmental and genetic risk factors

interact to evolve into a final common pathway of presynaptic D2 receptor-associated striatal
hyperdopaminergia. Furthermore, the pathway provides a framework linking the abnormal

neurochemical processes to symptoms and explains why many disparate risk factors and
functional and structural abnormalities are associated with schizophrenia. In addition to
guiding us through dopamine dysregulation, the multiple environmental and genetic risk
factors influence diagnosis by affecting other aspects of brain function that underlie negative
and cognitive symptoms. Schizophrenia is thus the result of dopamine dysregulation in the
context of a compromised brain.
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3. GLUTAMATE HYPOTHESIS OF SCHIZOPHRENIA

As discussed above, hypotheses of schizophrenia have progressed beyond the dopamine
hypothesis of overactive mesolimbic dopamine functions and underactive mesocortical
dopamine transmission. In a major breakthrough on the etiology of schizophrenia, it has been
proposed that numerous genetic risk factors, as discussed above, converge on the NMDA
receptors for the neurotransmitter glutamate. Theoretically, neurodevelopmental
abnormalities in glutamate synapse formation result in the hypofunction of NMDA receptors.
Indeed, genetic inactivation of NMDA receptors in ventral tegmental area dopamine neurons
results in specific behavioral modifications associated with drug-seeking behaviors and
significantly attenuates phasic dopamine release in the adult animals following the delivery of
a reward [154, 155]. Because NMDA receptors regulate dopamine neurons and dopamine
transmission, the hypofunction of NMDA receptors may be responsible for the abnormal
dopamine activity associated with the symptoms of schizophrenia.
3.1. The NMDA Receptor is Important for Cognitive Functions and Is

Subject to Dopaminergic Regulation
Glutamate receptors are classified into ionotropic and metabotropic receptors. The former
include NMDA, kainate, and AMPA subtypes whereas the metabotropic glutamate receptors
are composed of mGlu1-8 and modulate neurotransmission by activating G protein-coupled
synaptic transduction mechanisms. The NMDA receptor has long been associated with
learning and memory processes and with diseased states in psychiatric disorders [156]. The
NMDA receptor is a heteromeric complex formed by an assembly of subunits, with an
obligatory subunit NR1 and a combination of NR2A-D and/or NR3A-B subunits. It is
permeable to Ca2+ and is uniquely gated by both voltage and ligands (Figure 2A). The
biophysical and pharmacological properties of the NMDA receptor are greatly impacted by
the changes in NR2 and NR3 subunits and by binding sites of glycine, PCP, and Mg2+. It is
well known that the maturation of brain circuitry is usually coincident with the NMDA
receptor subunit switch that occurs at the onset of the critical period of cortical development
[157-160]. The differential regulation of NMDA receptor subtype expression with respect to
brain areas and cell types exerts an important function in the developmentally regulated
change of neuronal plasticity. The switch from 'young' to 'adult' forms of NMDA receptors
during corticolimbic development makes it extremely vulnerable to environmental or
epigenetic risk factors. The NMDA receptor subunit shift therefore marks the transition from
juvenile to “adult” neural processing in many brain regions [161, 162]. Multiple lines of
evidence suggest that the prepuberty period produces brain region-specific changes in NMDA
receptor activity and that NMDA receptor sensitivity to psychostimulants and stress reaches
the highest level [163, 164]. We propose that the normal expression of NMDA receptors in
the prefrontal and hippocampal neurons is critical for normal operation of cognitive functions.
Indeed, either overexpression or knockdown/knockout of individual NMDA receptor subunits
in the forebrain dramatically affects cognitive functions [160, 165-168].
Although the mechanisms involved in the switch of NR2B to NR2A remain unclear,
recent studies have provided interesting findings related to NMDA receptor subunit
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178 Wen-Jun Gao
trafficking, including phosphorylation, insertion (exocytosis), and internalization

(endocytosis). All of these processes are regulated by dopamine via activations of D1
stimulation in the striatal neurons [169-172]. Our recent data indicated that D1 activation
potentiates NMDA receptor-EPSCs by inserting NR2B subunits into the membrane surface in
the prefrontal neurons and that this action is PLCPKCSrc dependent [173, 174]. Because
NR2B subunits exhibit much higher surface mobility and endocytosis than other NMDA
receptor subunits [175-177], the higher proportion of NR2B expression in the prefrontal
neurons [178] makes the prefrontal synapses more plastic and vulnerable to detrimental
stimulation. Indeed, hyperdopamine stimulation initiates the switch by internalizing NR2B
subunits and thus affects neuronal communication through D2 receptor-mediated activation of
Akt-GSK-3β pathway in the prefrontal cortex [179]. In this context, the higher fraction of
NR2B appears to be a two-edged sword for the prefrontal neurons. On one hand, higher levels
of NR2B are needed for normal prefrontal functions [166] and for learning flexible behaviors;
on the other hand, neurons with more NR2B are more fragile and vulnerable to excess
glutamate release and dopamine stimulation [179-183] [173]. This intriguing hypothesis, yet
to be tested, seems to fit well with the vulnerability of prefrontal functions in the adolescent
brain. Indeed, increased levels of NR2B in the frontal cortex of juvenile mice affect anxiety-
and fear-related behaviors [184], whereas blockade of NR2B subunits in the cingulate cortex
impairs the formation of contextual memory [166].
Increasing evidence indicates that concurrent alterations of dopamine and NMDA
receptor function play a critical role in the pathophysiology of schizophrenia. The current
hypothesis postulates NMDA receptor hypofunction and cortical/subcortical dopamine
imbalance for the pathophysiology of schizophrenia [185]. However, evidence in support of
this hypothesis is still limited, and many of these speculations remain to be tested [127, 186].
Whether and how NMDA receptor hypofunction induces dysfunction of dopamine, or vice
versa, in the prefrontal cortex and other limbic brain regions remain to be tested.
3.2. Glutamatergic–GABAergic Interactions and NMDA Receptor

Hypofunction in Schizophrenia
In the past two decades, the abnormalities found in schizophrenia and the various models
of schizophrenia in humans and animals all point to an important contribution of the
glutamate and GABA system to the disease [187-189]. Accumulating studies have shown that
aberrant NMDA receptor function may underlie many aspects of molecular, cellular, and
behavioral abnormalities associated with schizophrenia [187, 190]. Recent evidence
increasingly supports the hypofunction of NMDA receptors in the limbic brain region in the
pathophysiology of schizophrenia [167, 187, 188, 190-194]. First, NMDA receptor
antagonists, such as PCP, MK-801, and ketamine, produce "schizophrenia like" symptoms in
healthy individuals [195-197]. Second, a majority of the genes that are associated with an
increased risk for schizophrenia can influence the function of NMDA receptors or related
receptor-interacting proteins and signal transduction pathways [14, 198]. Third, dysregulated
NMDA receptor subunits are usually seen in postmortem tissue from patients with
schizophrenia [199-201] and in animal models of NMDA receptor antagonism [202, 203].
Postmortem studies also show changes in glutamate receptor binding, transcription, and
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subunit protein expression in the prefrontal cortex, thalamus, and hippocampus of subjects
with schizophrenia [204]. These changes include decreased NR1, increased excitatory amino-
acid transporter, and altered NMDA receptor-affiliated intracellular proteins such as PSD95
and SAP102 in the prefrontal cortex and thalamus. Fourth, glutamatergic neurons also interact
with other neurons that have been strongly implicated in the pathophysiology of
schizophrenia, including morphologically altered GABAergic interneurons [205] and
antipsychotic drug-targeted dopamine neurons. Moreover, mice with reduced NMDA
receptor expression also display behaviors related to schizophrenia [167].
Figure 2. Hypothesis of NMDA receptor hypofunction. A, Schematic diagram of NMDA receptor

complex. B, NMDA receptor hypoactivity and glutamate neurotoxicity. PCP/MK801  NMDA
receptor hypofunction on GABAergic neurons  disinhibition of pyramidal neurons  more
glutamate release  AMPA/KA receptors excessively stimulated  excitotoxic damage.
On the basis of these observations, it has been postulated that the glutamatergic
disturbances may involve hypofunctioning of NMDA receptors on GABA interneurons [192,
202, 206, 207]. Compelling evidence has suggested that the NMDA receptor antagonist PCP
and its analogue compounds can produce a pattern of metabolic, neurochemical, and
behavioral changes that reproduce almost exactly those seen in patients with schizophrenia,
with remarkable regional specificity [208]. This finding has provided considerable insight
into the processes that lead to the development of the disease, emphasizing the potential
importance of NMDA receptor hypofunction. How might this be achieved? Activity in the
corticolimbothalamic circuit is strongly regulated by local GABAergic interneurons,
especially basket and chandelier cells. Output from the cortical pyramidal neurons is
suppressed and coordinated by GABAergic interneurons. These cells are activated by
recurrent collaterals from the pyramidal neurons and exert a powerful feedback inhibitory
action on pyramidal cells via synapses onto the soma and axon hillock (Figure 2B). Both
basket and chandelier cells are particularly important for restraining excessive pyramidal
neuron activity, which leads to dramatic disinhibition of the pyramidal neuron efferent
activity and elevated uncoordinated firing throughout the corticolimbic circuit (Figure 2B).
Considering the dysfunction of NMDA receptor subunits in patients with schizophrenia [200,
201, 204, 209-212], it has been speculated that NMDA receptor subunits, particularly the
NR2A subunit, distributed on interneurons may be responsible for NMDA receptor
hypofunction. The central pathological characteristics seem to be caused by NMDA receptor
hypofunction acting on GABAergic interneurons, followed by the disinhibition of
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180 Wen-Jun Gao
glutamatergic transmission and an overstimulation of non-NMDA receptors on pyramidal

neurons (Figure 2B) [192, 202, 206, 207].
The question is by what mechanism and when does the hypofunction of NMDA receptor
occur. It would be important to find out which neurons express altered glutamate receptor
subtypes, whether these neurons are inhibitory or excitatory, and how the circuitries are
affected. We propose a heuristic model for the pathophysiology of schizophrenia that
attempts to reconcile the neuropathological and neurocognitive features of the disorder. We
hypothesize that the hypofunction of the NMDA receptor on GABAergic interneurons
disrupts the functional integrity of the corticolimbic circuit, causing cognitive impairements
and negative symptoms. The resultant disinhibition of glutamatergic efferents and increased
glutamate release cause neurotoxicity and symptoms related to schizophrenia. The hypothesis
obviously depends on the differential NMDA receptor sensitivity to NMDA receptor
antagonists on the GABAergic interneurons. The result of these hypothesized mechanisms is
excessive stimulation of the glutamate receptor complexes. In turn, this excessive stimulation
could lead to disruption of ionic equilibrium across neuronal membranes, cell death, and other
mechanisms, particularly on the dopamine systems. The postulated existence of disinhibited
glutamatergic transmission and the subsequent cascade of excitotoxic events resulting from
NMDA receptor hypofunction, degeneration of GABAergic interneurons, or a combination of
both, have suggested diverse experimental therapeutic interventions for schizophrenia, such
as facilitation of NMDA receptor-mediated neurotransmission and potentiation of
GABAergic inhibition and antagonism of AMPARs [188, 213].
In summary, this review of previous studies has provided convincing evidence of
hypofunction of a subset of GABAergic interneurons and hypofunction of NMDA receptors
in the prefrontal cortex of patients with schizophrenia. Are these two separate and distinct
pathological mechanisms or can they be related to each other in some meaningful pattern?
Previous findings suggest a particular vulnerability of GABAergic interneurons to the effects
of NMDA receptor antagonists. On the basis of these findings, it is reasonable to speculate
that the NMDA receptors on frontal cortical and limbic GABAergic interneurons are most
sensitive to these antagonists and therefore may be an important site of pathology resulting in
NMDA receptor dysfunction. However, many questions remain to be answered. For example,
in the cortical circuitry consisting of both pyramidal cells and interneurons, why do PCP and
its analogue compounds selectively act on a subset of interneurons, i.e., parvalbumin-
containing, fast-spiking interneurons? Are NMDA receptor properties on interneurons
different from those on pyramidal cells? On the basis of the “disinhibition” hypothesis, does
PCP directly depress excitatory transmission between the pyramidal cell and the interneuron?
How does PCP affect the inhibitory transmission on pyramidal cells and what effect does it
have on different glutamatergic inputs such as corticocortical and thalamocortical synapses on
interneurons and pyramidal cells, respectively? Under the condition of hypothesized
hypofunctional NMDA receptors, such as subchronic or chronic PCP/MK-801 treatment, how

would the excessive release of glutamate lead to excitotoxicity in the interneurons? Would
dysfunction of NMDA receptors consequently result in dopamine dysfunction as proposed
above? Obviously, it is important to answer these questions as the first step, followed by
identification of their downstream signaling pathways [214] and their close relationships with
the possible involvement of dopamine D2 receptors. To address these questions, we recently
reported cell type-specific development of NR2 subunits in pyramidal neurons and
GABAergic interneurons of rat prefrontal cortex [178, 215]. NR2B levels remain high until
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adulthood, without significant NR2B-to-NR2A subunit switch, in layer 5 pyramidal neurons;

however, they are gradually replaced by NR2A subunits in fast-spiking interneurons [215].
These basic studies in NMDA receptor development in the prefrontal cortex have been
extremely useful in the formulation of an NMDA receptor hypofunction hypothesis.
As discussed above, we propose that dysfunction of NMDA receptors in the dopamine
neurons and GABAergic cells induces dopamine hyperactivity or GABA downregulation,
which in turn results in psychosis. Given that both dopamine and GABA systems are indeed
the targets of NMDA receptor disruption, our hypothesis is certainly plausible.
4. CONVERGENCE OF THE DOPAMINE HYPOTHESIS

WITH OTHER MAJOR HYPOTHESES IN SCHIZOPHRENIA
The original dopamine hypothesis of schizophrenia proposed that hyperactivity of

dopaminergic transmission leads to the symptoms of schizophrenia [3]. This hypothesis was
supported by the observation that all antipsychotic drugs blocked D2 receptors [4, 216].
Imaging studies provided more direct evidence for the dopamine hypothesis, suggesting
hyperfunction of the striatal dopamine system. These studies found increased uptake of L-
dopa, increased amphetamine-induced dopamine release, and increased occupancy and
density of D2 receptors in the striatum [8, 45, 49].
On the other hand, another deficit, a hypofunction of the prefrontal cortex, has been
associated with the cognitive symptoms. The nature of this hypofunction is unclear, but
hypofunctioning of both the dopamine system and the GABAergic system has been
postulated to account for this cognitive deficit [14, 127]. Although the evidence for
dopaminergic hypofunction in the prefrontal cortex is not yet substantial [15, 217-219],
several studies have found decreased expression of GABAergic markers in the cortex,
including the prefrontal cortex [205, 220-222]. These findings raise the important question:
Are cortical GABAergic hypofunction and subcortical dopaminergic hyperfunction related?
Indeed, they are. We recently found that overexpression of dopamine D2 receptors in the
striatum leads to decreased inhibitory GABAergic synaptic transmission and shifted
dopamine sensitivity in the mouse prefrontal cortex [223]. Furthermore, hypotheses of
schizophrenia have progressed beyond the dopamine hypothesis. An additional factor
supporting the evidence for dopamine dysregulation in schizophrenia is its plausibility in the
overall context of other transmitter systems that may be altered in schizophrenia, in particular
with the NMDA hypofunction hypothesis. More specifically, imaging studies have shown
that NMDA hypofunction can lead to dopamine alterations similar to those observed in
schizophrenia, namely, subcortical dopamine excess and cortical D1 upregulation [121, 224].
More recently, neurodevelopmental abnormalities in glutamate synapse formation have

resulted in the hypofunction of NMDA receptors, which in turn may result in abnormal
dopamine activity associated with psychosis and the symptoms of schizophrenia. Such
convergence suggests that the main neurochemical dysregulations described in schizophrenia
are not mutually exclusive. Glutamatergic and GABAergic alterations in schizophrenia could
be linked and could lead to or could be associated with inefficient control of cortical input
onto subcortical striatal dopamine and inefficient corticocortical connectivity and function.
Dopamine dysregulation may be the end point of a cascade of upstream events, an end point
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182 Wen-Jun Gao
for psychosis that is most directly associated with the major symptoms of the illness and their
treatment [57]. Therefore, future drug development should focus on the systems acting on the
signaling pathways or neurochemicals that lead to the final common dopamine pathway
instead of on the dopamine D2 receptor alone.
ACKNOWLEDGMENTS
This study was supported by grants R21MH232307 and R01MH232395 to W.-J Gao
from the National Institutes of Health.
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Dopamine Analysis in Neuroscience Research 97

the surface, a cross-linker (3) is commonly bound (glutaraldehyde, carbodiimide,

Figure 8. Polymeric immunoparticles with encapsulated antibodies.

6. DETECTION BASED ON MASS SPECTROMETRIC METHODS

AND COMPARISON WITH OTHERS

7. EXPERIMENTAL AND CLINICAL EXPERIENCE

Table 1. Limit of quantification (LOQ) for detection methods for Dapomine

Methods LOQ References

Measuring the changes in monoamine levels in discrete brain areas is currently

processes, in pathophysiology of various diseases and in investigations of drug actions on the

8. FUTURE PERSPECTIVE OF DOPAMINE MONITORING

Trimethylsilylation Derivatization and GC/MS Analysis. Analytical Chemistry, 82, 6447–

Kaluzna-Czaplinska, J.; Socha, E. & Rynkowski, J. (2010). Determination of homovanillic

serotonine in the presence of sodium ethylenediaminetetraacetic acid. Journal of

Barbara Pavan1* and Alessandro Dalpiaz2†

PHYSIOLOGICAL AND THERAPEUTICAL RELEVANCE

OF AC ISOFORMS IN THE CNS

The importance of AC in signal transduction of the CNS is highlighted by three special

TmACs contain 12 transmembrane helices arranged in two sets of six separated by a

EMBRYONIC STEM CELL NEURAL STEM CELLS ADULT STEM CELL

DOPAMINERGIC SYSTEM IN THE ADULT BRAIN

The neurotransmitter DA is a member of the catecholamine family and it is related to

PKA phosphorylates a number of proteins involved in signal transduction and regulation

CA2+- AND DA-RESPONSIVE ISOFORMS OF AC AS A PROSPECTIVE

(AC1) AC6 AC5

DOPAMINERGIC SYSTEM FAILURES

PD is one of the most common late-life neurodegenerative diseases, affecting

SCHIZOPHRENIA AS A PROTOTYPE OF DOPAMINERGIC OVERUSE

a consistent anxiolytic effect that was either increased or reduced by D1 agonists or

EXPERIMENTAL TOOLS AND THERAPEUTIC IMPLICATIONS

IN VITRO CELL LINES MODELS

DICTYOSTELIUM DISCOIDEUM: A POTENTIAL MODEL OF

D. discoideum could be furthermore functional to investigate the intriguing link between

DRUG-LIKE DIFFERENTIATING AGENTS:

MAKING DA NEURONS FROM NEURAL STEM CELL NICHE

CONCLUSION, INSIGHTS AND THE FUTURE

[6] Pavan, B; Biondi, C; Dalpiaz, A. Adenylyl cyclases as innovative therapeutics goals.

[37] Neve, KA; Seamans, JK; Trantham-Davidson, H. Dopamine receptor signaling. J.

[57] Komuro, H; Rakic, P. Orchestration of neuronal migration by activity of ion channels,

models of Parkinson's disease. Exp. Neurol., 2003, 184, 285–294.

[86] Kellendonk, C; Simpson, EH; Polan, HJ; Malleret, G; Vronskaya, S; Winiger, V;

MOLECULAR AND GENETIC BASIS OF THE

Shigeo Kitayama, Chiharu Sogawa and Norio Sogawa

regulation to the vulnerability of dopaminergic neurons to the putative toxicity of certain

1. CELL SURFACE EXPRESSION AND TRAFFICKING OF DAT

2. CELLULAR SIGNALING CASCADES REGULATING DAT EXPRESSION

Members of the nurotransmitter transporter family including DAT possess multiple

arginine abolished PKC-dependent internalization, suggesting that the ubiquitin moieties

2.3. PI3K, MAPK, other Protein Kinases

factor can mediate this type of rapid regulation.

system. This attractive hypothesis should be challenged in the future.

4. SYNTAXIN1A MODIFICATION OF DAT

5. SUBSTRATES AND INHIBITORS REGULATING DAT TRAFFICKING

5.1. Substrates Dopamine and Amphetamine

There is accumulating evidence indicating that substrates can produce down-regulation of

not required for amphetamine-induced down-regulation, while an increase in intracellular

6.  SYNYCLEIN, PARKIN AND GPR37 REGULATING

Parkinson‟s disease (PD) is a neurodegenerative disorder with characteristics of motor

Aggregation of -synuclein in protein inclusion, known as Lewy body, is characteristic

6.2. Parkin and Its Interaction with -Synuclein

Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to PD [81]. Although a

7. GENETIC BASIS OF THE DAT REGULATION

7.3. Transcriptional Regulation of DAT: Transcription Factors